03 Growthreproduction

Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Chapter 7
Microbial Growth and

Reproduction
1
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
2
Figure 7.1
3
Figure 7.2
4
Bacterial Cell Cycle

• cell cycle is sequence of events from
formation of new cell through the
next cell division
– most bacteria divide by binary fission
• two pathways function during cycle
– DNA replication and partition
– cytokinesis
5
Chromosome Replication and

Partitioning - 1
• most bacterial chromosomes are circular
• single origin of replication – site at which
replication begins
• terminus – site at which replication is
terminated, located opposite of the origin
• replisome – group of proteins needed for DNA
synthesis
• DNA replication proceeds in both directions
from the origin
• origins move to opposite ends of the cell
6
Figure 7.3
7
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
8
Figure 7.4
9
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
10
Figure 7.5
11
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
12
Z Ring Formation - Role in

Septation
• protein FtsZ
– tubulin homologue, found in most bacteria and
archaea
– polymerization forms Z ring, filaments of meshwork
• MinCDE system in E. coli limits the Z ring to the
center of the cell
– MinC, MinD, MinE oscillate from one side of cell to
other
– link Z ring to cell membrane
– Z ring constricts and cell wall synthesis of septal wall
13
Figure 7.6
14
Figure 7.7
15
Table 7.1
16
Cellular Growth and

Determination of Cell Shape
• determined by peptidoglycan synthesis
in bacteria
– penicillin binding proteins (PBPs) – link
peptidoglycan strands and catalyze
controlled degradation for new growth
– autolysins – PBP enzymes that degrade
peptidoglycan and site new units added
17
Figure 7.8
18
Cell Wall Growth and Cell

Shape Determination
• cocci divisome - new peptidoglycan forms
only at the central septum
– FtsZ determines site of cell wall growth
– FtsZ may recruit PBPs for synthesis of
septum
• rods are similar but elongate prior to
septation
– MreB determines cell diameter and
elongation as Z ring forms in center
19
Figure 7.9
20
Cell Wall Growth and Cell

Shape Determination
• vibrio (comma-shaped bacteria)
– FtsZ – forms Z ring
– MreB – helical polymerization throughout
cell
– crescentin – intermediate filament
homologue cytoskeletal protein
• localizes to short, curved side of cell
• asymmetric cell wall synthesis forms curve
21
Figure 7.10
22
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
23
The Growth Curve

• observed when microorganisms are
cultivated in batch culture
– culture incubated in a closed vessel with a
single batch of medium
• usually plotted as logarithm of cell
number versus time
• has four distinct phases
– lag, exponential, stationary, senescence, and
death
24
Figure 7.11
25
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
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
Balanced Growth
• during log phase, cells exhibit
balanced growth
– cellular constituents manufactured at
constant rates relative to each other
28
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
Figure 7.12
30
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
31
Possible Reasons for

Stationary Phase
• nutrient limitation
• limited oxygen availability
• toxic waste accumulation
• critical population density reached
32
Stationary Phase and

Starvation Response
• entry into stationary phase due to
starvation and other stressful conditions
activates survival strategy
– morphological changes
• e.g., endospore formation
– decrease in size, protoplast shrinkage, and
nucleoid condensation
– RpoS protein assists RNA polymerase in
transcribing genes for starvation proteins
33
Starvation Responses
• production of starvation proteins
– increase cross-linking in cell wall
– Dps protein protects DNA
– chaperone proteins prevent protein
damage
• cells are called persister cells
– long-term survival
– increased virulence
34
Senescence and Death Phase

• two alternative hypotheses
– cells are Viable But Not Culturable
(VBNC)
• cells alive, but dormant, capable of new
growth when conditions are right
• programmed cell death
– fraction of the population genetically
programmed to die (commit suicide)
35
Figure 7.13
36
Prolonged Decline in Growth
• bacterial population continually evolves

• process marked by successive waves of
genetically distinct variants
• natural selection occurs
37
Figure 7.14
38
The Mathematics of Growth

• generation (doubling) time
– time required for the population to
double in size
– varies depending on species of
microorganism and environmental
conditions
– range is from 10 minutes for some
bacteria to several days for some
eukaryotic microorganisms
39
Table 7.2
40
Exponential Population Growth
• population is
doubling Figure 7.15
every
generation
41
Measurement of Growth
Rate and Generation Time
Figure 7.16
42
Figure 7.17
43
Table 7.3
44
Measurement of
Microbial Growth
• can measure changes in number of
cells in a population
• can measure changes in mass of
population
45
Direct Measurement of Cell

Numbers
• direct cell counts
– counting chambers
– electronic counters – flow cytometry
– on membrane filters
46
Counting Chambers
• easy, inexpensive,
and quick Figure 7.18
• useful for
counting both
eukaryotes and
prokaryotes
• cannot
distinguish living
from dead cells
47
Direct Counts on Membrane

Filters
• cells filtered through special membrane
that provides dark background for
observing cells
• cells are stained with fluorescent dyes
• useful for counting bacteria
• with certain dyes, can distinguish living
from dead cells
48
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
Viable Counting Methods

• spread and pour plate techniques
– diluted sample of bacteria is spread over
solid agar surface or mixed with agar and
poured into Petri plate
– after incubation the numbers of organisms
are determined by counting the number of
colonies multiplied by the dilution factor
– results expressed as colony forming units
(CFU)
50

• membrane filter technique
– bacteria from aquatic samples are
trapped on membranes of known pore
size
– membrane soaked in culture media
– colonies grow on membrane
– colony count determines number of
bacteria in original sample
51
Figure 7.19
52
Figure 7.20
53

• if microbe cannot be cultured on
plate media
• dilutions are made and added to
suitable media
• turbidity determined to yield the
most probable number (MPN)
54
Measurement of Cell Mass

• dry weight
– time consuming and not very sensitive
• 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)
– quick, easy, and sensitive
55
Figure 7.21
56
The Continuous Culture of

Microorganisms
• growth in an open system
– continual provision of nutrients
– continual removal of wastes
• maintains cells in log phase at a
constant biomass concentration for
extended periods
• achieved using a continuous culture
system
57
Importance of Continuous
Culture Methods
• constant supply of cells in exponential
phase growing at a known rate
• study of microbial growth at very low
nutrient concentrations, close to those
present in natural environment
• study of interactions of microbes under
conditions resembling those in aquatic
environments
• food and industrial microbiology
58
The Chemostat
• rate of incoming
medium = rate of
removal of Figure 7.22
medium from
vessel
• an essential
nutrient is in
limiting quantities
59
Dilution Rate and Microbial

Growth
dilution rate – rate at
which medium flows
through vessel Figure 7.23
relative to vessel size
note: cell density

maintained at wide
range of dilution
rates and chemostat
operates best at low
dilution rate
60
The Turbidostat
• regulates the flow rate of media
through vessel to maintain a
predetermined turbidity or cell
density
• dilution rate varies
• no limiting nutrient
• turbidostat operates best at high
dilution rates
61
The Influence of
Environmental Factors on
Growth
• most organisms grow in fairly
moderate environmental conditions
• extremophiles
– grow under harsh conditions that
would kill most other organisms
62
Table 7.4
63
Solutes and Water Activity

• changes in osmotic concentrations in the
environment may affect microbial cells
– hypotonic solution (lower osmotic
concentration)
• water enters the cell
• cell swells may burst
– hypertonic (higher osmotic concentration)
• water leaves the cell
• membrane shrinks from the cell wall
(plasmolysis) may occur
64
Microbes Adapt to Changes in

Osmotic Concentrations
• reduce osmotic concentration of
cytoplasm in hypotonic solutions
– mechanosensitive (MS) channels in plasma
membrane allow solutes to leave
• increase internal solute concentration
with compatible solutes to increase their
internal osmotic concentration in
hypertonic solutions
– solutes compatible with metabolism and
growth
65
Extremely Adapted Microbes

• halophiles
– grow optimally in the presence of NaCl or
other salts at a concentration above about
0.2M
• extreme halophiles
– require salt concentrations of 2M and 6.2M
– extremely high concentrations of potassium
– cell wall, proteins, and plasma membrane
require high salt to maintain stability and
activity
66
Effects of NaCl on Microbial

Growth
Figure 7.24
• halophiles
– grow optimally at
>0.2 M
• extreme halophiles
– require >2 M
67
Solutes and Water Activity

• water activity (aw)
– amount of water available to organisms
– reduced by interaction with solute
molecules (osmotic effect)
higher [solute]  lower aw
– reduced by adsorption to surfaces
(matric effect)
68
Water Activity (aw)

• water activity of a solution is 1/100 the
relative humidity of solution
• also equal to ratio of solution’s vapor
pressure (Psoln) to that of pure water
(Pwater)
• Aw = Psoln/ Pwater
– Low water activity means most water is bound
• osmotolerant microbes can grow over
wide ranges of water activity
69
pH
• measure of the Figure 7.25
relative acidity
of a solution
• negative
logarithm of
the hydrogen
ion
concentration
70
pH
• acidophiles
– growth optimum between pH 0 and pH 5.5
• neutrophiles
– growth optimum between pH 5.5 and pH 7
• alkaliphiles (alkalophiles)
– growth optimum between pH 8.5 and pH 11.5
71
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
72
Temperature
• microbes cannot regulate their
internal temperature
• enzymes have optimal temperature
at which they function optimally
• high temperatures may inhibit
enzyme functioning and be lethal
73
Temperature
• organisms Figure 7.26
exhibit distinct
cardinal growth
temperatures
– minimal
– maximal
– optimal
74
Temperature Ranges for

Microbial Growth
• psychrophiles – 0o C to 20o C
• psychrotrophs – 0o C to 35o C
• mesophiles – 20o C to 45o C
• thermophiles – 55o C to 85o C
• hyperthermophiles – 85o C to 113o C
75
Table 7.5
76
Figure 7.27
77
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)
78
Oxygen Concentration
• growth in oxygen correlates with
microbes energy conserving
metabolic processes and the electron
transport chain (ETC) and nature of
terminal electron acceptor
79
Oxygen and Bacterial Growth

• aerobe
– grows in presence of atmospheric oxygen (O2)
which is 20% O2
• obligate aerobe – requires O2
• anaerobe
– grows in the absence of O2
• obligate anaerobe
– usually killed in presence of O2
80
Oxygen and Bacterial Growth
• microaerophiles
– requires 2–10% O2
• facultative anaerobes
– do not require O2 but grow better in its
presence
• aerotolerant anaerobes
– grow with or without O2
81
Figure 7.28
82
Basis of Different Oxygen

Sensitivities
• oxygen easily reduced to toxic reactive
oxygen species (ROS)
– superoxide radical
– hydrogen peroxide
– hydroxyl radical
• aerobes produce protective enzymes
– superoxide dismutase (SOD)
– catalase
– peroxidase
83
Strict Anaerobic Microbes

• all strict anaerobic microorganisms lack
or have very low quantities of
– superoxide dismutase
– catalase
• these microbes cannot tolerate O2
• anaerobes must be grown without O2
– work station with incubator
– gaspak anaerobic system
84
Figure 7.29
85
Figure 7.30
86
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
87
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
88
The Electromagnetic Spectrum
Figure 7.31
89
Radiation Damage
• ionizing radiation
– x-rays and gamma rays
– mutations  death (sterilization)
– disrupts chemical structure of many
molecules, including DNA
• damage may be repaired by DNA repair
mechanisms if small dose
– Deinococcus radiodurans
• extremely resistant to DNA damage
90
Radiation Damage…
• ultraviolet (UV) radiation
– wavelength most effectively absorbed by
DNA is 260 nm
– mutations  death
– causes formation of thymine dimers in DNA
– requires direct exposure on microbial surface
– DNA damage can be repaired by several
repair mechanisms
91
Radiation Damage…
• visible light
– at high intensities generates singlet
oxygen (1O2)
• powerful oxidizing agent
– carotenoid pigments
• protect many light-exposed
microorganisms from photooxidation
92
Microbial Growth in Natural

Environments
• microbial environments are
complex, constantly changing, often
contain low nutrient concentrations
(oligotrophic environment) and may
expose a microorganism to
overlapping gradients of nutrients
and environmental factors
93
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
94
Figure 7.34
95
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
96
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
97
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
98
Cell to Cell Communication

Within the Microbial Populations
• bacterial cells in biofilms communicate in
a density-dependent manner called
quorum sensing
• produce small proteins that increase in
concentration as microbes replicate and
convert a microbe to a competent state
– DNA uptake occurs, bacteriocins are
released
99
Quorum Sensing
• acylhomoserine lactone (AHL) is an
autoinducer molecule produced by
many gram-negative organisms
– diffuses across plasma membrane
– once inside the cell it induces
expression of target genes that regulate
a variety of functions
– many microbes produce effect
100
Figure 7.35
101
Quorum Sensing Systems

• processes regulated by quorum
sensing involve host-microbe
interactions
– symbiosis – Vibrio fischeri and
bioluminescence in squid
– pathogenicity and increased virulence
factor production
– DNA uptake for antibiotic resistance
genes
102
Figure 7.36
103

03 Growthreproduction

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03 Growthreproduction

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Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Microbial Growth and

Bacterial Cell Cycle

Chromosome Replication and

Z Ring Formation - Role in

Cellular Growth and

Cell Wall Growth and Cell

Cell Wall Growth and Cell

The Growth Curve

Possible Reasons for

Stationary Phase and

Senescence and Death Phase

Prolonged Decline in Growth

• bacterial population continually evolves

The Mathematics of Growth

Exponential Population Growth

Direct Measurement of Cell

Direct Counts on Membrane

Viable Counting Methods

Viable Counting Methods

Viable Counting Methods

Measurement of Cell Mass

The Continuous Culture of

Dilution Rate and Microbial

note: cell density

Solutes and Water Activity

Microbes Adapt to Changes in

Extremely Adapted Microbes

Effects of NaCl on Microbial

Solutes and Water Activity

Water Activity (aw)

Temperature Ranges for

Oxygen and Bacterial Growth

Oxygen and Bacterial Growth

Basis of Different Oxygen

Strict Anaerobic Microbes

The Electromagnetic Spectrum

Microbial Growth in Natural

Cell to Cell Communication

Quorum Sensing Systems

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