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BIU
2
INTELLIGENCE UNITS
Tracking Genetically-Engineered
Microorganisms
BIOTECHNOLOGY
INTELLIGENCE
UNIT 2
Tracking Genetically-Engineered
Microorganisms
Janet K. Jansson
Department of Biochemistry
Stockholm University
Stockholm, Sweden
Mark J. Bailey
National Environment Research Council
Oxford, U.K.
LANDES BIOSCIENCE
GEORGETOWN, TEXAS
U.S.A.
EUREKAH.COM
AUSTIN, TEXAS
U.S.A.
ISBN: 1-58706-009-4
99-056991
CONTENTS
1. Problems in Detecting Dormant (VBNC) Cells, and the Role
of DNA Elements in This Response ......................................................... 1
James D. Oliver
1.1. The Viable but Nonculturable State ............................................. 1
1.2. Assays Used to Determine Viability .............................................. 2
1.3. Characteristics of Cells in the VBNC State ................................... 3
1.4. Inducing Factors ............................................................................ 4
1.5. Bacteria Known to Enter the VBNC State .................................... 5
1.6. Resuscitation from the VBNC State .............................................. 5
1.7. In situ Evidence of the VBNC Response ....................................... 7
1.8. Use of PCR to Detect Cells in the VBNC State ............................ 9
1.9. Effect of Plasmids on Entry into the VBNC State ........................ 9
1.10. Significance of the VBNC State in the Release
of Genetically Modified Bacteria ................................................. 12
1.11. Conclusion ................................................................................... 13
2. The Use of Antibiotic Resistance Gene Markers
for Studying Bacterial Populations in Natural Environments ............. 17
Sharon Egan and Elizabeth M.H. Wellington
2.1. Introduction ................................................................................. 17
2.2. Cultivation Based Assays ............................................................. 18
2.3. Direct Molecular Monitoring Methods ...................................... 20
2.4. Choice of Antibiotic Resistance Gene Markers .......................... 20
2.5. Cross-Resistance .......................................................................... 21
2.6. Marking of Bacteria with Antibiotic Resistance Genes .............. 21
2.7. Use of Antibiotic Resistance Genes
to Monitor Gene Transfer in Soil ................................................ 22
2.8. Ethical Concerns .......................................................................... 23
2.9. Conclusion ................................................................................... 24
3. Extraction and Analysis of Microbial Community Nucleic Acids
from Environmental Matrices ................................................................ 29
Jan Dirk van Elsas, Kornelia Smalla and Christoph C. Tebbe
3.1. Introduction ................................................................................. 29
3.2. Extraction of Microbial Cells from Environmental Matrices .... 33
3.3. Cell Disruption ............................................................................. 35
3.4. Extraction and Purification of DNA ........................................... 36
3.5. Analysis of DNA and Detection of Specific Sequences .............. 38
3.6. Extraction and Purification of RNA ........................................... 39
3.7. Detection of RNA ......................................................................... 40
3.8. Quantification of Specific Targets
in Environmental Nucleic Acids ................................................. 42
3.9. Conclusion ................................................................................... 44
EDITORS
Janet K. Jansson
Department of Biochemistry
Stockholm University
Stockholm, Sweden
Chapter 7
Jan Dirk van Elsas
Research Institute for Plant Protection
Wageningen, The Netherlands
Chapter 3
Mark J. Bailey
National Environment Research Council
Institute of Virology and Environmental Microbiology
Oxford, U.K.
Chapter 8
CONTRIBUTORS
A. Vande Broek
F.A. Janssens Laboratory of Genetics
Kardinaal Mercierlaan 92
Heverlee, Belgium
Chapter 6
Sharon Egan
Department of Biological Sciences
University of Warwick
Coventry, U.K.
Chapter 2
F.J. de Bruijn
MSU-DOE Plant Research Laboratory
Department of Microbiology
NSF Center for Microbial Ecology
Michigan State University
East Lansing, Michigan, U.S.A.
Chapter 7
R.J. Ellis
Molecular Microbial Ecology
Laboratory
NERC, Institute of Virology
and Environmental Microbiology
Oxford, U.K.
Chapter 8
Milena Carlot
Dipartimento di Biotecnologie Agrarie
Universit di Padova
Legnaro, Italy
Chapter 10
Alessio Giacomini
Dipartimento di Biotecnologie Agrarie
Universit di Padova
Legnaro, Italy
Chapter 10
Viviana Corich
Dipartimento di Biotecnologie Agrarie
Universit di Padova
Legnaro, Italy
Chapter 10
Kersti Gustafsson
Microbiologist/Ecotoxicologist
National Chemicals Inspectorate
Solna, Sweden
Chapter 12
Philip J. Hill
School of Biological Sciences
University of Nottingham
Sutton Bonington Campus
Sutton Bonington, Leicestershire, U.K.
Chapter 5
Marco P. Nuti
Dipartimento di Chimicae
Biotecnologie Agrarie
Universit di Pisa
Pisa, Italy
Chapter 10
Penny R. Hirsch
Soil Science Department
IACR-Rothamsted
Harpenden, U.K.
Chapter 11
James D. Oliver
Professor of Biology
Director, Interdisciplinary
Biotechnology Program
University of North Carolina
Charlotte, North Carolina, U.S.A.
Chapter 1
Matti T. Karp
Department of Biotechnology
University of Turku
Tykistokatu
Turku, Finland
Chapter 5
M. Lambrecht
F.A. Janssens Laboratory of Genetics
Kardinaal Mercierlaan 92
Heverlee, Belgium
Chapter 6
A.K. Lilley
Molecular Microbial Ecology
Laboratory
NERC, Institute of Virology
and Environmental Microbiology
Oxford, U.K.
Chapter 8
Kristina Lindstrm
Department of Applied Chemistry
and Microbiology
Division of Microbiology
Viikki Biocenter
University of Helsinki
Helsinki, Finland
Chapter 4
Tom A. Mendum
Soil Science Department
IACR-Rothamsted
Harpenden, U.K.
Chapter 11
Antonio J. Palomares
Departamento de Microbiologia y
Parasitologia
Universidad de Sevilla
Sevilla, Spain
Chapter 5
James I. Prosser
Department of Molecular
and Cell Biology
University of Aberdeen
Institute of Medical Sciences,
Foresterhill
Aberdeen, Scotland
Chapter 5
Alfred Phler
Department of Genetics
University of Bielefeld
Bielefeld, Germany
Chapter 11
Werner Selbitschka
Department of Genetics
University of Bielefeld
Bielefeld, Germany
Chapter 11
Kornelia Smalla
Institut fuer Biochemie und
Pflanzenvirologie, Biologische
Bundesanstalt
Braunschweig, Germany
Chapter 3
Andrea Squartini
Dipartimento di Biotecnologie Agrarie
Universit di Padova
Legnaro, Italy
Chapter 10
J.R. Stoltzfus
MSU-DOE Plant Research Laboratory
Department of Microbiology
NSF Center for Microbial Ecology
Michigan State University
East Lansing, Michigan, U.S.A.
Chapter 7
va Tas
Department of Biosciences
Division of Genetics
Viikki Biocenter
University of Helsinki
Helsinki, Finland
Chapter 4
Christoph C. Tebbe
Institut fuer Agrarecologie, FAL
Braunschweig, Germany
Chapters 3, 9
Ian P. Thompson
Molecular Microbial Ecology
Laboratory
NERC, Institute of Virology
and Environmental Microbiology
Oxford, U.K.
Chapter 8
Jos Vanderleyden
F.A. Janssens Laboratory of Genetics
Kardinaal Mercierlaan 92
Heverlee, Belgium
Chapter 6
Elena Vendramin
Dipartimento di Biotecnologie Agrarie
Universit di Padova
Legnaro, Italy
Chapter 10
Patrizia Vian
Dipartimento di Biotecnologie Agrarie
Universit di Padova
Legnaro, Italy
Chapter 10
Elizabeth M.H. Wellington
Department of Biological Sciences
University of Warwick
Coventry, U.K.
Chapter 2
Tracey M. Timms-Wilson
Molecular Microbial Ecology
Laboratory
NERC, Institute of Virology
and Environmental Microbiology
Oxford, U.K.
Chapter 8
PREFACE
nvironmental microbiology is currently one of the most rapidly expanding areas of scientific research. Impetus for advanced investigations has been provided by the development and application of molecular techniques that facilitate the identification, characterization and
monitoring of microbes. These advances now allow detailed investigations, developed in the laboratory, to be undertaken in the natural environment. Such studies confirm the remarkable biological diversity
represented by microorganisms from their basic genetic structure to the
regulated communications that occur within and between communities
contributing to ecosystem function. However, while it is apparent that
microorganisms constitute the greater part of the planets biomass, and
are central in maintaining the biosphere, we remain essentially ignorant
of a great majority of the functions or processes they undertake. One of
the limiting factors in the study of their ecology, even within communities or populations, is that of scale. For instance, for soil it is very hard to
assess microbes and their activities at the level of each individual pore
where microbial soil inhabitants occur. Highly sensitive and specific tools
are, thus, required for such detailed investigations. However, there is a
paradox. Until a greater knowledge of the genetic and metabolic diversity
within the microbial environment is obtained it remains difficult to
investigate these complex communities or design relevant experiments
that target the role of individuals or specific genes and their products.
This situation is currently changing at an ever increasing rate. In this volume we have attempted to bring together a series of reviews of the approaches taken to study the ecology and functional activity of individual
microbial cells and populations in environmental habitats, with a special
focus on the use of marker/reporter genes for monitoring release strains.
Traditionally, microbial ecology has been limited to studies of
microbial processes, such as cycling of nitrogen or carbon, occurring by
uncharacterized species in a black box scenario. For example, denitrification, nitrification and nitrogen fixation processes have been assessed by
analyzing the specific nitrogen forms appearing as a result of these processes. On the other hand, the study of particular taxa was limited to those
for which suitable cultivation methods and laboratory growth media had
been devised. However, we now know that the majority of microorganisms in nature are not capable of growing on the available media under
standard laboratory conditions. Moreover, in some instances bacteria, that
have been successfully cultivated in the laboratory, may lose the ability to
grow on laboratory media after introduction into the environment, presumably as a result of a stress response. These bacteria may still be viable
or metabolically active in the environment and this apparently recalcitrant state must be considered when these organisms are studied. The
We intend this book to be of relevance for all those concerned with studies of the environmental fate of genetically modified or unmodified microorganisms. In particular, this volume will be of value to researchers developing
organisms intended for release, and to representatives of regulatory agencies
concerned with guiding experimental or commercial applications. This volume provides an up-to-date collection of data on the development, use and
assessment of biomarkers and bioreporters for the study of bacterial function
in the natural environment.
We would like to extend our thanks to all the authors for providing the
necessary text for this publication, and for the patience and understanding they
have shown during the editing process. We would also like to thank the editorial staff for their support.
Janet K. Jansson
Jan Dirk van Elsas
Mark J. Bailey
CHAPTER 1
icrobial ecologists have long recognized that large proportions of microbial populations inhabiting natural habitats appear to be nonculturable. Indeed, plate counts of
bacteria in soil, rivers and oceans typically indicate that far less than 1% of the total bacteria
observed by direct microscopic examination can be grown on culture media. It has also
long been known that certain portions of bacterial populations in natural environments
seem to disappear during certain seasons, only to reappear at other times. We now
understand that at least part of the explanation for these observations is not due to seasonal
die-off of the cells, but to their entry into what is most commonly called the viable but
nonculturable state.1
A bacterial cell in the viable but nonculturable (VBNC) state may be defined as one
which fails to grow on the routine bacteriological media on which it would normally grow
and develop into a colony, but which is in fact alive and metabolically active. Bacteria enter
into this dormant state in response to one or more environmental stresses which might
otherwise be ultimately lethal to the cell. Thus, the VBNC state should be considered a
means of cell survival. Eventually, when the inducing stress is removed, these cells are able
to emerge from the VBNC state, and again become culturable on routine media.
The typical VBNC response is illustrated in Figure 1.1, which shows the response of the
human pathogen, V. vulnificus, to exposure to low temperature (5C). Such a temperature is
below that at which this aquatic bacterium can grow and, if it were not for the VBNC
response, is a temperature which would eventually lead to death of the population.
As is evident from Figure 1.1, cells lose their ability to be cultured (shown by the open
squares) in a rather linear manner, eventually reaching a point where platings suggest a total
lack of any living cells. However, whereas death of a bacterial population generally leads to
lysis of the cells and loss of cell structure, direct examination of cells entering the VBNC
state indicates that the cells remain intact (as shown by the open circles of Fig. 1.1). Such
cells could, of course, have died, but simply not undergone lysis. The primary evidence that
such cells are alive, even if nonculturable, is from data obtained when one of the direct
viability assays is applied to such cultures. These assays (described below) allow the direct
Tracking Genetically-Engineered Microorganisms, edited by Janet K. Jansson, Jan Dirk van Elsas,
Mark J. Bailey. 2000 EUREKAH.COM.
Fig. 1.1. Entry of V. vulnificus into the VBNC state in an artificial sea water microcosm at 5oC.
Shown are plate counts () on HI agar in cfu/ml, total cell counts () by the acridine orange
staining method, and direct viable counts () by the substrate responsive method using yeast
extract and nalidixic acid. Reprinted with permission from: Whitesides MD, Oliver JD. Appl
Environ Microbiol 1997; 63:1002-1005.
determination of the viability of individual cells in a population, without the need for culture. As seen in Figure 1.1 (open circles), such assays often indicate that a large proportion
of the apparently dead population is indeed alive.
Fig. 1.2. Elongation of viable cells following addition of yeast extract and nalidixic acid by the
method of Kogure et al.2 Nonviable cells remain as small, coccoid cells.
Fig. 1.3. Macromolecular synthesis in V. vulnificus during entry into the VBNC state at 5C. Cells
were assayed for protein ( ), DNA ( ), and RNA () synthesis. Reprinted with permission
from: Oliver JD. In: Kjelleberg S, ed. Starvation in Bacteria. New York: Plenum Press 1993:239-272.
Cell wall synthesis, or at least metabolism of the constituents of these structures, also apparently continues, as addition of penicillin (an inhibitor of cell wall synthesis) to VBNC cells
has generally been observed to result in rapid cell death.1
Most studies have observed that, if a cell entering the VBNC state harbors plasmids
(extrachromosomal DNA elements which are able to control a variety of generally nonessential cell functions), then these plasmids are retained. This finding may prove to be highly
relevant to the VBNC state of genetically modified cells released to the environment, as will
be discussed later in this chapter. In contrast, it is becoming increasingly apparent that (possibly even major) changes in the cells chromosomal DNA may be occurring as cells enter
the VBNC state.1 This aspect may also be critical to release studies, as these changes bring
into question the ability to employ such powerful molecular techniques as the polymerase
chain reaction (PCR) to detect these otherwise undetectable cells. This concern is also dealt
with later in this chapter.
The time required for cells to enter the VBNC state varies markedly with the bacterium
and the inducing conditions. Reports of months being required are not uncommon, while
others have reported days for the same bacteria. In general, times of a few days to a month
seem typical. One factor which has been shown to have a dramatic effect on the time
required for lab-grown cells to become nonculturable is the age of the cells. We have shown
that, whereas V. vulnificus cells from the logarithmic phase of growth generally require less
than 10 days to become completely nonculturable at 5C, those taken from the stationary
phase require over a month. Indeed, a direct correlation between time required to become
nonculturable and the age of the population has been demonstrated.11
Salmonella enteritidis
S. typhimurium
Shigella dysenteriae
S. flexneri
S. sonnei
Vibrio anguillarum
V. campbellii
V. cholerae
V. fischeri
V. harveyi
V. mimicus
V. natriegens
V. parahaemolyticus
V. proteolytica
V. vulnificus (biotypes 1 and 2)
Fig. 1.4. Changes in culturable cell (plate) counts and cell morphology during temperature downshift to 5C and subsequent resuscitation of the nonculturable cells by incubation at ca. 22C.
Reprinted with permission from: Nilsson L et al. J Bacteriol 1991; 173:5054-5059.
While entry into a VBNC state has been described by many researchers and for many
different bacterial species, demonstrating resuscitation for these cells has not always been a
simple matter. Indeed, while some bacteria like V. vulnificus can be resuscitated by a simple
reversal of the inducing stress, in others it has been quite difficult to show. We now realize
that resuscitation may be an extremely complex event, one which may be quite difficult to
demonstrate in the lab. In the case of L. pneumophila, simple addition of nutrients to the
cells (which enter the VBNC state in response to nutrient deprivation) does not reverse the
dormancy. It was found, however, that the addition of certain amoebae, which are natural
hosts to this bacterium in the aquatic environment, does allow resuscitation of this causative agent of Legionnaires disease.14
Other problems also exist in demonstrating resuscitation from the VBNC state. It has
been difficult to overcome the argument that what was being observed in the name of resuscitation was, in fact, regrowth of a few culturable cells which had escaped detection
during plating of the population under study. However, we have recently presented very
strong evidence that, at least in the case of V. vulnificus, true resuscitation does occur.15 Our
studies employed extensively diluted populations of VBNC cells in which it was statistically
impossible that any culturable cells were present. Resuscitation of these populations occurred at such a rapid rate that, if it were due to regrowth of culturable cells, they would
have to have had a doubling time of approximately 6 minutes. This is clearly an impossible
generation time for cells incubated at a suboptimal temperature without nutrients or aeration (Fig. 1.5). We also observed that the presence of nutrients appears to inhibit (but not
kill) VBNC cells, and this may be the reason such cells are nonculturable when plated onto
the high organic nutrient media routinely employed for bacterial culture.
Fig. 1.5. Time required for resuscitation of VBNC V. vulnificus cells. Cells from a VBNC microcosm (< 3.3 x 101 cfu/ml) were shifted to room temperature and aliquots removed at hourly
intervals and plated onto HI agar. Reprinted with permission from: Whitesides MD, Oliver JD.
Appl Environ Microbiol 1997; 63:1002-1005.
Fig. 1.6. Entry into (Fig. 1.6A) and resuscitation from (Fig. 1.6B) the VBNC state by V. vulnificus
placed into membrane diffusion chambers in estuarine waters of the coast of North Carolina. A.
Cells were placed into water at a temperature of 10-15C. Shown are plate counts (), total
direct counts (), and direct viability assays (). B. Plate counts of both the encapsulated ()
and non-encapsulated () forms of V. vulnificus induced into the VBNC state in the laboratory
at 5C. Four days after entry into the VBNC state (day 11), resuscitation of the cells is seen when
placed into estuarine waters at a temperature of 16-19C. Reprinted with permission from: Oliver
JD, Hite MF, McDougald D et al. Appl Environ Microbiol 1995; 61:2624-2630.
10
Fig. 1.7. Randomly amplified polymorphic (RAPD) DNA analysis of V. vulnificus cells induced
into the VBNC state by incubation at 5C. Lanes 1, 5, 9, 11, and 12 contain a 123 bp ladder. Lanes
2-4 contain cells entering the VBNC state at 0, 1, and 2 days; lanes 6-8 contain cells entering the
VBNC state at 3, 4, and 5 days; lane 10 contains cells entering the VBNC state at 7 days.
Reprinted with permission from: Warner JM, Oliver JD. Appl Environ Microbiol 1998;
64:3025-3028.
Fig. 1.8. (opposite) Effect of plasmid FAC510 carriage in P. fluorescens on culturability in river
water at different temperatures. The same P. fluorescens strain was without the plasmid (, parent), with the plasmid (, plasmid), or with the entire plasmid inserted into the chromosome
(, chromosome). Cells incubated at 5C (A) or at 37C (B). Plate counts were performed on
L agar. Reprinted with permission from: Oliver JD, McDougald D, Barrett T et al. FEMS Microbiol
Ecol 1995; 17:229-238.
11
12
Fig. 1.9. P. fluorescens placed into soil microcosms at 5(), 25(), and 37C (). Shown are
plate counts for each temperature. S. Bunker and J.D. Oliver (unpublished).
13
nutrient deprivation, may also be important for the induction of the VBNC state in
pseudomonads. Wilson and Lindow17 have shown that cells of P. syringae which are actively
growing on leaves or which were only recently inoculated to leaf surfaces remain fully
culturable. However, their study showed that culturable counts of such cells, after 80 hours
of epiphytic growth under constant environmental conditions, were two- to four-fold less
than direct viable counts, indicating that up to 75% of the population had entered the VBNC
state. As these authors stated, This finding is particularly relevant to the monitoring and
detection of GEMS [genetically engineered microorganisms] in terrestrial ecosystems,
because if part of an epiphytic population has become metabolically inactive . . . the viable
bacterial population size will be underestimated by the plate count method. The plate count
may indicate that a GEM has disappeared from the ecosystem, whereas actually, the GEM is
still present in low numbers in a viable but nonculturable, quiescent state.
The presence of bacterial cells in the VBNC state presents a major new dilemma in that
the ability to monitor the persistence and spread of released GMOs is critical. The routine
methods (primarily platings) for detecting bacteria in natural environments can not be
employed for cells in the VBNC state. It also appears from our studies that alternate methods to detect such cells, such as PCR amplification, may not be practical alternatives. It must
also be asked whether cells in this state are able to either donate or take up genetic material
(plasmid or chromosomal DNA) to/from other bacteria which are present as part of the
normal microflora. If such gene exchange is possible (although this has not been shown to
date), then the problem is compounded, as not only would these GMOs be undetectable,
but they might take part in the development of previously non-existent strains of bacteria.
Most studies to date on VBNC cells from a variety of genera suggest that plasmids are not
lost during entry into this state, suggesting that the acquisition of any new genes would
likely take place in an otherwise relatively stable genetic environment.
To conclude on a more positive note, our recent studies using certain genetically-tagged
bacteria suggest some hope in our ability to detect bacteria present in natural environments
in the VBNC state. The green fluorescent protein (Chapter 7) is an extremely stable protein which is coded for by the gfp gene present in certain marine jellyfish, and which fluoresces when irradiated with ultraviolet light. The use of this protein is currently finding
considerable interest as a marker, following insertion of the gene into the DNA of bacteria
intended for environmental release. In collaboration with Dr. Janet Jansson and researchers
in her laboratory at the University of Stockholm, we have found that cells producing this
fluorescent protein continue to fluoresce even when induced into the VBNC state. Indeed,
as seen in Fig. 1.10, not only does a gfp-labeled strain of P. fluorescens continue to fluoresce
as it becomes nonculturable, but it appears to fluoresce brighter than logarithmic phase
(actively metabolizing) cells of the same gfp-labeled strain. If these results are corroborated
in the field, then the use of this marker for monitoring released cells may be of significant
value in providing a means of identifying released bacteria even when they are no longer
culturable. Such field studies are currently in progress in the authors laboratory.
1.11. Conclusion
Since the pioneering studies from Colwells laboratory,24,25 it has become evident that
many gram-negative bacteria enter into a state of dormancy in response to one or more of
a variety of environmental stresses, including both low and high temperatures. VBNC cells
remain metabolically active, although they can not be cultured by standard methods. Cells
undergo a variety of morphological, physiological, and biochemical changes as they enter
this state, all of which may allow the cells to survive what could otherwise be lethal conditions. On removal of the inducing stress, these cells can resuscitate from the VBNC state,
again becoming metabolically active.
14
Fig. 1.10. Fluorescence due to the green fluorescent protein in P. fluorescens, as detected by flow
cytometry. Shown is the percent of control cell fluorescence of cells incubated at 5C, 30C, and
37.5C for up to 19d. Cells incubated at 37.5C were completely nonculturable by day 15 in this
study. M. Lowder and J.D. Oliver (unpublished).
Unfortunately, it appears that cells in the VBNC state may undergo changes in their
chromosomal DNA which prevents their detection through PCR amplification. Further, it
is possible that VBNC cells may participate in genetic exchange with bacteria making up the
normal microflora of a release environment. Such an event could involve gain or loss of
plasmids, an event having potentially dramatic consequences on the conditions inducing
the VBNC state. Whether these dormant cells can be genetically transformed or transduced
is not know at this time, but if so, such events could lead to significant genetic modifications
in these nonculturable cells.
Thus, the VBNC state presents a number of potential concerns to the researcher and
regulator alike, making detection of the introduced cells difficult, and presenting a situation
wherein gene exchange could potentially occur, yet be undetectable. Whether or not new
technologies such as the use of the gfp marker gene will at least provide a means for monitoring such cells is currently under investigation.
Acknowledgments
J.D. Oliver is a member of the MAREP Concerted Action sponsored by the European
Commission Biotechnology Programme, DGXII.
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24. Roszak DB, Colwell RR. Survival strategies of bacteria in the natural environment. Microbiol
Rev 1987; 51:365-379
25. Xu HS, Roberts NC, Adams LB et al. Survival and viability of nonculturable Escherichia
coli and Vibrio cholerae in the estuarine and marine environment. Microb Ecol 1984;
8:313-323.
CHAPTER 2
2.1. Introduction
ntibiotic resistance genes have been used to mark bacteria by providing a readily selectable phenotype, which can be detected using selective growth media. Detection and monitoring is therefore culture-dependent. A wide range of resistance genes have been
characterised (Tables 2.1 and 2.2) which confer resistance to commercially available, inexpensive antibiotics. In addition, resistance genes have provided a valuable tool for cloning
and genetic manipulation procedures in both prokaryotes and eukaryotes. Many industrial
strains carry resistance genes which have resulted from cloning procedures, enabling selective maintenance of a construct or plasmid carrying the resistance gene by inclusion of the
respective antibiotic in the growth medium (For details see ref. 1).
The main environmental application for antibiotic resistance gene markers has been
the monitoring of gene transfer associated with plasmid or transposon mobility. In addition, the tracking of bacterial populations in soil, water or other environments, and determination of the extent of plant root or shoot colonisation has relied on antibiotic resistance
phenotypes.2-4 A distinction can be made between the use of antibiotic resistant mutants
and the introduction of cloned resistance genes. The former have been used in field releases
where it was necessary to avoid releasing antibiotic resistance genes into the environment.2
The selection provided by antibiotics has provided a powerful tool for isolation of marked
bacteria and cultivation based assays have allowed quantitative estimates of cell numbers.
Resistance genes have also been detected using molecular methods such as PCR, and this
approach provides confirmation of the phenotype. Due to ergotropic and clinical use, some
resistance genes have become widely disseminated in the environment and are less useful as
selective markers. The choice of resistance gene is therefore important to avoid certain common genotypes. For field releases ethical considerations are important to avoid the use of
any resistance gene which may become disseminated to the indigenous bacterial community and thus ultimately contribute to the clinical problems of antibiotic resistant pathogens. The aim of this chapter is to provide information useful for the selection and exploitation of resistance genes as markers for detection and enumeration of bacteria in natural
environments.
Tracking Genetically-Engineered Microorganisms, edited by Janet K. Jansson, Jan Dirk van Elsas,
Mark J. Bailey. 2000 EUREKAH.COM.
18
Table 2.1. Some examples of common bacterial resistance genes and their mode of
action
Gene
Resistance phenotype
Host
Mechanism of resistance
aph(3)-Iva
nptII
aphII
tsr
strA
ant(2)-I
ant(6)-Ia
ant(9)-Ia
aac(3)-I
aac(6)-Ih
aac(3)-Ia
mgt
tetA/B/C
tet(M)
ampC
otrA/B
kanamycin, neomycin
kanamycin, neomycin
neomycin
thiostrepton
streptomycin
gentamicin, sisomicin
streptomycin
spectinomycin
gentamicin
sisomicin, tobramycin
gentamicin, sisomicin
oleandomycin
tetracycline
tetracycline
-lactams
oxytetracycline
Bacillus circulans
Enterobacter
Streptomyces lividans
S. lividans
Enterobacter
Pseudomonas
E. faecalis
Staphylococcus aureus
Pseudomonas
Acinetobacter spp.
E. coli
Streptomyces antibioticus
Enterobacter
Streptococcus pneumoniae
gram-negative bacteria
Streptomyces rimosus
Phosphorylation
Phosphorylation
Phosphorylation
Target modification
Phosphorylation
Nucleotidyltransferase
Adenylylation
Adenylylation
Acetylation
Acetylation
Acetylation
Glycosyltransferase
Efflux
Ribosomal modification
Hydrolysis
Ribosomal modification/efflux
19
Gene/
transposon
Host
Detection1
Reference
kanamycin
aph/ nptII
Enterobacter
agglomerans
SP, PCR
Selenska et al14
Escherichia
coli
SP, MPN-PCR
Recorbet et al15
kanamycin
nptII
kanamycin
SP, probing
kanamycin
SP
Orvos et al34
kanamycin
nptII (Tn5)
Azospirillum sp.
SP, MPN
probing
Bentjen et al35
kanamycin
nptII (Tn5)
P.fluorescens
SP, MPN-PCR
van Overbeek
et al32
neomycin
nptII (Tn5)
R.leguminosarum
SP, probing
neomycin
nptII (Tn5)
Rhizobia
SP, probing
Armager and
Delgutte44
erythromycin
erm
Bacillus
SP
Wendt/Potthof
et al55
kanamycin
nptII
P. fluorescens
SP
Smit and
van Elsas24
gentamicin
aadB
chloramphenicol cam
B. subtilis
SP
McDonald et al46
kanamycin
Tn5
E.agglomerans
/E.coli
SP
Klingmller50
neomycin,
thiostrepton
viomycin
aphII
tsr
vph
Streptomyces lividans SP
Herron et al6
thiostrepton
tsr
S. lividans
SP, probing
Marsh and
Wellington31
tetracycline
unknown
B. subtilis
SP
Amner et al21
neomycin
aphII
Streptomyces
violaceolatus/
S. lividans
SP
Wellington et al16
thiostrepton
viomycin
tsr
vph
20
to selective components in the medium such as antibiotics. Under nutrient limitation some
bacteria have been demonstrated to be less susceptible to antibiotics.10,11
21
2.5. Cross-Resistance
Resistance to the same antibiotic can be achieved by different genes coding for
enzymes with various modes of action or the same gene can confer resistance to more than
one antibiotic (Table 2.2). Resistance to paromomycin is conferred by either a
phosphotransferase or an acetyltransferase.27 Other resistance gene products can confer a
cross-resistance to different antibiotics, for example, Skeggs et al28 have shown that an RNA
methylase aminoglycoside resistance determinant can confer resistance to kanamycin,
apramycin and gentamicin. The aac resistance gene which encodes an acetyltransferase in
Streptomyces fradiae and the phosphotransferase-encoding nptII from Tn5 confer resistance
to both neomycin and kanamycin.9,29 This functional diversity in resistance genes means
that the same phenotype can be due to quite different genes. Unequivocal identification is
only possible with selective isolation followed by a molecular method to confirm the presence of the specific resistance marker.17,30-32
22
for neomycin resistance on isolation media. This phenotype was not found in native rhizobia, which made it a suitable marker, particularly in combination with the spontaneous
chromosomally located rifampicin resistance mutation.
Antibiotic resistance gene markers have been inserted on multiple and single copy plasmids for monitoring the fate of inoculants and gene transfer in natural environments. The
choice of chromosomal or plasmid location is dependent on the requirements of the study,
but gene dose effects with multicopy plasmids will greatly improve molecular detection of
both DNA and mRNA. In addition, enhanced phenotypic resistance from multiple copies
of the resistance gene marker will aid selection.9 Both naturally occurring R-plasmids and
constructed plasmids with one or multiple resistance genes are readily available. However,
R-plasmids are mainly found in gram-negative hosts and many will not be able to replicate
in gram positive backgrounds. Plasmids from gram-negative and gram positive bacteria
have very different conjugation systems and genes involved in plasmid transfer, replication
and maintenance are very diverse.36 For gram positive bacteria, resistance plasmids have
been constructed (see next section 2.7). For example, Wipat et al 37 used tsr to mark a
multicopy streptomycete plasmid which was then introduced into Streptomyces lividans via
transformation of protoplasts. This allowed the monitoring of survival and spread of S.
lividans in soil microcosms.37
Many resistance marker genes can function in different bacterial species; for example,
the nptII gene has been used in many different genera (Table 2.2), but other marker systems
are restricted to specific groups. The host range of the vector must also be considered.
Although the nptII gene occurs frequently in diverse bacteria, it is often associated with
different transposons (Table 2.2).
The choice of promoters for marker genes is also an important consideration. The
nptII gene from Tn5 is expressed in Streptomyces, but the level of kanamycin resistance varies from 2-200 mg/ml, depending on promoter strength and copy number of the plasmid.9
23
the rhizosphere of different plants and also failed to detect transfer of Tn5 to other rhizobia
and even to introduced potential recipients.
Other antibiotic resistance gene markers have been used to study plasmid transfer in
the natural environment and to examine the extent of conjugation in soil. Cresswell monitored the transfer of plasmid pIJ673 containing neomycin (aphII), viomycin (vph) and
thiostrepton (tsr) resistance genes. The plasmid was a derivative of pIJ101.23 Putative
transconjugants were selected by growth on neomycin plus thiostrepton-containing agar plates
and plasmid presence confirmed by colony hybridisation with a labeled plasmid as a probe.
This technique was used to show inter-specific plasmid transfer in Streptomyces strains in
both sterile and non-sterile soil batch microcosms.6,30 High copy number plasmids also provide an advantage by effectively increasing the copy number of the gene, which can enhance
detection by molecular methods. In a study by Smit and van Elsas24 a plasmid and a chromosomally-inserted transposon were stably maintained in Pseudomonas fluorescens R2f in
soil for 7 days and a broad host range plasmid (IncQ) could be mobilised into several indigenous bacterial species. McDonald et al45 examined the persistence of a plasmid encoding
chloramphenicol resistance in B. subtilis and reported that plasmid stability in the inoculant population remained at 100% after 28 days. Other studies have shown that engineered
plasmids are not stably maintained by the hosts, particularly when released into the environment.46,47 It could be interpreted that in these cases the burden of maintaining multicopy plasmids is greater than the advantage conferred on the host by the marker gene
phenotype.48
Examination of intrageneric plasmid transfer in soil has shown that Tn5 labeled plasmids in both E. agglomerans and E. coli could transfer to homologous strains in soil, and the
transfer frequency was enhanced by the input of particular nutrients into the system.49 Herron
et al6 used pIJ673 (containing nptII, vph and tsr) to monitor the selective effect of antibiotics
on the plasmid transfer and survival of Streptomyces in soil microcosms. This study demonstrated that addition of neomycin and thiostrepton to soil microcosms increased the levels of
resistant transconjugants from 103 to 105.
The population dynamics of bacteriophage and their hosts in soil has also been studied
by marking phage with antibiotic resistance genes. Marsh and Wellington31 examined the
lysogenic infection of indigenous soil Streptomyces species by using a bacteriophage containing a thiostrepton resistance gene to mark bacteriophage and then select for host resistance in soil microcosms. The addition of tsr marked KC301 actinophage lysates to nonsterile
soil gave rise to thiostrepton resistant indigenous streptomycetes which were subsequently
shown to contain KC301 DNA by colony blots. Probing digested chromosomal DNA of
some of the lysogens showed that their chromosomes contained KC301 prophage.
24
of antibiotic resistance genes in enteric bacteria in the environment and within pathogenic
groups.53 These data demonstrate the horizontal transfer of genes in natural populations
and their selection under conditions of antibiotic administration. Therefore, selection pressure is the key problem. Certain antibiotic resistance gene markers have been proposed for
environmental use due to the lack of clinical application of the antibiotic used for selection,
thus reducing the chance of resistance development. However, it is often uncertain which
chemical classes of antibiotic will prove to be clinically useful in the future and how far cross
resistance will affect the use of chemically similar antibiotics. In the case of the antibiotic
avoparcin, a glycopeptide fed to pigs and poultry, there was not sufficient difference compared to vancomycin, the clinically important glycopeptide, to prevent cross resistance developing.53 As already discussed in Section 2.6, some field releases have been approved which
used antibiotic resistance gene markers for tracking genetically modified micro-organisms
(GMMs) and included Tn5 and the kanamycin resistance gene aph(3')-II (see Chapter 8).
The risk of a gene transferring from GMMs into the environmental gene pool is presumably
greater than for a transgenic crop plant marked with an antibiotic resistance gene. The only
GMM-based product containing an antibiotic resistance gene for marketing within the European Union is a kit consisting of Streptococcus thermophilus modified with the cat
(chloramphenicol acetyltransferase) gene. The cat gene is located on a plasmid, pMJ723,
harbouring the lux genes, and detects antibiotic residues in milk. The strain is not released
into to the environment, as it is contained in vials throughout the test. For transgenic crop
plants, the marker antibiotic resistance genes aph(3')-II and blaTEM-1, for kanamycin/neomycin resistance and -lactam resistance respectively, have been approved for marketing within
Europe. Future research should address the levels of transfer of these genes to indigenous
micro-organisms, as plant DNA containing aph(3')-II is known to successfully transform
suitable, competent, recipient bacteria. 54
2.9. Conclusion
Antibiotic resistance genes have a long history of reliability for use as highly selective,
versatile markers allowing detection and enumeration of bacteria in environmental samples.
Resistance genes found in non-antibiotic producing bacteria are readily expressed in a wide
range of host backgrounds. Examples include the neomycin resistance gene, nptII, which has
been used in gram-positive (high and low GC) and gram-negative bacteria, yeasts, plant
and animal cell cultures.
The resistance genes are highly mobile between bacterial groups and this may be a
reason for their versatility and often near universal expression. A wide range of genes have
been cloned and frequently used with their own promoter in diverse hosts. Levels of expression and strength of resistance may vary slightly from strain to strain but this is also related
to the nature of the construct used. Multiple copies of a resistance gene secure high levels of
resistance but only under defined conditions of growth and assay. No other type of marker
provides such an extensive range of genes for use with almost any bacterium, resulting in a
highly selective marker. Some bacteria now carry an extensive range of antibiotic resistance
genes resulting from the excessive use of antibiotics for over 50 years and this may reduce the
choice of genes useful for marking a strain within a population of close relatives. The background resistance of indigenous bacterial populations may also be a problem where certain
phenotypes, such as kanamycin resistance, are widespread in soil bacteria due to the use of
antibiotics in agriculture. This problem can be overcome by combining unique sets of resistance genes or by combining resistance genes with markers such as lux or gfp (see Chapters 5-7).
25
Acknowledgments
E.M.H. Wellington is a member of the MAREP Concerted Action sponsored by the
European Commission Biotechnology Programme, DGXII.
References
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20. Amner W, McCarthy AJ, Edwards C. Quantitative assessment of factors affecting the recovery of indigenous and released thermophilic bacteria from compost. Appl Environ
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26. McNaughton SJ. Rose DA, ODonnell AG. Growth and survival of genetically modified
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27. Zalacain M, Gonzalez A, Guerrero MC et al. Nucleotide-sequence of the hygromycin-
phosphotransferase gene from Streptomyces hygroscopicus. Nucleic Ac Res 1986; 14:
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28. Skeggs PA, Holmes DJ, Cundliffe E. Cloning of aminoglycoside-resistance determinants
from Streptomyces tenebrarius and comparison with related genes from other actinomycetes.
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29. Shaw KJ, Rather PN, Hare RS et al. Molecular genetics of aminoglycoside resistance genes
and familial relationships of the aminoglycoside-modifying enzymes. Microbiol Rev 1993;
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30. Wellington EMH, Cresswell N, Saunders V. Growth and survival of streptomycete inoculants and extent of plasmid transfer in sterile and non-sterile soil. Appl Environ Microbiol
1990; 56:1413-1419.
31. Marsh P, Wellington EMH. Phage-host interactions in soil. FEMS Microbiol Ecol 1994;
15:99-108.
32. van Overbeek LS, van Veen JA, van Elsas JD. Induced reporter gene activity, enhanced
stress resistance and competitive ability of a genetically modified Pseudomonas fluorescens
strain released into a plot planted with wheat. Appl Environ Microbiol 1997; 63:1965-1973.
33. Zeph LR, Stotzky G. Use of a biotinylated DNA probe to detect bacteria transduced by
bacteriophage P1 in soil. Appl Environ Microbiol 1989; 55:661-665.
34. Orvos DR, Lacy GH, Cairns J. Genetically engineered Erwinia carotovoraSurvival, intraspecific competition, and effects upon selected bacterial genera. Appl Environ Microbiol
1990; 56:1689-1694.
35. Bentjen SA, Fredrickson JK, van Voris P. Intact soil-core microcosms for evaluating the
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36. Macrin FL, Archer GL. Conjugation and broad host range plasmids in streptococci and
staphylococci. In: Clewell DB, ed. Bacterial Conjugation. London: Plenum Press, 1993:
313-330.
37. Wipat A, Wellington EMH, Saunders VA. Streptomyces marker plasmids for monitoring
survival and spread of streptomycetes in soil. Appl Environ Microbiol 1991; 57:3322-3330.
38. Tschpe H. The spread of plasmids as a function of bacterial adaptability. FEMS Microbiol
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39. Salyers AA, Shoemaker NB. Broad host range gene transfer: Plasmids and conjugative
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40. Sundin GW, Monks DE, Bender CL. Distribution of the streptomycin-resistance transposon
Tn5393 among phylloplane and soil bacteria from managed agricultural habitats. Can J
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27
41. Wiener P, Egan S, Huddleston, AS, Wellington EMH. Evidence for transfer of antibiotic
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43. Thomashow L, Weller DM, Bonsall RF et al. Production of the antibiotic phenazine-1carboxylic acid by fluorescent Pseudomonas species in the rhizosphere of wheat. Appl
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44. Amarger N and D. Delgutte. Assesment of gene transfer under field conditions in France.
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45. McDonald IR, Riley PW, Sharp RJ et al. Survival of plasmid-containing Bacillus subtilis
released into mushroom compost. Microbial Ecol 1998; 36:51-59.
46. Tokuda Y, Ano T, Shoda M. Survival of Bacillus subtilis Nb22, an antifungal-antibiotic
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47. Crowley DE, Brennerova MV, Irwin C. Rhizosphere effects on biodegradation of 2,5dichlorobenzoate by a bioluninescent strain of root-colonizing Pseudomonas fluorescens.
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48. Tang WZ, Pasternak JJ, Glick BR. Persistence in soil of the plant-growth promoting
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survival and gene transfer, as influenced by agricultural substrates. In: Stewart-Tull DES,
Sussman M, eds. The Release of Genetically Modified Microorganisms-REGEM 2. New
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Wellington EMH, van Elsas JD, eds. Genetic Interactions Among Microorganisms in the
Natural Environment. Oxford: Pergamon Press, 1992; 17-39.
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antibiotic resistance gene markers from genetically engineered Bacillus thuringiensis strains.
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beet DNA. Appl Environ Microbiol 1998; 64:1550-1554.
CHAPTER 3
3.1. Introduction
30
indirect, approach4,5). The second approach is based on the direct lysis of microbial cells in
the environmental matrix (e.g., soil), followed by separation of the nucleic acids from the
matrix and from cell debris and other impurities (Direct lysis approach6). Major differences
between the two strategies are the higher nucleic acid yields obtained with the direct lysis
approach, coupled to the co-extraction of larger amounts of contaminating substances7.
Moreover, the cell extraction/nucleic acid extraction approach in principle allows the removal of a large fraction of the extracellular nucleic acids from the bacterial cells prior to
lysis, thus allowing a better assessment of target DNA present inside the microbial cells in
the environment.
Since these pioneering studies on DNA extraction from soil,4-7 there has been a considerable methodological development, the main purpose of which was the omission of laborious purification steps, mainly hydroxyapatite column chromatography and cesium chloride (CsCl) gradient purifications, replacing these by faster approaches. Thus, the numerous
nucleic acid extraction protocols that are currently in use in different laboratories1-2,8-19 all
share a relatively small number of individual extraction and purification steps (Table 3.1).
Table 3.1. Some examples of frequently used steps in nucleic acid extraction and
purification protocols
Purpose
Step
Principle
Remarks
Cell lysis
Breakdown and
solubilization of cell
envelope components
Very diverse
requirements of
different cells (e.g.,
grampositives versus
gramnegatives.
Temperature shocks
combined with water
crystals, to destabilize
membranes
Recognized as
highly efficient in
lysing a wide range
of bacterial and
fungal/yeast cells
Microwave oven
Combined with
other lysis methods
Sonication
31
Purpose
Step
Principle
Remarks
Extraction
and
precipitation
Phenol
Standard method in
molecular biology
Chloroform
Denaturing action on
proteins and hydrophobes
Standard method in
molecular biology
Ethanol or isopropanol
precipitations
Standard method in
molecular biology
Polyethylene glycol
(PEG) precipitation
Precipitation/concentration
of DNA
CsCl precipitation
Removal of impurities by
salting-out effect
KAc/NH4Cl
precipitations
Glassmilk sorption
Sorption on glassmilk
beads, followed by
differential desorption
Highly efficient in
removing humic
compounds from
DNA
Elutip D
Chromatography
separation
Method repeated
on same extract
Powerful separation of
DNA from humics via
chromatography over
mini spin column
Sephadex
G50/G75/G200
Separation of
contaminants, e.g.,
humics, via gel filtration
Gel electrophoresis
PVPP* sorption
Selectively removes
humics from DNA/RNA
solutions
Hydroxy apatite
chromatography
Selective binding of
nucleic acids to HAP**,
column. Differential elution
of DNA or RNA by varying
phosphate concentrations
Theoretically very
good, but practically
difficult. Often low
recoveries
Purification
32
There have even been efforts to simplify and miniaturize soil DNA extraction protocols to a
level where their on site use becomes feasible.20 Most of the current protocols have been
shown to produce DNA and/or RNA suitable for the analysis of microbial diversity or
microbial fate. However, they often differ in the way they release and lyse microbial cells
from the environmental matrix, and such (qualitative and quantitative) differences are likely
to affect the final analyses performed. Hence, it is key to our understanding of microorganisms in their natural settings as described by molecular methods, that the possible biases
introduced by cell extraction and lysis methods are understood.
This chapter will review currently available strategies to recover and purify (1) microbial (bacterial) cells and (2) nucleic acids (DNA and RNA) from environmental matrices,
and will then briefly address the use of these nucleic acids in monitoring methods to assess
33
microbial diversity and inoculant fate. Figure 3.1 gives a general outline of these
methodologies.
34
Fig. 3.2. A protocol for the recovery of bacterial cells from soil.
An alternative method to disperse soil particles and dissociate microbial cells from soil,
sediment or root parts involves the use of cation exchange resins. Shaking of soil with, for
instance, Dowex39 or Chelex-10029,40,41 is used to remove the bivalent and polyvalent cations
responsible for electrostatic bonding between like-charged bacterial cells and soil particles.
Detergents are also used to overcome adhesive interactions. A comparison of five different
treatments (Fig. 3.2) showed that cell extraction could be improved by using Stomacher
blending instead of shaking. Consistent results and a fairly rapid treatment were possible
with the automated paddle action of the Stomacher. On the other hand, Lindahl and Bakken42
did not find any positive effect of the Chelex-100 treatment on cell extraction efficiency
and, thus, recommended simple, threefold repeated, blending with water. Shaking in lowelectrolyte concentrations, e.g. in distilled water, increases the interactive free energy
between like-charged soil particles. In a recent comparison of both methods with agricultural soil, the total cfu numbers obtained were found to be comparable between the methods (unpublished). However, the water-based protocol yielded a higher number of different
colony types, suggesting that the extraction protocol strongly affects the diversity of bacterial types obtained.
In particular for soils with high clay contents, separation of bacterial cells and soil
particles is necessary. This can be achieved by flotation in the density media Percoll or
35
Nycodenz,31,43 but not with simple centrifugation steps, because clay particles and bacterial
cells show similar sedimentation behavior. The efficiency of different cell extraction protocols thus depends on the soil type; hence, different protocols may prove suitable for different soils.43 However, even with the most optimized protocol, complete extraction of all bacterial cells, in particular of cells bound to soil particles, is probably impossible. Thus, for
studies on the diversity of microbial communities in soil, it seems reasonable that the cells
recovered represent, in their relative abundance, the surface-attached microbial community. As microbial cells may occur both at the outside of soil aggregates and in the interior
regions, the extent to which these soil aggregates are disrupted will determine the sites from
which the inhabiting bacterial cells are dislodged. To monitor the population dynamics of
bacterial inoculants, it is, thus, primordial to understand their localization, as the protocol
to be used and the limit of detection are affected by it. Moreover, inoculant cells may preferentially occur at different locations than indigenous microbes, and hence the requirements
with respect to extraction are different.
The bacterial cell fractions obtained can be used for the extraction of genomic DNA4,5
or for the parallel extraction of both DNA and RNA (Fig. 3.1).44,45 Furthermore, the cell
fractions can serve for assessments of cell numbers using fluorescent dyes or immunofluorescence enumeration. In general, nucleic acids recovered from soil/rhizosphere bacterial
cell fractions are less contaminated with coextracted humic acids, fulvic acids, polyphenols,
polysaccharides, or other plant-derived substances which can hinder molecular analyses,
than these recovered directly.7 For polluted soils, separation of bacterial cell fractions may
even be an absolute requirement. For instance, DNA extracted directly from a zinc-contaminated soil was not PCR amplifiable even after several purification steps, whereas DNA
extracted from the recovered bacterial fraction was amplifiable without any problems (Brim
et al, unpublished).
36
The strategies used in most protocols to achieve lysis of bacterial cells from environmental matrices, either in its presence or absence, are:
1. Enzymatic removal of cell wall material,
2. Chemical action such as solubilization of the cell membrane with detergents,
3. Physical action such as freezing/thawing, ultrasonication or microwave oven treatment, and
4. Mechanical action such as achieved via e.g., bead beating or mortar/pestle grinding.
Often, some of these strategies are combined. Key aspects are described below:
1. A range of enzymes that attack different cell wall components can be applied, such
as proteinase K, lysozyme, mutanolysin, lysostaphin, subtilisin or achromopeptidase.46 These enzymes are more effective when combined with EDTA.
2. Commonly used detergents are sodium dodecyl sulfate (SDS) and N-lauroylsarcosine
(Sarcosyl). Cetyltrimethyl ammonium bromide (CTAB) is also often used. Nonpolar detergents, such as Triton X, Tween or Nonidet P-40, which provide for milder
treatments, are less common.
3. Physical methods have mostly been combined with other (enzymatic or mechanical) methods, as there is doubt whether their single use will result in acceptable
levels of lysis.15,20 For instance, the application of freeze/thaw steps, even repeatedly,
has been shown to be inferior to bead beating.15,20 Moreover, it is difficult to achieve
high lysis rates by the single use of ultrasonication of soil slurries.
4. Of particular interest is the use of the bead beater (Braun cell homogenizer), which
is able to lyse even very recalcitrant cellular forms such as Bacillus spores, mycobacteria or fungal conidia.20,50,51 This method was proposed in a pioneering paper on
direct extraction of soil DNA7 and is still widely in use. Furthermore, a method in
which the abrasive action of mortar/pestle grinding was combined with liquid nitrogen snap-freezing was shown to be efficient in releasing fungal DNA.48,49,52
Table 3.1 lists the most o
c mmonly used steps of popular protocols. Combinations of
these approaches have also been used. For instance, Picard et al53 have shown that a combination of ultrasonication, microwave oven and enzymatical treatment resulted in a higher
degree of cell lysis and thus DNA yield from soil, than any of these steps performed in
separate. However, DNA shearing was a severe problem.
Problems with all these approaches are related to either the uneven distribution of
microbial cells throughout environmental matrices, resulting in their uneven exposure to
the action of lysis agents, or to differences in the resistance of different microbial types to
lysis. It is therefore prudent to state that complete lysis of cells in environmental matrices is
most often impossible to achieve. Hence, the data obtained on the basis of environmental
DNA (and/or RNA) should be regarded as descriptive of the populations recovered in a
relative sense, and rigorous checks on the nature of the possible bias introduced may be
necessary.
37
range of protocols for extraction of environmental nucleic acids is currently still in use.1,2
The main strategy is based on organic solvents which interact with the hydrophobic parts of
proteins and lipoproteins, resulting in their denaturation and extraction. Using, for instance,
phenolor chloroformwater phase separations, many proteins, lipoproteins and other
hydrophobic compounds concentrate in the organic phase, whereas nucleic acids will concentrate in the water phase. Phenol is known to be efficient in denaturing and extracting
proteins, whereas chloroform interacts with proteins and polysaccharidic material. Hence,
a combination of these two extractants is often employed. Chloroform also removes traces
of phenol still present in the aquatic phase. In addition to phenol/chloroform extractions,
ethanol or isopropanol precipitations of DNA, followed by washes, are often used to remove salts, SDS and other impurities.54
The solution produced in these initial steps, called the crude extract, will often be a
rather impure aqueous solution of nucleic acids. DNA and RNA show similar behavior and
may be both present in these crude extracts.
3.4.2. Purification
The aim of purification of the crude extract is to recover the nucleic acids from the
crude extract in a form ready for analyses by appropriate molecular techniques. Ideally, the
procedure is as rapid and simple as possible, and incurs minimal quantitative and/or qualitative loss of, or damage to, the nucleic acids. The different protocols currently in use in
different laboratories have been based on an array of different steps in various combinations. Table 3.1 presents a summary of purification methods employed in selected protocols.
There is ample evidence that the requirements, with respect to DNA purity, for restriction by different enzymes, cloning, PCR amplification, reverse transcription or direct
hybridization can be quite different. Moreover, different enzymes, e.g., thermostable DNA
polymerases, have different requirements as to DNA purity.14,55 Therefore, for each new
experimental system, the extent to which nucleic acid purification is needed has to be carefully established and controlled. However, a few trends in the purification of environmental DNA are worth mentioning. The powerful purification steps used in the early protocols,
such as CsCl gradients5 and hydroxyapatite chromatography,4,30 have been found to be
either very tedious or unpredictable with respect to yields. Gel electrophoresis has been
shown to offer a powerful (one-step) alternative strategy to obtain DNA pure enough for
molecular analyses via PCR.18,19,56, 57 Young et al58 even used polyvinylpolypyrrolidone in
their gels to enhance the selective removal of humics. However, gel electrophoresis may lead
to a bias when the DNA is subsequently used for PCR amplification and community fingerprinting.58 Moreover, the method can be tedious, and has therefore been superseded by
other approaches. Thus, there has been an extensive search for reliable alternative purification methods that were simple, fast and provided high throughput.8-12,15-19 Often, these protocols relied on a sequence of short, different, treatments, including e.g., KAc or NH4Ac
precipitations,8,11 CTAB extraction,10 hydroxyapatite mini-spincolumns12 and Sephadex gel
filtration.8,12,56,59,60
One rapid protocol that consistently yielded PCR amplifiable and restrictable DNA
from different soils, originally developed by Smalla et al,15 was based on a two- or three-step
purification consisting of sequential KAc precipitation, CsCl precipitation and (originally)
glass milk or Wizard spin column purification. In later work, it was shown that this protocol
was very flexible and adaptable to a wide range of soil types.61 In a parallel study, Zhou et
al 62 also described a protocol which contained four flexible purification routines applicable
to a range of different soils.
As previously mentioned, a recent development aimed at the on site extraction of nucleic
acids from soil. A very simple one-step purification over a Sephadex G-200 column was
38
found to yield DNA sufficiently pure for PCR amplification and detection of key microbial
groups.20 However, it is doubtful the DNA obtained was of a quality similar to that obtained
by more rigorous protocols.
39
to easily detectable or clonable quantities. The potential of this approach is tremendous, and it has revolutionized molecular microbial ecology. In accordance with
primer choice, specific target organisms or genes can be detected and quantified,50-51
or broader groups can be fingerprinted by techniques such as denaturing or temperature gradient gel electrophoresis (D/TGGE),67,68 terminal restriction fragment
length polymorphism (T-RFLP),69 single strand conformational polymorphism
(SSCP)70 or amplified ribosomal DNA restriction analysis (ARDRA). This field is
currently exploding, with new applications being reported almost monthly. However, due to problems inherent to mixed community PCR,71 the real value of the
resulting fingerprints for actual in situ microbial diversity remains to be assessed.
Furthermore, an important step forward in the analysis of D/TGGE profiles is the
use of probes based on the variable region 6 (V6 probes) of the 16S rDNA.72 This
highly variable region allows for the generation of highly specific probes which can
serve to identify bacterial types in gels, or to link the bands from D/TGGE gels to
either isolates or cloned 16S community amplicons.
40
community of a grassland soil on the basis of rRNA obtained from soil-extracted ribosomes. Duarte et al44 generated profiles of soil bacterial communities using reverse transcription and PCR amplification of RNA extracted from the bacterial fraction. The RNA
obtained from pre-extracted cells or ribosomes is likely to be less contaminated with coextracted material like humic acids.44,85 However, recent progress in optimizing PCR efficiencies even in the presence of a substantial amount of contaminating humic acids by a
considerate selection of heat-stable DNA polymerases and additives like single strand stabilizing proteins, suggests that partially purified RNA might be good targets for RT-PCR analysis.14,55,86 Generally the yield of ribosomal RNA has been reported to be higher with direct
or indirect extractions than with the ribosome isolation method.44,87 Early protocols to directly extract total RNA from soil could not detect intact ribosomal RNA molecules,59,88,89
but recently a rapid and direct extraction of intact 5S, 16S, 18S, 23S and 28S RNA, which
were amplifiable by RT-PCR has been reported.79
41
42
rRNA and DNA based techniques can be more significant, as shown by Duarte et al.44 DGGE
analysis of PCR or RT-PCR products generated with either DNA or RNA from the same
bacterial fraction showed that, next to similarities in the community profiles, there were
clear dissimilarities, suggesting that there were differences in the activity levels between the
various bacterial groups underlying the profiles.
3.8.1. MPN-PCR
The principle of MPN-PCR is similar to that in other MPN enumeration procedures.
Briefly, the sample material (extract) is serially diluted and amplified, in a number of replicates, to a level where detection is not possible anymore (outdilution). Theoretically, this
would be the case at the point of complete removal of all target sequences from the PCR
reaction mix. However, in practice the cutoff points can be slightly higher due to inefficiencies in PCR amplification of targets in environmental DNA. The data of all amplification
reactions are recorded, and the cutoff levels are determined in the replicates. A table based
on statistical principles will then indicate the most probable number of virtual amplifiable
targets (VAT) in the dilution assessed, and, thus, in the sample. If the minimal number of
targets amplifiable under the conditions used equals 1, then the VAT equals the actual
amplifiable numbers; if there is a discrepancy, a compensation factor should be included in
the calculations.
Nesme et al 119 successfully used MPN-PCR to quantify the number of pathogenic
Agrobacterium sp. strains in soils. MPN-PCR has further been employed, for instance, to
assess the numbers of targets of Mycobacterium chlorophenolicum, a pentachlorophenoldegrading bacterium, in soil.61 The method was specific and provided positive detection of
43
the target down to about 103 targets per g of soil. In addition, the fate of introduced
Paenibacillus azotofixans vegetative cells and spores in soil and wheat rhizosphere could be
successfully monitored in microcosms using specific MPN-PCR.51 However, an important
drawback of MPN-PCR is the excessive number of replicate PCR reactions needed, as well
as the careful controls that are required for PCR efficiency. This makes the method rather
laborious and expensive. MPN-PCR has now been superseeded by competive PCR methods as discussed below.
44
soil. cPCR was further found to be a very accurate method for quantitation of a genetically
modified cyanobacterium added to Baltic sea sediment.121 In these experiments, the competitor DNA was added to the sediment samples before DNA extraction and PCR amplification in order to account for variations in extraction efficiencies between samples.
However, as mentioned, competitive PCR based on amplification of DNA targets does
not assess viable cell numbers. Hence, if a focus on active cells rather than total cells is
required, the method should be combined with other methodology, viz microscopic assessments of viable cells. The activity (expression) of specific genes can be quantified by using
PCR preceded by a reverse transcription step (RT-PCR). This method is very sensitive for
the detection of transcripts, as shown with eukaryotic microorganisms;91,92 detection of
bacterial gene expression by RT-PCR in activated sludge was possible at a detection threshold
of 106 CFU g-1 soil.124 Competitive RT-PCR allows for quantitative determinations of gene
expression, as was shown in studies on the expression of peroxidases by Phanerochaete
chrysosporium in soil.93,94 Obviously, quantification on the basis of two enzymatic reactions
needs a series of controls to compensate for the putative biases of both processes.
45
suggest that lysis may well be confined to the larger cell-size fraction of the microbial community, since the numerous minute or dwarf cells present essentially escaped lysis. On the
other hand, the nature and significance of these dwarfs is currently under heavy debate, as
their putative role in natural environments is blurred. These considerations have to be taken
into account when DNA or RNA extraction protocols are to be used for microbial community structure or fate studies in soil. In addition, the majority of extraction protocols not
only yield bacterial, but also eukaryotic DNA, which may originate from a variety of source
organisms present in the system (e.g., fungi, protozoans, nematodes and/or plants). For
quantification purposes, a eubacterial probe is necessary to determine the proportion of
bacterial DNA in the total extracts obtained.
A major criticism on studies that were exclusively based on environmental DNA,
including those that identified novel microbial species by sequencing of cloned 16S ribosomal amplicons, has been the absence of significant evidence for the cellular nature of these
sequences. To bridge the gap between the evidence for novel types exclusively on the basis of
clone sequences and knowledge obtained from sound microbiological work in which
microbial functioning is central, a link to traditional, isolation-based microbiology has been
advocated.125 However, another strategy with potential to link the direct molecular information with that of microbial cells can be based on the detection of cells following their
reaction with a labeled probe, a process called whole cell hybridization. This procedure has
been applied successfully in aquatic environments, but it has met with problems of signal
weakness in soils.126 Recently, it was shown that whole cells from the environment can also
be used as targets for PCR (in situ PCR) or reverse transcription-PCR,127-129 and this approach has a potentially enhanced sensitivity. This method has been successfully applied to
aquatic systems, whereas its application to soil is still awaiting. Both methods have clear
potential as complementary tools to support findings obtained on the basis of the extractive
protocols. At this point in time, a major drawback may still be their sensitivity to false positive results, as well as the limitations with respect to minimal cell densities needed for successful observation of cells on microscopy slides. However, the combination of molecular
fingerprinting methods with the whole cell hybridization approach to monitor the potential enrichment and isolation of unculturable, VBNC or hithertoas yetuncultured
bacteria is a powerful tool which has so far been only sparsely explored.
Thus, albeit with a definite right of their own, molecular analyses on the basis of
extractive approaches clearly gain enormous scientific strength and meaning when they are
tied together with methods based on cultivation approaches. It is foreseeable that a polyphasic
strategy, in which the extractive, the whole cell hybridization and the cultivation-based
approaches are combined, ultimately offers the best perspective for future studies on the
fate and activity of microorganisms in their natural habitats.
Acknowledgments
This work was supported by grants from the EU-BIOTECH Programme. We thank
L.S. van Overbeek for critically reading the manuscript. J.D. van Elsas and C. C. Tebbe are
members of the MAREP Concerted Action sponsored by the European Commission Biotechnology Programme, DGXII.
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CHAPTER 4
Detection of Bacteria
by Their Intrinsic Markers
va Tas and Kristina Lindstrm
4.1. Introduction
he detection and identification of specific bacterial species and strains has long been of
interest for different applications in agriculture, the food industry, the bioleaching industry and the biological degradation of toxic wastes (bioremediation). In addition, basic
and applied sciences, such as microbial ecology, are investing more research into exploring
the components of microbial populations living in various ecological niches.
Classical microbial identification methods are primarily based on morphology, growth
characteristics and metabolic properties of the organism under study. For pathogens or
symbiotic organisms, host specificity is also an important feature. In addition, bacteriophage typing, antibiotic resistance, lipopolysaccharide and protein profiles, serological properties, and plasmid profiles of bacteria can be characterized. More advanced immunological
techniques include detection of antigens by enzyme-linked immunosorbent assay (ELISA)
or by fluorescent antibody labeling. The problem with most of these techniques is that they
require microbiological cultivation. Thus, for example, detection of released organisms may
not be possible when their numbers are low relative to those of the indigenous population.1
In addition, disadvantages of the immunological methods lay in possible cross-reactions
and nonspecific binding. The use of monoclonal antibodies can be a solution,2,3 but the
antigens to be detected can be variable depending on growth conditions.
Along with the traditional identification methods described above, molecular methods, including detection of specific nucleic acid sequences or profiles are now available.
Nucleic acid based detection techniques overcome some of the problems inherent in traditional identification methods (see above) and they are usually more rapid and sensitive
than traditional approaches. Detection of specific DNA or RNA sequences in environmental samples enables reliable detection of virtually any group of microorganisms, including
those in nonculturable states.4 In particular, DNA:DNA hybridization5 and the polymerase
chain reaction (PCR)6,7 are indispensable tools for detection of microorganisms in environmental samples. The reader is also referred to Chapter 3 for details on extraction and analysis of nucleic acids from environmental samples.
The targets for molecular identification methods can be intrinsic markers or introduced markers. An intrinsic marker can be defined as a natural (i.e., nonintroduced) DNA
sequence or phenotype that serves as a signature for a particular organism or group of
organisms. Detection of intrinsic markers is an important tool for identification of specific
Tracking Genetically-Engineered Microorganisms, edited by Janet K. Jansson, Jan Dirk van Elsas,
Mark J. Bailey. 2000 EUREKAH.COM.
54
microorganisms in the environment and complements the use of marker genes for genetic
tagging of microorganisms (see Chapters 5-7 describing introduced marker genes).
In this chapter we will introduce some examples of the use of intrinsic DNA markers
for monitoring of specific microorganisms. We will also describe methods for designing
specific nucleic acid probes and PCR primers, which are prerequisites for the application of
these detection techniques.
55
an antibiotic resistance marker carried by Tn5 was not expressed in some cells under high
temperature stress. Another example is when the marker is not a fully functional gene but a
recombinant fragment detectable by DNA techniques. Van Elsas et al28 used a small eukaryotic DNA fragment, pat from Solanum tuberosum, as a marker to study the fate of Pseudomonas fluorescens introduced into soil. This DNA sequence was not present in indigenous soil
bacteria, so problems with background were eliminated.31 Although not expressed as a phenotypic marker, the pat fragment was readily detected by DNA hybridization and by PCR.
56
57
rhizobial population. However, inoculation often fails because Rhizobium populations present
in the soil may outcompete the newly introduced strain.43 That is why rhizobia are among
the most important targets of genetic engineering, aiming to increase nitrogen fixation capacity or inoculation competitiveness.44 As a consequence of genetic engineering and environmental release, sensitive monitoring methods to detect rhizobia in the environment have
become essential. Examples of field releases of genetically modified rhizobia are given in
Chapters 8-11.
Rhizobium galegae is a recently described and well-studied species45 that nodulates Galega
officinalis and Galega orientalis (goats rue). The latter species is a perennial legume with
potential agricultural importance. R. galegae has not yet been genetically engineered, but
antibiotic resistant strains have been selected to facilitate laboratory studies. The species has
unique taxonomy and host specificity45-47 and it is not indigenous in Finnish soils.48 These
properties make it a suitable model organism for studying the fate of inoculant rhizobia in
the field in Finland. R. galegae species- and strain-specific probes and PCR primers were
developed in our laboratory. They allow specific detection of R. galegae from environmental samples as will be demonstrated in this chapter.
Fig. 4.1. Fragments of genomic DNA from R. galegae HAMBI 1174, which carry host specificity
genes, were analyzed for species specificity. The 2.4 kb EcoRI-fragment shown here was a good
candidate but it weakly hybridized with DNA from nontarget organisms. Its three subfragments
were also analyzed, and the 0.9 kb EcoRISalI fragment was species specific. Arrows show PCR
primers.
58
was verified in PCR reactions with purified genomic DNA from an assortment of strains of
R. galegae, other rhizobia and some other species.51 PCR amplification always produced an
850-bp specific fragment with R. galegae DNA. PCR-RFLP of this species-specific fragment
revealed a difference between R. galegae strains according to their host plant G. orientalis
and G. officinalis (Fig. 4.2).
59
Fig. 4.2. PCR-RFLP of the R. galegae-specific fragment from genomic DNA of different R. galegae
strains with the restriction enzymes HinfI and MspI. The host plant is shown above the samples.
Lanes: M, molecular weight marker; 1, reference strain, R. galegae HAMBI 1174; 2-15, other
R. galegae strains.
60
Fig. 4.3. Isolation of a strain-specific probe by subtraction hybridization. HAMBI 1174 and HAMBI
1207 are genetically closely related strains of R. galegae.
directly from crushed nodules (Fig. 4.4). The results were in good agreement with those of
dilution platings on selective media, in which the two strains could be counted.
The specific primers were also used for the amplification of DNA extracted from soil
and peat.61 After developing a suitable method for DNA extraction and purification from
the peat samples (for methods see Chapter 3), the PCR assay proved to be very useful for
quality control (i.e., confirmation of the inoculum strain) of commercial peat inoculants.
61
Fig. 4.4. PCR amplification of the R. galegae species-specific fragment (upper band) and the R.
galegae HAMBI 1174 strain-specific fragment (lower band) from crushed nodules. Lanes: ,
molecular weight marker; 1, R. galegae HAMBI 1174 genomic DNA; 2, R. galegae HAMBI 1207
genomic DNA; 3-26, nodules from G. orientalis inoculated with R. galegae; -, negative control
without template DNA. Reprinted with permission from Tas , Leinonen P, Saano A et al. Appl
Environ Microbiol 1996; 62:529-535.
62
Fig. 4.5. REP PCR fingerprint patterns of R. galegae DNA obtained by sonicating root nodules.
Lanes: S, lambda BstEII; 1, genomic DNA from reference strain R. galegae HAMBI 540; 2, field
nodule (R. galegae HAMBI 540); 3-8, nodules from G. orientalis inoculated with different R.
galegae strains; 9, negative control without template DNA. Reprinted with permission from Nick
G, Lindstrm K. System Appl Microbiol 1994; 17:265-273.
bial activities in the field (For a review see ref 65). The RT-PCR technique consists of synthesis of DNA from RNA by reverse transcription, and amplification of a specific DNA
fragment by PCR. The method is very sensitive, and by using appropriate controls the detection can be made quantitative. The first environmental applications included detection
of food-borne pathogens,66 and microorganisms used in bioremediation of contaminated
soils.65
For the selection of a suitable target gene for RT-PCR several characteristics should be
considered: the target gene should be abundantly expressed throughout the growth cycle of
the organism tested, and its expression should not be regulated at the transcriptional level.
Since bacterial mRNAs have a very short half-life, these methods discriminate living (i.e.,
metabolically active) cells from dead or dormant ones, although DNA from the latter ones
can still be detected by DNA-targeted Southern hybridization or amplified by conventional
PCR. (Detection of active contra dormant cells is discussed in more detail in Chapter 1.)
Some technical difficulties arise from the need for rapid isolation of undegraded mRNA.
However, in situ techniques are now under development for the amplification of gene transcripts inside microbial cells.18
In addition, the fingerprints achieved by the PCR-based applications mentioned in
section 4.2.1 can be used for identification of microorganisms in pure cultures or relatively
simple microbial communities. In highly diverse microbial systems such as soil, the banding
patterns are too complex to be assigned to specific organisms but a comparison of microbial communities (diversity as well as quantitative aspects)67 is possible. Particularly the
TGGE technique seems to be appropriate for monitoring effects of environmental disturbances such as pollution or release of new organisms.68
Hybridization of RNA and DNA on microarrayed chips (on glass or silica surfaces) is
another technique that will probably be broadly applied in the near future. Cheng et al
prepared nucleic acids of E. coli and other bacteria from human blood and hybridized them
with appropriate probes on microchips.69 Since the hybridization of RNA on microarrayed
63
chips allows monitoring of gene expression, this technique may prove to be useful in studying the dynamics of cell populations in environmental samples. Thus, microarrays have
great potential in medical diagnostics, food monitoring, water testing, and other fields.
New approaches based on specific molecular recognition mechanisms are also under
development, such as nucleic acid probes detecting proteins and other molecules,70 or synthetic DNA mimics that behave like DNA oligonucleotides but bind to nucleic acids with higher
specificity and affinity.71 Drolet et al have developed an ELISA-like assay using an oligonucleotide probe which binds to a protein target.70 A method called SELEX (systematic evolution of
ligands by exponential enrichment)72 allowed rapid selection of oligonucleotides that preferentially bind to the target molecule from a population of random sequences. High affinity
oligonucleotide ligands could be developed in this way for virtually any target molecule and
used, for example, as therapeutic or diagnostic agents.73 Nucleic acid ligands would have several advantages over antibodies.70 For example, they can be easily synthesized and thus
accurately replicated with the same binding properties, while antibodies may differ when generated in different laboratories and are also subject to animal-to-animal variation. Secondly,
animals are not required for the synthesis of oligonucleotide ligands. Also, the ligands can be
generated even for toxic or nonimmunogenic targets. Their small size can also be an advantage
for some applications. In the future, further DNA ligands will be developed for diagnostic
purposes and may be used for more general approaches. If successful, the method could challenge, for example, in situ hybridization and immune detection methods.
An interesting new approach is based on the use of a synthetic molecule peptide nucleic
acid (PNA). This molecule has recently been proposed to expand the applications of oligonucleotides.71 PNAs are DNA analogs with a polyamide backbone substituted with purine
and pyrimidine base sidechains. The specificity of PNA in binding to complementary nucleic
acids is higher than that of DNA strands, and the resulting PNA/DNA and PNA/RNA
duplexes have high stability compared to DNA/DNA and DNA/RNA duplexes. Similarly to
DNA, PNA can be labeled by biotin, fluorescein or reporter enzymes. Therefore, PNA is a
good candidate for hybridization studies and may, one day, share the burgeoning field of
DNA-based diagnostics with traditional nucleic acid probes.
Along with the above described sophisticated genetic approaches, phenotypic description of microbial communities is equally important. Metabolic fingerprints of individual
organisms or microbial communities can be determined by the BIOLOG system, which
colorimetrically detects the utilization of various carbon substrates. Phospholipid fatty acid
(PLFA) profiles are also increasingly being used to characterize the structure of microbial
communities.74,75
4.6. Conclusion
To assess the survival and behavior of deliberately released strains in the environment,
specific markers are required for monitoring the populations of both the released and the
indigenous strains. Intrinsic markers are of great value in these studies and their detection
complements the use of tagging genetically introduced marker genes. In addition, some
applications, e.g. detection of pathogens or food-borne microorganisms, do not allow the
use of genetically engineered markers. Therefore, detection techniques based on specific
intrinsic markers are indispensable. This chapter focused on these techniques and discussed
different methods for selection of suitable molecular probes and primers.
In addition to exploring previously described genes or ribosomal sequences, an alternative for probe selection is screening randomly cloned fragments. This approach can provide
specific probes also for organisms for which sequence data are limited. It was illustrated in this
chapter by the isolation of strain- and species-specific probes from R. galegae. The problem
with these random search methods is that the gene stability and genetic variability of the
64
fragments selected this way are unknown. However, in our experiments the data obtained
proved that our markers are stable and can be used for ecological studies.
Introduced marker genes may affect the fitness of the engineered organisms. In addition, gene transfer to nonmarked organisms can not be excluded. (Look for details in later
chapters of the book.) Therefore, intrinsic markers are generally considered to be more
stable than introduced markers, but further data is needed from comparative studies to
address this hypothesis.
Intrinsic markers can be detected with remarkable sensitivity, as illustrated by the fact
that detection of a single cell is possible from some samples. The performance of in situ
hybridization with fluorescent probes and the results of some PCR-based detection techniques are comparable in this sense with those of engineered marker genes, but extensive
optimization of assay conditions is required.
An early criticism on nucleic acid-based detection techniques was based on their incapability to distinguish active and dead cells, but this problem can be overcome by the use of
RNA-targeted probes. Detection of ribosomal RNA gives some insight into the dynamics of
a bacterial population since number of ribosomes correlates with the metabolic activity of
a cell. However, since rRNAs are extremely stable, they are not suitable targets for discriminating between living and nonliving cells. In contrast, detection of mRNA by hybridization
or RT-PCR allows description of the active population relative to the total number of cells.
Most of the molecular methods for detection of microorganisms based on intrinsic
markers do not require isolation and cultivation of organisms, rather they allow in situ
studies of individual members of complex microbial communities. By combined use of
several probes with different specificities and by use of fingerprinting methods, different
segments of a microbial population can be concurrently assessed. Therefore, the complementary use of several different methods will most likely be needed for accurate description
of complex natural and man-made microbial communities.
Acknowledgments
K. Lindstrm is a member of the MAREP Concerted Action sponsored by the European Commission Biotechnology Programme, DGXII.
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64:2173-2180.
CHAPTER 5
Luminescence-Based Microbial
Marker Systems and Their
Application in Microbial Ecology
James I. Prosser, Antonio J. Palomares, Matti T. Karp and Philip J. Hill
5.1. Introduction
olecular marker systems provide the ability to track specific microbial inocula in the
environment. This is a fundamental requirement for many ecological studies and is
particularly appropriate for the assessment of risks associated with the release of genetically
modified microorganisms. The efficiency of a marker system requires the absence of the
marker gene, or its phenotype, from the environment under study, stable insertion of the
marker genes into the host organism without loss of fitness and efficient and sensitive genotypic and/or phenotypic detection. Techniques for genotypic detection are similar for all marker
genes and distinctions therefore arise through differences in efficiency of phenotype detection
and the information that this provides. The major advantage of all marker systems over traditional techniques is the increased ability to distinguish specific organisms under study from the
indigenous population. Marker systems do, however, require laboratory cultivation of the host
strain and ecological studies utilizing marked strains must accept this limitation. This is not,
however, a limitation for applications to risk assessment of genetically modified microorganisms, which require growth in the laboratory before release.
This article focuses on luminescence-based techniques that involve marking with
prokaryotic or eukaryotic genes encoding luciferase, which catalyses light production. They,
like all marker systems, increase selective detection of inocula but provide several additional
advantages. In particular, they enable rapid and specific measurement of metabolic activity
of marked organisms, without cell extraction, and studies of their spatial distribution in
environmental samples.
70
reacts with molecular oxygen to form 4-hydroperoxy-FMNH (Intermediate II) (Fig. 5.1).
In the absence of aldehyde, this decays to generate FMN and hydrogen peroxide with no
light emission (dark decay). In the presence of aldehyde, Intermediate III (4 peroxyhemiacetal-FMNH) is formed, which decays to yield the aliphatic acid and an
excited emitter, proposed to be a 4-hydroxy-FMNH species,3 which undergoes a radiative
relaxation to emit light. After returning to the ground state, 4-hydroxy-FMNH decays to
generate water and oxidized FMN. Although the in vivo aldehyde substrate appears to be
tetradecanal, other long chain aldehydes (e.g., nonanal or decanal) may elicit a bioluminescent
response. Enzyme turnover rate is greatly affected by the nature of the aldehyde substrate used,
and may differ significantly and characteristically with luciferases from different species.
Fig. 5.1. The mechanism of bacterial bioluminescence. L represents luciferase, roman numerals show key intermediates, *
indicates excited intermediate.
strains studied,5 and gene luxH has been identified downstream of luxG in V. harveyi.8 These
genes are not essential for a bioluminescent phenotype but may be involved in the generation of reduced flavin substrate for the luminescence reaction.
All luxA and luxB genes sequenced (e.g., refs. 7,9) show a high degree of sequence
homology. The luxA gene shows greater conservation than luxB, consistent with the postulate that the subunit controls the kinetic properties of the enzyme. Sequence analysis
indicates that bacterial luciferases may be subdivided into two distinct classes, the first represented by those from V. fischeri and Photobacterium and the second by V. harveyi,
P. luminescens and a symbiont from the fish Kryptophanaron alfredi, the latter group comprising the less heat-labile enzymes. The high homology between and subunits both
within and between different species indicates that luxA and luxB arose by gene duplication.5 The subunit appears to be solely responsible for the catalytic activity of the enzyme
but the subunit is essential for functionality, and may be required to maintain the catalytically active conformation of residues.
72
74
Fig. 5.3. Plasmids constructed to study the expression of eukaryotic luciferases under the control
of different promoters in gram-negative bacteria. All of the plasmids were based on the RK2
derivative pRK293. Fragments with the represented fusions of luc and lucOR were cloned
in the sites of pRK293 indicated. B, BamHI; Bg, BglII; C, ClaI; H, HindIII S, SalI; X, XhoI.
Reprinted with permission from Cebolla A, Vzquez ME, Palomares AJ. Appl Environ
Microbiol 1995; 61:660-668.
higher levels of luminescence than other constructs.31 Use of PR::luc and P R::lucOR
enables distinction between Pseudomonas putida colonies emitting light of different wavelengths30 (Fig. 5.4).
5.4. Methodology
Luminescence-marked organisms can be monitored using several techniques. Lux and
luc genes may be detected by gene probing, but the major advantages of luminescence marker
systems lie in the ability to detect light.
Fig. 5.4. Luminescence emitted by two strains of Pseudomonas putida containing plasmids pAP2
and pACR3 with PR::luc and PR::lucOR fusions, respectively. Cells were incubated on nitrocellulose filters placed on solid medium, incubated for 30 h and photographed under normal illumination (A) and after 30 min exposure in the dark. Reprinted with permission from Cebolla A,
Vzquez ME, Palomares AJ. Appl Environ Microbiol 1995; 61:660-668.
beetle luciferase in E. coli cultures on nitrocellulose filters, with exposure for 10-15 minutes.30 Similar examination of E. coli streaks on solid media demonstrated the four colors of
bioluminescence emitted by different click beetle luciferases, with 2 s exposure time.33 This
approach has also been used to determine regions of plants infected with lux-marked pathogenic bacteria 34 and for detection of luciferase activity in alfalfa nodules induced by R. meliloti
containing a nifH::luc fusion.35
5.4.2. Luminometry
The major advantage of lux- and luc- marker systems lies in the relationship between
luminescence and cell activity enabling assessment of the activity of marked organisms by
quantification of luminescence, by luminometry. In organisms in which luciferase production is constitutive and where luminescence per cell is maximal, or constant, total light
emission is proportional to biomass concentration. This occurs in exponentially growing
cultures, with proportionality between luminescence and biomass concentration over several orders of magnitude.27,36 The convenience and validity of this approach depend on the
nature of the construct. For example, constitutive luciferase production may be achieved by
chromosomal marking with luxA and luxB genes, but luminescence requires addition of
dodecanal and incubation under optimal conditions. Cellular production of dodecanal is
achieved in cells additionally marked with luxCDE genes, but the additional genetic load
may affect the physiological characteristics of the marked organism. Increased luminescence may also be achieved by increasing lux-or luc- gene copy number, for example, in
plasmid marked strains, although this increases the likelihood of decreased host fitness and
stability.37 Finally, use of luminometry in samples with particulate material reduces sensitivity. Lower detection limits using luminome
try therefore depend on a number of factors.
For example, detection limits for chromosomally and plasmid-marked strains of P. fluorescens
in cell suspensions were 1.7 x 103 and 8.9 x 104 cells ml-1, respectively. Equivalent detection
limits in inoculated autoclaved sterile soil were 8.1 x 103 and 3.0 x 105 cell g-1, respectively,
76
while inoculation into nonsterile soil increased detection limits to 5.9 x 104 and 2.2 x 105
cells g-1, possibly through greater competition for nutrients.38 An added advantage of chromosomal marking is the frequent lack of apparent effect on host fitness.
In the environment, conditions are likely to be variable and sub-optimal for luminescence. Luminometry of untreated samples will therefore provide a measure of the activity
of marked organisms under nutritional and environmental conditions prevailing at the time
of sampling. Luminescence activity is equivalent to that measured using traditional techniques, e.g., dehydrogenase activity39 but luminometric measurements are considerably more
sensitive, convenient and rapid, with results within several minutes of sampling. More importantly, luminometry assesses the activity of the marked population only, in the presence
of high indigenous populations, while traditional activity techniques, e.g., enzyme assays,
substrate conversion rates, provide information on the total population. The technique is
also sufficiently sensitive to detect the activity of viable but nonculturable populations.40
Luminescence assays may be modified to assess biomass by amendment with nutrients and
incubation under optimal conditions.41 This measure of potential luminescence is equivalent to other measures of potential activity, e.g., the substrate induced respiration method.
5.4.3.Photon Imaging
Quantitative imaging of gene expression using luciferase based reporter constructs is
now possible due to the availability of extremely sensitive imaging equipment that can be
calibrated using light standards. These camera systems are usually based on image intensifiers coupled to video cameras, or cooled charge coupled device (CCD) technology. Intensified cameras have high sensitivity and the ability to watch the image form almost in realtime but, at high gain, suffer from reduction in spatial resolution. Cooled CCD systems rely
on capturing the image during a single long exposure. They have extremely low noise and
wide dynamic range and are increasingly available, leading to a reduction in cost of this
technology. The sensitivity of many cooled CCD cameras can be significantly increased by
pixel binning, although this reduces spatial resolution. Both systems may be configured for
microscopic analysis or imaging of macro samples. They are often supplied with flexible
image processing software that allows the user to count areas, quantify signals and superimpose images.
Examples of the power of this technology to assess gene expression, even in highly
complex environments, include assessment of mas gene (mannopine synthesis) expression
by Agrobacterium tumefaciens in different tobacco plant tissues during infection42 and the
coupling of luxAB with the nitrogenase promoters of nifD and nifH from Bradyrhizobium
japonicum, enabling imaging of luciferase expression in single soybean root nodules and,
indeed, in single bacteroid infected cells.43
5.5. Applications
5.5.1. Stable Tagging of Bacteria with Firefly Luciferase Gene
for Environmental Monitoring
Gram-negative strains engineered for in situ applications must meet a number of practical requirements for safe and efficient performance. These include not only the absence of
traits that may give them an advantage over nonengineered strains but also the ability to
retain the acquired genotype and phenotype in the absence of selection. Use of firefly luciferase addresses the first of these concerns, since D-luciferin is expected to be absent from
all prokaryotic cells and the enzyme is unlikely to provide any advantage over the wild type.
Stable inheritance of the marker gene, in the absence of selective pressure, can be achieved
by chromosomal marking using the mini-Tn5 system.44 A HindIII fragment containing only
the PR promoter and the luc genes was isolated and cloned into pUC18Not. The NotI fragment with the luc gene was cloned into the unique NotI site of pUT/mini-Tn5 Sm/Sp and
into pUTKm. The resulting plasmids, pTCR210 and pTCR240, were selected for studies of
transposition and luciferase expression in various gram-negative bacteria. 36 To tag bacteria
without the use of antibiotic resistance genes, the Km resistance gene may be replaced with
the lPR::luc fusion. To attenuate the expression in the environment, where the marked strain
must survive, and to allow sensitive detection, a DNA fragment containing the repressor
gene lacIq and a PR::lucOR fusion has been introduced on a suicide plasmid. PTEB510
(Fig. 5.5) is a representative of constructs able to express high luciferase levels only when
induced by IPTG. Transposition frequencies were suitable for marking purposes and the
levels of luminescence were sufficient for detection by the standard methods.
Fig. 5.5. Construction of suicide orange click beetle luciferase plasmids used for marking gramnegative bacteria. The selective markers are flanked by Tn5 19-bp terminal ends (I end and O
end). The IS50R tnp gene devoid of NotI sites (tnp*) is oriented divergently from the I end.
Restriction sites: E, EcoR1; Nt, NotI; Xb, XbaI; H, HindIII; B, BamHI; S, SalI; P, PstI; Bg, BglII.
Reprinted with permission from Vzquez ME, Cebolla A, Palomares, AJ. FEMS Microbiol Lett
1994; 121:11-18.
78
was inoculated into microcosms containing autoclaved or nonautoclaved soil after adjustment of matric potential to -30, -750 or -1500 kPa. Selective viable cell enumeration was
achieved by plate counting on media containing kanamycin and population activity was
measured by luminometry, radiorespirometry and dehydrogenase activity. Matric potential
did not significantly affect survival, as determined by plate counts, but survival was significantly greater in autoclaved soil at higher matric potentials. This may be due to reduced
competition and predation, as protozoan movement will be lower at the lowest matric potential. Luminescence activity decreased rapidly following inoculation, and at a greater rate
than viable cell counts, and was the most sensitive indicator of effects of matric potential
and autoclaving. It was also found to be significantly more sensitive, reproducible and convenient as a measure of population activity than traditional techniques of radiorespirometry
and dehydrogenase activity in autoclaved soil. More importantly, in nonautoclaved soil,
luminescence provided a measure of the marked organism only in the presence of a significant indigenous microbial population. Potential luminescence involved assessment of luminescence during incubation with nutrients and indicated the speed of recovery of starved
organisms. Early samples showed no increase in luminescence during the incubation period at the highest matric potentials, indicating little inactivation immediately following
inoculation, but significant increases were observed during incubation of samples from soil
at the lowest matric potential. The rate of recovery and final potential luminescence values
decreased with time spent by cells in the soil and with decreasing matric potential, and were
lowest in nonautoclaved soil. Samples taken from nonautoclaved soil at the lowest matric
potential after 28 days showed no recovery. This suggests that P. fluorescens, inoculated into
soil at this matric potential, would have negligible environmental impact within four weeks
of inoculation, as any available substrates would be rapidly utilized by competing indigenous organisms. Conversely, the lack of persistence of activity of this organism might question its suitability in performing the function for which it was originally designed. A combination of viable cell enumeration, luminescence and potential luminescence measurements
therefore provides information which is of relevance and importance to both risk assessment and the development of more efficient microbial inocula.
5.5.5. Predation
The ability to detect luminescence activity of specific marked strains has been exploited
to determine the effect of predation by protozoa on bacterial prey.48 Soil microcosms were
inoculated with lux-marked bacterial cells (P. fluorescens) at specific matric potentials to
ensure their location in small (neck size < 6 m) or intermediate size (neck size 6-30 m)
pores, while Colpoda steinii was introduced to large pores (neck size 30-60 m) (Fig. 5.6).
Viable cell concentrations of both populations were monitored and bacterial activity was
assessed by luminometry. Bacteria in intermediate pores were predated at a greater rate
than those introduced to small pores, and decreases in bacterial viable cell concentration
were associated with increases in the ciliate population. Small pores therefore appear to
provide protection from predation. Luminescence decreased, following inoculation, due to
loss of bacterial activity through lack of nutrients and this effect was greater in the presence
of the ciliate, due to predation. However, luminescence activity per cell was significantly
greater for cells introduced into intermediate-sized pores, where predation was greatest.
This suggests that predation, although reducing numbers of bacteria, results in the release
and turnover of nutrients by the ciliate such that surviving bacterial cells have greater activity. Demonstration of this effect using traditional activity techniques was not possible because of problems in distinguishing between bacterial and ciliate activity.
80
5.5.6. Biosensors
Use of living cells as biosensors offers several advantages over enzyme-based or other
biosensors. Most importantly, analytical systems that require a sequence of biochemical
reactions are greatly simplified by using cells within which all the reactions are conveniently
packaged, and carried out efficiently in an optimal environment. In addition, production of
bacterial-based biosensors is easily scaled-up, as only minor down-stream processing is
needed after bacterial cultivation.
Luminescence-based microbial biosensors for total toxicity have been developed and
commercialized using naturally luminescent bacteria 49 and measure toxicity as quenching
of luminescence following exposure of cells to a sample. Response times are short, ranging
from few seconds50 to tens of minutes and operation is simple and does not require highly
trained personnel or expensive instrumentation. Use of recombinant luminescent strains
for general toxicity testing is now under evaluation for commercial products.51 One
example is the assessment of inhibition of protein synthesis by strains of E. coli marked with
prokaryotic or eukaryotic luciferase, using the strong bacteriophage leftward promoter.52
This provides several-hundred-fold induction in less than ten minutes and incubation prior to
and after induction of protein synthesis enables distinction between effects on protein synthesis and direct metabolic inhibition. The test can be performed with lyophilized bacteria in less
than an hour and no bacterial cultivation is required, making it suitable for rapid and extremely sensitive detection of environmental samples.
82
5.7. Conclusion
All molecular marker systems enable the detection of tagged organisms through gene
probing and viable cell enumeration. The major additional advantages offered by luminescence markers lie in the ability to detect light emission by marked organisms. This provides
a degree of selective enumeration by colony counting, but is particularly valuable as a means
of assessing real time, in situ metabolic activity of marked organisms, for instance in studies
of spatial organization of organisms against background indigenous populations. Both
prokaryotic and eukaryotic systems may be exploited and a range of vectors for each is
available. Luminescence genes have also been used for assessment of general metabolic activity and as reporters of specific gene function. A range of relatively inexpensive methods is
available for detection and luminescence-based markers have been used to study a variety
of organisms and environments. The approach has particular advantages for monitoring of
genetically engineered microorganisms released into the environment and, increasingly, in
biosensor applications, and represents a powerful technique for fundamental studies of
microbial ecology.
Acknowledgments
MK acknowledges support from Maj and Tor Nessling Foundation. AJP acknowledges
support to Spanish Ministry of Education, Junta de Andalucia and Fundacin Ramn Areces.
All authors are members of the MAREP Concerted Action sponsored by the European Commission Biotechnology Programme, DGXII.
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number determination. Plasmid 1994; 32:336-341.
38. Amin-Hanjani S, Meikle A, Glover LA et al. Plasmid and chromosomally encoded luminescence marker systems for detection of Pseudomonas fluorescens in soil. Mol Ecol 1993;
2:47-54.
39. Meikle A, Killham K, Prosser JI et al. Luminometric measurement of population activity
of genetically modified Pseudomonas fluorescens in the soil. FEMS Microbiol Lett 1992;
99:217-220.
40. Duncan S, Glover LA, Killham K et al. Luminescence-based detection of activity of starved
and viable but nonculturable bacteria. Appl Environ Microbiol 1994; 60:1308-1316.
41. Meikle A, Glover LA, Killham K et al. Potential luminescence as an indicator of activation
of genetically modified Pseudomonas fluorescens in liquid culture and in soil. Soil Biol
Biochem 1994; 26:747-755.
42. Langridge WHR, Fitzgerald KJ, Koncz C et al. Dual promoter of Agrobacterium tumefaciens
mannopine synthase genes is regulated by plant growth hormones. Proc Natl Acad Sci
USA 1989; 86:3219-3223.
43. OKane DJ, Lingle WL, Wampler JE et al. Visualization of bioluminescence as a marker of
gene expression in Rhizobium infected soybean root nodules. Pl Mol Biol 1988; 10:387-399.
44. Herrero M, de Lorenzo V, Timmis K. Transposon vectors containing non antibiotic resistance selection markers for cloning and stable chromosomal insertion of cloned genes in
gram-negative bacteria. J Bacteriol 1990; 172:6557-6567.
45. Palomares AJ, Vzquez ME, Cebolla A. Determination of Rhizobium nodule occupancy by
luminescence. In: Hasting JW, Kricka LJ, Stanley PE eds. Bioluminescence and Chemiluminescence. Molecular Reporting with Photons. John Wiley and Sons, New York. 1997:
383-386.
46. Vzquez ME, Palomares AJ. Eukaryotic luciferase markers as monitoring methods for plasmid transfer analysis in soil environment In: Hasting JW, Kricka LJ, Stanley PE eds. Bioluminescence and Chemiluminescence. Molecular Reporting with Photons. John Wiley and
Sons, New York. 1997:387-390.
47. Meikle A, Amin-Hanjani S, Glover LA et al. The effect of matric potential on survival and
activity of a Pseudomonas fluorescens inoculum in soil. Soil Biol Biochem 1995; 27:881-892.
48. Wright D, Killham K, Glover LA et al. The role of pore size location in determining bacterial activity during protozoal predation. Appl Environ Microbiol 1995; 61:3537-3543.
49. Ribo JM, Kaiser KLE. Photobacterium phosphoreum, toxicity bioassay. Tox Assessm 1987;
2:305-323.
50. Lappalainen J, Juvonen R, Vaajasaari K et al. A new flash method for measuring the toxicity of solid and colored samples. 1998; Chemosphere:38,1069-1083.
51. Lampinen J, Virta M, Karp M. Use of controlled luciferase expression for monitoring of
chemicals affecting protein synthesis. Appl Environ Microbiol 1995; 61:2981-2989.
52. Lampinen, J, Virta M, Karp, M. Comparison of gram-negative and grampositive bacterial
strains cloned with different types of luciferase genes in bioluminescence cytotoxicity tests.
Environ Toxicol Wat Qual 1995; 10:157-166.
53. OHalloran TV. Transition metals in control of gene expression. Science 1993; 261:715-725.
54. Silver S, Walderhaug M. Gene regulation of plasmid- and chromosome-determined inorganic ion transport in bacteria. Microbiol Rev 1992; 56:195-228.
55. Summers AO. Organization, expression, and evolution of genes for mercury resistance.
Ann Rev Microbiol 1986; 40:607-34.
56. Silver SK, Budd KM, Leahy WV et al. Inducible plasmid-determined resistance to arsenate,
arsenite and antimony(III) in Escherichia coli and Staphylococcus aureus. J Bacteriol 1981;
146:983-996.
CHAPTER 6
6.1. Introduction
he most important criterion for an ideal marker/reporter system for rhizosphere bacteria is the absence of endogenous activity in the microbe of interest and in its natural
environment. Other criteria include inexpensive use of substrates and/or material necessary to detect the marker, the availability of simple though sensitive, nondestructive, and
histochemical assays to detect the strain or gene activity in planta, and unaffected fitness of
the host after introduction of the marker.1
Although lacZ,2 encoding -galactosidase, and phoA,3 encoding alkaline phosphatase,
are well suited as gene fusion markers and have been used in plant-interacting bacteria, a
high background in rhizosphere bacteria and higher plants often restricts their use in in
planta studies. Luciferase activity can be detected using sensitive and nondestructive assays
but needs costly materials and relies on the presence of oxygen and reduced cofactors in the
host cell.4 The major advantage of the bioluminescent system is the total absence of activity
in the plant-microbe background (see Chapter 5).5 The InaZ reporter (bacterial ice nucleation protein) does not need substrates nor sample processing for detection.6 Its major
advantage is a logarithmic relationship between ice nucleation activity and InaZ protein
concentration. However, it remains difficult to relate reporter activity to actual promoter
strength or gene transcriptional activity. The xylE gene product, catechol-2,3-dioxygenase,7
is detected by means of a soluble compound and therefore not suitable to detect bacteria
attached to plant tissue.8 Recently, the gfp gene, encoding green fluorescent protein, came
into use as a reporter gene in diverse biological systems (see Chapter 7).9
The E. coli gusA gene (previously also named uidA), encoding -glucuronidase, was
first used as a reporter gene in plants.10,11 It is currently being used in a wide variety of
biological systems, including plant-interacting bacteria. GUS activity is not present in most
higher plants12 and in bacteria of agricultural importance such as Meso-, Sino-, Azo-, Brady
Rhizobium, Agrobacterium, Azospirillum and Pseudomonas.13 In this chapter, the GUS
reporter system and its use to study bacterium-plant interactions are reviewed.
Tracking Genetically-Engineered Microorganisms, edited by Janet K. Jansson, Jan Dirk van Elsas,
Mark J. Bailey. 2000 EUREKAH.COM.
88
89
Table 6.1. Characteristics of Escherichia coli -glucuronidase (see text for ref.)
Characteristics
Description
Substrates
Exo-hydrolase
Molecular weight
pH range
Cofactors
Thermal stability
Special requirements
thermal inactivation with a half-life of two hours at 55C and about 15 minutes at 60C.
GUS is inhibited by some heavy divalent metal ions such as Cu2+ and Zn2+.11 Physical and
enzymatic characteristics of GUS are summarized in Table 6.1.
The enzyme can tolerate large amino-terminal additions for translational fusions. It is
not processed at the amino-terminus in E. coli and is found exclusively in the cytoplasm. It
is not subject to any post-transcriptional modification.
90
91
Chemical Name
Color of product
Company
X-GlcA
5-bromo-4-chloro-3indolyl--D-glucuronide
6-bromo-2-hydroxy-3naphthoyl-O-anisidine-D-glucuronide
5-bromo-6-chloro-3indolyl--D-glucuronide
6-chloro-3-indolyl-D-glucuronide
Indoxyl--D-glucuronide
6-chloro-3-indlyl--Dglucuronide
Blue
Naphtol ASBIglucuronide
Magenta-glcA
Salmon-glcA
Indoxyl-glcA
Red-Gluc
Depends on
choice of
coupling dye
Magenta
Biosynth AG
Salmon pink
Biosynth AG
Indigo blue
Red
Biosynth AG
Research Organics Inc.
92
Description
Properties
Ref.
Tn5-gusA1
Promoter probe
transposon
23
Tn5-gusA2
Promoter probe
transposon
gusA-expressing
transposon
gusA-expressing
transposon
mTn5SsgusA20
Tn5SSgusA21
mTn5SsgusA10
gusA-expressing
transposon
mTn5SsgusA11
gusA-expressing
transposon
gusA-expressing
transposon
mTn5SsgusA30
mTn5SsgusA31
gusA-expressing
transposon
mTn5SsgusA40
Promoter probe
transposon
GUS expression
vector
pRAJ289
pRAJ294
pKW117
pKW118
pKW119
pRG960
pRG970
pFAJ31
pWM2 (uidA1)
GUS expression
vector
Translation fusion
vectors
Broad host range
and promoter
selection cosmid
23
25
25
25
25
25
25
25
25
25
25
29
29
31
28
93
Name
Description
Properties
pWM3 (uidA2)
contains gusA
cassette for generating transcriptional fusions
contains gusA
cassette for generating transcriptional fusions
contains gusA
cassette for generating transcriptional fusions
contains gusA
cassette for generating transcriptional fusions
Promoter-probe
transposon
Promoter-probe
transposon
28
28
28
28
pWM4
(uidA2-cat)
pWM5
(uidA2-aadA)
pWM6
(uidA2-aph)
mTn5gfp-pgusA
mTn5gusA-pgfp11
mTn5gusA-pgfp12
mTn5gusA-pgfp21
mTn5gusA-pgfp22
Promoter probe
transposon
Promoter probe
transposon
Promoter probe
transposon
Ref.
Several researchers constructed reporter vectors based on gusA and lacZ, facilitating the
analysis of bidirectional promoter regions.29,30 All of these constructs can be used to monitor free-living populations of bacteria in soil and to monitor the colonization of plant roots
by bacteria. Additionally, a number of constructs have been specifically developed to monitor the colonization of plant roots by Azospirillum31,32 and Azoarcus.33
Recently, a series of promoter-probe transposons, mTn5gusA-pgfp, containing a
promoterless gusA gene and one or two constitutively expressed gfp genes has been constructed and found to be useful for gene expression studies in Rhizobium, Azospirillum and
Pseudomonas.34
94
spatial and temporal expression of the symbiotic genes were observed. The nod genes,
involved in nodule formation, are expressed in an early phase (from 36 hours after inoculation) on the root surface and nodule cortex and later (from 10 days after inoculation) especially at the nodule meristem. In contrast, the nif (encoding nitrogenase), fix (required for
nitrogen fixation) and syrM (a regulator of other symbiotic genes) are expressed 10 days
after inoculation in the active symbiotic zone of the alfalfa nodules spreading off to the rest
of the central region of the nodule. For histochemical assay of -glucuronidase, nodules
were embedded, fixed and stained with 20 g/ml 1-GlcA.
The transposons described by Wilson et al 25 were designed to monitor population
changes in the soil and the rhizosphere and to determine nodule occupancy in rhizobial
competition studies. All assay parameters to study rhizobial infection and nodule occupancy were optimized. The ideal concentration of the histochemical substrate X-Glc was
100 g/ml. Elevating the substrate concentrations to 250 or 500 g/ml elicited intrinsic and
inducible GUS activity from other soil bacteria. Rhizobial cells, marked with mTn5SSgusA10,
mTn5SSgusA11, mTn5SSgusA20 or mTn5SSgusA21, could be visualized especially on young
nodules. Rhizobia marked with mTn5SSgusA30 or mTn5SSgusA31, containing gusA expressed from a nifH promoter, could be visualized in older nodules with maximal expression in the central, nitrogen-fixing zone. In Figure 6.2, an X-GlcA stained section of an
alfalfa root nodule, infected with R. meliloti containing a nifH-gusA translational fusion, is
shown.35
Streit et al36 used a GUS+ strain of R. leguminosarum bv. phaseoli strain KIM5s to test
the nodulation competitiveness of 17 Rhizobium leguminosarum bv. phaseoli and 3 R. tropici
strains. They found that strains which could use simple aromatic compounds were generally more competitive. Kalinowski and Long37 used fusions between deletion derivatives of
a selected nod gene and gusA to monitor expression levels in free-living conditions and in
planta.
95
Fig. 6.2. Section of an X-GlucA
stained alfalfa nodule, infected
with R. meliloti (nifH-gusA)
ally revealed the presence of azospirilla in the epidermis and cortex layers. No bacterial
penetration through the endodermis or vascular tissue was observed.
In a second study, the colonization of wheat roots by a selection of A. brasilense
mutants impaired in motility, chemotaxis and plant root attachment, carrying
pFAJ31.13, was evaluated. Two nonmotile and a generally nonchemotactic mutant were
found to be impaired in the primary colonization of the wheat roots. It can be
hypothesized, that key compounds are specifically secreted by the root hair zone of
the wheat roots to which Azospirillum is attracted.40,41
The expression of a translational A. brasilense nifH-gusA fusion was studied in
free-living conditions and during wheat root association.31 Free-living nifH expression was shown to occur only under nitrogen-limiting microaerobic conditions, suggesting the presence of nitrogen and oxygen-dependent control mechanisms for nif
gene expression in Azospirillum. Analysis of nifH gene expression and nitrogenase
activity during the Azospirillum-wheat root association indicated that both oxygen
and the availability of carbon sources are limiting factors for associative nitrogenfixation.
96
Fig. 6.3. Primary colonization of the wheat hair root zone by Azospirillum brasilense Sp245
(pFAJ31.13).
97
gene cluster could be deduced. Since levels of expression were similar in minimal medium
and during the early stages of interaction with tobacco cells, it was suggested that nutritional conditions, rather than a particular plant factor, affect expression of the hrp genes in
P. syringae pv. syringae.
6.5 Conclusion
The GUS system is a precise and robust reporter for gene expression, allowing transcriptional and translational fusions between the gene of interest and the gusA gene. The
main advantages of GUS are the absence of endogenous activity in plants, the existence of a
large number of commercially available substrates and the number of constructs facilitating the use of the E. coli gusA gene.
GUS has been used both as a reporter and marker in studies with rhizosphere bacteria,
e.g., the Rhizobium-legume symbiosis, PGPR, endophytic and phytopathogenic bacteria.
Histochemical assays allow the assessment of the localization of GUS marked rhizosphere
bacteria on plant roots. Quantitative assays exist to count the number of GUS marked bacteria in the rhizosphere.
GUS-marked recombinant Azospirillum strains have been used in field release experiments in Italy in 1994 and 1995. The gusA gene, under the control of the nptII promoter,
was inserted in the genome of Azospirillum brasilense (wild-type and strains altered in auxin
biosynthesis). The engineered strains were used for inoculation of sweet sorghum by
Agronomica (Italy) in collaboration with the University of Padua, in the framework of an
EU project. The GUS marker allowed monitoring of survival and spread of the recombinant bacteria in soil samples with a detection limit of 102 CFUs of GUS-marked strains per
gram soil.
Acknowledgments
M.L. and A.V.B. are recipients of a predoctoral and postdoctoral fellowship of the Fund
for Scientific Research, Flanders, respectively. J. Vanderleyden is a member of the MAREP
Concerted Action sponsored by the European Commission Biotechnology Programme,
DGXII.
References
1. Wilson KJ. Molecular techniques for the study of rhizobial ecology in the field. Soil Biol
Biochem 1995; 27:501-514.
2. Silhavy TJ, Beckwith JR. Uses of lac fusions for the study of biological problems. Microbiol
Rev 1985; 49:398-418.
3. Reuber TL, Long, SL, Walker GC. Regulation of Rhizobium meliloti exo genes in free-living
cells and in planta examined using TnphoA fusions. J Bacteriol 1991; 173:426-434.
4. de Weger L, Dekkers LC, van der Bij AJ et al. Use of bioluminescence markers to detect
Pseudomonas spp. in the rhizosphere. Appl Environ Microbiol 1991; 57:3641-3644.
5. Cebolla A, Ruiz-Berrasuero F, Palomares AJ. Stable tagging of Rhizobium meliloti with the
firefly luciferase gene for environmental monitoring. Appl Environ Microbiol 1993; 59:25112519.
6. Lindgren PB, Frederic R, Govindarajan AG. An ice nucleation gene reporter gene system
identification of inducible pathogenicity genes in Pseudomonas syringae pv. phaseolicola.
EMBO J 1989; 8:1291-1301.
7. Winstanley C, Morgan JA, Pickup R et al. Use of a xylE marker gene to monitor survival
of recombinant Pseudomonas populations in lake water by culture on nonselective media.
Appl Environ Microbiol 1991; 57:1905-1913.
8. Buell CR, Anderson AJ. Expression of the aggA locus of Pseudomonas putida in vitro and
in planta as detected by the reporter gene xylE. Mol Plant-Microbe Int 1993; 6(3):331-340.
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9. Chalfie M, Tu Y, Euskirchen G et al. Green fluorescent protein as a marker for gene expression. Science 1994; 263:802-805.
10. Jefferson RA, Kavanagh TA, Bevan MW. GUS fusions : -glucuronidase as a sensitive and
versatile gene fusion marker in higher plants. EMBO J 1987; 6(13):3901-3907.
11. Jefferson RA. Assaying chimeric genes in plants : The GUS gene fusion system. Plant Molecular Biology Reporter 1987; 5:387-405.
12. Hu CH, Chee PP, Chesney RH et al. Intrinsic GUS-like activities in seed plants. Plant Cell
Reports 1990; 9:1-5.
13. Wilson K, Hughes SG, Jefferson RA. The Escherichia coli gus operon : introduction and
expression of the gus operon in E. coli and the occurrence and use of GUS in other bacteria. In : Gallagher SR, ed. GUS protocols. Using the GUS gene as a reporter of gene expression. San Diego : Academic Press, Inc. 1992:7-22.
14. Stoeber F. Etudes des proprits et de la biosynthse de la glucuronidase et de la glucuronide-permease chez Escherichia coli. Thse de Docteur des Sciences 1961; Universit de
Paris, France.
15. Jefferson RA, Burgess SM, Hirsh D. -glucuronidase from Escherichia coli as a gene-fusion
marker. Proc Natl Acad Sci USA 1986; 83:8447-8451.
16. Novel M, Novel G. Regulation of -glucuronidase synthesis in Escherichia coli K-12 : constitutive mutants specifically derepressed for uidA expression. J Bacteriol 1976; 127:406-417.
17. Novel M, Novel G. Regulation of -glucuronidase synthesis in Escherichia coli K-12 : pleiotropic constitutive mutations affecting uxu and uidA expression. J Bacteriol 1976;
127:418-432.
18. Tomasic J, Keglevic D. The kinetics of hydrolysis of synthetic glucuronic esters and glucuronic ethers by bovine liver and Escherichia coli -glucuronidase. Biochem J 1973; 133:789.
19. Taylor CB. Promoter fusion analysis: An insufficient measure of gene expression. The Plant
Cell 1997; 9:273-275.
20. Wilson KJ. gusA as a reporter gene to track microbes. In: Akkermans ADL, van Elsas JD,
de Bruijn FJ, eds. Molecular Microbial Ecology Manual. Dordrecht : The Netherlands, 1995.
21. Naleway JJ. Histochemical, spectrophotometric, and fluorometric GUS substrates. In:
Gallagher, SR, ed. GUS protocols. Using the GUS Gene as a Reporter of Gene Expression.
San Diego ; Academic Press, Inc. 1992: 61-76.
22. Fishman W, Nakajima Y, Anstiss C et al. Napthol AS-BI -D-glucuronidic acid: Its synthesis and suitability as a substrate for -glucuronidase. J Histochem Cytochem 1964;
12:298-305.
23. Sharma SB, Signer ER. Temporal and spatial regulation of the symbiotic genes of Rhizobium meliloti in planta revealed by transposon Tn5-gusA. Genes Dev 1990; 4:344-356.
24. Simon R, Quandt J, Klipp W. New derivatives of transposon Tn5 suitable for mobilization
of replicons, generation of operon fusions and indction of genes in gramnegative bacteria.
Gene 1989; 80:161-169.
25. Wilson KJ, Sessitsch A, Corbo J et al. -glucuronidase (GUS) transposons for ecological
and genetic studies of rhizobia and other gramnegative bacteria. Microbiology 1995;
141:1691-1705.
26. de Lorenzo V, Herrero M, Jakubzik U et al. Mini-Tn5 transposon derivatives for insertion
mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gramnegative
eubacteria. J Bacteriol 1990; 172:6568-6572.
27. Herrero M, de Lorenzo V, Timmis KT. Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosome insertion of foreign genes in
gramnegative bacteria. J Bacteriol 1990; 172:6557-6567.
28. Metcalf WW, Wanner BL. Construction of new -glucuronidase cassettes for makiing transcriptional fusions and their use with new methods for allele replacement. Gene 1993;
129:17-25.
29. Van den Eede G, Deblaere R, Goethals K et al. Broad host range and promoter selection
vectors for bacteria that interact with plants. Mol Plant-Microbe Interact 1992 ; 5(3):228-234.
30. Parry SK, Sharma SB, Terzaghi EA. Construction of a bidirectional promoter probe vector
and its use in analysing nod gene expression in Rhizobium loti. Gene 1994; 150:105-109.
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31. Vande Broek A, Michiels J, Van Gool A et al. Spatial-temporal colonization patterns of
Azospirillum brasilense on the wheat root surface and expression of the bacterial nifH gene
during association. Mol Plant-Microbe Interact 1993; 2:261-266.
32. Christiansen-Weniger C, Vanderleyden J. Ammonium-excreting Azospirillum sp. become
intracellularly established in maize (Zea mays) para-nodules. Biol Fertil Soils 1993; 17:1-8.
33. Hurek T, Reinhold-Hurek B, van Montagu M et al. Root colonization and systemic spreading of Azoarcus sp. strain BH72 in grasses. J Bacteriol 1994; 176:1913-1923.
34. Xi C, Lambrecht M, Vanderleyden J et al. Bi-functional gfp-and gusA containing mini-Tn5
transposon derivatives for combined gene expression and bacterial localization studies. J
Microbiol Meth 1999; 35:85-92.
35. Vande Broek A, Michiels J, de Faria SM et al. Transcription of the Azospirillum brasilense
nifH gene is positively regulated by NifA and NtrA and is negatively controlled by the
cellular nitrogen status. Mol Gen Genet 1992; 232:279-283.
36. Streit W, Kosch K, Werner D. Nodulation competitiveness of Rhizobium leguminosarum
bv. phaseoli and Rhizobium tropici strains measured by glucuronidase (gus) gene fusion.
Biol Fertil Soils 1992; 14:140-144.
37. Kalinowski G, Long SR. Deletion analysis of the 5 untranslated region of the Rhizobium
meliloti nodF gene. Mol Plant-Microbe Interact 1996; 9:869-873.
38. Beijerinck MW. ber ein Spirillum, welche freien Stikstoff binden kann ? Zentralbl Bakteriol
Parasitenkd Infektionskr Abt 1925; 63:353.
39. Okon Y, Vanderleyden J Root-associated Azospirillum species can stimulate plants. ASM
News 1997; 63:364-370.
40. Vande Broek A, Vanderleyden J. The role of bacterial motility, chemotaxis, and attachment in bacteria-plant interactions. Mol Plant-Microbe Interact 1995; 8:800-810.
41. Vande Broek A, Lambrecht M, Vanderleyden J. Bacterial chemotactic motility is important
for the initiation of wheat root colonization by Azospirillum brasilense. Microbiology 1998;
144:2599-2606.
42. Reinhold-Hurek B, Hurek T, Gillis M et al. Azoarcus gen. nov., nitrogen-fixing
proteobacteria associated with roots of Kallar grass (Leptochloa fusca (L.) Kunth), and
description of two species, Azoarcus indigens sp. nov., and Azoarcus communis sp. nov. Int
J Syst Bacteriol 1993; 43:574-584.
43. Smith SG, Greer Wilson TJ, Maxwell Dow J et al. A gene for superoxide dismutase from
Xanthomonas campestris pv. campestris and its expression during bacterial plant interactions. Mol Plant-Microbe Interact 1996 ; 9:584-593.
44. Xiao Y, Lu Y, Heu S et al. Organization and environmental regulation of the Pseudomonas
syringae pv. syringae 61 hrp cluster. J Bacteriol 1992; 174:1734-1741.
CHAPTER 7
7.1 Introduction
ince the cloning of the Green Fluorescent Protein (GFP) gene from the jellyfish Aequorea
victoria,1 and its expression in other organisms,2 GFP has rapidly become an important
biomarker/bioreporter in a wide variety of eukaryotic and prokaryotic organisms. The utility of GFP as a bioreporter/biomarker lies mainly in the simplicity of the biochemical reaction generating fluorescence. GFP expression and fluorescence does not depend on the
addition of co-factors or additional substrates, and only requires oxygen briefly for the autooxidation of GFP. When GFP is excited with light of the proper wavelength it autofluoresces.
Visualization of this fluorescence can be used to monitor gene expression, to localize proteins, to isolate novel genes, or to track tagged cells (For reviews see refs. 3-9). Because of
these advantages, GFP is replacing some of the traditional biomarkers/bioreporters used in
molecular microbial ecology, such as metabolic markers or luciferase markers which
require substrate addition and cellular energy reserves for visualization (For reviews see
refs. 6, 10, 11).
102
103
Table 7.1. GFP mutants. List of GFP variants giving the mutant name, amino acid
changes, excitation wavelength, emission wavelength, and relative intensity to
wtGFP
GFP name
Residues mutatated
Excitation
(nm)a
Emission
(nm)b
Relative
intensityc
wtGFP
None
395/470
509
H9
S202F
398
511
1.17
16
P9
I167V
471/396
502
1.66
16
P11
I167T
471/396
502
1.88
16
P4
Y66H
382
448
0.57
16
Y66W
458
480
ND
16
S65-T
S65T
490
510
20
Cycle 3
F100S, M145T,
V164A
385
510
18
25
RSGFP4
490
505
ND
22
GFPmut1
(EGFP)
F64L, S65T
488
507
30-50
24
GFPmut2
481
507
80-100
24
GFPmut3
S65G, S72A
501
511
70-80
24
GFPT203I
T203I
400
512
ND
23
GFPE222G
E222G
481
506
ND
23
GFPmut3*
gfp(AAV,
LVA, LAA,
or ASV)
S2R, S65G,S72A
GFPmut3* with
instability tail added
501
501
511
511
70-80
70-80
26
26
S65A
S65A
ND
505
70
S65G
S65G
ND
513
70
P4-3
Y66H, Y145F
381
445
21
W7
Y66W, N146I,
M153T, V163A,
N212K
433
475
21
V2
Y66W, I123V,
Y145H, H148R,
M153T, V163A,
N212K
432
480
21
P4-1
S65T, M153A,
K238E
504
514
ND
21
Ref.
104
and GFP222G, Table 7.1) were created using a strain of E. coli deficient in DNA polymerase
proofreading.23 One mutant was not fluorescent when excited with 395 nm light; the other
mutant was not fluorescent when excited with 470 nm light, although they were fluorescent
when excited at 400 or 481 nm, respectively. Neither of the corresponding mutations mapped
to the chromophore.
In another study, a codon-based mutagenesis scheme was used to mutate amino acid
residues in ositions 55-74 and a FACS machine to isolate red-shifted GFP mutants, mut1,
mut2 and mut3 (Table 7.1) with increased fluorescence intensity (30-100x wtGFP).24 These
authors found that 90-100% of the mutant protein was soluble, which probably contributes
to the enhanced level of emission. The authors also suggested that the mutations may result
in faster chromophore formation and increased 488 nm excitation. Using DNA shuffling, a
non-redshift mutant of GFP was isolated that remains soluble and has emission levels 18
times higher that of wtGFP.25 The different mutants described above have greatly increased
the utility of GFP as a biomarker/bioreporter in bacterial systems.
Variants that change the stability of GFP in bacteria have also been developed. Andersen
et al added short peptide sequences to GFP that cause bacterial proteases to degrade the
protein at different rates, generating GFP variants with shorter half lives.26 These mutant
GFPs allow studies of temporal gene expression.
The Gfp gene has also been specifically mutated to optimize expression in non-bacterial systems. Many of these mutants have codon usage optimized for a particular organism.
For example, GFP mutants have been developed for yeast, plants and mammalian cells (For
reviews see refs. 3,27). Therefore, it is now possible for researchers to select from a number
of GFP variants with different excitation and emission maxima and fluorescence levels (see
Table 7.1). Careful selection of a GFP variant suited for a particular application can maximize the utility of this marker.28
In some cases, there are problems that remain to be overcome, even when using the
more optimized mutant derivatives of GFP. For example, clonal populations of bacteria
expressing mutant derivatives of GFP may sometimes still exhibit variable levels of fluorescence.29 There have also been reports of no detectable fluorescence in bacteria containing
GFP, even when the Gfp gene is controlled by a promoter known to be active in that bacterium. For example, Kremer et al observed blue color in all E. coli cells harboring a plasmid
with the lacZ gene controlled by a heat shock promoter (hsp60).30 However, when the lacZ
gene in this plasmid was replaced with the Gfp gene, no fluorescence was observed. Egener
et al. (1998) observed fluorescence in Azoarcus sp BH72 harboring the gene encoding
GFPmut2 under the control of the nifH promoter.31 However, they could not detect fluorescence in bacteria harboring the genes encoding wtGFP or the P11 mutant GFP expressed
from the same promoter. Bacteria harboring a vector containing a kanamycin resistance
gene and the P11 Gfp gene, both expressed from a PpsbA promoter, were resistant to kanamycin but were not fluorescent (Stoltzfus and de Bruijn, unpublished data). The exact reasons for lack of detectable fluorescence have not been elucidated.
Toxicity of GFP can also be a problem when GFP is expressed at high levels in bacteria.
For example, E. coli cells harboring high copy-number plasmids strongly expressing
GFPmut1were found to be prone to cell lysis.32
However, it is now usually the case that by careful choice of mutant GFP, expression
levels and copy number (for example by chromosomal integration) most if not all of these
problems can be overcome.
105
situ analyses. GFP also allows detection of tagged bacteria regardless of the energy status of
the cells.17,33 A wide variety of detection methods suitable for analysis of bacteria expressing
GFP are available which enables optimization of the experimental design to address multiple relevant biological questions without the compromises sometimes associated with
biomarkers that require special detection protocols. These methods include the following:
1. visualization of bacterial colonies on plates using blue light illumination,
2. visualization of colonies or individual cells using an epifluorescence microscope,
3. visualization of single cells and three dimensional imaging of the pattern of cell
aggregation/colonization in samples using a laser-scanning confocal microscope,
4. visualization of gross aggregation/colonization patterns by tagged bacteria on
samples such as plant tissue using fluorescence stereomicroscopy,
5. counting fluorescent cells using flow cytometry,
6. screening bacterial collections using fluorescent microtiter plate readers, and
7. quantitation of fluorescence in suspensions using spectrofluorimetry. In some cases
the use of image enhancement software may be needed to distinguish the fluorescence of tagged bacteria from background autofluorescence, such as observed in
root or soil samples.17,34 Several reviews detailing these techniques are available (for
reviews see refs. 10,11, 35). An important development has been the development
of a filter set optimized for detection of GFP by epifluorescence microscopy.36
More specialized techniques for observing GFP-mediated fluorescence have also been
described. For example, scanning near-field optical/atomic force microscopy has been used
for determination of the spatial localization of tagged bacteria in liquid,37 and video-endoscopy has been employed to detect GFP expression in situ.38 Specialized software can create
three-dimensional images from optical sections made from samples containing GFP labeled bacteria using a laser scanning confocal microscope. Because GFP detection is noninvasive, real time and time-lapse videos can be used to track cells (or proteins) labeled with
GFP (For a review see 5). Studies using different GFP mutants have been carried out using
double and even triple labeling allowing simultaneous detection of multiple targets in the
same sample.21,39-41
Type of study
Localize Hbsu
Organism
Agrobacterium
tumefaciens A136
Alcaligenes eutrophus
Arthrobacter
chlorophenolicus A6
Azoarcus sp BH72
Bacillus megaterium
Bacillus subtilis
Bacillus subtilis
Bacillus subtilis
Bacillus subtilis
Bacillus subtilis
Bacillus subtilis
Bacillus subtilis
chromosomal
integration
chromosomal
integration
chromosomal
integration
chromosomal
integration
chromosomal
integration
chromosomal
integration
chromosomal
integration
chromosomal
integration
chromosomal
integration
mini-Tn5
mini-Tn5
plasmid
Type of Vector
pDL50B
pSG1141,pSG1147
pPK9C8
pF1
pSG1517
pSG1044
pCW28
pIIE-GFP-T
pBHFN35
pUTgfp
pAG408
pAEG3, pZEG3,
and related plasmids
Vector Name
S65-T
wtGFP
wtGFP
S65-T
S65-T
wtGFP
wtGFP
GFPmut2
P11
Cycle 3
GFPmut2
GFP mutant
64
72
63
60
62
59
58
57
31
46
68
56
Ref.
Table 7.2. GFP Marked Bacteria. List of bacteria in which GFP has been used as a biomarker/bioreporter. The table lists the genus
and species of bacteria used in the study, the type of vector, the name of the vector and the GFP variant carried on the vector
106
Tracking Genetically-Engineered Microorganisms
plasmid
plasmid
Bacillus subtilis
Bacillus subtilis
Escherichia coli
pGFP
pUTgfplux
plasmid
mini-Tn5
plasmid
Moraxella sp.
Mycobacterium avium
plasmid
plasmid
pWES4
pJBA28
pGFP
plasmid
mini-Tn5
pSopB-GFP
pSg20
pGFP
pAEG1, pZEG1,
and related plasmids
pURE-RD-GFP
plasmid
plasmid
plasmid
pK3G
pDR112,pDR123
Escherichia coli 01
Escherichia coli DH5a
plasmid
Escherichia coli
pCH50
pSG1902
pSG1151
Vector Name
Escherichia coli
Escherichia coli
phage
chromosomal
integration
chromosomal
integration
chromosomal
integration
Bacillus subtilis
Type of Vector
Type of study
Organism
wtGFP
GFP mut2
wtGFP
S65-T
wtGFP
GFPmut2
GFPmut2
P11
wtGFP
GFPmut2
GFPmut2
GFPmut2
S65-T
GFPmut1
wtGFP
GFP mutant
52
45
19
67
66
44
56
77
33
18
76
75
55
74
61
65
73
Ref.
Proteus mirabilis
HI4320
Pseudoaltermonas sp.
plasmid
plasmid
plasmid
plasmid
plasmid
plasmid
mini-Tn5
Pseudomonas
chlororaphis MA 342
plasmid
Mycobacterium
marinum
Mycobacterium
smegmatis
Mycobacterium
smegmatis
Mycobacterium bovis
BCG
plasmid
Mycobacterium bovis
BCG
Mycobacterium
smegmatis
plasmid
Mycobacterium bovis
BCG
chromosomal
integration
Mycobacterium avium
Type of Vector
Type of study
Organism
wtGFP
GFP mutant
PUTgfp2
pIVET-GFP
pSMC2
mini-Tn10-gfp-kan
pURE-RD-GFP
pGFM-12
P11
GFPmut3
GFPmut2
GFPmut2
GFPmut2
wtGFP
wtGFP
wtGFP
phsp60-gfp, pMlaphc-gfp,
pMtaphc-gfp, pmtra-gfp,
ptbprc3-gfp
pGFM-11
wtGFP
S65-T
wtGFP
pFPV2
pYL mutGFP
pGFM-11
phsp60-gfp,
wtGFP
pMlaphc-gfp, pMtaphc-gfp,
pmtra-gfp, ptbprc3-gfp
pMV306(hsp 60/gfp)
Vector Name
49
69
50
80
77
30
30
78
79
51
30
78
52
Ref.
108
Tracking Genetically-Engineered Microorganisms
plasmid
pFPV1
TOL-gfp (RP1::GFP)
plasmid
Yersinia
pseudotuberculosis
pFVP25
plasmid
pFPV1
plasmid
pUTgfp
Salmonella typhi
mini-Tn5
Sinorhizobium
meliloti MB501
pTB93F
pAG408
pAEG3, pZEG3,
and related plasmids
plasmid
plasmid
mini-Tn5
TOL-gfp (RP1::GFP)
plasmid
pJBA30, pJBA26
mini-Tn5
Tn5GFP1
pAG408
pRL765gfp
Tn5
derivative
mini-Tn5
Tn5
Pseudomonas sp B13
Sinorhizobium
meliloti 1021
Pseudomonas
Track movement of bacteria through
putida mt-2
a column of sand
Pseudomonas putida R1 Observe gene induction and bacterial
location in a bioreactor
Pseudomonas
Monitor plasmid transfer by conjugation
putida KT2442
pUTgfplux
mini-Tn5
Pseudomonas
fluorescens AS12
Pseudomonas
fluorescens SBW25
Pseudomonas
putida KT2442
TOL::gfpmut3b
plasmid
pGB5
Pseudomonas
fluorescens A506
plasmid
Vector Name
Pseudomonas
fluorescens WCS365
Type of Vector
Type of study
Organism
wtGFP
wtGFP
GFPmut3
wtGFP
wt GFP
GFPmut2
S65-T
Cycle 3
tGFP
GFPmut3b
wtGFP
Cycle 3
P11
GFPmut3b
P11
GFPmut2
GFP mutant
79
18
79
79
47
56
34
68
42
71
19
68
33
43
17
50
Ref.
110
from a constitutive promoter to tag P. fluorescens cells, and showed that the tagged cells
could be detected by flow cytometry, epifluorescent microcopy and laser-scanning confocal
microscopy. 17
In addition, dual tagging experiments using GFP and bacterial luciferase has allowed
simultaneous tracking of tagged bacteria cells and monitoring of their energy status (by
luciferase activity) in soil microcosms.33 Similarly, Arthrobacter chlorophenolicus cells were
tagged with either GFP or the firefly luciferase gene (luc) in order to monitor Arthrobacter
cells during bioremediation of 4-chlorophenol in soil.46 GFP-tagged cells could be enumerated in soil by flow cytometry, after extraction of the bacterial soil fraction by density gradient
centrifugation to remove soil particles and debris that would otherwise interfere with flow
cytometric measurements.33,46 GFP fluorescent A. chlorophenolicus 46 or P. fluorescens 33 cells
could be counted at relatively stable levels after inoculation to soil, compared to counts
based on CFU or luciferase expression, which decreased during incubation. These experiments demonstrate the utility of GFP as a stable marker for counting the total number of
tagged cells in environmental samples, regardless of their metabolic activity or culturability.
However, it is possible that some of the cells that were counted in such experiments are
dead.33,46 Recent studies in our laboratory and in others have shown that viable but nonculturable cells (see Chapter 1) can still retain GFP fluorescence.47,48
GFP can also be used to monitor transfer of plasmids between bacteria. Christensen et
al used a plasmid containing GFP controlled by an inducible promoter, PF10, to monitor
plasmid transfer on semi-solid surfaces.42 The P. putida donor strain did not contain the T7
RNA polymerase gene and therefore the T7 PF10 promoter was inactive and the bacteria
were not fluorescent. The recipient strain carried the T7 RNA polymerase gene integrated
in its chromosome. T7 RNA polymerase activates the PF10 promoter and hence any recipient that captured PF10-GFP became fluorescent. Thus, fluorescence resulted only when the
plasmid was transferred to the recipient strain. A similar system was used with GFP expression regulated by Plac to observe plasmid transfer in the phylloplane of bush beans and on
polycarbonate filters.43
111
Fig. 7.1. Confocal microscope projection of a stack of images of individual GFP-tagged Pseudomonas chlororaphis MA342G2 cells (white spots on this figure) distributed on the outer surface of a
barley seed coat or glume (large cells). A model LSM501 laser scanning confocal microscope
(Carl Zeiss, Jena, Germany) was used. The images are the result of pseudocolor merging of the
output of three channels. Three-dimensional rendering of the stack of images was obtained by
using the software 3D for LSM510, version 1.4 (Carl Zeiss).
confocal microscopy.18 In addition, Mycobacterium bovis cells were marked with GFP to
follow their infection of mice cells using flow cytometry.30,51 The infection of human macrophages and epithelial cells by Mycobacterium avium marked with GFP has also been observed,52 and Valdivia et al used GFP to visualize infection of live mammalian cells by Salmonella typhimurium, Yersinia pseudotuberculosis and Mycobacterium marinum.53
112
The localization of proteins involved in chromosome behavior has also been studied
using GFP. 62-64 Webb et al used a GFP-LacI fusion to localize specific regions of the B. subtilis
chromosome in vegetative and sporulating cells by inserting a lacO cassette at specific sites
in the chromosome.65 The same system was used to compare chromosome and plasmid
behavior during replication of E. coli.66,67
Promoterless GFP constructs can also be used to screen for promoters that are induced
under a specific set of physiological conditions. For example, Kremer et al inserted fragments of the M. bovis genome in front of the Gfp gene and found that 3 to 5% of the chimeric genes showed differing levels of fluorescence on agar plates.30 Suarez et al. (1997)
developed both plasmid and mini-Tn5-based promoterless GFP vectors.68 P. putida,
Pseudomonas sp B13 and Alcaligenes eutrophus were mutated with mini-Tn5::GFP. Up to
5% of the bacteria with chromosomal integration of the mini-Tn5 were fluorescent, displaying varied levels of expression. Moreover, a promoterless GFP vector was developed
that allows rapid selection for active promoters by fusing aspartate -semialdehyde dehydrogenase with GFP and using it as a selectable marker in Asd- strains.69 Another example
of the utility of GFP as a bioreporter is the construction of pGreenscript.70 In this vector
GFP replaces the -galactosidase (lacZ gene) as the marker for insertion of DNA in the
multiple cloning site of the popular cloning vector pBluescript.
Biomarking can be combined with bioreporting to obtain spatial and temporal information on gene expression in situ. For example, a nifH promoter-GFP fusion has been used
to monitor expression of nitrogen-fixing genes in Azoarcus sp. BH72 in soil and on rice
roots.31 Moreover, Mller et al used P. putida cells containing the Gfp gene expressed from
promoters controlling the expression of proteins involved in the biodegradation of toluene
to visualize gene expression in flow chambers and observed the effects of various community members on gene induction.71 These studies demonstrate that key activities of microbial communities in environmental samples can be specifically investigated in situ using
GFP as a reporter.
7.7. Conclusion
The large number of studies employing GFP as a biomarker/bioreporter clearly demonstrate the value and versatility of GFP. GFP is particularly useful for visualization of single
cells or cell aggregates in environmental samples without the need for any substrate addition. Cells tagged with GFP can be monitored by a range of fluorescence detection methods
including flow cytometry and fluorescence microscopy. GFP also can be used to quantitate
the number of cells of a particular cell population, regardless of the energy status of the
cells. This is in contrast to most of the other existing markers available, such as luciferase
markers, that require the cells to be metabolically active to be detected. While problems
with GFP as a biomarker/bioreporter in bacteria have been encountered, many have been
overcome by using mutant versions of GFP, adding appropriate translation initiation sites,
or adding flexible linkers to protein fusions. The many successful studies using GFP and the
continued development of new techniques incorporating GFP will undoubtedly lead to the
use of GFP in more species of bacteria to answer diverse questions in molecular microbial
ecology.
Acknowledgments
Jon R. Stoltzfus and Frans J. de Bruijn gratefully acknowledge support for studies carried out at MSU from the NSF Center for Microbial Ecology (CME; grant no. DEB9120006),
from the International Rice Research Institute (IRRI) and from the Department of Energy
(DOE; grant no. DE-FG02-91ER20021). Janet K. Jansson gratefully acknowledges support
from the Swedish Council for Engineering Science, the Carl Tryggers Foundation the Swed-
113
ish Foundation for Environmental Research and the Swedish Foundation for Strategic Research. Both F.J. de Bruijn and J.K. Jansson are partners of the MAREP Concerted Action
sponsored by the European Commission Biotechnology Programme, DGXII.
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green fluorescent protein as an indicator. Gene 1997; 189(2):159-162.
71. Mller S, Sternberg C, Andersen JB et al. In situ gene expression in mixed-culture biofilms:
evidence of metabolic interactions between community members. Appl Environ Microbiol
1998; 64(2):721-732.
72. Lewis PJ and Errington J. Use of green fluorescent protein for detection of cell-specific
gene expression and subcellular protein localization during sporulation in Bacillus subtilis.
Microbiology 1996; 142(4):733-740.
73. Webb CD, Decatur A, Teleman A et al. Use of green fluorescent protein for visualization
of cell-specific gene expression and subcellular protein localization during sporulation in
Bacillus subtilis. J Bacteriol 1995; 177(20):5906-5911.
74. Hale CA and de Boer PA. Direct binding of FtsZ to ZipA, an essential component of the
septal ring structure that mediates cell division in E. coli. Cell 1997; 88(2):175-185.
75. Raskin DM and de Boer PA. The MinE ring: An FtsZ-independent cell structure required
for selection of the correct division site in E. coli. Cell 1997; 91(5):685-694.
76. Yu XC, Tran AH, Sun Q et al. Localization of cell division protein FtsK to the Escherichia
coli septum and identification of a potential N-terminal targeting domain. J Bacteriol 1998;
180(5):1296-1304.
77. Zhao H, Thompson RB, Lockatell V et al. Use of green fluorescent protein to assess urease
gene expression by uropathogenic Proteus mirabilis during experimental ascending urinary
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78. Dhandayuthapani S, Via LE, Thomas CA et al. Green fluorescent protein as a marker for
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79. Valdivia RH and Falkow S. Bacterial genetics by flow cytometry: Rapid isolation of Salmonella typhimurium acid-inducible promoters by differential fluorescence induction. Mol
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64:2554-2559.
CHAPTER 8
8.1. Introduction
he selection of Genetically Modified Microorganisms (GMMs), or other candidate bacteria, for environmental release should be carefully considered.1 This is particularly
relevant when developing inocula with known functional traits, such as for biological control, Since it is likely that the most effective inocula derive from the intended target habitat.2,3
However, prior to the field release of a GMM, it remains essential to study the organisms
autecology using enclosed facilities. Of particular importance in such studies is the capacity
of the GMM to persist and cause an impact, features that are dependent on fitness and
competitive ability.4-6 To some extent, these factors can be predicted from knowledge of
closely related, indigenous populations.7 Data from our own releases in glasshouse and field
environments confirm those of previous investigations, i.e., any impacts caused by GMMs
are likely to be short lived and probably no more ecologically significant8-10 than those caused
by wild type bacterial inocula.11,12 It is worthwhile noting that the apparent competitiveness of the GMM used in our studies may have been exaggerated by the artificiality of glasshouse conditions.13 We observed that bacterial diversity in the phyllosphere of glasshouse
plants was in most cases significantly lower than that detected in the field. The calculated
Shannon diversity index (H) for the community colonizing immature leaves of 133 day old
glasshouse grown sugar beet plants was 0.04 (4 taxa) compared to 1.114 (20 taxa) for field
grown plants grown for approximately the same period. This study demonstrated that the
behavior of GMMs in protected environments such as glasshouses is unlikely to be representative of the environment and can only be addressed by field studies.
The aims of the investigations outlined below have been to provide appropriate information for the assessment of impact and survival of GMMs in the environment. We have undertaken a number of investigations, following a stepwise approach, to confirm the suitability of
the candidate bacterium Pseudomonas fluorescens SBW25, and its genetic modification.14 These
studies have been divided into three phases comprised of:
Tracking Genetically-Engineered Microorganisms, edited by Janet K. Jansson, Jan Dirk van Elsas,
Mark J. Bailey. 2000 EUREKAH.COM.
118
119
Table 8.1. Percentage of total colony forming units, growing on tryptic soy broth
agar, identified as P. fluorescens SBW25EeZY6KX isolated from the phytosphere
of glasshouse grown plants
Days after Sugar beet plant habitat sampled after seed inoculation with ca. 1 x 107 cfu GMM
sowing
29
50
64
71
92
103
126
133
190
220
231
531
Immature leaf
Mature leaf
Rhizosphere
Rhizoplane
Root cortex
18.6
64.4
-x
79.0
65.0
54.6
80.7
65.3
38.1
94.1
65.3
22.8
36.4
3.7
10.0
11.9
0.03
22.6
0.07
0.01
0.30
0.02
0.40
0.05
11.8
nd
Notes, nd = no GMMs detected (limit of detection 100 cfu g-1), = not determined. Total counts
estimated by serial dilution of plant tissue homogenates spread onto Trypic Soy broth agar; GMM
counts confirmed Pseudomonad selective agar (Oxoid) supplemented with 100 g ml-1
kanamycin, and 0.01% X-gal, method and table after Thompson IP, Ellis RJ, Bailey MJ. FEMS
Microbiol Ecol 1995; 17:1-14.
120
The GMM was applied as a seed dressing (7 x 106 cfu seed-1) to commercially pelleted
sugar beet (Beta vulgaris var. Amethyst) in April 1993.15 The field plots were planted in 10
by 10 row arrays surrounded by three rows of untreated plants in Evesham series clay soil at
the University Farm, Wytham, UK. Nine individual 5 m2 plots of sugar beet were established. Following a Latin square design three plots were planted with untreated seeds, three
with seeds inoculated with wild type P. fluorescens SBW25, and the remaining plots planted
with seed inoculated with P. fluorescens SBW25EeZY6KX. The survival and persistence of
the organism, together with its dispersal, were monitored until the crop was harvested in
January 1994. A number of plants were left in each plot so that the persistence of the GMM
on over-wintered sugar beet could be determined. Triplicate samples of soil and weed plants
were also collected on a regular basis. Leaves were removed from over-wintering sugar beet
plants in early March and were sampled further as new tissue developed. In April 1994 and
April 1995, untreated sugar beet seeds were sown in half the area of each of the plots and
monitored for colonization by the GMM. The presence of the GMM on the leaves and roots
of indigenous volunteer weed species was also investigated. Weed plant samples which included Creeping Buttercup (Ranunculus repens L.), Fat hen (Chenopodium album L.), Knot
grass (Polygonum avicular L.) and Thistle (Cardus teniflorus Curt.) were excavated from the
ground (Table 8.2). Root and leaf samples were examined for the presence of recombinants
as described for sugar beet samples. The GMM persisted very poorly in bulk soil and was
not detected in samples collected over the winter period following release to March 1994
(Table 8.2). The GMM (1.9 x 102 cfu g-1) was isolated from only one soil sample (October
1994) from all of the bimonthly samples collected from each plot between March 1994 until
December 1996 (n= 144).
Leaf
Root Cortex
Rhizoplane
Rhizosphere soil
Secondary leaf
Immature leaf
Mature leaf
Senescent leaf
Rhizoplane
Rhizosphere soil
Leaf
Root
Bulk soil
*
*
*
*
*
*
*
*
*
March 94
+b
++c
*
*
*
*
*
*
*
*
May 94
*
*
*
*
*
*
*
*
+
*
June 94
*
*
*
*
*
*
+
-
August 94
*
*
*
*
*
+
+
October 94
*
*
*
*
*
-
December 94
*
*
*
*
*
-
March 95
isolated from any sugar beet planted in April 1995, or from weed or bulk soil sample collected April 1995 - December 1996. The bacterium, P. fluorescens
SBW25 was isolated from the leaf surface of sugar beet grown at the field site, Wytham UK, and by site directed homologous recombination was
chromosomally marked, one locus carrying lacZY (lactose utilisation and ability to turn X-gal blue) and at another locus with aph-xylE (kanamycin resistance
and the ability to turn catechol yellow). In combination these novel phenotypes provided unique and highly sensitive isolation and molecular methods for strain
and marker gene detection.
Except for weeds, triplicate samples were taken. Limits of detection 5 cfu g-1
aWeeds: Creeping Buttercup (Ranunculus repens L.), Fat hen (Chenopodium album L.,) Knotgrass (Polygonum aviculare L.) and Thistle (Carduus tenuiflorus
Curt.). - No GMM detected in any replicate. *Not determined. bGMM detected in one replicate. cGMM detected in more than one replicate. GMM was not
Weeds a
Habitat
Plant
Table 8.2. Detection of a GMM, Pseudomonas fluorescens SBW25EeZY6KX, in the sugar beet release plots Wytham, 1994.
Inocula applied once as a seed dressing April 1993.
122
taken from replanted sugar beet leaves or roots, volunteer weeds or soil samples collected in
1995 or 1996 (Table 8.2).
123
tions would have identified mutants or indicated the selection of transconjugants as the
kanamycin resistance-catechol 2,3 dioxygenase genes were linked and inserted at the same
chromosomal site in P. fluorescens SBW25EeZY6KX.
Small populations of the GMM probably survived under particular conditions as colony
forming units were detected at highly variable densities on a few samples collected from
new plant growth and rhizosphere soil samples. However these populations were transient
and failed to become established. The loss of the inocula in a viable state from the release
site was confirmed in all subsequent surveys conducted in 1995 and 1996. The absence of
the introduced bacteria, as determined by plate counts assay and PCR screening of total
extracted soil DNA,14,24 or any evidence for gene transfer, confirms a lack of sustained activity in the environment. In the absence of activity it can be assumed that negligible or no risk
occurred to the environment as a result of these investigations.
So what are the risks associated with GMMs entering the environment? Should they be
considered more or less hazardous than the current usage of chemical and biological
reagents? To make such assessments, we must provide data drawn from a sound basic scientific understanding, which addresses concerns, real or potential, from which accurate
predictions can be made in order to gain public confidence and (if possible) identify
and quantify hazards.
8.8. Conclusion
Irrespective of the origin of the inoculum, the introduction of a large number of
organisms to any habitat is likely to have some effect (impact), even if only short term. This
inoculum effect has been demonstrated with both the introduction of unmodified bacteria and GMMs. Unmodified microbes have been released into the environment for many
decades, particularly in the agricultural practices of the biocontrol of pathogens and inoculation of crop plants with nitrogen fixing micro-organisms. Few studies have attempted to
determine the effect of these introductions on indigenous microbial communities. Assessments of the risks from GMMs have been made in situ in field releases where inocula have
been released into the natural environment.13,15,25,28,39,30 Particular consideration has been
given to the potential impact GMMs may have on ecosystems by disturbing natural communities or by exchanging genetic material.28,31 The plant surface and rhizosphere are the
target of a number of releases and have been the focus of many studies, including those of
fluorescent pseudomonads in the rhizosphere of a number of crops.15,18,19,21,25,30,32 Typically, GMMs contain introduced antibiotic resistance genes which facilitate their detection
against the background of indigenous strains sampled form the environment, and as a consequence of the technical approaches used to generate the recombinants.
It should be emphasized that the use of antibiotic resistance genes in GMMs does not
in itself pose an unacceptable generic risk. Potential harm may only arise from the transfer
of the introduced marker genes to other bacterial populations where the incidence or likelihood of external selective pressures (e.g., antibiotics) are encountered by the GMMs and
indigenous suitable recipient populations. Obviously, the use of marker genes that impart
resistance to clinically important antibiotics would not be advised, but the use of a marker
resistance in a GMM, for example kanamycin or ampicillin, where the specific phenotype is
comparatively common in soils and other natural environments, would not be considered
as an additional serious hazard. The possibility of selection through the use of antibiotics in
animal health or growth promotion could be a factor (Chapter 2). This may be compounded
by the application of untreated sewage or slurry to land where the GMM has been applied
or might be encountered. As with current practices, all GMM releases should be taken on a
case-by-case basis to evaluate their consequences on entering the environment, a part of
which is the consideration of the construct and the nature of the introduced genes themselves.
124
The nature of the introduced genes and the resulting phenotype is relevant as this will influence the degree of risk. The advantage of selective markers is that they can be used to readily
track and monitor the fate, persistence and activity of released GMMs. The facility to track
inocula, and therefore the fate of the genes is an important safety consideration.
Manipulated genes can be transferred horizontally to or from a GMM where they may
persist and be expressed. Although the frequencies of horizontal transfer will usually be low,
the location of manipulated genes at carefully chosen chromosomal sites can considerably
reduce these events further. However, it should not be necessary to prescribe the chromosomal location of introduced genes unless minimizing the chance of gene transfer is preferred. Investigations with marked mobile elements will provide data to evaluate gene flow
in the environment to ascertain whether restrictions to the chromosome, avoiding all known
mobile genetic elements is appropriate. The nature of the construct will dictate the probability of gene transfer, but perhaps the primary consideration must remain the phenotype
of the introduced genes and how the ecology and activity of the modified host is altered.
However, any introduced genes, in order to persist and proliferate, require at the very least
periodic amplification (selection) of their host. With the decline in concentration of the
manipulated genes in the environment, the potential for the future proliferation and spread
of these genes is considerably reduced. Precise prediction of the changes in behavior arising
from the acquisition of new genes is especially difficult because microbial populations are
engaged in highly complex interactions. Assessment of risks arising from the transfer of
manipulated genes requires the nature and activity of the genes selected for use in a GMM
to be considered on a case by case basis.
From the results presented in this chapter it would appear that the persistence of the
released GMM, P. fluorescens SBW25EeZY6KX, in the environment is limited. Even when
the GMM was detected it was present at very low levels and not in all replicate samples. This
lack of consistency may reflect that the levels of GMM were close to the limits of detection.
Lateral dispersal of the GMM during the release was limited so it was probable that the
GMM was confined to a relatively small area of soil around the plant root. Given this scenario, it was unlikely that colonization of plant roots would occur unless seeds were sown in
the exact position where an inoculated plant had grown the previous season. Thus, the
numbers of GMMs detected in 1994/5 were not considered to be ecologically significant.
This view was supported by our inability to detect the GMM in 1996. This implies that, as P.
fluorescens SBW25EeZY6KX does not appear to be able to persist in soil, it will not have any
long-term effects upon the site to which it is released.
Acknowledgments
These investigations were supported by the UK Department of the Environment and
the EU-Biotech program. Mark J. Bailey is a member of the MAREP Concerted Action sponsored by the European Commission Biotechnology Programme, DGXII.
References
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a population of leaf-associated pseudomonads. FEMS Microbiol Ecol 1999; 28:345-356.
2. Thompson IP, Bailey MJ, Fenlon JS et al. Quantitative and qualitative seasonal changes in
the microbial community from the phyllosphere of sugar beet (Beta vulgaris). Plant and
Soil 1993; 150:177-191.
3. Rainey PB, Bailey MJ, Thompson IP. Phenotypic and genotypic diversity of fluorescent
pseudomonads isolated from field grown sugar beet. Microbiol 1994; 140:2315-2331.
4. Lilley AK, Bailey MJ. The acquisition of indigenous plasmids by a genetically marked
pseudomonad population colonizing the phytosphere of sugar beet is related to local environmental conditions. Appl Environ Microbiol 1997; 63:1577-1583.
125
5. Lilley AK, Bailey MJ. Impact of pQBR103 acquisition and carriage on the phytosphere
fitness of Pseudomonas fluorescens SBW25: burden and benefit. Appl Environ Microbiol
1997; 63:1584-1587.
6. Lilley AK, Young JP, Bailey MJ. Bacterial population dynamics: plasmids a genetic mechanism for maintaining diversity and adaptation. In: Thomas CM ed. Chapman and Hall.
1999:In Press.
7. Lenski RE. Quantifying fitness and gene stability on micro-organisms. In: Ginzburg LR.
Ed. Assessing risks of biotechnology. Butterworth-Heineman, Boston. 1991:173-192.
8. Ellis RJ, Thompson IP, Bailey MJ. Metabolic profiling as a means of characterising plantassociated microbial communities. FEMS Microbiol Ecol 1995;16:9-18.
9. De Leij FAAM, Sutton EJ, Whipps JM et al. Effect of a genetically modified Pseudomonas
aureofaciens on indigenous microbial populations of wheat. FEMS Microbiol Ecol 1994;
13:249-258.
10. Doyle JD, Stotzky G. Methods for detection of changes in the microbial ecology of soil
caused by the introduction of micro-organisms. Microbial Releases 1993; 2:63-72.
11. Weller DM. Colonisation of wheat root by fluorescent pseudomonads suppressive to takeall. Phytopathol 1993; 73:1548-1553.
12. Yuen GY, Schroth MN. Interactions of Pseudomonas fluorescens strain E6 with ornamental
plants and its effect on the composition of root colonizing microflora. Phytopath 1986;
76:176-180.
13. Thompson IP, Ellis RJ, Bailey MJ. Autecology of a genetically modified fluorescent
pseudomonad on sugar beet. FEMS Microbiol Ecol 1995; 17:1-14.
14. Bailey MJ, Lilley AK, Thompson IP et al. Site directed chromosomal marking of a fluorescent pseudomonad isolated from the phytosphere of sugar beet; stability and potential for
marker gene transfer. Molec Ecol 1995; 4:755-764.
15. Thompson IP, Lilley AK, Ellis RJ et al. Survival, colonisation and dispersal of genetically
modified Pseudomonas fluorescens SBW25 in the phytosphere of field grown sugar beet.
Bio/Technology. 1995; 13:1493-1497.
16. Rainey PB, Bailey MJ. Physical and genetic map of the Pseudomonas fluorescens SBW25
chromosome. Mol Microbiol 1996; 19:521-533.
17. De Leij FAAM, Bailey MJ, Lynch JM et al. A simple most probable number technique for
the sensitive recovery of a genetically engineered Pseudomonas aureofaciens from soil. Lett
Appl Microbiol 1993; 16:307-310.
18. Bailey MJ, Lilley AK, Ellis RJ et al. Microbial ecology, inocula distribution and gene flux
within populations of bacteria colonizing the surface of plants: Case study of a GMM field
release in the UK. In: Van Elsas JD, Trevors JT, Wellington EMM eds. Modern Soil Microbiology. Marcel Dekker, New York. 1997:479-500.
19. Drahos DJ, Barry GF, Hemming BC et al. Spread and survival of genetically marked bacteria in soil. In: Fry JC, Day MJ, eds. Release of genetically engineered and other microorganisms. Cambridge University Press, Cambridge. 1992:147-159.
20. De Leij FAAM, Sutton EJ, Whipps JM et al. Spread and survival of a genetically modified
Pseudomonas aureofaciens on the phytosphere of wheat and soil. Appl Soil Ecol 1994;
1:207-218.
21. De Leij FAAM, Sutton EJ, Whipps JM et al. Field release of a genetically modified Pseudomonas fluorescens on wheat: Establishment, survival and dissemination. Bio/technology 1995;
13:1488-1992.
22. Lilley AK, Hails RS, Cory JS et al. The dispersal and establishment of pseudomonad populations in the phyllosphere of sugar beet by phytophagous caterpillars. FEMS Microbiol
Ecol 1997; 24:151-158.
23. Bailey MJ, Thompson IP. Detection systems for phylloplane pseudomonads. In: Wellington
EMH, van Elsas JD, eds. Genetic interactions between micro-organisms in the environment. Pergamon Press 1992:126-141.
24. Bramwell PA, Barallon RV, Rogers HJ et al. Extraction and PCR amplification of DNA
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25. Kluepfel DA, Kline EL, Skipper HD et al. The release and tracking of genetically engineered bacteria in the environment. Phytopathol 1991; 81:348-352.
26. Hirsch P, Spokes JD. Survival and dispersion of genetically modified rhizobia in the field
and genetic interactions with native strains. FEMS Microbiol Ecol 1994; 15:147-160.
27. Denning N, Morgan J, Whipps JM et al. The flagellin gene as a stable marker for detection
of Pseudomonas fluorescens SBW25. Lett Appl Microbiol 1997; 24:198-202.
28. Bailey MJ, Lilley AK, Thompson IP et al. Deliberate release of Recombinant Micro-organisms. In: Demain AL, Davies JE, eds. Manual of Industrial Microbiology and Biotechnology 2nd edition. American Society Microbiology, Washington. 1999:693-703.
29. Cory JS, Hirst ML, Williams T et al. Field trial of a genetically improved baculovirus insecticide. Nature 1994; 370:138-140.
30. Wilson M, Lindow SE. Release of recombinant micro-organisms. Ann Rev Microbiol 1993;
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31. Tiedje JM, Colwell RK, Grossman YL et al. The planned introduction of genetically engineered organismsEcological considerations and recommendations. Ecology 1989;
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Microbiol 1995; 61:3443-3453.
CHAPTER 9
9.1. Introduction
128
one reason for the lack of success,4,5 but the main reason seems to be the unpredictable
impact of environmental factors (e.g., temperature, humidity, nutrient availability) upon
the released bacterial cells, or the presence of other plant roots or indigenous competing
strains of the same species as the inoculant.6 In most cases of unsuccessful field applications, it is not known whether the lack of plant growth promotion can be attributed to the
poor survival of the inoculant, the loss of its competitive capability for nodulation, or to
other factors.7 To evaluate the reason for such failures it would be helpful to analyze the
rates of survival of such inoculants.
Although several methods for reisolation, identification and quantification of rhizobia
from soil are available, most of them require extensive laboratory studies, e.g., nodulation
of host plants in the laboratory or greenhouse followed by genetic fingerprinting, to distinguish inoculated cells from indigenous cells of the same species.8-10 Due to the methodological efforts, the number of samples which can be analyzed in one laboratory is rather
small and may not be sufficient for the evaluation of a specific inoculant in field release
experiments. Marker gene technology can provide the ideal tools to perform such evaluation studies, since an inoculant can be tagged specifically with a quickly detectable and
unambiguous trait.11,12 The characteristics, potentials and limitations of these tools are
described in detail in Chapters 5-7. Depending on the marker gene, strains can be directly
detected by microscopy (without cultivation), by PCR amplification of such genes from
DNA directly extracted from microbial communities (see also Chapter 3), or by quantification after growth (colony formation) on selective or nonselective growth media (Chapter
2). An important point to consider before using marker gene -tagged strains for evaluation
as inoculants is, that the insertion of a marker gene or its expression may affect the fitness of
a strain and thereby decrease the capacity to compete with indigenous bacteria.13-15 Another
potential disadvantage for field applications of marker gene-tagged strains is, that they are
genetically engineered (genetically modified microorganisms; GMMs) and, thus, their
release requires thorough risk assessment studies and permission of competent national
authorities. In fact, the case study reported in this chapter included the first two field
releases of GMMs in Germany. These releases were conducted in a collaboration between
the University of Bielefeld (M. Keller, A. Phler) and the FAL (Forschungsansalt fuer
Landwirtschaft; C.C. Tebbe, J.C. Munch).
129
sion in a film cassette. It is important to notice, that, due to its oxygen demand, luciferase
gene expression could only be detected when air was permitted to diffuse between the attached colonies and the film material. This was achieved by inserting a distance holder of
0.5 cm into the film cassettes.
In strain S. meliloti L1, the combined nptII-luc gene construct was chromosomally inserted into the recA gene. This gene is involved in homologous recombination of DNA and
also in DNA repair mechanisms, such as induced after UV exposure.17 Since the luc gene
cassette was inserted into this gene, the gene sequence was disrupted and, therefore, strain
L1 exhibited a RecA- phenotype.16 Microcosm studies in the laboratory demonstrated that
in the absence of its host plant, alfalfa, the recA-mutation reduced the ability of strain L1 to
persist in bulk soil.18 The other strain selected for our field release study was an isogenic
strain of S. meliloti 2011. This strain, designated L33, contained the same reporter gene
cassette as S. meliloti L1, but its chromosomal insertion was downstream of the recA gene,
and, thus, not disrupting it (Fig. 9.1). The strain was indistinguishable from its parent strain
2011, except for the expression of the marker gene.19
Fig. 9.1. Genetic maps of the chromosomal recA regions in the S. meliloti (former name: Rhizobium meliloti) strains 2011 (wild-type), L33 and L1 and positions of primers, which allow to
differentiate L1 and L33 by size of the PCR product. Reprinted with permission from: DammannKalinowski T, Niemann S, Keller M et al. Appl Microbiol Biotechnol 1996; 45:509-512.
130
Table 9.1. Comparison of microcosms, lysimeters and field plots used to study the
survival and ecological interactions of Sinorhizobium meliloti strains in soil
Parameter
Field lysimeters
Field plots
Size
diameter: 30 cm
depth: 65 cm
diameter: 30 cm
depth: 65 cm
3 m x 3 m squares
no depth limitation
soil horizons
reconstructed,
equilibrated for 2 month
reconstructed,
equilibrated for 1 year
natural horizons
(plough layer 25 cm
depth)
number of
3
replicates for
each treatment
total surface
0.4 m2
area inoculated
with each strain
0.6 m2
45 m2
total number
approx. 3 x 1011
of genetically
engineered cells
released
approx. 1 x 1011
approx. 2 x 1013
inoculation
technique
spraying of cell
suspension onto the
soil surface
Monitoring
parameters
* survival of GMMs
* recombinant gene
persistence
* nodulation efficiency
* GMMs colonization of
different soil horizons
* effect on plant growth
* effect on organic carbon
and nitrogen concentrations in soil
* microbial biomass
* quantification of selected
culturable bacterial populations
* immediate
metabolic response
(Biolog-method)
* survival of GMMs
*
* nodulation efficiency *
* GMMs colonization
of different soil horizons
* vertical transport of
*
GMMs
* effect on plant growth
* effect on organic car- *
bon and nitrogen concentrations in soil
*
* microbial biomass
* quantification of selected culturable bacterial
populations
*
survival of GMMs;
horizontal
transport of
GMMs
rood nodule
occupancy in the
field
nodulation
efficiency
colonization of
rhizospheres from
host plants and
weed
impact of GMMs
and the microbial
diversity found in
plant rhizospheres
* ingestion and
transport of
GMMs by soil
invertebrates
131
Parameter
Field lysimeters
Field plots
stability of
ecological
parameters
Low
Medium
High
maximum
length of
monitoring
1.5 years
2 years
> 3 years
Problems
* soil compaction
* crevices in soil columns
and rim effects
* limited amount of
sampling dates
* limited amount of
sampling dates
* refilling holes after
auger insertions and
its impact on soil
structure
* large scale
production of
inoculants
* inoculation of
field plots without
aerial spread of
GMMs
* spread into
neighboring plots
with host plants
Reference
Schwieger et al23
Schwieger et al24
Dresing et al25
Schwieger and
Tebbe26
The major difference between soil columns in the greenhouse and field lysimeters were
the environmental factors acting on both systems. In the greenhouse, soil columns were
wetted to allow plant growth. However, in order to study active movement of the GMM
strains, columns were not saturated with water and, thus, no flow-through water could be
collected. In order to protect the columns from frost damage, the minimum temperature in
the greenhouse was not permitted to be below 4C. Moreover, the maximum temperature
was not above 30C. In contrast, field lysimeters were exposed to the natural conditions,
including periods with temperatures below 0C and above 30C. Wind exposure in the field
resulted in a rather quick drying of the soil in the lysimeters, even after heavy rain periods.
On the other hand, large amounts of flow-through rain water could be collected; 42.5 l per
lysimeter over a period of 18 months. The soil surface inoculated in the field plot investigation was approx. 100 times larger than that of the greenhouse columns or field lysimeters.
This allowed the removal of more material for analyses during this investigation. Also, holes
created by auger insertions in the plot experiment during sampling were not refilled, as in
greenhouse or lysimeter studies, since vertical transport was not analyzed and such holes
were not much different in size compared to those, naturally produced by mice in the field.
The selection of parameters monitored along with the assessment of the survival of the
inoculated S. meliloti strains L1 and L33, respectively, depended on the characteristic specificities of each model system. Since the model systems were studied subsequently, we could
optimize our monitoring methods and omit parameters that proved to be insensitive or
remained unaffected. For instance, the immediate metabolic response of soil extracted
microbial communities20,21 (community level physiological profiles, CLPP) did not detect
any differences between the treatments (inoculation, noninoculated controls). Therefore,
this parameter was not further included in the field monitoring. Transport by rain water
could not be analyzed on the field plots, but on the other hand, the plots were ideal for study
of the colonization of rhizospheres of host plants and weed plants growing between the
host plants. Also, only from the field plots, a large variety of soil insects could be collected
132
during the growing season. The gut contents of several of these insects were analyzed for the
occurrence of marker gene tagged, bioluminescent cells.22
Fig. 9.2. Three model systems used to study the survival and microbial ecology of bioluminescent
S. meliloti strains (GMMs) in soil. Left, soil columns in the greenhouse, allowing to study bacterial colonization of different soil horizons; field lysimeters (middle) of the same design, and field
plot inoculation (right).
133
cells initially introduced into each model system. Due to the relatively low amount of microbial cells added, it was not surprising to see that general parameters, like microbial biomass, organic carbon or total nitrogen (with variances of at least 10%), did not respond to
this treatment at all. Other parameters, like population sizes of culturable heterotrophic
bacterial communities on cellulose, glucose or aromatic compounds, also did not respond
to the inoculation procedure. Thus, these parameters, which were suspected to be indicative
for nonintended, dramatic changes within the soil microbial community, were only monitored during the first two stages of this investigation (soil columns and field lysimeters).
134
Fig. 9.4. PCR mediated detection of the recombinant luc-marker gene in DNA, directly extracted
from soil. PCR products of strain L33 (415 base pairs; lanes 3, 5, 7, 9, 11, 13, 16) and L1 (1011
base pairs; lanes 2, 4, 6, 8, 10, 12 and 15) are clearly distinguishable. DNA was extracted from soil
columns (0-25 cm depth) after a day (lanes 2, 3), 2.1 weeks (4, 5), 4.1 weeks (6, 7), 8.1 weeks (8,
9), 16.1 weeks (10, 11), and 24.1 weeks (12,13). Other lanes show size standards and controls.
Reprinted with permission from: Schwieger F, Willke B, Munch JC et al. Biol Fertil Soils 1997;
25:340-348.
remained in the range between 2 x 103 and 7 x 104 cfu g-1 soil, with a seasonal impact on the
population sizes. Interestingly, in the first two years, the populations of L1 were below those
of L33 in Fall and Winter but above L33 in Spring, as observed in the two following years.25
This suggested that there was an ecological significance of the recA mutation. However, in
the third year, this phenomenon was not significant. Even though it is extremely difficult to
determine the reason for significant differences between recA- and recA+, the majority of
sampling dates did not yield such differences and, thus, we can conclude that the recA gene
was not crucial for S. meliloti to successfully colonize the field plots with alfalfa. Due to the
high persistence of both strains, L1 and L33, we can also conclude that the luciferase marker
gene did not interfere with the environmental fitness of S. meliloti. This clearly supports the
assumption that in contrast to some other marker genes, such as luxCDABE, luc has a rather
low impact on fitness.
Vertical transport of surface soil inoculated cells was studied in greenhouse columns
and field lysimeters, but only the latter system yielded reliable data. In the greenhouse, more
than 98% of the inoculated cells were recovered in the upper 10 cm in three of four soil
columns analyzed after 85 weeks of incubation.23 However, in one column, layers below 20
cm soil depth were almost homogeneously colonized with titers of 104 to 105 cfu g-1 soil.
Rim effects and crevices in the soil column probably led to transport of the surface inoculated cells in that column. Additionally, oxygen diffusion from the bottom of the soil columns, which could not completely be sealed from the greenhouse atmosphere and a
homogenous temperature of the soil column, may have promoted growth in such deeper
soil layers. In the lysimeters, the temperature and the gas atmosphere were more similar to
conditions in the surrounding field soil environment. With this, more realistic system, monitoring of the inoculated cells showed no migration of the GMMs into layers below 20 cm
depth (threshold of detection 100 cfu g-1 soil) and flow-through rain water did not transport any detectable bioluminescent cells through the 65 cm soil profile (threshold of detection 10 cfu ml-1). Thus, it could be concluded that no risk of vertical migration of GMMs on
the field site for the subsequent field plot inoculation existed. By selection of the field site, at
135
which the groundwater table was 20 m below the soil surface, unintentional spread into
ground water could be excluded.
Horizontal spread of inoculated cells could only be analyzed in the field plot experiment. The experimental field consisted of 20 plots, each a square of 9 m2 in 4 x 5 rows, with
each plot separated from the other by 3-meter noninoculated strips, seeded with grass. Plots
were inoculated in block randomized order with S. meliloti wild-type, L33, L1, or not inoculated. Already 12 weeks after the field release, bioluminescent cells were detected in the rhizosphere of alfalfa growing on noninoculated plots.26 Two weeks later, when we analyzed the
titer of bioluminescent cells in bulk soil, recombinant cells were detected on the
noninoculated control plots with an average titer of 2.2 x 101 cfu g-1 soil. This titer increased
further throughout the 3 year monitoring period to numbers only one order of magnitude
below those on the inoculated plots.25 Mixed populations of strain 2011, L33 and L1 were
found on wild-type-inoculated plots. Thus, inoculation of S. meliloti 2011 did not completely inhibit the colonization by the GMMs. Sampling outside of the alfalfa seeded plots
never resulted in detection of any significant amounts of bioluminescent cells.25 The horizontal spread of the GMMs was obviously restricted to the presence of alfalfa roots.
Unintentionally, a large number of different weed plants (approx. 20 different species)
grew on the alfalfa-seeded plots during the first vegetation period after inoculation. Several
of such weed plants were sampled concomitantly with alfalfa plants to study their rhizosphere colonization by bioluminescent cells. As expected, the rhizosphere of alfalfa plants
provided a well suited habitat for S. meliloti and was densely colonized by bioluminescent
cells (> 105 cfu g-1 root material). Some weed plants, e.g., Capsella bursa-pastoris, did not
enrich for any bioluminescent rhizobial cells, but for the weed Chenopodium album, we
found approx. 103 cfu g-1 root material on inoculated and 101 cfu g-1 on noninoculated
control plots, 12 weeks after the field release.
A total of approx. 1,200 pure culture colonies were isolated on growth media adapted
to the isolation of rhizosphere bacteria. These isolates were obtained from rhizospheres of
alfalfa and C. album plants, grown on inoculated and noninoculated plots 12 weeks after the
release of the GMMs. The species richness, as detected by restriction fragment length polymorphism of PCR amplified 16S rRNA genes (ARDRA) was higher in rhizospheres of alfalfa than of C. album. The diversity of isolates was characterized at the phylogenetic level
using restriction fragment length polymorphisms of PCR amplified 16S rRNA genes. The
number of ARDRA pattern types, which correlated with species richness and diversity (expressed as the Shannon Index), was larger in rhizospheres of alfalfa than in that of C. album.
The species richness was unaffected by inoculation in rhizospheres of C. album but
increased in rhizospheres of alfalfa.26 Possibly, the S. meliloti inoculation increased the
nutritional status of the early developing alfalfa plants and concomitantly resulted in the
release of rhizosphere exudates stimulating the growth of a larger variety of soil bacteria.
9.6. Conclusion
The results of this still ongoing investigation on the ecology of a genetically-tagged
Sinorhizobium sp. in the field demonstrate the advantages and disadvantages of different
model detection systems for such studies. In general, greenhouse soil columns were useful
to demonstrate that the model strain, S. meliloti, did not cause any dramatic alterations in
the soil microbial community. However, to study vertical translocation and long term
effects of inoculated bacterial cells in the soil, more sophisticated systems with temperature
gradients, freeze thawing cycles to prevent soil compaction, and control of the soil atmosphere would be needed. Such factors were intrinsically provided in our field lysimeter
investigation. The major limitation of both soil columns and lysimeters compared to the
field plot experiment was that the number of samples which could be taken for analysis was
136
limited due to the small size of both systems. Field plots were also found to be extremely
useful for observing the impact of the nondeliberately controlled biological parameters,
namely soil invertebrates and weed plants, on the survival of the released GMMs. Interactions with both compartments, which serve as habitats of a large, mainly uncharacterized,
diversity of microorganisms, could also be studied.
It can be expected, that future environmental applications of recombinant bacterial
inoculants will not only include strains for biofertilization like S. meliloti in alfalfa fields,
but also bacterial strains for biocontrol or biodegradation. In general, it would be ecologically sound if any GMM construct is eliminated from the environment after its job has been
done. A continued persistence potentially includes ecological risks, e.g., exclusion of other
indigenous microorganisms, spread into neighboring nontarget ecosystems, transfer of
recombinant genes to indigenous microorganisms and noncontrolled establishment of new
properties in microbial communities, such as enhanced emergence of resistances. In order
to develop GMMs with improved ecological properties, long-term studies on the environmental fate of GMMs are needed. In our own studies, the luc-tagged GMMs were persistent
for at least four years after their field release and it can be concluded that this recombinant
trait did not dramatically effect the environmental fitness of the strains. Thus, for longterm monitoring of field released microorganisms, the luc marker gene should be an ideal
tool.
Acknowledgments
I would like to thank Frank Schwieger, Birgit Willke and Rona Miethling for their collaboration which was essential for the success of the project. The co-operation with our
colleagues from the University of Bielefeld, especially Mathias Keller, Werner Selbitschka
and Alfred Phler is gratefully acknowledged. Excellent technical assistance was provided
by Simone Dose and Phan Tuong Nguyen. The experimental work was supported by funds
from the Bundesministerium fr Bildung, Wissenschaft, Forschung und Technologie (grants
BEO 0310549A, BEO 0310664, and BEO 0311203). C. Tebbe is a member of the MAREP
Concerted Action sponsored by the European Commission Biotechnology Programme,
DGXII.
References
1. Young, JPW. Phylogeny and taxonomy of rhizobia. Plant and Soil 1995; 186:45-52.
2. Maier RJ, Triplett EW. Toward more productive, efficient, and competitive nitrogen-fixing
symbiotic bacteria. Crit Rev Plant Sci 1996; 15:191-234.
3. Olsen PE, Rice WA, Bordeleau LM et al. Levels and identities of nonrhizobial microorganisms found in commercial legume inoculant made with nonsterile peat carrier. Can J
Microbiol 1996; 42:72-75.
4. Paau AS. Improvement of Rhizobium inoculants. Appl Environ Microbiol 1989; 55:862-865.
5. Van Elsas JD, Heijnen CE. Methods for the introduction of bacteria into soil: A review.
Biol Fertil Soils 1990; 10:127-133.
6. Stacey G. The Rhizobium experience. In: Halvorson O, Pramer D, Rogul M, eds. Engineered organisms in the environment: Scientific issues. Washington: ASM Press,
1985:109-120.
7. Lowendorf HS. Factors affecting the survival of Rhizobium in soil. In: Alexander M, ed.
Advances in Microbial Ecology. New York: Plenum Press, Vol.4, 1977:87-124.
8. Handley BA, Hedges AJ, Beringer JE. Importance of the host plants for detecting the population diversity of Rhizobium leguminosarum biovar viciae in soil. Soil Biol Biochem 1998;
30:241-249.
9. Laguerre G, Bardin M, Amarger N. Isolation from soil of symbiotic and nonsymbiotic
R. leguminosarum by DNA hybridisation. Can J Microbiol 1993; 39:1142-1149.
137
CHAPTER 10
10.1. Introduction
his brief account will summarize the experiences gathered over an eight year period
(1989-1996), devoted to assessing the potential risks associated with the construction
and use of genetically modified microorganisms. As part of the BAP, BRIDGE and IMPACT
I European projects, these studies fall primarily in the category of prenormative research; the
aims of which were to provide information to competent authorities and protection agencies and to contribute to the build up of case histories for the validation of environmentally
safe guidelines. With this respect, the experimental schemes adopted have been focused on
addressing a simple and somewhat rhetorical question: whether or not a neutral genetic
modification, not conferring advantages to the bacteria, could perturb the environment
into which they are released?
140
synthetic promoter fulfilled expectations, conferring levels of -gal activity that more than
doubled those of the tac promoter cloned in an isogenic vector-background. Interestingly,
the regulated version of the cassette behaved differently in the two tested species: in E. coli
an IPTG-induced regulated tac promoter was stronger than the regulated synthetic promoter while in Rhizobium the situation was reversed. The cassette proved therefore efficient
for monitoring of rhizobia. An additional advantage was the facility of performing highlyspecific fragment amplification from environmental samples ensured by a DNA sequence
virtually unique in nature.
141
Table 10.1. Expression level of the introduced genes: -galactosidase activity and
resistance to mercury chloride
Strain
1003 (wt)
1110
1111
1112
IPTG-induced
uninduced
MIC (g/ml) of
HgCl2
38
6.849
12.982
718
40
260
15.165
243
<1
10
10
5
three different carrier formulations, namely; sterile liquid medium, -irradiated sterile
vermiculite, and non sterile peat. The latter was a suitable environment to assess possible
gene transfer to indigenous peat microflora. The three GMMs, and the parent strain, were
inoculated into the three different microcosms. Strain survival and marker stability were
monitored by periodic sampling from stored packages for over 500 days. Results are shown
in Figure 10.2. The liquid medium supported high viability during the first 100 days whereas
vermiculite appeared more suitable for long term survival. The chromosomal integration
was extremely stable and allowed a survival rate comparable to that of the parent. Strain
1110, carrying the regulated plasmid-borne cassette, demonstrated weaker persistence. By
contrast, strain 1111 rapidly lost the plasmid carrying the unregulated construct, but plasmid free segregates survived as well as the wild type. Marker loss was easily detected by
plating bacteria on X-gal plates. A typical feature of the highly expressed lac genes in strain
1111 was the small size of its dark blue colonies compared to the larger and paler revertants.
Horizontally-acquired marker genes were sought among peat indigenous bacteria but the
background levels of lactose utilization and of HgCl2 resistance (respectively 20% and 0.07%
of the total aerobic bacterial peat population) hindered such analysis. However, when 1650
HgR colonies were screened for lac phenotypes, 139 colonies showed both marker phenotypes and were tested by hybridization with probes from pDG3, (mercury resistance genes
and the 59-mer synthetic promoter). Two colonies hybridized with the first probe and two
different ones with the second one. When the isolates with homology to the synthetic promoter probe were checked with a lacZ probe no signal was detected. These fragmentary
homologies may suggest that transformation by liberated DNA portions had occurred, but
absolute proof was not obtained for gene transfer or exchange.
142
Fig.10.2. Survival (A-B-C) and marker stability (D-E-F) of Rhizobium strains in inoculant packages. A, D, liquid medium; B, E, sterile vermiculite; C, F, unsterilized peat. Symbols: , strain
1003; , strain 1110; , strain 1111; , strain 1112. Each point represents the mean of data
obtained from three different packages. Standard deviations were within the ranges 4% to 41%
for liquid, 8% to 59% for vermiculite, and 21% to 68% for peat samples (redrawn with
modifications from Corich V, Bosco F, Giacomini A et al. J Appl Bacteriol 1996; 81:319-328.).
Nodulation was correspondingly low for all introduced strains, less than 1.4% out of a
sampled population of 1229 nodules. The detection limit inherent to the markers used was
in the order of 102 cfu g-1 soil dry weight.
In parallel, the possible impact on resident biota was measured by plate counts of total
aerobic culturable bacteria, spore forming bacteria, microfungi, streptomycetes, fluorescent
143
10.6. Field trial II; Fate and Impact of a GMM Native to the Site
The experience gained by field release of an allochtonous strain prompted the consideration as to what would have happened if a competitive, indigenous Rhizobium engineered
with the same genetic modification had been used. To address this question we had a set of
data available from RAPD fingerprinting analyses of > 250 nodule occupants from the first
release site. The Rhizobium leguminosarum bv. viciae natural local population showed more
than 30 different RAPD profiles some of which were present in a large number of nodules.
Nodulation tests on an alternative host, Vicia faba subsp. minor, also contributed data to
identify the profile of the dominant strain which occupied over half of the nodules on both
plants (profile D on Table 10.2).
A spontaneous rifampicin resistant derivative of this strain was genetically modified by
introducing plasmid pDG3, yielding the equivalent of strain 1110 used in the first release,
but in a background strain native of the chosen habitat and successful on its symbiotic
hosts. The strain was named 1114. The performance of this GMM lived up to the standards
of its parent whose competitive traits were maintained in terms of nodulation and persistence in the habitat. Despite the active colonization and infection of strain 1114, again the
resident microbiota did not show significant alterations, as determined by plate counting,
indicating that a GMM extensively invading its environment, does not necessarily alter the
measured soil populations.6 An analysis of heterospecific but related species such as
Sinorhizobium meliloti was carried out from pea rhizosphere using MPN counts by means
of the homologous host alfalfa. The GMM did not alter S. meliloti numbers. Moreover, their
growth appeared not to be stimulated in the rhizosphere of the heterologous pea plant. In
terms of marker/reporter genes efficiency in monitoring, differences were noticed between
the first released GMM, based on strain 1003, and 1114, the dominant profile D strain.
The former, despite carrying mercury resistant genes, was difficult to grow on mercury con-
Table 10.2. Frequency of the most abundant RAPD profiles shown by nodule
occupants on pea (P. sativum) and vetch (V. faba) in the soil of the release
P. sativum
(250 nodules)
V. faba
(32 nodules)
10%
13%
9%
19%
11%
6%
55%
44%
< 1%
9%
144
taining media when plated straight from soil suspension, although it would grow well if
restreaked from agar plates or liquid cultures. This is presumably due to the necessity for
induction of mercury resistance. The latter strain, 1114 did not present such a limitation,
allowing a faster screening. Conversely, the lacZ marker driven to high expression by the
synthetic promoter, conferred a distinct phenotype in the 1003 background, while in strain
1114 a higher endogenous -galactosidase activity made the reporter less distinctive when
monitoring the GMMs.
10.7. Conclusion
In essence, the experiments described here indicate that the efficiency of detoxifying
and catabolic marker genes can not be generalized and is dependent on the physiology of
each bacterial strain. Their stability instead, is correlated with both gene expression and
genomic location. As for field release practice, data suggest that the use of an allochtonous
strain does not warrant successful colonization, and that on the contrary, strong autochtonous
isolates can yield backgrounds for a more promising field exploitation. GMM impact on
resident life is not necessarily associated with niche occupation and will therefore be dependent on the nature of the introduced genetic modification.
Acknowledgment
M.P Nuti is a member of the MAREP Concerted Action sponsored by the European
Commission Biotechnology Programme, DGXII.
References
1. Giacomini A, Ollero FJ, Squartini A et al. Construction of multipurpose gene cartridges
based on a novel synthetic promoter for high-level gene expression in gram-negative bacteria. Gene 1994; 144:17-24.
2. Corich V, Bosco F, Giacomini A et al. Fate of genetically modified Rhizobium leguminosarum
biovar viciae during long-term storage of commercial inoculants. J Appl Bacteriol 1996;
81:319-328.
3. Elhai J and Wolk CP. A versatile class of positive-selection vectors based on the nonviability
of palindrome-containing plasmids that allow cloning into long polylinkers. Gene 1988;
68:119-138.
4. Robinson JB, and Tuovinen OH. Mechanisms of microbial resistance and detoxification of
mercury and organomercury compounds: Physiological, biochemical and genetic analyses.
Microbiol Rev 1984; 48:95-124.
5. Corich V, Giacomini A, Concheri G et al. Environmental impact of genetically modified
Azospirillum brasilense, Pseudomonas fluorescens and Rhizobium leguminosarum released as
soil/seed inoculants. In: Jones D, ed. Biosafety Results of Field Tests with Genetically Modified Plants and Microorganisms. Oakland, University of California Division of Agriculture
and Natural Resources 1995: 371-388.
6. Nuti MP, Basaglia M, Bonfante P et al. Field release of genetically modified biofertilizers
and phytostimulators. In: Matsui S, Miyazaki S, and Kasamo K, eds. The Biosafety Results
of Field Tests of Genetically Modified Plants and Microorganisms. Tsukuba, Ibaraki, Japan: JIRCAS Publisher, 1996: 101-111.
CHAPTER 11
11.1. Introduction
he population genetics of soil bacteria provides strong evidence that genes are exchanged
in the natural environment (For examples, see refs 1-3). However, there is little information on the frequency and time scale of such events. There are concerns that the deliberate
release of beneficial genetically modified microorganisms (GMM) as plant inoculants could
result in the generation of undesirable hybrids if novel genes are transferred from the GMMs
to the native population. Conversely, genes from native strains could be transferred into the
inoculant, affecting its performance or persistence. There is extensive information on the
survival of rhizobial inoculants,4 which have been widely used for over 100 years in agriculture to form dinitrogen-fixing symbioses with leguminous plants. In recent years there have
been several releases of GM rhizobia (see refs 5-7 and Chapters 9 and 10). Also evidence has
been obtained for the transfer of a conjugative transposon from an inoculant to native rhizobia,8,9 but there are no reports of genes being transferred into rhizobial inoculants.
To investigate this possibility, Selbitschka et al10 marked a strain of Rhizobium
leguminosarum biovar viciae which had been cured of its symbiotic plasmid and consequently had lost the ability to form nodules on pea plants, by insertion of the Escherichia
coli uidA (gusA) gene into the chromosome. This gene conferred -glucosidase (GUS) activity, not normally present in rhiziobia or plants (see Chapter 6). When the strain, CT0370,
re-acquired a conjugative symbiotic plasmid, it regained nodulation ability and formed
nodules with GUS activity. Thus, the GUS marker not only facilitated the monitoring of the
survival of CT0370 after field release, but it also provided a simple screen: any pea root
nodules containing rhizobia with GUS activity would indicate symbiotic gene acquisition
by CT0370. To increase the potential for the detection of transfer events, the release site
selected contained an established population, released seven years previously by Hirsch and
Spokes,6 of R. leguminosarum biovar viciae strain RSM2004 containing a conjugative symbiotic plasmid that had been shown to confer nodulation ability on strain CT0370.
146
sequence between the chromosomal recA and alaS genes, reported by Selbitschka et al12 In
order to accomplish integration of the gus gene cassette, Selbitschka et al10 introduced an
XhoI restriction site by site-directed mutagenesis in the recA-alaS region, immediately adjacent to the putative rho-independent terminator of the recA gene. The chromosomal site
had been chosen since previous characterization of Sinorhizobium meliloti strain L33 which
carried the bioluminescence mediating firefly luc gene in the analogous recA-alaS intergenic
region had shown that an insertion in the target site per se did not adversely affect the
strains fitness. Selbitschka et al13 reported that strain L33 behaved like its parent strain with
respect to vegetative and symbiotic properties such as growth rate, growth competitive abilities or symbiotic performance (see also Chapter 9).
The recA-alaS region was cloned in E. coli using a vector which could not replicate
autonomously in Rhizobium, and contained sacRB (sucrose sensitivity) and aacC1 (gentamicin resistance) markers. Following mobilization into strain LRS39401, gentamicin
resistant transconjugants were assumed to have undergone a chromosomal insertion of the
vector in the target site by homologous recombination. Following selection on sucrose, some
transconjugants had lost the gentamicin and sucrose-sensitivity markers of the vector but
retained a GUS phenotype. These were tested for vector sequences by extracting DNA,
digesting with BamH1, EcoR1 and NruI, and probing gel blots with vector DNA. PCR with
primers designed to identify a single GUS gene cassette insertion in the predicted position
was used to confirm the structure (Fig. 11.1). Primers were: recA forward, uidA reverse,
uidA forward, and alaS reverse. PCR products arose only from reactions with recA forward,
uidA reverse and uidA forwards, alaS reverse. Spontaneous mutation to spectinomycin
resistance was selected in one such construct, producing strain CT0370.
147
from each section of the noninoculated buffer zones) were collected and pooled, sieved
through a 5 mm mesh, 10 g resuspended in 100 ml sterile distilled water, and 100 l plated
on five replicate plates. Total nodulating rhizobia were enumerated using Vicia hirsuta
infection tests with most probable number (MPN) estimation as described by Hirsch and
Skinner.14
Strains CT0370 and RSM2004 could be detected in DNA extracted directly from soil
using PCR with primers specific for the GUS insert and Tn5, respectively, as reported by
Cullen et al.15
148
The release took place in July 1994: inoculant granules were placed in drills (10 cm
apart), into which 840 untreated Avola peas (PGRO, Peterbourgh, U.K) coated in inoculant
were planted at 10 cm intervals. A total of 662 g of peat inoculant containing 9 x 1011 live
cells was applied; initial soil samples thus contained 4.9 x 105 cfu CT0370 g-1 soil. Samples
were taken immediately before and after the release, then weekly for 10 weeks and then
fortnightly. Peas were again planted in May 1995 and July 1995, but without further inoculation. An initial ten-fold drop in CT0370 cfu numbers was observed during the first 10
weeks, to 5 x 104 culturable cells g-1 soil (Fig. 11.3). Subsequently numbers remained around
104, similar to the numbers of native R. leguminosarum in the soil (Fig. 11.3). The inoculant
appeared to survive better than RSM2004 which had rapidly dropped to 102 cfu g-1 soil
although it subsequently persisted at this level. No CT0370 was detected in any buffer zone
samples: the major dispersion mechanism in arable fields is via soil cultivation6 and the
buffer zones were not cultivated.
Fig. 11.3. Survival of strain CT0370 and other rhizobia in the field
149
plates. The mixed growth was suspended in 1 ml H2O by vortexing and serial dilutions were
plated onto selective media. Sterile field soil was prepared by drying and milling, passing it
through a 5 mm sieve and autoclaving it three times at 121C for 1 hr with 24 hr intervals
between sterilizations. Nonsterile conjugations were performed in fresh field soil
(7.5% w/w H2O) previously passed through a 5mm sieve. Samples from late log phase cultures of CT0370 and RSM2004 were mixed, centrifuged, and resuspended in 500 l/H2O.
Then, 5 g of soil were added, the mixture was vortexed and incubated for 4 days at 28C.
Samples of parental cultures, and of soil after 2 and 4 days (resuspended as described for
field soil) were diluted and plated onto selective media. To determine how plasmid transfer
rate was affected by the presence of pea plants, and of introducing inoculant in peat granules, field soil in pots was mixed with peat inoculant to give RSM2004 and CT0370 levels of
approximately 106 cfu g-1 soil, and peas were planted. Control pots contained the parents
alone or strain CT0370 carrying pSym2004. Samples of the inoculant granules and of rhizosphere soil after one and two weeks were resuspended and plated as described for field
samples. Root nodules were screened for GUS activity as described previously.
The highest transfer frequency of the pSym plasmid from RSM2004 to CT0370, i.e.,
9 x 10-5 transconjugants per recipient, was observed in laboratory matings on filters. In sterile soil microcosms with 106 to 107 cfu of each parent strain per g soil, the highest frequency
was 2.6 x 10-6 transconjugants per recipient cell; in nonsterile soil it was 2.3 x 10-7. In pot
experiments with peat inoculant containing both parents, providing 5 x 106 cfu g-1 soil, no
transconjugants were found, indicating that in field soil with only 102-103 RSM2004 and
104-105 CT0370 cfu g-1 soil, the frequency would most likely be too low to detect.
CT0370 colonies reisolated from soil were screened for the acquisition of other plasmids from the soil population by subjecting them to PCR with primers designed to amplify
two rhizobial plasmid replication origins identified in the field population.16,17 More than
1000 colonies were screened, pooled in groups of 10 for DNA extraction and tested by PCR.
No positive results were obtained, although control reactions in which one colony of a positive field isolate was included did give the expected band.
11.8. Conclusion
Although strain CT0370 lacked symbiotic genes, it survived well in field soil after release and remained at a level similar to that of the native rhizobial population for five years
of sampling. This compares to the prior release of RSM2004 at the same site, where numbers declined by two orders of magnitude in the six months following release, but subsequently stabilized at around 102 cfu g soil-1.4,6 However, RSM2004 does not persist in all
soils and it is probable that survival is influenced by both soil type and bacterial strain.4 The
GUS marker alone would not have facilitated counting of CT0370 because of GUS activity
in the indigenous population of heterotrophic soil bacteria. However, in conjunction with
the chromosomal antibiotic resistances of CT0370, the gus gene made it possible to detect
the strain in soil against the background population, without effecting the enumeration of
RSM2004 (which contains different antibiotic resistances, neomycin and rifampicin). The
GUS marker offered a very sensitive detection system for identifying root nodules with
containing rhizobia with GUS activity and facilitated the screening of many more nodules
than would have been feasible with markers. The lack of transconjugants with GUS activity
in the field indicates that plasmid transfer does not occur at elevated levels in the field compared with laboratory studies. Symbiotic plasmid transfer from RSM2004 to CT0370 could
be detected in soil microcosms with parental densities of 106-107 cfu g soil-1, much higher
than those in field soil. The population density of rhizobia on pea roots is much higher than
in bulk soil but did not appear to be sufficient to enable conjugation to occur at a detectable
level and no evidence was found for acquisition of symbiotic or other plasmids from native
150
Acknowledgments
This work was supported in part by EC Biotechnology Programmes (BIO2 CT92-0370
and BIO4 CT96-0434). IACR-Rothamsted receives grant-aided support from the Biotechnology and Biological Sciences Research Council of the UK. A. Phler and W. Selbitschka
are members of the MAREP Concerted Action sponsored by the European Commission
Biotechnology Programme, DGXII.
References
1. Young JPW, Wexler M. Sym plasmid and chromosomal genotypes are correlated in the
field populations of Rhizobium leguminosarum. J Gen Microbiol 1988; 134:2731-2739.
2. Lilley AK, Bailey MJ. The acquisition of indigenous plasmids by a genetically marked
pseudomonad population colonising the phytosphere of sugar beet is related to local environmental conditions. Appl Environ Microbiol 1997; 63:1577-1583.
3. Villadas PJ, Burgos P, Rodriguez-Navarro DN et al. Characterization of rhizobia homologues of Sinorhizobium meliloti insertion elements ISRm3 and ISRm4. FEMS Microbiol
Ecol 1998; 25:341-348.
4. Hirsch PR. Population dynamics of indigenous and genetically modified rhizobia in the
field. New Phytol 1996; 133:159-171.
5. Bosworth AH, Williams MK, Albrecht K et al. Alfalfa yield response to inoculation with
recombinant strains of Rhizobium meliloti with an extra copy of dctABD and/or modified
nifA expression. Appl Environ Microbiol 1994; 60:3815-3832.
6. Hirsch PR, Spokes JD. Survival and dispersion of genetically modified rhizobia in the field
and genetic interactions with native strains. FEMS Microbiol Ecol 1994; 15:147-160.
7. OFlaherty S, Monne-Loccoz Y, Boesten B et al. Greenhouse and field evaluations of an
autoselective system based on an essential thymidylate synthase gene for improved maintenance of plasmid vectors in modified Rhizobium meliloti. Appl Environ Microbiol 1995;
61:4051-4056.
8. Sullivan JT, Patrick HN, Lowther WL et al. Nodulating strains of Rhizobium loti arise
through chromosomal symbiotic gene transfer in the environment. Proc Natl Acad Sci
USA 1995; 92:8985-8989.
9. Sullivan JT, Ronson CW. Evolution of rhizobia by acquisition of a 500-kb symbiosis island
that integrates into a phe-tRNA gene. Proc Natl Acad Sci USA 1998; 95:5145-5149.
10. Selbitschka W, Jording D, Nieman S et al. Construction and characterisation of a Rhizobium leguminosarum biovar viciae strain designed to assess horizontal gene transfer in the
environment. FEMS Microbiol Lett 1995; 128:255-263.
11. Hynes MF, McGregor NF. Two plasmids other than the nodulation plasmid are necessary
for formation of nitrogen-fixing nodules by Rhizobium leguminosarum. Mol Microbiol 1990;
4:567-574.
12. Selbitschka W, Arnold W, Priefer UB et al. Characterization of recA genes and recA mutants of Rhizobium meliloti and Rhizobium leguminosarum biovar viciae. Mol Gen Genet
1991; 229:86-95.
13. Selbitschka W, Dresing U, Hagen M et al. A biological containment system for Rhizobium
meliloti based on the use of recombination-deficient (recA-) strains. FEMS Microbiol Ecol
1995; 16:223-232.
151
14. Hirsch PR, Skinner, F.A. The identification and classification of Rhizobium and
Bradyrhizobium. In: Board RG, Jones K, Skinner FA, eds. Identification Methods in Applied and Environmental Microbiology Blackwell Scientific Publications, Oxford. 1992:45-65.
15. Cullen DW, Nicholson PS, Mendum TA et al. Monitoring genetically-modified rhizobia in
field soils using the polymerase chain reaction. J Appl Bacteriol 1998; 84:1025-1034.
16. Turner SL, Rigottier-Gois L, Power RS et al. Diversity of repC plasmid-replication sequences
in Rhizobium leguminosarum. Microbiology 1996; 142:1705-1713.
17. Rigottier-Gois L, Turner SLT, Young JPW et al. Distribution of repC plasmid-replication
sequences among plasmids and isolates of Rhizobium leguminosarum bv. viciae from field
populations. Microbiology 1998; 144:771-780.
CHAPTER 12
Regulatory Aspects
Kersti Gustafsson
12.1. Introduction
any countries have chosen to regulate GMOs (genetically modified organisms) for deliberate release into the environment. Reasons for regulation are the lack of knowledge
on the fate and behavior of GMOs and inserted genomes deliberately released into the environment and lack of experience of accompanying risks. Another reason is the concern of
people in general about releases of GMOs into the environment. Some countries are of the
opinion that ethical aspects of these matters are important. Regulation and control
facilitate increased general knowledge concerning the deliberate release of GMOs and hopefully contribute to a safe and acceptable development of the area.
This chapter deals mainly with GMM (genetically modified organisms), however, much
of the regulation is developed for the whole group of GMOs.
The United States has chosen to regulate GMOs under the existing legislation, while
the European Union (EU) has chosen to introduce new directives. Sweden has an act regulating GMOs and the responsibility among authorities is divided according to existing legislation. Since much is dependent on the vector of the gene modification, i.e., the organism,
a divided responsibility among different experts must be relevant. The use of biotechnology
in industrial applications as well as for research and development purposes has increased.
This is also a reason to have a divided responsibility for the control of the use of GMOs.
154
International Standards Organization (ISO); Food and Agriculture Organization (FAO); Microbial
Strains Data Network (MSDN).
2 Case-by-case means an individual review of a proposal against assessment criteria which are
relevant to the particular proposal; this is not intended to imply that every case will require review
by a national or other authority since various classes of proposals may be excluded.
Regulatory Aspects
155
small-scale field research with GMOs.2 The key safety factors in determining the safety of
any experiment were: i) the characteristics of the organism(s) used, including the introduced genome; ii) the characteristics of the research site and the surrounding environment;
and iii) the use of appropriate experimental conditions. The characteristics of importance
for the microorganism included dispersal, survival, multiplication and the potential for gene
transfer. The document also considered the mode of action of the GMM, including persistence and degradation of any newly acquired toxic metabolite. Additional important characteristics of the GMM, including interactions with other species and/or biological systems,
were also considered.
In 1995, the OECD initiated an Expert Group to manage the implementation of OECDs
Programme for the Harmonisation of Regulatory Oversight in Biotechnology. In 1998, the
group was renamed as a Working Group. The Group is currently made up of experts
involved in the regulation of biotechnology who have been nominated by their Member
State. The Working Group is a subsidiary body to the Joint Meeting of the Chemicals Group
and Management Committee of the Special Programme on the Control of Chemicals within
the OECD. The main focus of the Work Programme is the international harmonization of
regulatory oversight in biotechnology which will ensure that environmental health and safety
aspects are properly evaluated, while avoiding nontariff trade barriers to products of the
technology. The main areas of work are: i) the development of Consensus Documents on
specific scientific issues related to biotechnology; ii) outreach activities, including the development and maintenance of Biotrack Online, that makes information on the
Harmonisation Programme available to anyone interested; and iii) general issues associated
with harmonization of biotechnology regulation.a The regulatory developments in Member States can be found via Biotrack Online.b
12.3. The European Union and the European Free Trade Association
The European Union has chosen to regulate GMOs mainly via a directive on the contained use of genetically modified microorganisms3 and a directive on the deliberate release
into the environment of genetically modified organisms.4 The directives were modeled on
the chemicals notification directives.c These GMO directives also have impact on the regulation of GMMs within the European Free Trade Association (EFTA).
The responsible institution for Horizontal Legislation concerning GMOs within the
EU is Directorate-General XI: Environment, Nuclear Safety and Civil Protection.d For specific product legislation, the responsibility is shared by other Directorates as follows: DG III
(Industry) and DG VI (Agriculture). Other Directorates involved in regulation of GMOs
are DG VII (Transport)responsible for the safe transport of GMO, and DG XII (Science,
Research and Development) and The European Commission Joint Research Centre (Institute for Systems, Informatics and Safety) are responsible for information on research and
development of GMOs.
Apart from the above mentioned directives, other relevant regulations concern the protection of workers from the risks of exposure to biological agents (Council Directives
90/679/EEC and 93/88/EEC). For some products, there are other relevant legislations: for
additives in feeding stuffs (Council Directive 93/114/EEC); for medicinal products (Council
http://www.oecd.org/ehs/service.htm
http://www.oecd.org/ehs/country.htm
c
http://europa.eu.int/en/comm/dg11/guide/part2g.htm
d
http://www.oecd.org/ehs/cecreg.htm
b
156
Directive 93/41/EEC); and for novel food (European Parliament and Councils Regulation
No 258/97).
Directive 90/219/EEC lays down common measures for the contained use of GMMs
with a view to protecting human health and the environment. Directive 90/219/EEC was
amended in October 1998.5
The objective of Directive 90/220/EEC is to approximate the laws, regulations and administrative provisions of the EU Member States and to protect human health and the environment:
when carrying out deliberate releases of GMOs into the environment for research
and development purposes - Part B;
when placing products on the market that contain, or consist of, GMOs intended
for subsequent deliberate release into the environmentPart C.
Since disparity between rules concerning GMOs in the Member States may cause unequal conditions of competition or trade barriers it is necessary to approximate the laws in
the different Member States. Although the objective of the directive is the establishment
and maintenance of the internal market, nevertheless health and environmental safety aspects, as well as consumer protection should be at a high level of consideration.
Before a deliberate release of a GMO into the environment is performed, the responsible person must submit a notification to the competent authority in the EU Member State
where the release is going to take place. Such research and development experiments are
notified according to Part B of directive 90/220/EEC. The notification must also be circulated among other Member States and EFTA states via the European Commission (further
on referred to as the Commission). All countries are allowed to comment on the circulated
summary notification information format (SNIF), although the comments from other states
do not have a decisive effect on the decision. It has to be noted that the competent national
authority takes its decision independently. The authority giving the consent must have evaluated the information and have made itself sure that the release will be safe for human health
and the environment.
A company that wants to place a product containing a GMM(s) on the market has to
notify the product. The notification can be sent to a competent authority in any of the EU
Member States before the product is for the first time placed on the EU market. Products
are notified under Part C of directive 90/220/EEC. The notification must be circulated among
other Member States and EFTA states via the Commission. All countries can comment on
the circulated summary notification information format (SNIF). A joint decision is thereafter taken among EU Member States according to specified rules in the directive and the
decision taken will be valid in all EU Member States and EFTA states.
The Commission has now presented a proposal to the European Council concerning
amendments of directive 90/220/EEC.6 Some main improvements suggested include:
i. simplified administrative procedures;
ii. the possibility for the Commission to consult a scientific committee concerning
ethical consequences of biotechnology;
iii. mutual principles on risk assessment;
iv. monitoring of products placed on the market; and
v. marketing approvals for a fixed time period. The Economic and Social Committee
of the European Commission has given its diversified opinion on the proposal.7
12.4. Canada
The Canadian Environmental Protection Act (CEPA) requires that all new substances,
including those that are living organisms, are to be assessed for their potential to harm the
e
http://www.ec.gc.ca/cceb1/eng/97brochuree.html
Regulatory Aspects
157
environment, the environment on which life depends, or human life or health, prior to
being imported or manufactured in Canada.e The CEPA New Substances Notifications
(NSN) Regulations describe the information that must be provided to Environment Canada
in order for this assessment to be done. The NSN regulations covering substances like chemicals or polymers, have been in effect since 1994 and the 1997 amendment covers substances
that are organisms, or products of microorganisms (biochemicals and biopolymers). The
amendment was published in the Canada Gazette, Part II, on March 5, 1997 and came into
effect on September 1, 1997.
12.5. Switzerland
On July 1, 1997, a series of amendments to the Federal Law on Environmental Protection and to the Federal Law on Epidemics were enforced concerning the use of organisms
used in modern biotechnology.f The amendments provide the legal basis for specific regulations of environmental health and safety issues associated with GMOs. The new legislation covers all types of organisms and applications on the basis of the self-responsibility of
the user. For the environmental use of GMOs and/or pathogenic organisms, special provisions, including risk assessment and notification or authorization procedures are requested.
The legislation can be applied directly. However, regulations are needed to fully establish
the new procedures and to define the role and responsibilities of the various authorities. A
regulation has been proposed by the government on the use of organisms in the environment. The regulation is harmonized with the corresponding directive of the European Union
90/220/EEC.
http://www.oecd.org/ehs/swireg.htm
http://www.oecd.org/ehs/usareg.htm
h
http://www.epa.gov/opptintr/biotech/biorule.htm
g
158
Act; Final Rule and was published in the Federal Register on April 11, 1997. An Internet
website has been created to allow more efficient public, governmental and educational
access to the TSCA Biotechnology Program.h
The final regulation of microbial products of biotechnology under the TSCA regard
commercial biotechnology research and development activities using intergeneric microorganisms for e.g., biofertilizers, biosensors, biotechnology reagents, commodity or specialty chemical production, energy applications, waste treatment or pollutant degradation
and commercial biotechnology products.
Persons that intend to use intergeneric microorganisms (microorganisms formed by
combining genetic material from organisms in different genera) for commercial purposes
in the United States should submit a Microbial Commercial Activity Notice (MCAN) to the
EPA at least 90 days before such use. To test new microorganisms for commercial research
and development purposes in the environment, a TSCA Experimental Release Application
(TERA) should be submitted to the EPA 60 days prior to initiating field trials.
New microorganisms include intergeneric microorganisms. The EPA believes that
intergeneric microorganisms have a sufficiently high likelihood of expressing new traits or
new combinations of traits to be termed new and warrant review. Microorganisms that
are not intergeneric would therefore not be new and thus would not be subject to reporting under Section 5 of TSCA.
In 1993, a comparison was made between some selected risk assessment strategies.8
The necessary information generally demanded for an ecological risk assessment could be
generalized as:
Biology of the organism and characteristics of the genome
Fate of the organism and the genome in the ecosystem
Impact of the organism and the genome on the ecosystem
Effects of the organism and the genome on other organisms in the ecosystem
By including humans as organisms in the ecosystems, the above generalizations are
also valid for effects and impact on humans.
With regard to chemicals, the discussions on risks and assessment of risk started mainly
after disasters had started to occur. With regard to radiation, the discussions on risks have
occurred basically parallel with the development of the technique. However, with modern
biotechnology and the use of GMOs, the risk discussions were initiated before the full
impact of the technique was known. Therefore the discussions on risks of GMOs sometimes lack connection to real examples and technique development within the area of
research. The development might actually be slowed down due to the pro-active risk discussions. With continuous risk discussions during the notification process of field trials and
GMO products, the research and development rate might be slowed down. However, obvious
risk aspects can easier be discovered due to the risk discussions and should be managed
early in the research and development process, and this will be of benefit for the companies,
the scientists and the public.
In October 1998, a workshop was held in Stockholm, Sweden, which focussed on the
scientific basis for risk assessment of microbiological plant protection products charged by
the European Commission, DG VI.i,9 There was a general apprehension that risk assessment of GMMs and risk assessment of microbiological plant protection products have very
much in common. Microorganisms intended for deliberate release are regulated in different ways according to the purpose of the release; nevertheless, the risk assessments should
benefit from general multidisciplinary discussions.
Regulatory Aspects
159
http://www.kemi.se
160
knowledge on risks from other areas. GMMs must often be used in connection with chemicals, e.g., as above due to selectable marker genes or for post-release cleanup. Methyl bromide has been suggested for post-release clean up. The use of very hazardous chemicals
should in general be avoided in post-release treatment after field experiments.
The proposal for a new EU-directive on the deliberate release of GMOs6 opens up the
possibility for the Commission to consult scientific committees concerning ethical consequences of biotechnology. In the scope of the Swedish legislation, it is claimed that ethical
concerns shall be taken into account in assessments of deliberate releases of GMMs.
There seems to be a general public concern about GMOs, but the concern is dependent
on the product and its usefulness. There is, in general, little concern about improved medicinal drugs, the potential benefit is obvious (for example recombinant insulin). However,
there is generally a lot of concern with GMO food and a lack of usefulness is often claimed.
It is difficult to foresee how GMM products for biodegradation etc. will be received. The
pros and cons for the public, for the companies and/or for science, of using GMMs for
different purposes must be weighed against the use of other technologies.
In 1998, the Swiss voted on an initiative concerning the prohibition of the use of
transgenic animals, of the release of genetically engineered plants into the environment and
of patenting of plants and animals. The result was not in favor of the prohibition, however
the matter illustrates some major concerns about biotechnology.
Acknowledgment
K. Gustafsson is a member of the MAREP Concerted Action sponsored by the European
Commission Biotechnology Programme, DGXII.
http://www.olis.oecd.org/biotrack.nsf
http://www.olis.oecd.org/bioprod.nsf
l
http://biotech.jrc.it/GMO.htm
m
http://www.cordis.lu/biotech/src/projects.htm
k
Regulatory Aspects
161
Table 12.2. Summary of deliberate field trials in the European Union notified
under part B of Directive 90/220/EEC 21 October 199115 March 19991
47 releases with 7 bacterial genera, 1 yeast and 2 viruses have been performed in 7
EU Member States
Country
GMM
Year
Main trait/Purpose
Finland
Streptococcus sp
1995
France
Bacillus sp.
Bacillus sp.
Pseudomonas sp.
Rhizobium sp.
1993
1993
1992, 1994
1995
Germany
Rhizobium sp.
1994, 1997
marker system
Italy
Azospirillum sp.
Pseudomonas sp.
Pseudomonas sp.
Pseudomonas sp.
1994, 1995,
1998, 1999
1994, 1995
1997
1998
Rhizobium sp.
Rhizobium sp.
1994
1994, 1995
Escherichia coli
Pseudomonas sp.
Pseudomonas sp.
1994
1994, 1995
1998
Pseudomonas sp.
Rhizobium sp.
Rhizobium sp.
Rhizobium sp.
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Sinorhizobium sp.
1998
1997
1997
1998
1997
1997
lactose metabolism
1998
The
Pseudomonas sp.
Netherlands Pseudomonas sp.
1994
1996
Pseudomonas sp.
1998
marker system
chitinase synthesis; phenazine-1carboxylic
acid synthesis
2,4 diacetyl-phloroglucinol synthesis;
phenazine-1-carboxylic acid synthesis
Autographa
californica nuclear
polyhedrosis virus
1993, 1994
efficacy
Bacteriophage M13
Pseudomonas sp.
Rhizobium sp.
1994
1993, 1994
1993, 1994
pollution tracing
survival, persistence and dispersal
marker system
Spain
United
Kingdom
162
References
1. Organisation for Economic Co-operation and Development. Recombinant DNA Safety
Considerations. 1986; 69. Paris: OECD Publications.
2. Organisation for Economic Co-operation and Development. Safety considerations for biotechnology. 1992; 50. Paris: OECD Publications.
3. The Council of the European Communities. Council Directive of 23 April 1990 on the
contained use of genetically modified microorganisms (90/219/EEC). Official Journal of
the European Communities 1990; L(117):1-14.
4. The Council of the European Communities. Council Directive of 23 April 1990 on the
deliberate release into the environment of genetically modified organisms (90/220/EEC).
Official Journal of the European Communities 1990; L(117):15-27.
5. The Council of the European Union. Council Directive 98/81/EC of 26 October 1998
amending Directive 90/219/EEC on the contained use of genetically modified microorganisms. Official Journal of the European Communities 1998; L(330):13-31.
6. Commission of the European Communities. Proposal for a European Parliament and Council Directive amending Directive 90/220/EEC on the deliberate release into the environment of genetically. Official Journal of the European Communities 1998; C(139):1-24.
7. Economic and Social Committee. Opinion of the Economic and Social Committee on the
Proposal for a European Parliament and Council Directive amending Directive 90/220/
EEC on the deliberate release into the environment of genetically modified organisms.
Official Journal of the European Communities 1998; C(407):1-6.
8. Gustafsson K, Jansson JK. Ecological risk assessment of the deliberate release of genetically
modified microorganisms. Ambio 1993; 22(4):236-242.
9. KemI. Proceedings Microbiological Plant Protection Products - Workshop on the Scientific Basis for Risk Assessment. 1999:1-77. Swedish National Chemicals Inspectorate, Solna.
10. Nielsen KM et al. Horizontal gene transfer from transgenic plants to terrestrial bacteria
a rare event? FEMS Microbiology Reviews 1998; 22:79-103.
11. Ljungquist S, Andrn R, Fermr C. Risk att resistens mot antibiotika sprids frn GMO?
1999; 99(1):1. 25. Kemikalieinspektionen, Stockholm.
12. Kruse H, Jansson J. The use of antibiotic resistance genes as marker. 1997; 97(3):1-47.
Norwegian Pollution Control Authority, Oslo, Norway.
Index
A
B
-galactosidase 141, 144
-glucuronidase 87-90, 94
Biocontrol 123
Bioluminescence 70, 72, 75, 81
Biomarker 101, 102, 104-106, 112
Bioreporter 101, 102, 104, 106, 111, 112
Biosensors 80, 81, 85
Biotechnology 153, 155-158, 160
H
Heavy metal sensor 81
C
Cell extraction 29, 30, 32, 34, 35, 39
Confocal microscopy 102, 110, 111
Cross-resistance 21
Culturable cells 1, 5, 6, 9
I
Intrinsic markers 53, 63, 64
Kanamycin 20-22, 24
E
Environmental monitoring 29
European Commission 155, 156, 158, 160
F
Field lysimeters 129-134
Field release 117, 118, 122, 123, 128, 129,
133, 135, 136, 139, 143-145, 147
Field trials 141, 143, 158, 160, 161
Fingerprinting method 54, 55, 64
M
Mercury resistance 139-141, 144
Metabolically active cells 1, 13
N
Neomycin 18, 21-24
Nucleic acid 29, 30, 32, 33, 35, 37-39, 42-44
164
O
OECD 153-155, 160
P
PCR amplification 37, 38, 40, 42-44
Plant-pathogenic bacteria 96
Plasmid transfer 148-150
Promoter probe transposon 89, 91
Pseudomonas fluorescens SBW25 117
Quantitative PCR 41
Regulation 153-159
REP-PCR 61
Reporter gene 139, 143
Reporter system 87, 89, 90, 93
Resuscitation 5-8
Rhizobia 127, 128, 132, 135, 140, 141, 143,
145-150
V
V. vulnificus 1
VBNC (viable but nonculturable) 1-10,
12-14