Bacterial Growth, Environmental Effects and Strategies
Microbial Physiology Module 2
Aims and Objectives
By the end of this modules you should
Understand the processes of bacterial growth Be able to describe the phases of bacterial growth Be able to distinguish between methods of determining bacterial growth Understand the effects of
Nutrient levels Temperature Oxygen Osmotic pressure pH
on bacterial growth
Bacterial Growth
Bacterial growth equates to cell reproduction
Compare growth of multicellular vs unicellular organisms
Multicellular: increase in the size of the organisms Unicellular: increase in the number of individuals in the population
Changes in cell number are used to monitor bacterial growth
Reproduction of Bacterial Cells
Binary fission
Assexual reproduction A cell divides to produce two identical progeny cells
Binary Fission
Binary fission involves 3 processes
Elongation DNA replication Cell division
Compare this arrangement to the G1 S G2 M C cell cycle in eukaryotic cells
Cell Elongation
Biosynthesis of a new cell wall and membrane
Begins at specific sites located at the poles of cocci cells (eg. Enterococcus)
As new cell wall material is synthesised, is forced away from the site resulting in an elongated cell
For Gram-negative rods (such as E. coli)
Cell wall is added all around the the cylindrical region Outer membrane material is inserted at specific adhesion sites between the cytoplasmic and outer membrane
Cell Elongation Diagrams
DNA Replication
For molecular details, see Module 1 Duplication of the cells chromosome E. coli cells reproduce every 20 minutes Replication of the E. coli chromosome takes 40 minutes
A new round of DNA replication is initiated prior to the completion of the previous round Multiple replication forks Dividing cells inherit one complete copy of the genome plus additional material produced as a result of subsequent replication
DNA Replication
Chromosome duplication also offers a mechanism of regulation
DNA replication always begins at the origin Genes closest to the origin are duplicated first This effectively increases the copy number of these genes
Increases the products from these genes
Genes associated with cell wall / membrane synthesis are located near the origin
Septum Formation
Partioning of chromosome and cytoplasmic components Formation of a crosswall between the two cells Chromosomes are separated by associations with the cytoplasmic membrane Septum is formed by invaginations of the cell membrane, followed by the cell wall
Growth Rate
Time it takes a bacterial cell to reproduce
generation time or doubling time denoted by k
reciprocal of the doubling time see Atlas page 418 for full details
is characteristic of bacterial species is defined by other factors
temperature media conditions pH
Growth Rate: Some Examples
Organism Bacillus stearothermophilus Escherichia coli Pseudomonas putida Vibrio marinus Mycobacterium tuberculosis Treponema pallidum Temp (C) 60 37 30 15 37 37 Generation Time (min) 11 20 45 80 360 1980
Physiological Effects of Growth
Cell mass increases
cells become larger during periods of rapid growth increase in cell components is required
increase in DNA, RNA and protein
There is a relative increase in RNA levels compared to DNA and protein at higher growth rates
due to increase in the number of ribosomes as a result of cells requirement for more protein
Effects of Bacterial Growth on Macromolecular Composition of Cell
100 90 80 70 60 50 40 30 20 10 0 0 0.5 1 1.5 2 2.5
No. Doublings / hr
% of mass
Protein RNA DNA
Genetic Adaptations for Increased Growth Rates
Bacteria have evolved adaptations for increased gene expression for increased growth rates
in E. coli, genes encoding essential structures (eg OMP) are located near the origin of replication effectively increasing the dosage of these genes during replication OMP mRNAs also have a longer half-life than mRNAs for genes involved in catabolism increasing the time they are available for translation
Population Growth Phases
Unrestricted growth
growth that occurs when there are no limiting factors on the population
eg. Nutrients waste product accumulation pH
Balanced growth
synthesis of all cell constituents in a balanced manner
Bacterial Growth Curve
Lag
Log viable bacteria / mL
Exponential
Stationary
Death
Time
Lag Phase
Lag
Occurs when organisms are transferred to new medium Little increase in cell number Bacteria are transporting nutrients inside cell from the new medium Cells are preparing for replication and division Individual cells increase in size growth is generally unbalanced and unrestricted
Exponential (or log) Phase
Lag Exponential
Bacterial cell division begins
proceeds as a geometric progression cell numbers increase as an exponential function of time
Growth is unrestricted, but balanced
the concentration of all macromolecules within the cells are increasing at the same rate
Stationary Phase
Lag Exponential Stationary
No net increase in cell numbers
growth rate = death rate
Brought about by exhaustion of nutrients, waste product buildup or changes in physical conditions Metabolic rate decreases
Feedback mechanisms regulate enzymes involved in key metabolic steps
Stationary Phase
Cells are more resistant to environmental stresses Significant physiological changes can occur between cells in log phase and those in stationary phase
eg. Arthobacter cells change from rod-shaped cells (log phase) to cocci (stationary phase)
Growth is unbalanced
various cellular components are synthesised disproportionately
Death Phase
Lag Exponential Stationary Death
Decline in cell numbers Brought about when toxins or waste products reach a threshold concentration Bacterial growth is restricted and unbalanced
cells cannot obtain all requirements for growth or replication
Measurement of Bacterial Growth
Direct method
cell counts
either via a microscope or cell counter
Indirect methods
colony counts (viable cell counts) Spectrophotometry (turbidity) Weight (dry vs wet) ATP determination
Turbidity vs Viable Cell Count
Log10 Optical cfu/mL Density
9.0 1.0 0.75 8.0 0.5 7.0 6.0 0.25 5.0
Lag
Exponential
Stationary
Death
Turbidity Viable cell counts
Time
Death phase is less obvious when measuring turbidity as non-viable cells have similar absorption properties to viable cells
Viable Cell Counts
1 mL
1/10
1/100
1/103
1/104
1/105
1 mL
9 mL Broth
1 mL
1 mL
1 mL
Plate 1 mL, incubate and count colonies
Lots
Lots
159
17
Growth of Bacterial Cultures
In nature
conditions cannot be controlled many species co-exist changes in conditions may cause population shifts as conditions favour certain members of the population over others
In the lab
conditions can be controlled established to favour a particular organism important in industrial processes for the accumulation of desired metabolic products
Batch Cultures
produce bacterial growth curves just discussed inoculation of fresh media with bacteria nutrients are expended metabolic products accumulate closed environment
eg. Inoculating 100 mL of a rich media in a 1 L flask with E. coli
Continuous Culture
Fresh medium replaces spent medium
continuous replenishing of nutrients and removal of waste products permits continuous growth of culture
Continuous culturing can be controlled by
Turbidostat
monitors turbidity and cycles media as required
Chemostat
constant flow rate continuously cycles media keeps cells in log phase
Synchronous Culture
Synchronous growth
all cells divide at the same time can be achieved by altering environmental conditions
repeatedly changing the temperature adding fresh media to cells entering stationary phase
can only be maintained for a few generations
Effects of Nutrient Concentration on Bacterial Growth
Nutrients
obtained from the environment used for energy and biosynthesis of macromolecules
Cell solids components (H, O, C, N, P and S) Cations (K, Mg, Ca, Fe, Mn, Co, Cu, Mo and Zn) Anions (Cl) Vitamins
Most natural ecosystems are characterised by low nutrient levels
bacteria must be able to survive periods of starvation
General Strategies for Coping with Low Nutrient Levels
Starvation Factor
Amino acids Ammonia
System
Stringent response Ntr system
Genetic Control
relA (stringent factor) spoT (ppGpp degradation) glnA (Glutamine synthetase) glnG (NRI: response regulator) glnL (NRII: histidine kinase) cya (adenylate cyclase) crp (catabolite repression protein) phoBRUA
Glucose Phosphate
Catabolite repression Pho system
Stringent Response
Response to depleted amino acid pool
amino acid starvation
Mechanism for controlling the transcription of specific operons that code for rRNA and tRNA Reduces the rate of protein synthesis by decreasing the synthesis of rRNA
shuts down a number of energy-draining activities as a single response
How the Stringent Response Works
Amino acid starvation results in the expression of relA (stringent factor) stringent factor associated ribosomes allow uncharged tRNA to bind to the A site stringent factor catalyses the pyrophosphorylation of GTP (to pppGpp) or GDP (to ppGpp) ppGpp may
inhibit transcription of tRNA and rRNA genes cause stalling of translation and premature termination
Stringent Response
Ammonia Starvation
Low levels of nitrogen Ntr System
scavenging system
turns on genes for ammonia production from other nitrogen sources genes for glutamine synthetase are also induced
ATP-dependent assimilation of glutamine from low levels of ammonia glutamine amino nitrogen can be transferred to other amino acids ammonia supply for the cell
Ntr System in E. coli
glnA-glnL-glnG
glnA: glnL: glnG: glutamine synthetase NRII (a histidine kinase) NRI (a response regulator)
two-component regulatory system
discussed further in Module 4 phosphorylation of the histidine kinase transfer of the phosphate to response regulator response regulator acts as DNA binding protein and regulates transcription
regulation of gln gene transcription is via 54
Phosphate Starvation
Response to low levels of inorganic phosphate The Pho system
utilises phosphates from sources other than inorganic phosphates involves over 100 proteins over production of alkaline phosphatase utilisation of phosphate from organic sources
Specific Strategies for Survival in Conditions of Low Nutrients
Oligotrophs
bacteria that preferentially grow at low nutrient levels generally have slow growth rates can acquire substrates against steep concentration gradients conserve available resources generally small cells produce prosthecae which increases the surface area to volume ratio
Endospore-Forming Bacteria
Differentiation to a non-reproducing form Endospore formation is repressed by glucose and other growth substances Energy for sporulation comes from cellular protein and poly--hydroxybutyrate Spore-forming organisms have specific genes responsible for spore formation
Expression is controlled by sigma factors
Genetics of Endospore Formation
Endospore properties
Can withstand adverse conditions of dessication and elevated temperatures
Can remain viable almost indefinitely
Under permissive conditions of water and nutrient availability and acceptable temperature, spores germinate
Spore swells Breaks out of spore coat Elongates Returns to a vegetative cell
Effects of Temperature
One of the most important factors influencing bacterial growth Specific cells growth within well-difined temperature growth ranges
Difined by
Minimum temp.
Metabolic inactivity below this temp
Maximum temp
Cells dont grow above this temperature
Optimal temperature
Highest growth rate
Growth Ranges
Optimum Temperature
Relative growth rate
Minimum Temperature
Maximum Temperature
10
20
30
40
50
60
70
80
90
Temperature (C)
Growth Ranges
Extreme Thermophiles
(optimal growth >80C)
Thermophiles
Relative growth rate
Mesophiles
(optimal growth 20-40C)
(optimal growth >40C)
Psychrophiles
(optimal growth <20C)
10
20
30
40
50
60
70
80
90
Temperature (C)
Psychrophiles
Optimal growth below 20C Can grow below 0C (if liquid water is available) Found in Artic, Antarctic and ocean environments Can be found in refrigerators
Cause food spoilage
Psychrophile Physiology
Enzymes and ribosomes are active at low temps
Can be inactivated at moderate temps (~25C)
More lipids with unsaturated or short chain fatty acids in psychrophile membranes
Membranes more semifluid in the cold Under higher temps, membranes become leaky
Limit of psychrophile growth and metabolism may be the availability of liquid water
Mesophiles
Optimal growth between 20-40C
Many have optimal temps of 37C
Physiological temperature for humans
Normal body flora and most pathogens are mesophiles
Grow rapidly and optimally in the human body Eg. E. coli
Thermophiles and Extreme Thermophiles
Optimal growth for thermophiles: >40C
many have optima between 55-60C
Optimal growth for extreme thermophiles: > 80C Sources
hot springs hydrothermal vents
Many thermophiles can survive low temperatures
viable thermophilic bacteria found in frozen Antarctic soil
Physiology of Thermophiles
Enzymes are not readily denatured at high temps
possibly due to specific primary sequences
Membranes posses a major proportion of high molecular weight and branched fatty acids
membranes remain semipermeable at high temps
Temperature Growth Ranges: Examples
Temperature (C)
-20 -10 0 10 20 30 40 50 60 70 80 90 100 110
Vibrio marinus Pseudomonas acanae Escherichia coli Vibrio cholerae Staphylococcus aureus Streptococcus pyogenes B. stearothermophilus Thermus acquaticus Pyrococcus furiosus
Heat Shock Response
Occurs when organisms are shifted to a higher temperature
evolutionarily conserved response
Results in the production of a set of heat shock proteins (Hsps)
24 proteins in E. coli transcription of 20 of these is under the control of 32
encoded by rpoH
Roles of HSPS
Protect proteins against degradation at elevated temperatures
denatured proteins aggregate and become nonfunctional
Involved in all growth-related processes
cell division replication transcription and translation protein folding membrane function
Lethal Effects of Temperature
Lethal effects of heat
Heat sterilisation
121C for 15 minutes for steam sterilisation 180C for 180 minutes for dry heat sterilisation
Lethal effects of cold
if >0C, but less than minimum growth, bacteria will lose viability due to absence of growth formation of ice crystals can destroy cells
freeze fracturing
Effects of Oxygen
Classification of microorganisms based on O2 tolerance or requirement Aerobes
Obligate aerobes
absolute requirement for molecular O2 carry out aerobic respiration
Microaerophiles
grow only in low concentrations of O2 O2 is an absolute requirement
Anaerobes
Anaerobes
Obligate anaerobes
O2 is inhibitory to microbial growth carry out fermentation
Strict anaerobes
very sensitive to O2 die even with a short exposure
Facultative anaerobes
grow in the presence or absence of O2 +O2: aerobic respiration -O2: fermentation
Oxygen Toxicity
Relationship between organisms and O2 can be more than metabolic other factors include the formation of toxic O2 products and the availability (or absence) of enzymes to deal with these products
catalase and peroxidase degrade peroxides superoxide dismutase degrades superoxides
Reduced O2 may arise from reduced flavoproteins (and other electron acceptors) and as a result of radiation
Anaerobiosis
Ability of some facultative anaerobes (such as E. coli) to carry out aerobic respiration using oxygen at the terminal electron acceptor when molecular oxygen is available Can also use anaerobic respiration using nitrate (or other terminal electron acceptors) when oxygen levels are depleted Regulated by the Arc and Fnr systems
Osmotic Pressure and Salinity
Osmotic pressure
diffusion of water across cell membranes in response to solute concentrations often associated with saline environments
Hypertonic solutions can lead to cell shrinkage and dessication Hypotonic solutions can result in cell bursting Osmotolerant
organisms that can withstand high osmotic pressures
Osmophiles
require high solute concentrations for growth
Halophiles
Salinity has important effect on osmotic pressure Halophiles
require NaCl moderate halophiles (3% NaCl) extreme halophiles (upto 25% NaCl)
High NaCl concentrations normally disrupt membrane transport systems and denature proteins
some organisms, such as Halobacterium, possess unusual plasma membranes and many unusual enzymes
Hydrostatic Pressure
The pressure exerted by a column of water as a result of the wieght Each 10m water depth = 1 atm. Hydrostatic pressures > 200 atm generally inactivate enzymes and disrupt membrane processes barotolerant
can grow at high hydrostatic pressures
barophiles
grow best at high pressures
Effects of pH
pH 0 Increasing Acidity
Bacteria exhibit various tolerances to pH pH effects are largely based on changes in the nature of proteins
Effects charge interactions within and between polypeptides
Neutral pH 7 Increasing Alkalinity
Effect secondary and tertiary structure of proteins Most grow in a range between 6.0 and 9.0
Neutralophiles
Grow best under neutral conditions
pH 14
Acidophiles
Restricted to growth at low pH Acidophilic membranes cannot function under neutral conditions Physiology
Internal pH of all cells is relatively neutral If pH of environment is less than the pH of the cytoplasm (i.e. a large pH), it will be hard to generate the PMF required for ATP synthesis Many acidophiles have a reverse membrane potential (, charge separation across the membrane)
Outside of membrane is negatively charged Inside of membrane is positively charged
Combination of large pH and reversed generate ATP via PMF
Alkaliphiles
Bacteria with high pH requirements for growth
Can be in the range of 9 to 11
Physiology
Generation of PMF
Reversed pH across the membrane
7 to 9 internally, 9 to 11 extrenally
Alkaliphiles use Na+/H+ or K+/H+ antiporters to maintain sufficiently high to drive the PMF
pH Tolerances of Various Bacteria
Organism Thiobacillus thiooxidans Lactobacillus acidophilus Escherichia coli Clostridium sporogenes Nitrobacter spp. Nitrosomonas spp. Minimum pH 1.0 4.0-4.6 4.4 5.0-5.8 6.6 7.0-7.6 Optimum Maximum pH pH 2.0-2.8 5.8-6.6 6.0-7.0 6.0-7.6 6.6-8.6 8.0-8.8 4.0-6.0 6.8 9.0 8.0 10.0 9.4
Effects of Light
Light is required by photosynthetic bacteria to generate ATP Function optimally at specific light intensities and utilise specific wavelengths Some can move through their environment in response to light (phototaxis) Light (in particular UV light) can have detrimental effects
Some organisms synthesise carotenoids and pigments that absorb harmful wavelengths before they can cause damage
Learning Exercises
Read Principles of Microbiology (Atlas) Chapter 9 Find examples for each group discussed in this module
Eg. Alkaphiles, barophile, barotolerant etc
Next Week Module 3
Genetic Adaptation
