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Chapter 7
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Reproductive Strategies
• the reproductive strategies of
eukaryotic microbes
– asexual and sexual, haploid or diploid
• Bacteria and Archaea
– haploid only, asexual - binary fission,
budding, filamentous
– all must replicate and segregate the
genome prior to division
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Figure 7.1
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Figure 7.2
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Figure 7.3
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Chromosome Partitioning
• replisome pushes, or condensation of,
daughter chromosomes to opposite ends
• MreB (murein cluster B) – an actin
homolog, plays role in determination of
cell shape as spiral inside cell periphery,
and chromosome segregation
– new origins associate with MreB tracks
– if MreB is mutated, chromosomes do not
segregate
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Figure 7.4
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Plasmid Segregation
• plasmids replicate independently and
carry proteins necessary for segregation
• E. coli R1 plasmid produces three
proteins essential for its inheritance
– ParM – similar to MreB, actin homolog
forms long filaments
– ParR (repressor) and ParC (centromere-
like) both bind to origins and link to ParM
– ParM filaments elongate and separate
plasmids to opposite ends of cell
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Figure 7.5
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Cytokinesis - Septation
• septation – formation of cross walls
between daughter cells
• several steps
– selection of site for septum formation
– assembly of Z ring
– linkage of Z ring to plasma membrane (cell
wall)
– assembly of cell wall synthesizing machinery
– constriction of cell and septum formation
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Figure 7.6
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Figure 7.7
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Table 7.1
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Figure 7.8
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Figure 7.9
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Figure 7.10
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Growth
• increase in cellular constituents
that may result in:
– increase in cell number
– increase in cell size
• growth refers to population
growth rather than growth of
individual cells
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Figure 7.11
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Lag Phase
• cell synthesizing new components
– e.g., to replenish spent materials
– e.g., to adapt to new medium or other
conditions
• varies in length
– in some cases can be very short or even
absent
26
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Exponential Phase
• also called log phase
• rate of growth and division is
constant and maximal
• population is most uniform in terms
of chemical and physical properties
during this phase
27
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Balanced Growth
• during log phase, cells exhibit
balanced growth
– cellular constituents manufactured at
constant rates relative to each other
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Unbalanced Growth
• rates of synthesis of cell components
vary relative to each other
• occurs under a variety of conditions
– change in nutrient levels
• shift-up (poor medium to rich medium)
• shift-down (rich medium to poor medium)
– change in environmental conditions
29
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Figure 7.12
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Stationary Phase
• closed system population growth
eventually ceases, total number of
viable cells remains constant
– active cells stop reproducing or
reproductive rate is balanced by death
rate
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Death Phase
It was argued that when cells are in the decline phase, they are dead,
however, there is a debate over this statement. There are two alternative
hypotheses explaining the phenomenon;
– Cells are Viable But Not Culturable (VBNC)
• Cells alive, but dormant. Cells were genetically
programmed to survive. They will not be grown under
laboratory conditions but in animal passage they might
grow again.
– Programmed cell death
• Fraction of the population genetically programmed to die (commit
suicide). Those will leak their nutrients to be consumed by other
surviving bacteria that will not lyse and might grow later when
cells are passed through an animal.
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Loss of Viability
Figure 6.8 In all cases, the survivors will not be able to grow under
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regular laboratory conditions.
Loss of viability
• A; cells leave stationary phase due to starvation and
accumulation of toxic waste and inter decline phase.
• C; some part of the culture inters the state of viable but non-
culturable due to starvation. They can only grow through
passage through animals.
37
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Why it happen?
• bacterial population continually evolves as
they are better able to use released nutrients
upon the death of other cells.
• process marked by successive waves of
genetically distinct variants with more
capability of survival
• natural selection occurs
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Prolonged Decline in Growth
Prolonged growth experiments reveal that an exponential decline
phase is some times replaced by gradual decline in the number
of culturable cells which can last for months or years. The most
strong cells will utilize nutrients released from dead cells and
tolerate toxic waste. i.e natural selection occurs
• bacterial population continually evolves
• process marked by successive waves of genetically distinct
variants
Figure 6.9
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Figure 7.16
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Table 7.2
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Exponential microbial
growth as drawn from
data in table 7.2.
population is Logarithmic
doubling every
generation
Direct plot
Figure 7.15
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Table 7.3
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Measurement of
Microbial Growth
• can measure changes in number of
cells in a population
• can measure changes in mass of
population
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Measurement of Cell Numbers
• Most obvious method is direct cell counts
– Counting chambers; Petroff-Hausser counting chamber
can be used for counting prokaryotic cells. While
hemocytometers can be used for counting both prokaryotic
and eukaryotic cells.
– Electronic counters; such as Coulter counters are used for
direct counting of protists and yeast or cells can be counted
by flow cytometer.
– Membrane filters; used mainly for counting bacteria from
large liquid volumes.
• Viable cell counts
– plating methods
– membrane filtration methods
46
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Counting Chambers
• easy,
inexpensive, and Figure 7.18
quick
• useful for
counting both
eukaryotes and
prokaryotes
• cannot
distinguish living
from dead cells
47
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Flow Cytometry
• microbial suspension forced through
small orifice with a laser light beam
• movement of microbe through orifice
impacts electric current that flows
through orifice
• instances of disruption of current are
counted
• specific antibodies can be used to
determine size and internal complexity
49
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Figure 7.19
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Figure 7.20
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Measurement of Cell Mass
An increase in the cell growth is accompanied by an increase in
cell mass.
Dry weight is the most direct method of cell mass estimation
– time consuming and not very sensitive
• Measuring the quantity of a particular cell constituent
– e.g., protein, DNA, ATP, or chlorophyll
– useful if amount of substance in each cell is constant
• Turbidometric measures (light scattering) of cultures
– Amount of scattered light is directly proportional to the
biomass of the cells. High absorbance means low
transmittance = high microbial growth
– quick, easy, and sensitive
55
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Figure 7.21
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The Continuous Culture of Microorganisms
Growing bacteria in a flask is a closed system as no addition of
new nutrients and no removal of waste therefore the log phase
only stays for short period of time.
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The Chemostat
58 Figure 6.16
Dilution Rate and Microbial Growth
dilution rate D, is the rate at which medium flows through vessel relative to vessel
size. F is the flow rate, v is the vessel volume. D = f/v
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• Dilution rate reflects the amount of new nutrients added to the
media, so at first, when dilution rates are low the
microorganisms grow well because of the accumulation of the
nutrients but soon the essential nutrient will be depleted.
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Importance of continuous culture methods
Industrial
1. Constant supply of cells in exponential phase growing at a
known rate. This could be beneficial if the organism is used
for vaccine production.
2. Food and industrial microbiology benefits. i.e production of
nutrients such as vitamins or certain proteins that is used as
drugs …etc.
Research
1. Study of microbial growth at very low nutrient
concentrations, close to those present in natural environment
2. Study of interactions of microbes under conditions resembling
those in aquatic environments
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The Influence of Environmental Factors on
Growth
• Most organisms grow in fairly moderate environmental
conditions however some procaryotes can live in very harsh
environments. Example;
• Extremophiles
– grow under harsh conditions that would kill most other
organisms. e.g some bacteria can live under 1.5 miles
below earth surface, no O2 and temperature > 60˚ C.
63
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Table 7.4
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Solutes and Water Activity…con
• Water activity (aw) or water potential
– amount of water available to organisms
– reduced by interaction with solute molecules (osmotic
effect)
higher [solute] lower aw
– reduced also by adsorption to surfaces (matrix effect)
It is important that bacteria are able to respond to changes in
their osmotic concentrations in their environment. Example
microbes in hypotonic environment can reduce osmotic
concentrations in their cytoplasm by the use of inclusion
bodies (collecting the small proteins in large bulk) or some
other organisms will stretch the pores in their membranes and
allow solutes to leave to prevent water from accumulating
inside. Or some protists use contractile vacuoles to expel water
outside such as the Paramesium.
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Water Activity (aw)
• water activity of a solution can be expressed quantitatively
and is equal to 1/100 of the relative humidity of solution.
• also equal to ratio of solution’s vapor pressure (Psoln) to that
of pure water (Pwater)
• Aw = Psoln/ Pwater
67
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pH
• measure of the Figure 7.25
relative acidity
of a solution
• negative
logarithm of
the hydrogen
ion
concentration
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pH
• Acidophiles
– growth optimum between pH 0 and pH 5.5. Most fungi
prefer pH 4-6
• Neutrophiles
– growth optimum between pH 5.5 and pH 7. most bacteria
and protists.
• Alkalophiles
– growth optimum between pH 8.5 and pH 11.5
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pH
Microorganisms respond to external pH changes using
different mechanisms;
– Most acidophiles and alkalophiles maintain an internal pH
near neutrality
• Their plasma membrane is impermeable to protons
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pH
• most microbes maintain an internal pH near
neutrality
– the plasma membrane is impermeable to proton
– exchange potassium for protons
• acidic tolerance response
– pump protons out of the cell
– some synthesize acid and heat shock proteins that
protect proteins
• many microorganisms change the pH of their
habitat by producing acidic or basic waste
products
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Effect of temperature
• The most important factor influencing the effect of
temperature is the temperature sensitivity of the enzyme-
catalyzed reactions with the speed of the enzyme reactions
double for every 10° C rise. Above certain temperatures the
speed slows and very high temperature become lethal as this
temperature will affect both structure and function aspects
of the microbe
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Temperature
• Organisms exhibit
distinct cardinal growth
temperatures
– minimal
– maximal
– Optimal
• These cardinal
temperatures are not
fixed, i.e based on
certain changes in the
media they might be
changed. Table 6-5
shows some of these
Temperatures.
77 Figure 6.20
Classes of organisms based on temperature
• Psychrophiles; grow well at 0 and have optimum at about 15 ° C
and maximum at 20 C .The cell membranes of the bacteria living
in this environment contains high amount of unsaturated fatty
acids that keeps the membrane semi-fluid.
• Psychrotrophs or facultative psycrophiles; grow at 0-7° C with
optimum between 20-30 ° C and maximum at about 35 ° C. this
type of bacteria and fungi are major food spoilage organisms.
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Table 7.5
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Figure 7.27
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Adaptations of Thermophiles
• protein structure stabilized by a variety
of means
– e.g., more H bonds
– e.g., more proline
– e.g., chaperones
• histone-like proteins stabilize DNA
• membrane stabilized by variety of means
– e.g., more saturated, more branched and
higher molecular weight lipids
– e.g., ether linkages (archaeal membranes)
82
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Oxygen Concentration
• growth in oxygen correlates with
microbes energy conserving
metabolic processes and the electron
transport chain (ETC) and nature of
terminal electron acceptor
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• microaerophiles
– requires 2–10% O2
• facultative anaerobes
– do not require O2 but grow better in its
presence
• aerotolerant anaerobes
– grow with or without O2
85
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Figure 7.28
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Oxygen Concentration
87 Figure 6.22
Classification of organisms based on O2
requirements
• Obligate aerobes; contain SOD and Catalase and can not live
without O2. Oxygen serves as the terminal electron acceptor
for electron transport chain. It participates in sterol and fatty
acid synthesis in multicellular eukaryotes.
• Facultative anaerobes; contain SOD and catalase. do not
require O2 for growth but grow better in its presence.
• Aerotolerant; contains SOD but not catalase. Ignores the
presence of O2 and grow equally well whether its present or
not.
• Strict or obligate anerobe; does not contain SOD or catalase.
Can not live in its presence. Thus they obtain their energy
through fermentation.
• Microaerophiles; contain SOD and low levels of catalase.
Need 2-10% O2 not regular 20 %.
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Figure 7.30
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Pressure
• microbes that live on land and water
surface live at 1 atmosphere (atm)
• some Bacteria and Archaea live in
deep sea with very high hydrostatic
pressures
93
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Pressure
• barotolerant
– adversely affected by increased
pressure, but not as severely as
nontolerant organisms
• barophilic (peizophilic) organisms
– require or grow more rapidly in the
presence of increased pressure
– change membrane fatty acids to adapt
to high pressures
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The Electromagnetic Spectrum
Figure 7;31
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Radiation
Radiation Damage
• Ionizing radiation; is very harmful to microorganisms. It has
very short wavelength and high energy.
• There are two major forms of ionizing radiation;
– x rays and gamma rays. Low levels of these radiation will
cause mutations death
– Radiation disrupts chemical structure of many molecules,
including DNA. It destroys;
– Hydrogen bonds and oxidize double bonds.
– Destroys ring structures and polymerize some molecules
• Oxygen enhances these changes by producing hydroxyl fee radicals
– damage may be repaired by DNA repair mechanisms
however, higher levels are lethal. Therefore,
– Ionizing radiation is used for sterilization.
– Deinococcus radiodurans
96 • extremely resistant to DNA damage
Radiation Damage…
• Ultraviolet (UV) radiation; can kill all types of
microorganisms due to its short wavelength (10-400) and high
energy with the most lethal UV radiation is 260 nm because
this wavelength is the most absorbed by DNA.
– mutations death.
– UV light causes formation of thymine dimers in DNA which
inhibits DNA replication and function.
– DNA damage can be repaired by several repair
mechanisms.
– Longer UV wavelengths (325-400) are also harmful by
inducing the breakdown of tryptophan to toxic products.
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Radiation damage…
• Visible light is so important but at high intensities it become
harmful by the action of pigments (chlorophyll, flavins,
bacteriochlorophyll and cytochromes) which absorb light and
get excited and act as photosensitizers. These will then
transfer its energy to O2 generating the singlet oxygen (1O2)
• It is powerful oxidizing agent and will quickly destroy a
cell. This is also employed by phagocytic cells to destroy
bacteria after engulfing.
• Many microorganisms avoid the harmful damage of extensive
light by carotenoid pigments.
• This pigment protect many light-exposed
microorganisms from photooxidation by quenching
singlet O2 by converting its energy back to the
unexcited ground state.
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Growth Limitation by Environmental
Factors
• Leibig’s law of the minimum
– total biomass of organism determined by nutrient present
at lowest concentration. An increase in limiting essential
nutrient such as phosphate will increase the population
until some other nutrient become limiting.
• Shelford’s law of tolerance
– above or below certain environmental limits, a
microorganism will not grow, regardless of the nutrient
supply. Such as extreme temperature or pH, O2 level,
pressure or the presence of other inhibitory substances.
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Responses to low nutrient levels (oligotrophic
environments)
100
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Biofilms
• most microbes grow attached to surfaces
(sessile) rather than free floating
(planktonic)
• these attached microbes are members of
complex, slime enclosed communities
called a biofilm
• biofilms are ubiquitous in nature in water
• can be formed on any conditioned surface
101
Figure 7;33
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Biofilm Formation
• microbes reversibly attach to
conditioned surface and release
polysaccharides, proteins, and DNA
to form the extracellular polymeric
substance (EPS)
• additional polymers are produced as
microbes reproduce and biofilm
matures
104
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Biofilms
• a mature biofilm is a complex, dynamic
community of microorganisms
• heterogeneity is differences in metabolic
activity and locations of microbes
• interactions occur among the attached
organisms
– exchanges take place metabolically, DNA
uptake and communication
105
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Biofilm Microorganisms
• the EPS and change in attached
organisms’ physiology protects microbes
from harmful agents
– UV light, antibiotics, antimicrobials
• when formed on medical devices, such as
implants, often lead to illness
• sloughing off of organisms can result in
contamination of water phase above the
biofilm such as in a drinking water
system
106
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Quorum Sensing
• acylhomoserine lactone (AHL) is an
autoinducer molecule produced by many gram-
negative organisms to regulate biofilm
formation and increase virulence factors
– diffuses across plasma membrane
– once inside the cell it induces expression of
target genes that regulate a variety of
functions
– many microbes produce effect
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Figure 7.36
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