Debora Frigi Rodrigues

Mentor: Dr. James Tiedje

 Outline
• Importance of studying cold-adapted
microorganisms

• Distribution and diversity of two cold-adapted

genera on our planet

• Characterization and identification of species

from a cold-adapted genus

• Architecture of thermal adaptation in cold-

adapted species
Why study cold adapted organisms?
• Ecology: nutrient cycling, greenhouse gas
production and consumption…

• Biotechnology: industry, foods,

bioremediation (new enzymes)…

• Evolution: microbial life a million years ago

• Astrobiology: strategies for life on

cryoplanets; biomarkers for life detection
 Cold temperatures on Earth

❧80% of Earth’s
biosphere is Alaska 85%
permanently
cold China 20%
Russia &
❧20% of Earth’s
Canada
land is
55%
permafrost

❧Permafrost:
Soil, sand,
sediment at or
below 0°C for 2
or more years

Map provided by NSDIC

 Low temperature environments
Permafrost T= 0 to -14°C
20% of land surface

Sea Ice T= - 2 to - 35°C

13% of Earths’ surface ~80% of
the Earth’s
surface! Seasonal Ice and Snow
3% of Earth’s surface
© 2005 AECOM

Deep Sea T<4°C Glaciers T= 0 to - 40°C

>95% of ocean 10% of land surface
volume JLM Visuals
Kolyma Lowland, Siberia
Why study the Siberian permafrost?
Because of lots of environmental
stresses, such as…

• Cold temperatures, but stable

-10 to -12°C

Depth below ground

• Low water activity
aw = 0.90
• Low nutrients
frozen matrix
• Radiation
0.03 Mrad/ year for 3 M years
Dr. Rivkina
 Sampling soil in the permafrost
• Kolyma Lowland, Northeast Eurasia

– Cold arid arctic climate

– Mean annual air temperature of -14oC
– Upper soil layers thaw and freeze
each year

•permafrost cores remain •“natural freezer”

frozen and uncontaminated
•samples remained frozen
•surface of extracted cores during transport
was trimmed away
•cores were stored at -20oC
•slow rotary drill •cores were divided into in the lab
•no solutions or sections and placed in pre-
drilling mud sterilized aluminum cans
Bacterial colonies grown from permafrost

Dr. Hinsa
Isolates characterized from Arctic permafrost
Soil
oldest Growth
# of isolates age -2.5C
Actinobacteria
Arthrobacter (17) 3m 15:17
Microbacteriaceae (5) 3m 5:5
Firmicutes
Exiguobacterium (4) 3m 4:4
Planococcus (5) 30k 3:5
Bacteroidetes
Flavobacterium (4) 30k 1:4
Proteobacteria
γ - Psychrobacter (4) 30k 3:4
α - Sphingomonas (2) 3m 2:2

Blue = Gram positive; Red = Gram negative; m= million

years; k= thousand years
 Psychrobacter genus

• Gram negative
Psychrobacter arcticus 273-4
• Gamma Proteobacteria
• Predominantly isolated from
cold or salty environments
– Shallow and deep marine
water
– Sea ice
Dr. Ponder – Salty Foods
– Intestine of fish
• Grow at temperatures down to
-10oC
 Exiguobacterium genus
• Gram-positive
• Found in a variety of Exiguobacterium sibiricum 255-15
environments:
– Antarctica, Siberian
permafrost
– Alkaline environments
– Food processing effluents
– Hot springs
• Defining characteristics of
genus
– Peritrichous flagella
– Facultatively anaerobic
 Work design
Biogeography, diversity and
abundance of Exiguobacterium
and Psychrobacter genus
Characterization and Identification
of Exiguobacterium isolates from
Siberian permafrost
Identification of genes related to
cold and heat acclimation in
Exiguobacterium 255-15
- 54 soil/sediment DNA
extraction from: Michigan, Iowa,
Brazil, Puerto Rico, Hawaii,
Siberia and Antarctica
-Q-PCR and 16S rRNA clone
libraries
-Computational analyses of
Biodiversity
- Polyphasic approach
-Genome analysis
-Transcriptome analysis
 Samples for my study

Iowa and Michigan

Siberia

Hawaii Puerto Rico

Brazil

Antarctica
 Quantitative
real-time PCR
with SYBR
green
SYBR green dye attaches
only in double strand
DNA and fluoresce.

The more DNA target, the

more fluorescence is
seen
 1 2 3 4 5 6 7 1 2 3 4 5 6 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 8 9 101121314151618192021222324
Hawaii

8
Iowa

Puerto Rico Brazil Michigan Antarctica Siberian permafrost

Samples
 Why patchy distribution?
• Environmental factors? pH, salinity, organic
matter, micro and macronutrients…

• Principal component analysis (PCA):

– Look at relationships between variables
– Group large data set into small artificial variables
(PC) that will account for most variance in the
large data set
– Goal: Reduce redundancy in those variables =
correlate with one another
Principal component analysis of the
different samples
Correlation bi-plot of physico-
chemical and Q-PCR results
Variables (axes F1 and F2: 76.60 %)
1

0.75
Moisture
Psychrobacter
0.5 OM pH
Salinity
Exiguobacterium
0.25
K
F2 (15.80 %)

Cu
CEC
0

-0.25 Mg

Ca
-0.5

-0.75

-1
-1 -0.75 -0.5 -0.25 0 0.25 0.5 0.75 1
F1 (60.81 %)
 1 2 3 4 5 6 7 1 2 3 4 5 6 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 8 9 101121314151618192021222324
Hawaii

8
Iowa

Puerto Rico Brazil Michigan Antarctica Siberian permafrost

Samples
 Sites studied for Psychrobacter spp.
Comandante Ferraz – KBS – Mid- Mangrove “Las
marine sediment successional Cabezas de San
community Juan”

Samples for clone libraries

Botany point – marine MSU – Baker Cave system
sediment Woodlot “Las
Carmelitas”

Sites studied for Exiguobacterium spp.

Clone library construction and analysis
Bacterial Sequence each clone
community to identify OTUs
DNA extraction (Operational
Taxonomic Unit,
Gene of interest i.e.“species”)
amplified by PCR with
specific primer

PCR product

Insert PCR product

into plasmids 2 OTUs
4 OTUs
7 OTUs
Clone the plasmid
into E.coli cells Measure Diversity Find closest
Richness and evenness relative

Select clones
with plasmids
 Diversity concepts
• Richness: number of • Evenness: the frequency
species per sample of different species
occurrence

Community 1 Community 2
Community 1 Community 2
2 species = 1 individual = 5 individuals
4 species or OTUs
or OTUs = 9 individuals
= 6 individuals = 5 individuals
= 3 individuals
Community 1 is richer than community 2 Community 1 has less evenness than
community 2
A community dominated by 1 or 2 species is considered to be less diverse
than one in which several different species have a similar abundance
 E.aurantiacum
E. indicum K22-26
64
Michigan A04
Exiguobacterium sp. 8N
E.homiense
E.aestuarii strain TF-16

Exiguobacterium spp. 57
E.lactigenes
Exiguobacterium sp. A1
E.taiwanense
Exiguobacterium sp. India orange

clone library results

45
Exiguobacterium sp. M37
Exiguobacterium sp. AT4
54
Exiguobacterium sp. AT1b
52 Antarctica C04
E.marinum strain TF-80
66
Antarctica E12
Antarctica D10
52 Antarctica B08
51 Antarctica C05a
Antarctica D02
99 Antarctica B12
78 Antarctica D11
Antarctica G05
E.acetylicum

100%
65 E.sibiricum strain 255-15
G4 B3 91
Exiguobacterium sp. strain 5138
47 Exiguobacterium sp. India Stream
E.sibiricum strain 7-3
D5 66
Antarctica D05
undance

56 Puerto Rico E07

D2 90% 70
98
Puerto Rico B12
E.oxidotolerans
Puerto Rico H06
Puerto Rico G06
81 E.undae
72

80%
E.undae strain 190-11
Antarctica G04
C4 C7 H6 96
Michigan C07
90 E.antarcticum
80 Michigan B03
B.subtilis

0.005
 P. fozii
49 Michigan H11

100%
P. okhotskensis
P. luti LMG 21276
Puerto Rico G03
B3 87 Michigan H02
60 Puerto Rico H02
F7
H10
90%
P. arcticus 273-4
Puerto Rico B03
A12
of OTUs abundance
G10 E12 53
Puerto Rico A07
87
P. glacialis DD43
H2 H11 68
P. cryohalolentis K5

H3a
80%
C9 B4
46
Antarctica G10

49
89 Michigan E07
Antarctica C07
73
Antarctica H02
Antarctica E07

70% 76
74 P. cibarius JG-220
Michigan C02
P. immobilis DSM 7229T
Antarctica H10
ercentage)

Psychrobacter
Michigan A12

60%
69
67 Puerto Rico E12
Michigan F07
Antarctica H03a

spp. clone
52
Antarctica H03
P. faecalis OS-38
50% 98 P. pulmonis CECT 5989T

library results
87 Psychrobacter sp. ikaite
Puerto Rico D03
Michigan C09

40%
Puerto Rico B04
Moraxella atlantae

0.01
 Diversity analysis
• Measuring diversity within a community
• Shannon index (H)
Accounts for both richness (number of species) and evenness
(proportion) of the species. The index increases either by
having more unique species, or by having a greater species
evenness. More sensitive for impact of rare species. Sampling
sensitive.

• Chao1 estimator
Chao1 index, a nonparametric estimator suitable for microbial
diversity analysis. It estimate the richness of a community.

• Simpson index (D)

Accounts for both richness and evenness of the species. D varies
from 0 to 1. When (D) is close to 0, the diversity is high. More
sensitive for impact of common/abundant species, less for rare
species. Sampling insensitive.
 Results Diversity
Samples Genus studied Total No of No. Of Shannon Chao 1 Simpson
sequences OTUs Index estimator Index
(D)

Ant. Psychrobacter 129 21 2.60 21.43 0.09

MI Psychrobacter 150 14 2.22 17.00 0.13
PR Psychrobacter 137 11 1.84 11.50 0.21
Ant. Exiguobacterium 149 7 1.53 7 0.26
MI Exiguobacterium 109 4 0.34 5 0.84
PR Exiguobacterium 135 1 0 1 1

Ant.= Antarctica; MI= Michigan; PR= Puerto Rico

 Conclusions
• Exiguobacterium is more widely distributed on
our planet, but less diverse than
Psychrobacter

• Neither Psychrobacter nor Exiguobacterium

are cosmopolitan due to environmental
factors: salinity, pH and Cu

• Both genera seems to preferentially inhabit

polar regions
 Work design
Biogeography, diversity and
abundance of Exiguobacterium
and Psychrobacter genus
Characterization and Identification
of Exiguobacterium isolates from
Siberian permafrost
Identification of genes related to
cold and heat acclimation in
Exiguobacterium 255-15
- 54 soil/sediment DNA
extraction from: Michigan, Iowa,
Brazil, Puerto Rico, Hawaii,
Siberia and Antarctica
-Q-PCR and 16S rRNA clone
libraries
-Computational analyses of
Biodiversity
- Polyphasic approach
-Genome analysis
-Transcriptome analysis
 Exiguobacterium strains from Siberian
permafrost

~250 mile
s
Pliocene Pleistocene Holocene
Quaternary

20-30 tds 7-3

200-600 tds 190-11

2-3Mi 255-15 E. sibiricum 7-3 E. undae 190-11 E. sibiricum 255-15,

Tertiary

pleiromorphic rods. Borehole motile short rods.

rods. Borehole 5/94 (1994), 5.5 m Borehole 2/94 (1994),
6Kh-Yu (1989), 43.6 m
8m
 Polyphasic approach
* Phenotypic analyses:
– Chemotaxonomic markers
– Metabolic characteristics
– FAME
– Cell morphology at different temperatures
* Genotypic analyses:
– rep- PCR
– DNA- DNA percent similarity
– Multi-loci sequence typing
 Summary of phenotypic results
Strains Peptidoglycan Quinones Polar Lipids Carbon FAMES
source
255-15     
7-3     
190-11     
E. undae     
E. antarcticum     
E. acetylicum    
E. aurantiacum     

A3 α L-Lys-Gly MK7 major Major:

PG,DPG ,PE

The same symbol means very similar/identical traits

 Summary of genotypic clusters
Strains BOX-PCR DNA-DNA 16S rpoB recA hsp70 gyrB citC
similarity rRNA
255-15        
7-3        
190-11        
E. undae        
E. antarcticum        
E. acetylicum        
E. aurantiacum        

The same symbols means very similar trait or clustered strains

 Conclusions

• The permafrost isolate 190-11 (200 K) is

the same species as E. undae DSM 14481T
– Type strain is isolated from warmer
environment (Germany)
• The permafrost isolates 255-15 (2-3 Mi)
and 7-3 (20-40 K) are the same species
even though they have different cell
morphology
 Work design
Biogeography, diversity and
abundance of Exiguobacterium
and Psychrobacter genus
Characterization and Identification
of Exiguobacterium isolates from
Siberian permafrost
Identification of genes related to
cold and heat acclimation in
Exiguobacterium 255-15
- 54 soil/sediment DNA
extraction from: Michigan, Iowa,
Brazil, Puerto Rico, Hawaii,
Siberia and Antarctica
-Q-PCR and 16S rRNA clone
libraries
-Computational analyses of
Biodiversity
- Polyphasic approach
-Genome analysis
-Transcriptome analysis
 Genome analysis on
Exiguobacterium sibiricum 255-15

• General features

– 2.9 Megabase genome

– 47.8 % GC content
– 2,978 candidate ORFs
– 138 Major contigs
– 65 Minor contigs
Natalia Ivanova, Lawrence Berkeley National Labs
 Exiguobacterium
E x ig u o b a c t e r biuipmh a s ic A rrh e n iu ssibiricum
S tra in 2 5 5 -1 5
P lo t

3
biphasic arrhenius plot
2

0
3 .1 3 .2 3 .3 3 .4 3 .5 3 .6 3 .7
LN gen/hr

-1
2
R = 0 .9 4 0 7
-2

-3
2
-4
R = 0 .9 8 0 4
39oC 28oC
10oC -2.5oC
-5
o
1 0 0 0 /TK( )
 Experimental design
39oC 28oC 10oC -2.5oC

6 biological
replicates
0.1 ≤
O.D.600 ≤ 0.3

100mL for RNA

extraction

Dye swaps
Technical
Replicates:
Indirect labelling
cy3 cy5 cy3 cy5 cy3 cy5 cy3 cy5

6 hybridizations for each comparison

 Direct comparisons of the samples
• 70 mer probes
• Duplicate spots and negative controls: 10 Human and
10 Arabidopsis
• 2931 gene-specific probes
• 6 group-specific probes for 25 ORFs
• Data analysis by limma within CarmaWeb environment
• Differential expression when P-value < 0.01

39oC 28oC

10oC -2.5oC
 Conclusions
• Exiguobacterium is adapted to cold
temperatures: not many differences in
gene expression at 4oC, 10oC and 28oC.

• Genes believed to be expressed under

heat- and cold-shock conditions are also
expressed under continuous growth at the
higher extremity of the arrhenius profile
and at subzero temperatures, respectively.
 Take home messages
• Microorganisms successful in the poles are
not confined to the poles

• Environmental factors affect presence and

diversity of microorganisms

• Several molecular and physiological

adaptations seem to be necessary for
survival and growth at the higher extremity of
growth range as well as at subzero
temperatures
 ACKNOWLEGEMENTS
My Committee members: Especial thanks:
• Dr. Goris
• Dr.Tiedje • Dr. Ramette
• Dr.Thomashow • Dr. Gilichinsky
• Dr. Kathariou • Dr. Pellizari
• Dr. Ivannova
• Dr.Bagdasarian • Dr. Zhou
• Dr.Britton • Dr. He
Astrobiology group: • Dr. Gantner
• Dr.Ayala-del-Río • Chia-Ju Lin
• Dr. Bergholz • Ederson Jesus
• Dr. Bakermans • All tiedje lab
• Dr. Hinsa • My friends
• My husband: Murillo Xavier
• Dr. Vishnivetskaya