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Prokaryotes either bacteria or archaea

o Differ from eukaryotes in size and simplicity
o Most lack internal membrane systems
Can see shape and general morphological features under a light microscope
Shape and Arrangement
o Cocci - spheres
o Diplococci pairs eg Neisseria
o Streptococci chains
o Staphylococci grape like clusters
o Tetrads 4 cocci in a square
o Sarcinae cubic configuration of 8 cocci
o Bacilli rods
o Coccobacilli very short rods
o Vibrios resemble rods, comma shaped
o Spirilla rigid helices
o Spirochetes flexible helices
o Mycelium network of long multinucleate filaments
o Pleomorphic organisms that are variable in shape depending on outside factors
eg environment
Size
o Oscillatoria RBC e. coli streptococcus poxvirus influenza virus -
o Smallest 0.3 m (Mycoplasma)
average rod 1.1 - 1.5 x 2 6 m (E. coli coccobacilli)
o Largest - Epulopiscium fishelsoni: isolated from the intestinal tract of a brown
surgeon fish from the Red Sea
Size: 600X80 um (more than a million times larger in vol than E. coli
o Even larger (100X)
Thiomargarita nambiensis (Ocean sediment off the coast of Namibia)
Common Bacterial structures and function
o Plasma membrane selective permeable barrier, mechanical boundary of cell,
nutrient and waste transport, location of may metabolic processes (respiration,
photosynthesis), detection of environmental cues for chemotaxis
o Gas vacuole an inclusion that provides buoyancy for floating in aquatic
environments
o Ribosomes protein
o Inclusions storage of carbon, phosphate and other substances
o Nucleoid localization of genetic material (DNA)
o Periplasmic space in typical gram-negative bacteria contains hydrolytic enzymes
and binding proteins for nutrient processing and uptake; in typical gram-positive
bacteria may be smaller or absent
o Cell wall protection from osmotic stress, helps maintain cell shape
o Capsules and slime layers resistance to phagocytosis, adherence to surfaces
 o Fimbriae and pili attachment to surfaces, bacterial conjugation and transformation,
twitching and gliding motility
o Flagella swimming and swarming motility
o Endospore survival under harsh environmental conditions
Bacterial Cell envelope
o Plasma membrane
Similar to eukaryotic plasma membrane
Fluid mosaic model of membrane structure proteins float within the
membrane
Peripheral membrane protein on surface easily removed
Integral protein inside the membrane transporters
Glycoproteins attached to surface
o Bacterial Lipids
Saturation levels of membrane lipids reflect environmental conditions such
as temperature
At cold temp have unsaturated fatty acids so the membrane remains fluid,
saturated fatty acids at high temps
Bacterial membranes lack sterols but do contain sterol-like molecules,
hopanoids they stabilize membrane, also found in petroleum
o Function
Encompasses the cytoplasm
Selectively permeable barrier
Interacts with external environment
Receptors for detection of and response to chemicals in
surroundings
Transport systems
Metabolic processes
o Bacterial Cell wall
Peptidoglycan (murein)
rigid structure that lies just outside the
cell plasma membrane
two types based on Gram stain
o Gram-positive: stain purple; thick
peptidoglycan
o Gram-negative: stain pink or red;
thin peptidoglycan and outer
membrane
 Cell wall functions
o Maintains shape of the bacterium almost all bacteria have one
o Helps protect cell from osmotic lysis
o Helps protect from toxic materials
o May contribute to pathogenicity
Peptidoglycan structure - peptidoglycan only found in bacteria
o Mesh like polymer of identical subunits forming long strands
Two alternating sugars
N-acetylglucosamine (NAG)
N- acetylmuramic acid
Alternating D- and L- amino acids
D amino acids are unique to peptidoglycan, they make the cell wall
very stable and degradation resistance against common peptidases
Cysteine and methionine contain sulphur
o Strands are crosslinked
Peptidoglycan strands have a helical shape
Peptidoglycan chains are crosslinked by peptides for strength
interbridges may form
peptidoglycan sacs interconnected networks
various structures occur
Gram-Positive cell walls
o Composed primarily of peptidoglycan quite porous so large molecules can pass
through
o May also contain teichoic acids (negatively charged)
Helps maintain cell envelope
Protect from environmental substances
May bind to host cells
o Some gram positive bacteria have layer of proteins on surface of peptidoglycan

 Gram negative cell walls

o More complex than gram-positive
o Consist of a thin layer of peptidoglycan surrounded by an outer membrane
o Outer membrane composed of lipids, lipoproteins and lipopolysaccharaide (LPS)
o No teichoic acids
LPS
o Three parts
Lipid A
Core polysaccharide
O side chain (O antigen) extends outside the cell
o Lipid A embedded in outer membrane
o Core polysaccharide, O side chain extend out from the cell
o contributes to negative charge on cell surface
o helps stabilize outer membrane structure
o may contribute to attachment to surfaces and biofilm formation plaque is a kind of
biofilm
o creates a permeability barrier
o protection from host defences (O antigen)
can actually change O antigen to help avoid host immune system
o can act as an endotoxin (lipid A) can cause septic shock syndrome if it gets in the
blood stream
Gram negative outer membrane permeability
o More permeable than plasma membrane due to presence of porin proteins and
transporter proteins
o porin proteins form channels to let small
molecules (600 700 daltons) pass
Mechanism of Gram stain Reaction
o Gram stain reaction due to nature of cell wall
o shrinkage of the pores of peptidoglycan layer
of Gram positive cells
constriction prevents loss of crystal
violet during decolourisation step
o thinner peptidoglycan layer and larger pores
of Gram negative bacteria does not prevent
loss of crystal violet
Osmotic Protection
o Hypotonic environments
Solute concentration outside the cell
is less than inside the cell
water moves into cell and cell swells
cell wall protects from lysis
o Hypertonic environments
solute concentration outside the cell is greater than inside
 water leaves the cell
plasmolysis occurs
Evidence of protective nature of the cell wall
o lysozyme breaks the bond between N-acetyl glucosamine and N-acetylmuramic acid
o penicillin inhibits peptidoglycan synthesis
o if cells are treated with either of the above they will lyse if they are in a hypotonic
solution

Cells that lose a cell wall may survive in isotonic environments

o Protoplasts
o Spheroplasts
o Mycoplasma
No cell wall
But plasma membrane more resistant to osmotic pressure
Archaea
o Highly diverse with respect to morphology, physiology, reproduction and ecology
o Best known for growth in anaerobic, hypersaline, pH extremes, and high-
temperature habitats
o Also found in marine arctic temperature and tropical waters
o Many features in common with Eukarya
genes encoding protein: replication, transcription, translation
o Features in common with Bacteria
genes for metabolism
o Other elements are unique to Archaea
unique rRNA gene structure
capable of methanogenesis (the
production of methane)
Archaeal Cell Envelopes
o Differ from bacterial envelopes in the molecular
makeup and organization
S layer may be only component outside
plasma membrane
some lack cell wall
capsules and slime layers are rare
Archaeal Membranes
o Composed of unique lipids
isoprene units (five carbon, branched)
ether linkages rather than ester linkages to glycerol
 o Some have a monolayer structure instead of a bilayer structure
Archaeal Cell walls lots of diversity
o Lack peptidoglycan

Translocation
o movement of proteins from cytoplasm to plasma membrane or periplasmic space
May be posttranslational
May be cotranslational (occurs during translation)
include transport proteins, ETC proteins, proteins involved in chemotaxis
and cell wall synthesis, enzymes
Secretion
o movement of proteins from the cytoplasm to external environment
hydrolytic enzymes for nutrient break down
Common translocation and secretion systems
o Sec-dependent pathway
Major pathway for all bacteria for transporting proteins across the plasma
membrane
o Gram-negative bacteria
may use Sec system
also must cross the outer membrane using Types I, II, III, IV, V systems
o All pathways require energy
Sec-dependent
o Posttranslational
o Allows the protein to be translocated when protein is unfolded
o Sec A binds to the signal peptide and inserts into the channel created by SecYEG
proteins
 o ATP hydrolysis by SecA translocates the protein through the channel
o Cotranslational see slides

Tat (twin-arginine translocase) System

o Protein translocation system in Bacteria and some Archaea
o Moves across plasma membrane
o Tat pathway translocates folded proteins with twin arginine residues in their signal
sequence
o Works with Type II secretion system
Type 1 secretion systems
o related to ABC transport systems
o Gram-positive/Gram-negative bacteria, and Archaea
o Secretion of toxins, proteases, other proteins
o transports proteins from cytoplasm across both plasma membrane and outer
membrane
Type II pathways transport proteins across outer membrane that were first translocated
across plasma membrane by Sec-dependent pathway
Types III is sec independent
o forms injectisomes (a needle like structure)
o transports virulence factors and other proteins
o machinery is used to pierce host cell and inject proteins into the host cell, can be
involved with rearranging the host cell cytoskeleton.
Type IV secretion system
o Also has a needle like structure
o secrete proteins
o transfer DNA from donor to recipient bacterium during conjugation
o found in both Gram-positive and Gram-negative
Type V are sec-dependent
o Autotransporters (transport themselves out)
o 3 domains
1st recognized by sec system
2nd forms a hole in the outer membrane
3rd domain transportation through the hole formed by 2nd domain
 Types I and IV also in Gram-positives
Types II, III, and V are unique to Gram-negatives
o most secrete virulence factors
Components outside of the cell wall
o Outermost layer in the cell envelope
o Glycocalyx
capsules and slime layers not as organized as capsules
S layers a protein layer
o Aid in attachment to solid surfaces e.g., biofilms in plants and animals
Capsules
o Usually composed of polysaccharides
o Well organized and not easily removed from cell
o Visible in light microscope
o Protective advantages
resistant to phagocytosis
protect from desiccation as its a moist, sticky surface outside cell
exclude viruses and detergents
o if knock out genes for capsules, those without capsules are not pathogenic eg
streptococcus pneumoniae
Slime layer
o similar to capsules except diffuse, unorganized and easily removed
o slime may aid in motility, use the slime to glide along a surface
S layers
o Regularly structured layers of protein or glycoprotein that self-assemble
in Gram-negative bacteria the S layer adheres to outer membrane
in Gram-positive bacteria it is associated with the peptidoglycan surface
o Protect from ion and pH fluctuations, osmotic stress, enzymes, and predation
o Maintains shape and rigidity
o Promotes adhesion to surfaces
o Protects from host defences like capsule to some extent
o May function as a receptor for bacteriophage
External Structures
o Extend beyond the cell envelope in bacteria
o Function in protection, attachment to surfaces, horizontal gene transfer, cell
movement
pili and fimbriae
flagella
pili and fimbriae
o short, thin, hairlike, proteinaceous appendages (up to 1,000/cell)
o can mediate attachment to surfaces and motility
o help bacteria to attach to solid surfaces
sex pili
o longer, thicker, and less numerous (1-10/cell)
o genes for formation found on plasmids
 o required for conjugation
Flagella
o Threadlike, locomotor appendages extending outward from plasma membrane and
cell wall
o Thin, rigid protein structures that cannot be observed with bright-field microscope
unless specially stained
o Functions
Motility allows them to swim towards food/away from danger
attachment to surfaces
may be virulence factors in some bacteria eg Helicobacter pylori
Flagella Distribution
o Monotrichous one flagellum - pseudomonas
o Polar flagellum flagellum
at end of cell
o Amphitrichous one
flagellum at each end of cell
o Lophotrichous cluster of
flagella at one or both ends
o Peritrichous spread over
entire surface of cell
3 parts of flagella
o Filament
o Hook
o Basal body
Much simplier in gram positive (on
right) as no outer membrane
About 10 proteins for forming basal body and hook, a single protein for the filament
(flagellin, a polymer)
o Grows at the tip of the filament not at the base
o Protein gets folded out through the hollow core and then polymerised
Archaeal Flagella
o Flagella thinner
o More than one type of flagellin protein
o Flagellum is not hollo
o Hook and basal body difficult to distinguish
o More related to type IV pili
o Growth occurs at the base, not the end
Motility
o Bacteria and Archaea have directed movement
o Chemotaxis move toward chemical attractants such as nutrients, away from
harmful substances
o Move in response to temperature, light, oxygen and osmotic pressure
Bacterial Flagellar Movement
o Flagellum rotates like a propeller
very rapid rotation up to 1100 revolutions/sec
 in general, counterclockwise (CCW)
rotation causes forward motion (run)
in general, clockwise rotation (CW)
disrupts run causing cell to stop and
tumble
Mechanism of Flagellar Movement
o Like a motor
o MotA and MotB creat a channel through
which protons can flow. This causes flagellum
to rotate
o Protons moving through the Mot protein
exert electrostatic forces on helically arranged
charges on the rotor proteins
o Default mode is counter clockwise
o A protein which controls direction of rotation, once phsphorylated it binds to the
motor protein and the change in charges makes the motor to rotate clockwise, but
within a few seconds the phosphate is removed by another protein, and rotation
goes back to anticlockwise
Twitching and Gliding Motility
o May involve type IV pili and slime
o Twitching
pili at ends of cell
short, intermittent, jerky motions
pili alternately extend and retract
o Gliding
smooth movements
due to slime layer
Chemotaxis
o Movement toward a chemical attractant or away from a chemical repellent
o Changing concentrations of chemical attractants and chemical repellents bind
chemoreceptors of chemosensing system
o Shown using semi solid agar plates, which allow the bacteria to move more
o Positive chemotaxis moving toward nutrients etc
o Negative chemotaxis moving away from stuff
o Tumble = change of direction
o Run = movement forward
o Normally its random, with many tumbles, but in the presence of an attractant,
tumbling frequency is intermittently reduced and runs in direction of the attractant
are longer
o Behavior of bacterium is altered by temporal concentration of chemical
o Chemotaxis away from repellent involves similar but opposite responses
Bacterial Cytoplasmic Structures
o Cytoskeleton
o Intracytoplasmic membranes
o Inclusions granules also known as inclusion bodies
 o Ribosomes
o Nucleoid and plasmids
Cytoskeleton
o Homologues of all 3 cytoskeletal elements have been identified in bacteria
Tubulin, actin and intermediate filaments
o Functions are similar as in eukaryotes
Role in cell division, protein localization, and determination of cell shape

Best studied examples

o FtsZ many bacteria forms ring during septum
formation in cell division
o MreB many rods maintains shape by positioning
peptidoglycan synthesis machinery
o CreS rare, maintains curve shape
Intracytoplasmic Membranes
o Plasma membrane infoldings
observed in many photosynthetic bacteria
observed in many bacteria with high
respiratory activity
Inclusions
o Granules of organic or inorganic material that are
stockpiled by the cell for future use
o Some lie free in the cytoplasm while some are
enclosed by a single-layered membrane
membranes vary in composition
some made of proteins; others contain lipids
 may be referred to as microcompartments
Storage Inclusions
o Storage of nutrients, metabolic end products, energy, building blocks
o Glycogen inclusions
o Carbon storage poly--hydroxybutyrate (PHB)
o Phosphate - Polyphosphate (Volutin)
o Amino acids - cyanophycin granules (store extra nitrogen)
Microcompartments
o Not bound by membranes but compartmentalized for a specific function (protein
shell)
o Carboxysomes - CO2 fixing bacteria
contain the enzyme ribulose-1,5,-bisphosphate carboxylase (Rubisco), for
CO2 fixation
Gas vacuoles
o Protein cylinders
o found in aquatic, photosynthetic bacteria and archaea
o provide buoyancy in gas vesicles
o allow bacteria to float near the surface of water so they can absorb sunlight
Magnetosomes
o found in aquatic bacteria
o magnetite particles for orientation in Earths magnetic field so they can swim down
to the nutrient rich sediments
o cytoskeletal protein MamK helps form magnetosome chain
Ribosomes
o In cytoplasm
o Complex protein/RNA structures
sites of protein synthesis
bacterial and archaea ribosome = 70S
eukaryotic (80S) S = Svedburg unit
o Bacterial ribosomal RNA
16S small subunit
23S and 5S in large subunit
The nucleoid
o Where DNA and genetic material is
o Usually not membrane bound (few exceptions)
o Usually 1 closed circular, double-stranded DNA molecule
o Supercoiling and nucleoid proteins (different from histones) aid in folding
Plasmids
o Extrachromosomal DNA
found in bacteria and archaea
usually small, closed circular DNA molecules
o Exist and replicate independently of chromosome
episomes may integrate into chromosome
inherited during cell division
The Bacterial Endospore
 o Complex, dormant structure formed by some bacteria
o Various locations within the cell
o Highly Resistant to numerous environmental conditions
Heat, radiation, chemicals, desiccation
o Mainly belongs to bacteria of genus
Bacillius (anthrax, if inhale the spores can germinate in the lungs and cause
anthrax)
Clostridium (one species cause tetanus) (another causes food poisoning as
has the botchulinum toxin)
Sporosarcina
Most soil bacteria
Most are gram positive bacteria
o When conditions are right, the spores can germinate and give rise to a cell which can
then start dividing
o When sterilising, its not sterile until you get rid of the endospores too, Autoclaved
to get rid of them, kinda like a big pressure cooker
o These spores can live for thousands of years, possibly even millions of years, paper
recently claimed to have found one 25-40 million yrs ago, found in gut of an extinct
bee which got trapped in amber.
o Another paper claimed 250 million yrs, as spores were found trapped in salt crystals
Endospore structure from inside to outside
o Core, - DNA, Ribosome and very little water
o Inner membrane highly impermeable
o Germ cell wall
o Cortex, peptide of lycan
o Outer membrane
o Coat composed of 70 different proteins which are highly cross linked
o Exosporium
Why so resistant
o Calcium (complexed with dipicolinic acid) stabilizes DNA
o Small, acid-soluble, DNA-binding proteins (SASPs) stabilizes the DNA within the
core
o Dehydrated core
o Spore coat and exosporium protect
Sporulation
o 200 genes involved
o Process of endospore formation
o Occurs in hours (up to 10 hours)
o Normally commences when growth ceases because of lack of nutrients
o Complex multistage process
 Formation of Vegetative Cell
o Activation
prepares spores for germination
often results from treatments like heating (70-80 degrees)
o Germination
environmental nutrients are detected
spore swelling and rupture of absorption of spore coat
increased metabolic activity
o Outgrowth
emergence of vegetative cell
o this occurs fairly quickly, comes out of the endospore, and then divides, depending
on germination time
Archaeal vs. Bacterial cytoplasm
o Very similar lack of membrane-enclosed organelles
o May contain inclusion bodies (e.g. gas vesicles for buoyancy control)
o All the usual components ribosomes nucleoid region inclusion bodies
o Some structures different
 Bacterial Cell Structure Lectures Questions
o Compare and contrast the cell walls of typical Gram-positive and Gramnegative
bacteria.
o Relate bacterial cell wall structure to the Gram-staining reaction.
o Distinguish pili (fimbriae) and flagella, and describe the ultrastructure of of both
Gram-positive and Gram-negative bacterial flagella.
o Describe in general terms the process of sporulation and discuss those properties of
endospores that are thought to contribute to its resistance to environmental
stresses.
o Compare and contrast the major characteristics of bacterial and archaeal cells.
o Describe the major characteristics and functions of the protein secretion systems
described in the lecture.
Microbial nutrition
Common Nutrient Requirements
o macroelements (macronutrients)
C, O, H, N, S, P, K, Ca, Mg,
and Fe
required in relatively large
amounts
o micronutrients (trace elements
Mn, Zn, Co, Mo, Ni, and Cu
required in trace amounts
often supplied in water or
in media componenets

Requirements for Carbon

o Heterotrophs
use organic molecules as carbon sources which often also serve as energy
source
o autotrophs
use carbon dioxide as their sole or principal carbon source
 Requirements for Nitrogen, Phosphorus and Sulfur
o needed for synthesis of important molecules (e.g., amino acids, nucleic acids)
o N - organic molecules, ammonia (inorganic), nitrate (inorganic) via assimilatory
nitrate reduction, nitrogen gas via nitrogen fixation
o Phosphorus - most organisms use inorganic phosphorus which is directly
incorporated into their cells
Needed for nucleic acids, phospholipids etc
o Sulfur - most organisms use sulfate and reduce it by assimilatory sulfate reduction
In methionine and cysteine
Found in vitamins as a core factor, eg vitamin b1
Some organisms have sulfur granules in the cytoplasm
Nutritional Types of organisms
o Based on energy source
Phototrophs use light - photosynthesis
Chemotrophs obtain energy from oxidation of chemical compounds
Most bacteria are chemotrophs
o Based on electron source
Lithotrophs use reduced inorganic substances
Organotrophs obtain electrons from organic compounds
Growth factors amino acids, purines and pyrimidines and vitamins etc
o Organic compounds
o Essential cell componenets (or their precursors) that the cell cannot synthesize
o Must be supplied by environment if cell is to survive and reproduce
o Auxotrophs
A mutant organism that requires a particular additional nutrient which the
normal strain does not
A mutant that lacks a growth factor, so that factor must be added to the
growth medium in order for it to grow
o Autotrophs
Wild type organism which can synthesize all the growth factors
Culture Media
o Need to grow, transport, and store microorganisms in the laboratory
o Culture media is solid or liquid
preparation
o Must contain all the nutrients
required by the organism for
growth
o Classification -------
Selective only allow
growth of a particular type
Chemical and Physical Types of Culture Media
o Defined or synthetic media
All components and their concentrations are known
o Complex Media
Contain some ingredients of unknown composition and/or concentration
 Some media components
o Peptones protein hydrolysates prepared by partial digestion of various protein
sources
o Extracts aqueous extracts, usually of beef or yeast
o Agar sulfated polysaccharide used to solidify liquid media; most microorganisms
cannot degrade it
Functional Types of Media
o Supportive or general purpose media (e.g. Tryptic Soy Agar)
support the growth of many microorganisms
o Enriched media (e.g. blood agar)
general purpose media supplemented by blood or other special nutrients
some bacteria need the blood to be heated, which turns the agar chocolate
colour so called chocolate argar
Selective Media
o favour the growth of some microorganisms and inhibit growth of others
o e.g., MacConkey agar selects for Gram-negative bacteria
bile salts inhibit growth of gram-positive bacteria and promote growth of
gram negative bacteria
Differential Media
o Distinguish between different groups of microorganisms based on their biological
characteristics
o e.g., blood agar haemolytic versus nonhaemolytic bacteria
beta-hemolytic streptococcus needs treatment???
o e.g., MacConkey agar lactose fermenters versus nonfermenters
can tell difference between e. coli and salmonella due to colour on plate

Isolation of Pure cultures

o Population of cells arising from a single cell developed by Robert Koch
o Allows for the study of single type of microorganism in mixed culture
o Spread plate, streak plate, and pour plate are techniques used to isolate pure cultures
 Streak Plate
o Involves technique of spreading a mixture of cells on an agar surface so that individual
cells are well separated from each
other
involves use of
bacteriological loop
o Each cell can reproduce to form a
separate colony (visible growth or
cluster of microorganisms)
Spread Plate and Pour Plate
o CFU/mL colonie forming unit, 20 CFU = 20 cells
o Spread plate
small volume of diluted mixture containing approximately 30300 cells is
transferred
spread evenly over surface with a sterile bent rod
o Pour plate
sample is serially diluted
diluted samples are mixed with liquid agar
mixture of cells and agar are poured into sterile culture dishes
o Both may be used to determine the number of viable microorganisms in an original
sample

o
o Here if we had 50 colonies then the original sample would be 50*10^3*5=25*10^5/mL
Microbial Growth on Solid Surfaces
o Colony characteristics that develop when microorganisms are grown on agar surfaces
aid in identification
o Differences in growth rate from edges to center is due to
oxygen, nutrients, and toxic products
 cells may be dead in some areas
pick a colony from the edge, as they are more metabolically active than those in
the centre when plating out
Microbial Growth in Natural Environments
o Most microbes grow attached to surfaces (sessile) rather than free floating (planktonic)
o These attached microbes are members of complex, slime enclosed communities called a
biofilm
o Biofilms are ubiquitous in nature in water
o Can be formed on any conditioned surface
Biofilm Formation
o Microbes reversibly attach to conditioned surface and release polysaccharides, proteins,
and DNA to form the extracellular polymeric substance (EPS)
o Additional polymers are produced as microbes reproduce and biofilm matures

Biofilm Heterogeneity
 Once a biofilm forms, the equipment must be changed as they are in area where antibiotics
cant reach, and its also hard to get rid of the persister cells, which have the ability to
repopulate the bacteria living inside the biofilm.
Why bacteria form Biofilms?
Microbial Nutrition Questions
o 1. Compare and contrast supportive, enriched, selective, and differential media,
listing examples of each and describing how each is used.
o 2. What are pure cultures and why are they important?
o 3. Describe the formation of biofilms and summarize their importance in natural
environments, industrial settings, and medicine.
Microbial Growth
Growth
o An increase in cell number
o Microbiologists usually study population growth rather than growth of individual
cells
Binary Fission

o
Bacterial Cell Cycyle
o Cell cycle is sequence of events from formation of new cell through the next cell
division
most bacteria divide by binary fission
o Two pathways function during cycle
Replication and partition of DNA
Cytokinesis formation of the septum and progeny cells
 Cell Cycle of E. coli

Chromosome Partitioning An Example

o Replisome pushes, or leads to condensation of, daughter chromosomes to opposite
ends
o ParA/ParB proteins in C. crescentus
ParA polymerizes to form filaments
ParB binds DNA at parS site near origin of replication; technically, ParB binds
2 copies of parS site since DNA has been replicated
ParA interaction with 1 of the 2 ParB/parS complexes causes it to
depolymerize, pulling one copy of the DNA away
parS site found in almost 70% of 400 sequenced bacterial genomes
more work remains to understand partitioning in bacteria

But other mechanisms are present aswell, as if you knock out these genes, partitioning still
occurs
Cytokinesis Septation
o Septation formation of cross walls between daughter cells
 o Several steps
selection of site for septum formation
assembly of Z ring (composed of protein FtsZ)
assembly of cell wall synthesizing machinery
constriction of cell and septum formation
Z Ring Formation - Role in Septation
o Protein FtsZ
tubulin homologue, found in most bacteria and archaea
polymerization forms Z ring, filaments of meshwork
o MinCDE system in E. coli limits Z ring to cell center
MinC, MinD, MinE oscillate from one side of cell to other
high concentration of MinC at poles prevents formation of Z ring at those
locations
o Anchoring proteins link Z ring to the plasma membrane
o Cell wall-synthesizing machinery assembled (for peptidoglycan etc)
o Constriction of the Z ring, invagination of the plasma membrane, and synthesis of
septal wall complete division
Cell Wall Growth and Cell Shape Determination
o Importance of MreB in determining cell shape:
MreB depletion in rod-shaped bacteria assume a spherical shape.
All rod-shaped bacteria and archaea have at least one homologue of MreB,
coccoid-shaped cells lack proteins in MreB family.
Sperical/Coccoid
o After division one hemisphere is new and one is old
Rod (common)
o One new pole and one old one
 Vibrio Cell Wall Growth and Cell Shape Determination
o Vibrio (comma-shaped bacteria)
FtsZ forms Z ring
MreB helical polymerization throughout
cell
crescentin intermediate filament
homologue
localizes to short, curved side of
cell
asymmetric cell wall synthesis
forms curve
slows down peptidoglycan synthesis on one side, caused the curve
Archaeal Cell Cycle Information
o Information obtained so far has been produced from a limited number of archaeal
species
o Archaeal Cell Cycles Resemble Eukaryal Cell Cycles Except in Segregation of
Chromosomes
o Even then, contradictions exist
o Diversity in archaeal cell cycles is obvious and abundant, and more research needs to be
performed to understand the systems more thoroughly
The Growth Curve
o Observed when microorganisms are
cultivated in batch culture
o Usually plotted as logarithm of cell number
vs. time
o Has five distinct phases
Lag Phase
o Cell synthesizing new components
e.g., to replenish spent materials
e.g., to adapt to new medium or other conditions
o Varies in length in some cases can be very short or even absent
o When taken from phases other than log phase
Exponential Phase (Log Phase)
o Rate of growth and division is constant and maximal
o Population is doubling every generation
Stationary Phase
o Closed system population growth eventually ceases, total number of viable cells remains
constant
o active cells stop reproducing or reproductive rate is balanced by death rate
o possible reasons for this phase
Nutrient limitation, Limited oxygen availability, Toxic waste accumulation,
Critical population density reached
 Death Phase and Prolonged Decline in Growth
o Death Phase: The number of viable cells declines exponentially
o 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 population genetically programmed to die
(suicide)
o Population size remains more or less constant (can last months to years)
o Bacterial population continually evolves
o Process marked by successive waves of genetically distinct variants (natural selection
occuring)
The mathematics of growth
o 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
g=t/n
t=hrs or min of exp growth
n=number of generations
n=log(N)-log(N0)/log2
o N=the final cell number
o N0=the initial cell number
Or use a graph look at where pop. Doubles
and go along and then go down to time.
Measurement of Microbial Growth
o Can measure changes in number of cells in a population
o Can measure changes in mass of population
Direct Measurement of Cell Numbers
o Direct cell counts
counting chambers can only total number of cells,
so some may be dead. Also need 10^6 cells/ml for it
to be accurate
electronic counters
flow cytometry
on membrane filters
 Direct Counts on Membrane Filters
o Aquatic samples are filtered through special membrane that
provides dark background for observing cells, where the cells stay
on the top
o Cells are stained with fluorescent dyes
o Useful for counting bacteria
o With certain dyes, can distinguish living from dead cells
Flow Cytometry
o Microbial suspension is forced through small orifice with a laser
light beam
o Movement of microbe through orifice impacts electric current
that flows through orifice
o Instances of disruption of current are counted
Viable counting: Alive or dead?
o Whether or not a cell is alive or dead isnt always clear cut in
microbiology
o cells can exist in a variety of states between fully viable and
actually dead
Viable Counting Methods
o 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)
o Membrane filter technique

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)
o In samples which cant be cultured, or are solid, too big so mask others
o The last highest dilution which shows growth will have between 1-10 cells, the last
dilution should have no growths
o Commonly used in water samples

 Measurement of Cell Mass

o Dry weight time consuming and not very sensitive
o Quantity of a particular cell constituent
E.g., protein, ATP, or chlorophyll
useful if amount of substance in each cell is constant
o Turbidometric measures (light scattering) quick, easy, and sensitive
The Influence of Environmental Factors on Growth
o Most organisms grow in fairly moderate environmental conditions
o Extremophiles grow under harsh conditions that would kill most other organisms
Influence of Environmental Factors on growth
o 1. Solutes
o 2. pH
o 3. Temp
o 4. O2
o 5. Pressure
o 6. Radiation
Solutes and Water Activity
o Microbes Adapt to Changes in Osmotic Concentrations
o Reduce osmotic concentration of cytoplasm in hypotonic solutions
mechanosensitive (MS) channels in plasma membrane allow solutes to leave
o Increase internal solute concentration with compatible solutes to increase their internal
osmotic concentration in hypertonic solutions - plasmolysis
solutes compatible with metabolism and growth (KCl and organic solutes such as
choline, proline and glutamic acid)
o most bacteria keep the osmotic concs of their cytoplasm a bit higher than the
environment
Extremely Adapted Microbes
o Halophiles
grow optimally in the presence of NaCl or other
salts at a concentration above about 0.2M
o 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
Solutes and Water Activity (aw)
o water activity (aw)
amount of water available to organisms
reduced by interaction with solute molecules
(osmotic effect)
o higher [solute] lower aw
o Also equal to ratio of solutions vapour pressure (Psoln) to that of pure water (Pwater)
o Aw = Psoln/ Pwater
low water activity means most water is bound (dH2O=1; milk=0.97; sat salt
sol=0.75; dried fruits=0.5)
 o Osmotolerant microbes can grow over wide ranges of water activity
o Preserve food by adding salt and sugar to take water away, so microbes cant grow and
food doesnt spoil
pH

regardless of living in acidic or basic conditions, the cytoplasm stays close to neutral
acidophiles
o growth optimum between pH 0 and pH 5.5 (Transport K ions into the cell, thus decreasing
the movement of H ions into the cell; Proton transporters)
neutrophils
o growth optimum between pH 5.5 and pH 7 (exchange K for protons using antiport
transport system) If pH reaches below 4.5
o eg E. coli
alkaliphiles (alkalophiles)
o growth optimum between pH 8.5 and pH 11.5 (by exchanging internal Na ions for
external protons)
 some survive by turning on acid shock and heat shock proteins within the cell. Not long term
living
Temperature
o Microbes cannot regulate their internal
temperature
o Organisms exhibit distinct cardinal growth
temperatures
minimal
maximal
optimal
o 5 different groups
psychrophiles 0 C to 20 C
membrane high % of unsaturated
fatty acids
Produce solutes
psychrotrophs 0 C to 35 C
mesophiles 20 C to 45 C
bacteria which make us sick
thermophiles 55 C to 85 C
hyperthermophiles 85 C to 113 C
in hot springs, steam vents
Adaptations of Thermophiles
o Protein structure stabilized by a variety of means
more H bonds
more prolin
chaperones
o Histone-like proteins stabilize DNA
o Membrane stabilized by variety of means
more saturated, more branched and higher
molecular weight lipids
ether linkages (archaeal membranes)
some have single layer of carbon membrane
which provides more protection
Oxygen and Bacterial Growth
o Aerobe grows in presence of atmospheric oxygen (O2)
which is 20% O2
o Obligate aerobe requires O2
o Anaerobe grows in the absence of O2
o Obligate anaerobe usually killed in presence of O2
o Microaerophiles requires 210% O2
o Facultative anaerobes do not require O2 but grow
better in its presence
o Aerotolerant anaerobes grow with or without O2
 Basis of different oxygen
sensitivities
o oxygen easily reduced to toxic
products
superoxide radical
hydrogen peroxide
hydroxyl radical
o aerobes produce protective
enzymes
superoxide
dismutase (SOD)
catalase and
peroxidase
no enzyme to take
care of hydroxyl
radical, just have to
minmise its
production
Strict Anaerobic Microbes
o lack or have very low
quantities of
superoxide
dismutase
catalase
o These microbes cannot tolerate O
o Anaerobes must be grown without O2
work station with incubator
gaspak anaerobic system
 Growing Anaerobes
o Special anaerobic media containing
reducing agents (thioglycollate or cysteine)
o Pumped out oxygen and flush with CO2
and nitrogen
o Gaspak jars
o Candle jars
Pressure
o Microbes that live on land and water
surface live at 1 atmosphere (atm) 1 for
every 10 meters
down
o Some Bacteria and
Archaea live in deep
sea with very high
hydrostatic
pressures (barophile
or piezophile)
Radiation Damage
o 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
very efficient damage repair systems
o Ultraviolet (UV) radiation
wavelength most effectively absorbed by DNA is 260 nm
mutations death
causes formation of thymine dimers in DNA
DNA damage can be repaired by several repair mechanisms
o Visible light
at high intensities generates singlet oxygen (1O2)
powerful oxidizing agent
carotenoid pigment
protect many light-exposed microorganisms from photooxidation
Return singlet oxygen back to its ground state
Cell to Cell Communication Within the Microbial Populations
o Bacterial cells using molecular signals to communicate with each other in a density
dependent manner called Quorum Sensing
o Once they grow to a certain density, certain genes turn on
 Bacteriocin produced by competent cells which kills
non competent cells so the competent cells can take up
their bacteria
o Bacteria then becomes more virulent
Acylhomoserine lactone (AHL) is an autoinducer
molecule produced by many Gram-negative organisms
o diffuses across plasma membrane
o once inside the cell, induces expression of target
genes regulating a variety of functions, controlled
by quorum sensing systems, eg virulence, biofilm
formation, bioluminescence, DNA uptake
Processes regulated by quorum sensing involve host-
microbe interactions
o symbiosis Vibrio fischeri and bioluminescence in
squid
o pathogenicity and increased virulence factor production
o Biofilm formation
o DNA uptake
Interdomain communication, eg nitrogen fixing bacteria
o Flavonoids produced by plant
o Once flavonoids taken up, chemicals/Nod factors produced by bacteria
o bacteria enter the plant as the nod factors work on the roots to allow uptake
Microbial Growth Questions
o Describe the functions of cytoskeletal proteins in a typical bacterial cell cycle and in
determining cell shape.
o Describe the function of the enzymes observed in microbes that protect them against toxic
O2 products.
o Define quorum sensing and provide examples of cellular processes regulated by quorum
sensing.
o Describe the five phases of a microbial growth
curve observed when microbes are grown in a
batch culture.
o Evaluate direct cell counts, viable counting
methods, and cell mass measurements for
determining population size.
Microbial Genetics
DNA transfer
DNA as Genetic Material
o Griffith in 1928 observed the change of
nonvirulent organisms into virulent ones as a
result of transformation
o Avery, MacLeod and McCarty in 1944 showed
that the transforming principle was DNA
 Showed that the cells were transformed and rough and smooth phenotypes combined
 Bacterial Transformation
o Uptake of naked DNA by a competent cell (a cell
which can take in DNA) followed by incorporation
of the DNA into the recipient cells genome
o Griffth used streptococcus
o Bacilus
o Haemophilus
o Neisseria
o Others have this ability to be transformed naturally
Natural Transformation in S. pneumoniae
DNA Uptake in Bacterial Transformation

Artificial Transformation
o transformation done in laboratory with species that are not normally competent
(e.g., E. coli)
o variety of techniques used to make cells temporarily competent
e.g., calcium chloride treatment
makes cells more permeable to DNA
1*10^5 transformans/mg DNA
o Electroporation
High voltage pulses are delivered
High efficiency 1*10^10 transformans/ mg DNA
Electrical field makes pores in the membrane, so any DNA can get entry into
the cell
Transfection
o Bacteriophage, isolate its DNA and put it in bacteria
o Transformation is any DNA, Transfection is phage genome (from bacteriophage)
For eukaryotes
o Transfection - any foreign DNA going0 into a cell
o Transformation where the normal cells become malignant
Transduction
o The transfer of bacterial genes by viruses
o Viruses (bacteriophages) can carry out the lytic cycle (host cell is destroyed) or viral
DNA
integrates
into the host
genome
(becoming a
latent
prophage)
 o Virulent phage lytic cycle
o Temperate phage Lysogenic cycle or lytic
cycle
o Lysogeny, then the phage DNA is integrated
into the host genome
o Lysogen, bacterial cell in which a phage exists
as DNA in its dormant state (prophage). A
prophage is either integrated into the host
bacteria's chromosome or more rarely exists
as a stable plasmid within the host cell.
Transduction is using the whole Phage particle
Transfection is only using the isolated DNA from a
phage particle
Phages are specific to hosts
Generalized Transduction
o Any part of bacterial genome can be
transferred
o Occurs during lytic cycle of virulent phage
o During viral assembly, fragments of host DNA
mistakenly packaged into phage head
generalized transducing particle
efficiency is quite high
can use the phage to add
mutations to other cells
Specialised transduction
o Carried out only by temperate
phages that have established
lysogeny
o Only specific portion of bacterial
genome is transferred
o Occurs when prophage is
incorrectly excised
 The Mechanism of Transduction for Phage Lambda and E. coli

Phage Conversion
o Conversion of nontoxin-producing strains of Corynebacterium diphtheriae to toxin-
producing strains
Gene came into bacteria with help from a bacteriophage
o Change in the structure of LPS on the cell surface upon lysogenisation
o O-antigen modification (serotype conversion) in Shigella flexneri
o Genes are being moved around by bacteriapages over evolution, which creates
different phenotypes, so some may be detected by immune system and some may
not for example in S. flexneri
o Hard to make vaccines as have lots of different phenotypes.
Plasmids
o Small, autonomously replicating DNA molecules that can exist independently or
integrate reversibly into the host chromosome
o Circular DNA elements
o Replicate independently of chromosomes
o Carry wide variety of genes
Toxin production
Antibiotic resistance
Metabolic genes, allow to degrade or grow on organic stuff
o Can integrate and deintegrate from chromosomes (episomes)
Structure
o Small, double-stranded, usually circular DNA molecules
o Supercoiled molecules will run faster on gel
o Open-circular duplex run a little slower, happens if there is a nick
 o Linear duplex even slower, if there is nick in the same spot on both strands
Isolation of Plasmid DNA
o Bacterial lysis, lysozyme and EDTA (also act as chelating agent, stops function of
nucleases) add sucrose after cell wall is gone to the suspension to prevent
immediate lysis of the cell, otherwise the cell will go into osmotic lysis, cell will burst
if under hypotonic conditions. Then lyse the cell under controlled and gentle
conditions by adding detergent like sds. Then cell will open up and DNA will come
out
o Removal of cell debris and larger fragments of chromosome, part of the
chromosome remain attached to cell wall, so removing debris will bring down
chromosomal DNA as well, then take supernatant which will contain plasmid DNA
which you can then precipitate it with ethanol or can do density gradient
configuration to palate down the DNA
o Separation of plasmid DNA
Replication
o Very similar to chromosomal replication
o Origin of replication, every plasmid has one
o As plasmid so small, happens very quickly
o Plasmids use cellular enzymes used for chromosomal DNA as they dont have their
own
Copy number is how many copies of a plasmid a cell has, it is determined by the genes on
the plasmid and also interaction between host and plasmid DNA
So need to have lots of copies of a plasmid when u want to express it
Curing of Plasmids
o Can be eliminated from a cell very easily
o Spontaneous curing over generations just lose a plasmid
o Can be induced, by using DNA binding DICE, which bind to DNA and will block the
replication of the plasmid, dont have much effect on chromosomal replication, but
will stop plasmid
Incompatibility
o Many bacteria will have 2 or more plasmids present at the same time in a bacterial
cell and they are stably maintained, however some pairs cant live together, called
incompatible
o Always seen when genetically distinguishable pairs of the same plasmid are used
o Controlled by genes on the plasmid
If you have selection pressure, eg grow on minimalistic growth medium (only 1 source of
carbon) then if you put the plasmids in, only the ones with the plasmids will grow and will be
selected for. When you take away the selection pressure, eg grow on nutrient agar, will see
that some cells will have lost the plasmids or some of them. Need the selection pressure
otherwise the plasmids are incompatible
The F plasmid
o Conjugative (or fertility plasmids) (F plasmid) can transfer copies of themselves to
other bacteria during conjugation
o Usually only find 1 copy per cell, almost of plasmid is genes for transfer, donor
cells can transfer F plasmid into cells which dont have it, due to this gene cluster
 F plasmid integration
Have insertion elements (IS
elements), elements which are
present on the chromosome as
well, due to this homology,
they can integrate via
recombination at different
locations
Bacterial Conjugation
o J. Lederberg and E.
Tatum demonstrated
the transfer of genes
between bacteria that
depends on
o Plasmid codes for sex
pilus, which makes
contact with recipient
cells which are always
F-, and transfers F plasmid to recipient. The
recipient is now F+ and can donate to others.
direct cell to cell contact mediated by
the F pilus
unidirectional DNA transfer from
donor to recipient
Evidence for Bacterial Conjugation and the U-Tube
Experiment (on right), left side bacteria cant make
 contact with right side, so when plated out there was no growth on minimal medium
F+ x F- Mating

Type IV secretion system encoded by F

factor
o Pilus is a single protein
o TraA (pilin) forms pilus
o TraD codes coupling factor
o TraA is helicasewhich binds to
5 prime end of DNA and puts it
in TraD and then translocase
comes in and pumps the DNA
into the recipient cell
 Rolling-Circle Replication (on right)
o Complementary strand synthesis
occurs in the recipient as its going in
+ -
F x F Mating
o A copy of the F factor is transferred to
the recipient and does not integrate
into the host chromosome
o Donor genes usually not transferred
o F factor codes for sex pilus Type IV
secretion system that makes contact
between cells
o Plasmid is replicated by rolling circle
method
o Happens very quickly
o Orientation of F factor determines
which order the genes will enter the
recipient

Mating Experiment

Plate out on glucose minimal and streptomycin plates

 Resistance Factors
o R factors (plasmids)
o have genes for resistance
to antibiotics
o some are conjugative
o usually do not integrate
into chromosome
Mechanisms of antibiotic
resistance
o Altering the target site of
23s RNA molecule
(erythromycin)
Binds to the
adenine and
blocks protein
synthesis
Gene alters
target site so the antibiotic doesnt work
o Modifying the antibiotic so it is no longer active (chloramphenicol,
penicillin/ampicillin)
Single enzyme beta lactamase which hydrolyses the beta lactam ring (part of
the above antibiotics)
Chloramphenicol acetyltransferase destroys chloramphenicol
o Preventing the antibiotic from entering the cell (tetracycline)
Resistance gene changes permeability of the cell so tetracycline cant enter
o Specifying an enzyme which provides a substitute for a host-specified enzyme which
is the target of the antibiotic (sulpha drugs)
Sulfanilamide and Its Relationship to Folic Acid Structure
o Sulpha drugs get incorporated instead of
p-aminobenzoic acid and stops the
synthesis of folic acid
Col plasmids
o encode colicin (an antimicrobial protein)
kills E. coli
a type of bacteriocin
protein that destroys
other bacteria, usually
closely related species
o some are conjugative
o some carry resistance genes
Other Types of Plasmids
o virulence plasmids
carry virulence genes
e.g., genes that confer resistance to host defence mechanisms
e.g., genes that encode toxins
 o metabolic plasmids
carry genes for metabolic processes
e.g., genes encoding degradative enzymes for pesticides
e.g., genes for nitrogen fixation
Questions: Plasmids and Bacterial Conjugation
o Describe four mechanisms used to confer antibiotic resistance in bacteria.
o Describe the features of the F factor that allow it to (1) transfer itself to a new host
cell and (2) integrate into a host cells chromosome.
o Outline the events that occur when an Hfr cell encounters an Fcell.
o Design an experiment to demonstrate the role of F pilus in conjugation.
Hfr (high frequency of recombination) Conjugation
o donor Hfr cell has F factor integrated into its chromosome
o donor genes are transferred to recipient cell
o a complete copy of the F factor is usually not transferred
 by doing a interrupted mating experiment, a time course
experiment, and record genes in recipient cell, you can
sequence the genes in the donor strain. The longer you
leave it go, the more genes get transferred.
As a whole copy of F factor is not transferred, the recipient
remains F-.
3 types of mating
o F+ x F- get F+ and F+
o Hfr x F- get Hfr and F-
o F x F- get F and F
F Conjugation
o Result when the F factor incorrectly leaves the host
chromosome
o Some of the F factor is left behind in the host
chromosome
o Some host genes have been removed along with
some of the F factor
these genes can be transferred to a second
host cell by conjugation
o recipient gets whole copy of F
Mechanisms of Genetic Variation
Mutations and Mutagenesis
o Mutations
stable, heritable change in nucleotide
sequence
may or may not have an effect on the phenotype of an organism
o The genotype designation
hisA
His+
Mutations and Mutagenesis How mutations arise
o Spontaneously
develop in absence of any added agent
usually thought to arise randomly
o induced
develop after exposure to a mutagen
Spontaneous Mutations
o Arise without exposure to external agents
o May result from errors in DNA replication
due to base tautomerization resulting in transition and transversion
mutations
due to insertion or deletion of nucleotides
o May also result from the action of mobile genetic elements such as transposons
 Transition and Tautomerization
Mutations
o Rare forms of amino acids cause
incorrect paring
Insertions and Deletions

DNA lesions such as Depurination

o Transversion mutation example below
 Induced mutations
o Caused by agents that directly damage DNA
base analogues
structurally similar to
normal bases
mistakes occur when they
are incorporated into
growing polynucleotide
chain
DNA modifying agents
alter a base causing it to
mispair
intercalating agents
distort DNA to induce single
nucleotide pair insertions
and deletions
Base Analogue 5-Bromouracil
 Effects of Mutations
o Wild type most prevalent form of gene
o Forward mutation wild type mutant form
o Reversion mutation mutant phenotype wild type phenotype
o suppressor mutation occurs when the second mutation is at a different site than the
original mutation
 Mutant Detection and Selection
o Mutations are generally rare
o Observation of changes in phenotype
o Replica plating technique used to detect auxotrophic mutants
o Use of environmental condition in which only desired mutant will grow e.g., selection
for revertants
from auxotrophy
to prototrophy
 Carcinogenicity Testing (Ames Test)
o Based on observation that most carcinogens
are also mutagens
o Tests for mutagenicity are used as screen for
carcinogenic potential
reversion rate in presence of
suspected carcinogen > reversion
rate in absence of suspected
carcinogen
then, agent is a mutagen, and may
be carcinogen, only an indication coz
its on bacteria
o Original strain should have a point mutation,
so that the reversion can occur
o Small amount of histidine to support growth
to allow for replication to get mutation, not enough to have a colony grow
o To make the test more powerful:
use of strains lacking DNA repair enzymes
use of liver enzyme preparations to convert the chemicals into their active
mutagenic forms
o Questions: Mechanisms of Genetic Variation
Design an experiment to isolate mutant bacteria that are threonine auxotrophs.
Propose an experiment to isolate revertants of a threonine auxotroph and
predict the types of mutations that might lead to the revertant phenotype.
Explain how the Ames test is used to screen for potential carcinogens and
evaluate its effectiveness.
Prescots microbiology
Microbial Metabolism
o Bacterial growth requires energy
o Metabolic Processes yield energy and reducing power that are used for growth and
survival
From an industrial perspective
o Making yogurt, sour cream and cheese from milk
o Ethanol production, beer, wine, sauerkraut (preserved foods)
Microbes living in gut produce short chain fatty acids
Microbes can help plants to grow
o Bacteria inside stems eg nitrogen fixing bacteria in sugar chain
o Fungi in and around roots help with nutrient uptake
o Bacteria on root surface
o Bacteria inside nodules bacteria fix nitrogen from the air and exchange it to the plant
for carbon
Environmental perspective
o Important components of the atmosphere are involved in their production
Eg CO2 and methane
 Bacterial metabolism
o Provides the energy and reducing power required for bacterial growth and survival
o Plays an important role
Industrial production of food, therapeutics, etc
Human/Animal/Plant health and disease
Environmental cycling of various compounds

Catabolism
o Breaking down things
 o Food broken down into ATP, strip electrons off it to make more ATP and carry out
other reactions, create waste product and subunits used to make other important
stuff, eg amino acids, nucleotides and lipids.
Anabolism
o Using subunits to make stuff, eg cell
structures
Nutrient (C) sources for bacteria
o Inorganic C source (CO2)
Autotroph
o Organic and inorganic C source
Mixotroph
o Organic C source
Heterotroph
Electron sources for bacteria
o Organotroph from organic material
o Lithotroph (stone eaters) from
inorganic material
Energy Sources for Bacteria
o Light
phototroph
o Chemical energy
Chemotroph
From organic material chemoorganotroph
From inorganic material chemolithotroph
Summary of bacterial metabolism

We are chemoorganoheterotrophs
Plants are photolithoautotroph

Nutrients, electrons and energy sources are required for bacterial metabolic processes
 o Bacteria can use different energy sources (light, inorganic chemicals, organic
chemicals)
o Bacteria can use different metabolic processes (fermentation, respiration,
photosynthesis) to transform energy sources into ATP, the general energy currency
in all cells
How does bacterial metabolism occur
o Nutrients and energy sources (organic/inorganic compounds) are transported into
the cell
o Energy, electrons and carbon are converted to ATP, reduction equivalents (reduced
electron carriers) and metabolites for anabolism
Light is used to power electron
movement used to generate ATP
Respiration and fermentation, differ in
whether the cell has a functioning
electron transport chain or not.
o Oxygen is an electron acceptor in
the chain and is converted to
water
o Fermentation is a shorter
pathway, but much less efficient,
doesnt use the chain
What happens to the carbon source
o Use ATP to fix carbon to turn
inorganic carbon (CO2) to organic C eg
glucose. Plants do this
o Use organic C source for ATP and produce
CO2 and other c intermediates for
anabolism
Electron Source

Redox Definitions
o Loss of electrons oxidation
o Gain of electrons reduction
o Electron donors become oxidised
o Electron acceptors become reduced
 Electron transport chain
o Electron transfer is an
energetically favorable process
Electron carriers
o NAD+
o FAD
o Heme group usually iron
o Iron-sulfur proteins
o Ubiquinone
o All vary in the tendency to attract
electrons
Redox reactions (see tables)
o Organised in order of their
reduction potential (tendency to
gain electrons)
o Electrons will move from electron donors at the top of the list to electron acceptors at
the bottom of the list
Electron transport chains are arranged in order of redox
potential
A proton motive force is generating ATP

Electron donors in the chain can generate protons

ATP can also be produced by substrate phosphorylation
 Energy source is transported into the bacterial cell
Compound is oxidised in progressive steps by bacterial enzymes and/or the electron transport
chain/proton motive force to release energy that is captured in ATP
Electrons/protons released are captured by cofactors
The ATP, electrons/protons, and intermediates generated in the process of catabolism are used
in anabolic processes required for bacterial growth and survival
ATP is used to support the energy requirements of the cell (i.e. motility, transport)
Where does bacterial metabolism occur
o Cytoplasm
o Cell membrane
o (chlorosome photosynthesis in green sulfur bacteria)
Respiration and Fermentation

o Aerobic or anaerobic
o Aerobic requires oxygen which acts as the final electron acceptor
o Anaerobic have different electron acceptors, eg sulfate, nitrate, CO2, fumarate etc
o Start with an organic source (glucose etc), get electrons from it, use them to generate
ATP via electron transport chain, or use in synthesis reactions???
o Fermentation is a lot shorter, doesnt operate with an electron transfer chain,
alternative method for breaking down glucose when O2 is not available.
o Produce lactate in muscles when not enough O2 available or provided fast enough, eg
sprinting
Chemoorgantroph, heterotroph and organotrophs use respiration and fermentation
 Respiration I breaking down the carbon
source
o Glucose (6C) broken into pyruvate
(3C) glycolysis
Produces some ATP, NADH
and H+
o Then pyruvate enters TCA cycle and
gets futher broken down into CO2,
making ATP, FADH, NADH and H+.
Fermentation I breaking down the carbon
source
o first part is the same
o Pyruvate doesnt enter TCA
cycle and is broken down,
creating other by-products, eg
lactic acid, ethanol, propionic
acid and butyric acid.
Producing energy from reducing
equivalents (respiration only)
o Electron donors (eg NADH,
FADH produced during
glycolysis) through ETC
proton motive force to
oxidative phosphorylation
ATP
 Get lots of reduction
equivalents which are
used to make ATP
Electrons then get
passed through different
complexes to O2.
 Producing energy from reducing equivalents electron transport chain in mitochondria

NADH donates electrons and protons to FMN and protons are donated to other side of
membrane
FAD doesnt produce as much ATP, it donates to 2nd complex
3rd complex transfers H+ over membrane moving the elctrons through the membrane
During aerobic respiration, oxygen is used as the terminal electron acceptor
During anaerobic respiration, some organisms use other electron acceptors, e.g. sulfate,
nitrate, CO2, Fe3+.
In bacteria
o Principal is the same
o Electrons enter in on a carrier, transfer of electrons causes a proton gradient to form
 Fermentative metabolism
o C source growth in absence of oxygen acids, ethanol, gas
o Many bacteria/fungi that grow (facultatively) anaerobically
o Eg yeast and lactic acid bacteria
Pathways
o During respiration (presence of oxygen), the NADH+H+ would be removed in the ETC
o When the ETC is not working (fermentation, in absence of oxygen) NADH+H+ needs
to be removed in other reactions or glycolysis will stop

o Cell will take NADH + H+ and use it to make NAD+ producing lactate, recycling the
NAD+.
 Fate of Pyruvate in Fermentations

Cells have invented ways to turn pyruvate into other substances to recycle NAD+
Cheese fermentations
o Milk/cream either pasteurizated or not in vat, then inoculate with lactic acid
bacteria, lactococcus.
Lactose fermentation
o Depending on what bacteria is used,
gives different types of cheese.
Add renin
o An enzyme which causes milk curd to
form and coagulate, so can remove the
solids (fats etc) used in cheese from the
water
Curds cut, whey released
Pressing salting
Ripening
o Important in flavour development
Variations in chesses
o Type, form of milk/cream used
Cow, goat, sheep etc
General processes involved in Cheese fermentations
(Variations continued)
Starter cultures used in addition to Lactococcus:
o Mixed cultures of homo- and hetero- (CO2 production, texture affects) fermenters
 o Geotriclium candidum, Pencillium spp.
typically used to inoculate the surface of the
cheese
Ex. Brie
o Penicillium roqueforti used to inoculate the
cheese
Ex. Blue cheese
o Combination of both
Ex. Cambozola
General processes involved in beer fermentations
o Malted grain is crushed and mixed with hot
water until enzymes degrade they starch into
sugars
o The resulting mash goes to the mash tun for
further enzyme activity

Variations in the process

o Types of malt n Light malt
ex. Pilsner
o Medium malt
ex. Traditional
o Dark malt
ex. Guiness
o Hops
Type of hops used
When hops are added
Ex. India Pale Ale
Types of yeast used
o Lagers fermented by Saccharomyces carlsbergensis
Bottom fermentor
5-12C for 7 days
o Ales fermented by Saccharomyces cerevisiae
 Top fermentor
15-25C for 2-5 days
Points to remember
o During respiration, organic C sources can be broken down completely to CO2 during
glycolysis and TCA cycle, producing ATP, NADH and FADH.
o NADH and FADH produce ATP in the electron transport chain. Oxygen is used as the
terminal electron acceptor in aerobic respiration.
o During fermentation (low or no oxygen), the carbon source is only partially broken
down to pyruvate. This produces less ATP and NADH than during complete
breakdown to CO2.
o Pyruvate can be converted to a number of interesting products by both bacteria and
yeast cells e.g. ethanol (beer, wine), lactic acid (cheese products).
Photosynthesis in microorganisms
Where does microbial photosynthesis occur?
o Plants
o At least half of photosynthesizing organisms are bacteria, specifically aquatic
bacteria (cyanobacteria, algae and bacteria) in both salt and fresh water
o Not in middle of oceans coz need nutrients specifically nitrogen, so mostly on land
and colder areas.
Photosynthesizing algae and cyanobacteria
Some photosynthesizing bacteria
o Cyanobacteria
o Often filamentous, not single celled
o Sometimes form colonies
o Nostoc has cells in which nitrogen fixation takes place
Purple photosynthetic sulfur bacteria
Photosynthesis
 Phototrophy converting light into ATP
o Electrons come to chain from organic
molecules and that can produce ATP
o During photosynthesis that energy and
source of electrons have a different source.
Light is basically what produces the energy,
it transports electrons to a more negative
redox potential, to also travel along an ETC
which produces a proton motive force to
produce ATP.
Classification of Bacteria based on their Energy
Sources
o Chlorophyll-based phototrophy

o Light excites an electron in a chlorophyll pigment and enables those electrons to be

transported along an ETC to NAD(P)+. During this a PMF is created and when
protons flow back through an ATPase ATP is produced.
o Rhodopsin are related to pigment in your eyes and do something similar to
chlorophyll
Photosynthesis Overview
o Plants, algae (eukaryotes) and cyanobacteria (prokaryotes) conduct oxygenic
photosynthesis
 o Chloroplast came from a cyanobacterium invading another cell to produce a plant
cell. Ie chloroplasts inside plants are basically ancient cyanobacterium
o During photosynthesis, the cell needs reducing power, carbon and energy. In this
case the energy comes from light and light is used to produce ATP. That ATP is used
to fix an inorganic carbon source into an organic carbon molecule (a sugar). Needs a
source of electrons to fix that carbon usually water. A water molecule is split into
Oxygen which diffuses out and then the electrons and protons are taken off and the
electrons are bound to a carrier which is used to fix carbon. So reducing power
comes from water and produces oxygen.
Purple and Green bacteria conduct anoxygenic photosynthesis (prokaryotes)
o Light is used to generate ATP
o Reducing power is generated from different electron donors, usually H2S.
o Basically dont use water and dont produce oxygen.

o These bacteria usually live in sea beds

Chemical Basis of Photosynthesis Chlorophyll

o Chlorophyll has 4 porphyrin rings, a central magnesium atom, some very
hydrophobic side chains (a long fatty acid) which anchors the pigment into a
membrane.
o Depending on side chains, absorption spectrum changes slightly, which changes
colour of pigment
o Similar to a haem group, but has mg instead of iron.
 Accessory Pigments

o Help chlorophyll to capture light

o Chlorophyll doesnt absorb well in the green spectrum
so need other pigments, usually molecules with
conjugated double bonds, which can capture light in
regions where chlorophyll cant so more light energy
can be captured.
Different pigments absorb different wavelengths
Photosynthetic pigments can determine the colour of bacteria
o Due to variation of pigments and accessory proteins
depending on where they live.
o Deep in ocean, red light is removed first, so only blue
light gets in
Photosystem
o Many of the accessory pigments and chlorophyll dont
take part in the reaction, they are just part of an
antenna complex.
o A lot of pigments are aggregated together in a so called
antenna, acts to concentrate light into the centre
molecule (reaction centre) which actually is involved in
the reaction.
 Photosynthetic processes take place on a membrane
o Light instigates electron flow down the electron transport chain, establishes the
proton motive force, which produces ATP

protons travel in opposite direction to one saw last time

there are 2 reaction centres where the energy from photons from light energy are used to
energise electrons for this ETC. Photosystem 1 and 2. Main difference is that they capture light
at different wave lengths. Photosystem 1 mainly at 700nm and photosystems 2 at 680nm. Each
reaction centre has chlorophyll in them, light excites the chlorophyll and will make one of these
electrons move essentially from water through chlorophyll onto some electron carriers. So
electrons from the water molecules are lost and oxygen is produced. Electrons flow along the
electron transport chain, to another photosystem where the electrons are excited again making
the redox potential more negative so they can flow at the end to a ferredoxin and FAD complex.
In the end the electrons are transported from water to an electron carrier which is used in
biosynthesis. Basically energy comes from light and electrons come from water.
 Noncyclic photophosphorylation

Really bad to start with as the electrons are at a positive redux potential where they want to
be at. Light in the photosystem makes the electrons go to a more negative redox potential
so that they can then flow onto a number of electron carriers. So the electron flows from
the first photosystem to the next one where it goes to a more negative redox potential again
and from there it flows onto NADP to produce NADPH and H+.
Prokaryotic ETC and reducing power purple nonsulfur bacteria

o Usually only have 1 photosystem containing bacterial chlorophyll.

o Very short ETC, which is driven by light, and excited at a much longer wavelenght
(870nm). The electron is propeeled to a more negative redox potential and then
flows onto a number of different electron carriers which produces a proton gradient,
which then produces ATP.
 o These organisms need to synthesise reduced electron carriers which is not done
during this. The electrons just cycle from the chlorophyll molecule back to the
chlorophyll molecule and dont use water as a source of electrons, but can use
energy to push electrons onto NAD+ in a reaction called reverse electron flow. So
use energy to propell electrons to a more negative redox potential of NAD+ needed
for biosynthesis. The electrons drawn out of this cycle are replaced from electrons
from organic molecules, eg saxonate or fumarate.
Prokaryotic ETC and Reducing Power - Green Sulphur Bacteria

o Use other inorganic sources for electrons such as sulfur, Have an electron transport
chain, have bacterial chlorophyl, light energy excite electrons and the electrons flow
through a number of electron carriers producing ATP. It can either be cyclic so the
same electrons can cycle through producing energy or the electrons can be
transferred onto NAD+ to produce NADH which the cell can then use for other
reactions but then the electrons in the cycle have to be replaced. In oxygenic
photosynthesis they are replaced by water, but in these organisms they are replaced
by H2S or sulfite, producing sulfer or sulfate.
Specialised structures for prokaryotic photosynthetic processes
o Chlorosomes on plasma membrane
The Calvin cycle for carbon (CO2) fixation
o Basic principals are cell uses ATP and NADH to fix carbon onto an organic molecule
using an enzyme called rubisco (ribulose 1,5-bisphosphate carboxylase/oxygenase).
Most abundant enzyme on planet. Takes carbon from atmosphere and puts on an
organic sugar molecule molecule, ribulose 1,5 bisphosphate and produces fructose-
6-phosphate. Goes from a 5 carbon sugar to a 6 carbon from the addition from CO2.
Its a cyclic reaction which uses ATP and NADH gained from photosynthesis.
The reductive TCA cycle
o Takes CO2 and adds it onto TCA cycle intermediates to produce a bigger carbon
molecule.
Photoautotrophs use carbon from CO2
Photoheterotrophs use carbon from organic sources.
 The Importance of Nitrogen
In biological systems (humans, animals, plants) nitrogen is the principal component
o Amino acids and proteins
o Nucleic acids n Bacteria contain ~ 13% N
Plant nitrogen is the underlying source of all human nutritional nitrogen - production of
fertilizer requires 2-4% of earths natural gas yearly output (non-renewable)
Plant sequestered N is the source, directly and indirectly, of all human nutritional N.
Lack of N fertiliser availability in the developing world limits crop production.
Overuse of N fertilisers in developed world poses environmental threats.
Nitrogen makes up 0.1% of the Earths crust but 80% of the atmosphere.
Atmospheric nitrogen is primarily gaseous N2, which is hard to access because of the highly
stable triple bond: N N.
N must be solubilised as NH3 before it can be used by cells.
What is nitrogen fixation?
o The conversion of atmospheric nitrogen into ammonium
o by nitrogen fixing bacteria
o Industrially
o Through lightning
The nitrogen Cycle

Industrial Nitrogen Fixation The Haber Bosch Process

N2 + 3H2 2NH3
o Need.
o ~500 atmosphere
o pressure 600C
o catalyst
 Biological Nitrogen Fixation nitrogenase enzyme

Ferredoxin is an electron donor to the Fe protein.

The Fe protein hydrolyses ATP and reduces the MoFe protein.
Uses 16 ATP for the reaction, when ATP binds to the enzyme it lowers the redox potential of
the enzyme which makes its possible for the enzyme to transfer electrons to the MoFe
protein, where it is then transferred to nitrogen, where ammonia and nitrogen gas is
produced.
The MoFe protein then reduces N2 to NH3.
Nitrogen fixation by nitrogenase
o Nitrogen fixation is catalysed in bacteria by the enzyme complex nitrogenase:
o N2 + 8e- + 8H+ + 16ATP 2NH3 + H2 + 16ADP + 16Pi
o The nitrogenase enzyme is energetically expensive and catalytically slow.
o A legume consumes 12 g of fixed carbon (from photosynthesis) for every 1 g of
nitrogen fixed.
o Nitrogenase is irreversibly inactivated by oxygen. Different organisms have evolved
strategies to protect nitrogenase from oxygen.
 Assimilation of ammonia by glutamate dehydrogenase

o Precursor is an organic acid, the ammonium is added to the keto group making
gluamate.
o Transaminases can then take this ammonia off glutamate and put it onto other
amino acids.
Assimilation of ammonia by glutamine synthetase

o NADPH is an electron carrier which takes place in biosynthesis reactions, where is

NADH is used in break down of organic molecules
 Nitrogen fixing bacteria - examples

Only bacteria can fix nitrogen.

Nitrogenase is inhibited by oxygen, so cant do photosynthesis
and nitrogen fixing at same place
Cyanobacteria
o Heterocysts are thick walled cells with an anaerobic inner
environment for nitrogen fixation.
o The heterocyst in the filament of Anabaena allows
fixation to occur in aerobic conditions.
o Vegetative cells carry out photosynthesis.
Symbiotic Cyanobacteria
o Symbiotic relationship between
o Water fern Azolla, and
o Cyanobacteria Anabaena azollae
o Azolla fern in rice patties is colonised by bacteria which
fix nitrogen. When the fern decomposes it provides
nitrogen to the rice.
Some cyanobacteria are partners in lichens
o Lichens: Symbiosis between fungus and algae (C fixation)
or cyanobacteria (C and N fixation)
Some free-living nitrogen-fixers: archaebacterial
o Eg. Methanococcus.
o Living in hot springs at >60C.
o Nitrogenase is heat stable.
Acetobacter-sugarcane symbiosis
o Acetobacter living inside sugarcane in the intercellular spaces.
o The sugar provides the ATP for nitrogen fixation
 Nitrogen-fixing symbioses in legumes
o $30-50 billion value
o 35% of world oil (soybean + peanut)
o 33% of human N requirements (66% in some areas)
o Beef, wool, and milk production dependent
o 3rd largest plant family (17,000 species)
o Symbiotic N2 fixation (Rhizobia)
o Mycorrhizal symbiosis
o Paradigm plant-microbe interactions
o Critical for sustainable agriculture
Nitrogen-fixing symbioses in legumes
o The most sophisticated N-fixing symbiosis.
o The nodule is a unique root organ designed to support the rhizobia.
o The rhizobia differentiate into bacteriods, surrounded by a specialized plant
membrane
o Bacteria in vesicles inside nodules called biosomes.
Leghemoglobin protects nitrogenase from oxygen Leghemoglobin is pink, and related to
haemoglobin in humans
o In the nodule
o Its very good at binding oxygen, pulls O2 out of the atmosphere inside the cell and
delivers it to the ETC of the bacteria.
o Need some oxygen but cell tries to reduce it as much as possible to produce
nitrogen
Signal exchange between legumes and rhizobia
o Legume hosts exude specific flavonoids with a specific structure recognised by
rhizobia. A transcription factor binds to it, which causes expression of genes
required for symbiosis
o Rhizobia produce species specific Nod factors necessary for nodulation. Exact
structure determine species specificity. Its recognised by a receptor on the right
host and initiates the symbiosis
Rhizobia invade root hairs of their hosts

Rhizobia re-initiate cell divisions in the root which later form nodules.
 Rhizobia invade the nodule where they fix nitrogen

What goes on inside the bacteroid (modified bacterial cell)?

Need oxygen captured by the leghemoglobin to produce ATP which is used by nitrogenase to
get nitrogen used in amino acids which are then transported to the plant. The plant in
return provides lots of fixed carbon to the bacteria.
Nitrogen carbon and sulfur cycles in the bioshpere
Microbes in agriculture have a large emmision of green house gasses
 Nitrogen cycle
o Nitrogen reaches the soil and is converted into organic matter, once they
decompose it returns to the soil as ammonium, where it can be taken up by plants
or oxidised by bacteria by a process called nitrification. The products can then be
denitrified and turned into gasses which are then sent to the atmosphere or plant
consumes them.
On terrestrial systems, N-fixation and denitrification almost equal themselves out. Most
excess nitrogen leaks into waterways, where N-fixation can also occur, but most is lost by
denitrification.
Chemolithotrophs the mavericks of recycling
o They use inorganic molecules for a source of energy and electron source. Most of
them but not all are autotrophs.
Nitrogen cycle

Denitrification use nitrate as an electron acceptor in anaerobic respiration

o Eg Pseudomonas, Bacillus

o Useful in cleaning up excess nitrogen in waste waters meg in soils, wetlands, sewage
plants (generally low oxygen environments)
 During denitrification, nitrate acts as an electron acceptor in anaerobic respiration

This produces a lot of nitrous oxide which is in the atmosphere and is due to lots of nitrogen
in soils etc, so its used in this process
Difference in redox poten9als between NAD+/NADH and NO3 - /N2 is smaller than the
difference between NAD+/NADH and O2/H2O, but its still fairly reasonable
A lot of these organisms can switch, so if there is O2 available then it uses O2, but uses N2 if
it instead is available
Nitrifying bacteria
o Very diverse chemolithoautotrophs, aerobic
o nitrification gain electrons from oxidation of
ammonium to nitrate or nitrite
nitrite further oxidized to nitrate
o Nitrosomaonas species do the first step, but Nitrobacter species used for second
step
 Oxidation of NH3 to nitrite

These can use inorganic compounds as donors for the ETC. In the first step the ammonia
reacts with oxygen to produce hydroxylamine. This then donates electrons to this ETC and
moved to cytochromes, and eventually passed onto oxygen. They use nitrogen stuff as the
donor and oxygen as the acceptor. The first step of oxidation of ammonia to hydroxylamine
requires electrons, so some of the electrons produced are used to do this, so not all
electrons produced are available for use. These organisms use a reduced form of nitrogen
as a donor in the ETC.
Oxidation of nitrite to nitrate
o Reverse electron flow to make NADH
 o
Reverse electron flow is necessary to reduce NAD+ if the electron donor redox pair has a
more positive redox potential than NAD+/NADH
Nitrogen cycle
 Electron acceptors in aerobic and anaerobic respiration
o NO3-, SO42-, CO2, S
The sulfur cycle
In volcanic activity and hydrothermal vents
Sulfur in the atmosphere from fossil fuel combustion, which turns to acid rain H2SO4.
Its not as abundant in organisms, but needed for systeine, methionine, coenzyme A, biotin
etc.

It can occur in the most reduced form and most oxidised form.
1) Oxidation of S compounds by chemolithotrophs, i.e. using reduced sulfur compounds as
electron donors in the ETC
o Can use reduced sulfur as donors in the ETC. These bacteria are aerobic and use O2
as the final electron acceotor.
o It converts a reduced form of sulfur into a very oxidised form of it.
 o Some electrons are used in a reverse electron flow to produce NADH.

Organisms which use oxidation of reduced S

compounds
o Bacteria living around hydrothermal
vents can oxidise H2S
o Some of these bacteria live as
symbionts inside tube worms near
hydrothermal vents
The ultimate electron transport chain
o Some bacteria (Desulfobulbacae) living
in deep water mud have abundant
electron sources as H2S but no good
electron acceptor bacteria form a long
(cm range) chain to transport the
electrons to oxygen in upper water
layers!
Reduction of S compounds
o SO42- H2S
o (Assimilatory sulfate reduction: sulfate is reduced and then incorporated into
organic molecules)
o Dissimilatory sulfate reduction: sulfate acts as an electron acceptor in anaerobic
respiration (similar to nitrate)
Dissimilatory sulfate reduction (e.g. Desulfovibrio)
o Sulfate is converted to APS (adenosine phosphosulfate), which acts as the electron
acceptor during anaerobic respiration
o Source of electrons come from organic molecules. The electrons first go to a
hydrogenase which then go to cytrochromes and eventually go onto sulfur
 compounds. First sulfate reacts with ATP to
form APS so get sulphite, then its reduced to
sulfur.
o Using sulfate as an electron acceptor is
energetically inefficient
Sulfate reduction happens in the shallow subsurface
biosphere
Sulfate reducing bacteria are important in cleaning
up mining waste water
o Mine drainage often contains pyrite (Fe2S),
which is oxidised to sulfate, producing
sulfuric acid. Often toxic metals are present
as well.
o Desulfovibrio can reduce sulfate to H2S (gas),
removing sulfate and increasing the pH. H2S
can also react with toxic metals to produce
stable metal sulfides.
Summary S cycle

Important points
o Both N and S can occur in a number of more oxidised or more reduced forms
o The reduced forms can donate electrons to the ETC in chemolithotrophs
o The oxidised forms can be electron acceptors during anaerobic respiration
o Both elements are incorporated into organic compounds by bacteria: N and S
assimilation, leading to the formation of amino acids
The carbon cycle
o Carbon source comes from CO2, decomposing organic matter. CO2 can be reduced
to methane, or methane can be oxidised to CO2.
 Methanotrophs oxidise methane back to CO2.
CO2 in the atmosphere as a gas, but its also dissolved in water, which is a problem as it
causes acidification (carbonic acid). So the more CO2 in the atmosphere and more in water,
then the more the oceans get acidified. CO2 can get into the water from atmosphere but
also from decomposing life in the water. Same can happen on land.
Release of CO2 during the TCA cycle. Its produced during the break down of pyruvate, and
basically pyruvate is a product of glycolysis, and that pyruvate is broken down further to
NAD and NAPH, and during this procures pyruvate is completely decarboxylated to CO2.
The Calcin cycle fixation of CO2 to form sugars.
o Done by plants and other photosynthetic bacteria
o Enzyme RUBISCO
Takes and organic carbon molecule, a C5 sugar, and the CO2 reacts with the
sugar to produce 2 intermediates. Then using ATP and NAD it leads to the
production of Fructose. So this removes CO2 out of the atmosphere and
converts it into organic sugars.
Methane emissions and CO2 are trending upwards at the moment, increasing temperatuers.
Sources of methane
o In general anoxic environments
o Peat bogs
o Land fill
o Termites
o Ruminant animals (cows)
Methane clathrate
o Methane surrounded by water molecules. Stable at low temperatures, a lot of these
can be found in Siberia in the tundra and down deep in the ocean. They are mined
as they can be used as gas to burn, but with rising temperatures, currently in the
 artic areas, where it was stable, the warming of them causes the methane to come
out and warm the atmosphere, so it could accelerate global warming.
Wetlands are some of the biggest sources of methane emission
A fair bit from termites, more than from ocean
Anthropogenic emissions (what we produce), so burning of fossil fuels, ruminants (livestock)
make a huge amount of methane. Overall we are producing a lot more methane than that is
used up.
Methanogens bacteria that produce methane from CO2
Methanotrophs - bacteria that convert (oxidise) methane into CO2
Methanotrophs (living off methane)

Methane is a very reduced molecule, so its a good donor, so its used as a donor in the ETC.
In this case, they dont directly donate electrons to the chain, but are in a number of
reactions, which form acid and transport the electrons onto NADH. The prinicipal is they are
a source of electrons for the ETC, and the carrier is NADH. They are aerobes and use oxygen
as the final acceptor.
Methanogens anaerobic habitats
o Mainly archaea, but lots of them can be
o Usually have methano in their name
o Digestive tracts
Hindgut fermentors including humans, insects
Ruminants (mainly)
o Anoxic sediments marshes, swamps, landfills
o Geothermal sources of H2 and CO2 hydrothermal vents
o Biodegradation facili9es sewage
o Methanogens produced methane from CO2.
 The rumen of a cow
o Where a lot of the fermentation reactions take place
Fermentation and methanogenesis in the rumen

Have glycolysis, but no TCA cycle

Produce acetate, propionate, butyrate which the cow uses.
Some of the bacteria in the rumen produce hydrogen and CO2. The methanogens use the
hydrogen as a source of electrons, and the CO2 is used as an electron acceptor to produce
methane. Then that methane is burped out by the cow. Results from digestion and
fermentation of sugars from cellulose.
Methanogenesis from CO2 and H2
o Basically a small ETC which use H has a source of electrons, which are transported
onto ferredoxin, which are then transported onto CO2 in a stepwise reaction. The
CO2 is reduced, to formyl which is then reduced to methylene and then methyl.
Reduction of methyl group to methane.
o Energetically this is not very good, as CO2 is an energetically very inefficient electron
acceptor.
Significance of methanogenesis in the rumen
o CH4 or methane is considered a greenhouse gas that contributes to global warming
o The effect of ruminant livestock on greenhouse gas emissions is significant
Australia - 12% total greenhouse gas emissions New Zealand - 60% total
greenhouse gas emissions
o Loss of energy in methane emission by ruminants is estimated to be between 2 and
15% of the gross energy intake
 o There is a great deal of interest in reducing or eliminating methanogens in the
rumen microflora To reduce greenhouse gas emissions To increase production
efficiency
o Strategies to reduce/eliminate methanogens in the rumen microflora Changing the
composition of the rumen microflora
o Biological control directed at methanogens and associated organisms
o Vaccination so cow will kill the bacteria
o Establishment of effective acetogenic and bacteriocin producing populations
Acetogenesis H2 + CO2 Acetate
Summary of the carbon cycle
o CO2 is produced in the TCA cycle during respiration
o CO2 fixation occurs via the Calvin cycle in photosynthetic organisms, producing
sugars
o CO2 can be converted to methane by methanogenic
bacteria (archaea) using CO2 as an electron acceptor in
anaerobic respiration
o Methanogenesis in ruminant animals and anoxic
environments is substantial
o Methanotrophs use methane as an energy source, this
produces NADH (e- donor to the ETC) and CO2
Electric bacteria eg geobacter
Bacterial fuel cells
o Bacterial fuel cell bacteria transfer electrons straight onto
a metal anode, i.e. bacteria generate electricity (using
mud/organic waste as C and electron source)
o Bacteria directly touch the anode by forming nanowires
many bacteria combine inside biofilms that act like tiny
wires conducting electricity the next biocomputer
innovation?
o Dont produce a lot of current, but as they are so small can
use to make tiny circuits in computers.
Some questions- respiration and fermentation
o What do many heterotrophic organisms use oxygen for?
Act as the final electron acceptor in the ETC
o Why do facultative anaerobes prefer to live aerobically if oxygen is present?
O2 is a better acceptor, so can produce more ATP, than from anaerobic
methods. TCA cycle will produce more ATP than from fermentation
o What is the difference between fermentation and anaerobic respiration?
Anaerobic respiration still has a function ETC, but dont use O2 as the
acceptor, use something else. It produces less ATP as the other acceptors
have worse reduction potentials
o Name some important electron acceptors in anaerobic respiration
Sulfate, nitrate
o Name some important products of fermentation
Lactic acid, butyrate, ethanol
 Some questions photosynthesis
o What is the source of oxygen in photosynthetic organisms?
Electrons initially come from chlorophyll and get put onto NADH. From
water, water is split up, oxygen is a product of this. Electrons come from
water and oxygen is a bi-product of this.
o Where do photosynthetic organisms source the electrons from?
Chlorophyll absorbs in red a blue wavelengths
o Why have different photosynthetic organisms evolved a range of photosynthetic
pigments?
If grow with not much light need other pigments to capture light
o What is the carbon source in photosynthetic organisms?
CO2
Some questions -nitrogen fixation
o What is the product of nitrogen fixation?
ammonium
o Why does nitrogen fixation require a lot of ATP?
Need to break the triple bond of N2
o How is ATP produced for nitrogen fixation in rhizobia?
Given sugars and organic acids by host plant, which are metabolised through
the TCA cycle which produces electron carriers which take the electrons to
the ETC, to produced ATP.
o What is the role of leghemoglobin?
Binds oxygen to make the environment more anaerobic to protect
nitrogenase
o How do photosynthetic cyanobacteria solve the problem of protecting nitrogenase
from oxygen?
Have special cells, one cell is aerobic for photosynthesis, one is anaerobic to
do nitrogen fixation called heterocyst.
Some questions N, S, and C cycle
o Name three sources of electrons in chemolithotrophs
Methane, H2S, ammonia are electron donors
o Explain why nitrate is a preferable electron acceptor compared with sulfate
Different redox potentials, the bigger the difference in reduction potential
between donor and acceptor the more ATP can be made.
o What is the biggest input of anthropogenic nitrogen into the biosphere?
Fertilizer, harber bosch process
o Why does sulfate reduction take place under anoxic conditions? not in exam
o What is the carbon source for methane production in the rumen?
Carbon dioxide
Genomics
o Study of molecular organization of genomes, their information content, and gene
products they encode
network of interconnected circuits
 window into entire microbial communities metagenomics you want to
find out what type of microbes live in an environment, looking at microbial
communities in an environment
evolutionary insights can look at phylogenetic relationships
DNA sequencing
o Sanger DNA Sequencing you clone the piece of DNA into a vector, then you do
random sequencing, once you get the sequence, then you fill the gaps. Have to
make sure every base your reading is correct, its quite a laborious job. Its
expensive and time consuming.
o Next generation DNA sequencing
Bioinformatics
o Analysis of genome data using computers
o Generates data on genome content, structure, and arrangement
o Also provides data on protein structure and function
o Uses annotation to determine location of genes on newly sequenced genome
o Further examination carried out using in silico analysis
Genome annotation
o Have DNA and translate it in all 6 possible reading frames.
o 2nd frame has no stop codon, so is the open reading frame.
o You have to find all the open reading frames, to figure out the protein coding
sequences

o Also have to look at the start sequences for translation the shine delgarno
sequence and start codon. If they arent there then its not a potential open reading
frame.
Blast (basic local alignment search tool) software
o Take a gene and do a base by base comparison of 2 or more gene sequences, then if
you find similarity between your sequence and the data base sequence, then you
can say that this open reading frame may be doing that function.
o Lots of similarity between MinD.
 o
o Blast gives idea of what the function might be, whereas annotation only tells what
open reading frames might be.
o Conserved hypothetical proteins function has not been found, but the DNA
sequence has some similarity in the data base
o Proteins of unknown functions proteins unique to a particular organism, ie no
similarity to genes in the data base
Functional genomics
o Determination of how genome works
o Uses physical maps of location of genes
o Provides info on
Metabolic pathways
Transport mechanisms
Regulatory and signal transduction mechanisms
Approach to identifying genes of unknown function
o Knock out approach. Knock the gene out and then look at what happens to the
phenotype
o Has been done with saccharomyces cerevisiae
Construction of mutant strains, each with a deletion in a specific ORF of
unknown function
These used in studies to reveal their mutant phenotype
Mutants phenotype used to assign tentative function for gene
Only difference is that only 1 gene has been knocked out, so know that
genes function if phenotypes differ when assayed
Transcriptome analysis
o Can determine which genes are expressed at a specific time or have changed
expression in response to environmental changes
o By directly sequencing total cellular mRNA (RNA-seq)
cellular mRNA is converted to cDNA with reverse transcriptase
adapter sequences are added to the ends of the cDNA fragments
each fragment is sequenced
sequence data can be used in two ways
 identify sequences by alignment with known (reference) genome
sequences if you already have a genome sequenced strain, then
its the reference
convert sequences to amino acid sequences and compare to
databases of known protein sequences
o take mRNA from different conditions, and then see which genes are upregulated or
down regulated, eg 1 from high pH and 1 from low pH.
o No cloning required
o Can also do knock out stuff with it
Proteomics
o The study of the proteome the entire collection of proteins that an organism
produces
o Provides information about genome function not available from mRNA studies
o Information determines what is actually happening in cells is referred to as
functional proteomics
Analysis of proteome
o Proteome often analyzed by two dimensional gel electrophoresis
first dimension
isoelectric focusing - pH gradient determines isoelectric point
second dimension
electrophoresis (SDS-PAGE) and separation by molecular weight
o load sample onto a pH strip to separate the proteins based off their isoelectric point
(the pH where the net charge on the protein becomes 0). So once you load the
protein, when they hit a particular pH and their net charge becomes 0 and will stop
migrating. So you get separated bands. Then you take the strip and do an
electrophoresis, and separate the proteins by molecular weight, with biggest at the
top and smallest at the bottom, so you can separate all the proteins on the band.
Bands may contain multiple proteins, with either similar or different sizes.
o Can look at expression, by how much protein you get from each band compared to
the wild type.
Further Proteome analysis
o Tandem Mass Spectrometry
unknown spot from 2-D gel is cut and cleaved
fragments are analyzed by mass spectrometer
mass of fragments is plotted
protein tentatively identified from probable amino acid composition
Comparative Genomics
o Set of analyses by which gene function and evolution can be inferred by studying
similar nucleotide and amino acid sequences found among organisms
o Comparisons of genomes
Relative sizes of microbial genomes
Smallest genomes belong to parasitic microbes, lose genes coz not
needed as use the hosts
Genome size reflects metabolic and morphological complexity
 o Horizontal gene transfer (HGT)
o Genomic islands (permanently integrated mobile genetic elements)
Chunk of DNA from another thing eg phage DNA integrated into
chromosome, transposons, plasmids etc
o Pathogenicity islands (when the genomic islands code for virulence proteins) look
at ratios of base pairs. In e. coli about 50% is GC, if the sequence your looking at say
only has 30% GC then you know it somehow came from outside. can also see
phage DNA there in islands, as they are left over after phage DNA integration into
the DNA
o Phylogenic relationships between microbes can be studied by synteny order of
genes on genome on graph if there is straight line then order of genes between
species are similar and thus phylogenetically related
Pathogen virulence genes
o pseudogenes non-functional genes in non-pathogen relatives
Reverse vaccinology
o development of new vaccines using only specific proteins of pathogens
M. bovis can cause TB in different animals
M. leprae causes leprosy 3.3 Mb
M. tuberculosis (4.4Mb) and M. bovis have different hosts, has M. tuberculosis has deletions
so it can only infect humans
In M. leprae has about 1000 pseudogenes. Lost them coz they are intracellular for long
times and can use the cells stuff, and grow very slowly. Doubling time for M. leprae is about
2 weeks
Reerse Vaccinology
o Start from protein not gene
o Take a pathogen and look at antigenic proteins on cell.

o Find the protein, then find the genes, then express them
Metagenomics
o Environmental genomics
cultivation-independent technique
 used to learn more about the diversity and metabolic potential of microbial
communities
takes a census of microbial populations and can determine the presence and
level of classes of genes
utilized to produce the Genomic Encyclopedia of Bacteria and Archaea
project
improves reference databases by sequencing genomes of a wide
variety of cultured microorganisms
basically take a census of the bacteria living in an environment
good as only 2% of bacteria can be cultured so if you plated out you would
miss a lot
so can discover lots of new genes, metabolic pathways etc
collect sample of the ecosystem, then sequence it.

Get large gaps in the genome from fragmentation techniques, but if it is a known gene then
you can fill them in with a reference sequence to figure out what it does
Recombinant DNA technology
o Steps in cloning a gene
Isolate DNA
Cut it (PCR)
Transform into a vector
Put it into another genome
 o Restriction enzymes
o Gel electrophoresis
o Southern blotting (Western)
Want to identify a gene present in your fragment. Take a genome, digest it,
run on a gel, and want to know if the gene is present in those fragments
Run DNA on gel has to be single stranded, then blot it onto a membrane.
Then use probe such as labelled HisA with radioactive phosphate, then you
hybridise it with the DNA on the membrane. If it finds a match it will light
up, so you know that fragment contains the gene.
Western blotting is similar but uses proteins for the gel and antigens as
probes. So if a band lights up its the protein you are looking for.
o PCR
Cloning Vectors and Creating Recombinant DNA
o there are four types of cloning vectors plasmids (most commonly used) phages
and viruses cosmids (hybrid between plasmids and phage) artificial
chromosomes
o each type of cloning vector generally has
an origin of replication
a selectable marker
multicloning site or polylinker allows you to insert DNA
Plasmids as Cloning Vectors (pUC19 and YEp24)

Can replicate in ecoli and in yeast

lacZ is used to screen for recombinants
o multicloning site is in lacZ gene, so it disrupts the lacZ gene. So the clones that dont
have inserted DNA will be blue in the presences of x-gal plates as lacZ is functional,
so you can get rid of the blue colonies so they only have the recombinants.
Phage Vectors
 o are engineered phage genomes previously genetically modified to include restriction
sites.
o after insertion of foreign DNA the recombinant phage genome is packaged into the
capsid and used to infect host cells
o use as a delivery vehicle to inject DNA into a host
o take empty heads and put recombinant DNA into them. Then plate recombinant
phages with host bacteria to obtane plaques
Cosmids
o Do not exist in nature
o These vectors have been constructed to contain features from both phages and
plasmids
they have a selectable marker, multiple cloning sites from plasmids, and a
cos site from phage
phage then introduces recombinant DNA into E. coli
o Cos site are like restriction sites. Terminase recognises this sequence and makes
sticky ends.
o Once DNA is injected into the cell the DNA becomes circular due to the cos sites.
o Each lambda particle receives a complete genome unit due to cos sites, so you can
control how much DNA goes in based on placement of cos sites.
o Once injected it becomes a plasmid, lacking phage genome apart from cos sites.
o Allows for cloning of 47kb as thats how much phage can package
Artificial Chromosomes
o Used when large fragments of DNA must be cloned
o Bacterial artificial chromosomes (BACs) (Designed using F plamids) played
important role in the human genome project
o Yeast artificial chromosomes (YACs) may be unstable, for very large DNA
fragments
Finding the right clone
o By colony hybridisation with a radioactive probe when gene is not expressed
o By use of specific antibody to detect the production of protein when gene is
expressed and protein can be isolated
o Complementation
o Phenotype rescue
Expressing Foreign Genes in Host Cells
o Cloned genes, in the new host cell, are called heterologous genes and may not be
expressed unless they are modified
recombinant gene must have a promoter that host RNA polymerase
recognizes
differences exist in eukaryotic and bacterial hosts
o Expression vectors are used to overcome problems with expression of recombinant
genes in host cells
o Contain inducible promoters that result in high-level transcription
Expression vectors
o Factors affecting the expression of cloned genes in bacteria
o Number of copies, more copies = more expression
 o Strength of promoter, how tightly RNA poly binds to promotor makes more
expression
o RBS ribosome binding site???
o Codon usage, some codons are used more frequently, so Histidine is used more in
E.coli, so if you have a rare codon it will change expression.
o Fate of the protein, protect protein against other proteins, secrete it after its
production
Role of regulatory switches
o Many proteins produced in high amounts are toxic, inducible system is used.
Microbial Control Methods
o Physical agents
o Chemical agents
o Mechanical removal methods
o Biological agents
o Disinfection: The destruction or removal of vegetative pathogens but not bacterial
endospores. Usually used only on inanimate objects
o Sterilization: The complete removal or destruction of all viable microorgnisms. Used
on inanimate objects
o Antisepsis: Chemicals applied to body surfaces to destroy or inhibit vegetative
pathogens
o Chemotherapy: Chemicals used internally to kill or inhibit growth of microorganisms
within host tissues.
Definition of Frequently Used Terms
o Sterilization destruction or removal of all viable organisms
o Disinfection killing, inhibition, or removal of disease causing (pathogenic)
organisms
disinfectants - agents, usually chemical, used for disinfection, usually used
on inanimate objects
o Sanitization reduction of microbial population to levels deemed safe (based on
public health standards)
o Antisepsis prevention of infection of living tissue by microorganisms
Antiseptics - chemical agents that kill or inhibit growth of microorganisms
when applied to tissue
Antimicrobial Agents
o Chemotherapy use of chemicals to kill or inhibit growth of microorganisms within
host tissue
o Agents that kill microorganisms or inhibit their growth
o -cide suffix indicating that agent kills
Germicide include bactericides, fungicides, algicides, and viricides
o -static suffix indicating that agent inhibits growth
include bacteriostatic and fungistatic
The Pattern of Microbial Death
o decimal reduction time (D value) time to kill 90% must be sure that persister cells
(viable but nonculturable (VBNC) condition) are dead
Conditions influencing the effectiveness of antimicrobial agent activity
 o Population size
The larger the population the longer it takes to kill them
o Population composition
Different microbes have different susceptibility to killing agents, eg E. coli
easier to kill than mycobacterium due to cell wall differences
Actively growing cultures are more susceptible than stationary phases as
they have a greater intake of chemicals
o Concentration or intensity of an antimicrobial agent
80% ethanol is more effective than absolute as the water content allows
entry into the cell. Absolute ethanol will dehydrate the cell and preserve the
cell by taking all the water out
o Duration of exposure
o Temperature as temperature rises to optimum for growth of bacteria, the bacteria
grow faster and so the bacteria can more easily take up the chemical and it has a
higher efficacy. EG 11% Phenol at 10 degrees takes 2.5 hours to kill, increase to 20
degrees takes <60 minutes
o Local environment
pH (easier to treat if acidic), viscosity (can effect effectiveness, less water
means need higher temperatures), concentration of organic matter, etc.
organisms in biofilms are less susceptible to many antimicrobial agents
Mechanical Removal Methods
o Filtration
Reduces microbial population or sterilizes materials by removing
microorganisms
What kind of material?
Also used to reduce microbial populations in air
Filtering liquids
o Membrane filters porous membranes with defined pore sizes that remove
microorganisms primarily by physical screening
Filtering Air
o High-efficiency particulate air (HEPA) filters used in laminar flow biological safety
cabinets
o Air is filtered through filters and put back to the other air
o Face masks restrict the entry of pathogens
Physical control methods
o Heat
o Radiation
Moist heat
o Destroys viruses, fungi, and bacteria
o Boiling: Many pathogens are readily destroyed by boiling for a few min
o Will it destroy spores? Does it sterilize? Spores will not be destroyed, only the
microbial load will be reduced.
o Degrades nucleic acids, denatures proteins, and disrupts membranes
Steam Sterilization
o Carried out above 100 degrees which requires saturated steam under pressure
 o 15 psi 121 degrees
o Uses an autoclave
o Effective against all types of microorganisms
o Does it kill spores? At 15 psi it kills endospores
o Quality control includes strips with spores of Geobacillus stearothermophilus
Pasteurization
o Controlled heating at temperatures well below boiling
o Used for milk, beer, and other beverages
o Process does not sterilize but does kill pathogens present and slows spoilage by
reducing the total load of organisms present
o flash pasteurization (high temperature short term HTST)
72C for 15 seconds then rapid cooling
o ultrahigh-temperature (UHT) sterilization
140 to 150C for 1 to 3 seconds
Dry Heat Sterilization
o Less effective than moist heat sterilization, requiring higher temperatures and
longer exposure times
items subjected to 160170oC for 2 to 3 hours
o Oxidizes cell constituents and denatures proteins
o Usually done on glass ware or powders which need to stay dry
Dry Heat incineration
o Flaming equipment
o Burning bodies of infected animals
Ultraviolet (UV) radiation
o Wavelength of 260 is most bactericidal (DNA absorbs)
o Causes pyrimidine dimers to form
o UV limited to surface sterilization because it does not penetrate glass, dirt films,
water, and other substances
o Has been used for water treatment
Ionizing Radiation
o Gamma radiation penetrates deep into objects
o Destroys bacterial endospores
o Used for sterilization and pasteurization of antibiotics, hormones, sutures, plastic
disposable supplies, and food
o Use radioactive cobalt 60 as a gamma radiation source to sterilise fruits, vegetables,
meat, fish and spices.
Phenol Coefficient Test
o Potency of a disinfectant is compared to that of phenol
o Phenol Coefficient is expressed as the ratio of the effectiveness of the test germicide
to that of phenol against a test organism.
o Highest dilution 1:250 dilution of test germicide kills same amount as highest
dilution 1:50 Phenol, so the test germicide is 5 times more effective.
Phenolics
o Commonly used as laboratory and hospital disinfectants
o Act by denaturing proteins and disrupting cell membranes
 o Tuberculocidal, effective in presence of organic material, and long lasting
o Disagreeable
odour and can
cause skin
irritation
o Endospores are
not killed by
phenolics
Alcohols
o Ethanol and isopropanol mainly
o Among the most widely used disinfectants and antiseptics
o Two most common are ethanol and isopropanol
o Bactericidal, fungicidal, but not sporicidal
o Inactivate some viruses
o Denature proteins and possibly dissolve membrane lipids
Halogens
o Any of five elements: fluorine, chlorine, bromine, iodine, and
astatine
o Important antimicrobial agents
o Iodine
Skin antiseptic
Oxidizes cell constituents and iodinates proteins
At high concentrations may kill spores
Skin damage, staining (due to yellow colour), and allergies can be a problem
Iodophore
iodine complexed with organic carrier
released slowly to minimize skin burns
o Chlorine
Oxidizes cell constituents
Important in disinfection of water supplies and swimming pools, used in
dairy and food industries, effective household disinfectant (bleach)
Destroys vegetative bacteria and fungi
Chlorine gas is sporicidal (kills endospores)
Can react with organic matter to form carcinogenic compounds
o Heavy Metals
e.g., ions of mercury, silver, arsenic, zinc, and copper
copper sulphate is a very good algicide, to control algal growth in lakes or
ponds
Effective but usually toxic
Combine with and inactivate proteins; may also precipitate proteins
o Quaternary Ammonium Compounds
cationic detergents are effective disinfectants
kill most bacteria, but
not M. tuberculosis or
endospores
 safe and easy to use, inactivated by hard water and soap
o Aldehydes
Highly reactive molecules
Sporicidal and can be used as chemical sterilants
Combine with and inactivate nucleic acids and proteins

37% solution of formaldehyde gas is called formalin

2% glutaraldehyde
o Sterilizing Gases
Used to sterilize heat-sensitive materials
Microbicidal and sporicidal
Ethylene oxide sterilization is carried out in equipment resembling an
autoclave
Betapropiolactone and vaporized hydrogen peroxide
Combine with and inactivate DNA and proteins

Are quite toxic, so have to leave the materials after they are sterilezed for a
while before use
o Chemotherapeutic Agents (antibiotics)
Chemical agents used to treat disease
Destroy pathogenic microbes or inhibit their growth within host
Most are antibiotics
microbial products or their derivatives that kill susceptible microbes
or inhibit their growth
The Development of Chemotherapy
o Paul Ehrlich (1904) developed concept of selective toxicity identified dyes that
effectively treated African sleeping sickness
o Sahachiro Hato (1910) working with Ehrlich, identified arsenic compounds that
effectively treated syphilis
o Gerhard Domagk, and Jacques and Therese Trefouel (1935) discovered
sulfonamides and sulfa drugs
Sulfonamides or Sulfa Drugs
o Prontosil red dye gets turned into sulfanilamide
o structurally related to sulfanilamide, a para aminobenzoic acid (PABA) analog
o PABA used for the synthesis of folic acid and is made by many pathogens
unlike humans, these pathogens cannot take up PABA, they need to
synthesise it, whereas humans get it from diet.
 Need to synthesise
folic acids to survive
inside a host
sulfa drugs are
selectively toxic for
these pathogens
because they
compete with PABA
for the active site of
an enzyme involved
in folic acid synthesis,
resulting in a decline
in folic acid concentration
Drug has no effect on host as we dont synthesise folic acid.
Penicillin
o First discovered by Ernest Duchesne (1896), but discovery lost
o Accidentally discovered by Alexander Fleming (1928)
observed penicillin activity on contaminated plate
did not think could be developed further
o Effectiveness demonstrated by Florey, Chain, and Heatley (1939)
o Fleming, Florey, and Chain received Nobel Prize in 1945 for discovery and production
of penicillin
o Controls gram positive bacterial infection
Later Discoveries
o Streptomycin, an antibiotic active against tuberculosis, was discovered by Selman
Waksman (1944)
Nobel Prize was awarded to Waksman in 1952 for this discovery
o By 1953 chloramphenicol, terramycin, neomycin, and tetracycline isolated
General Characteristics of Antimicrobial Drugs
o Selective toxicity ability of drug to kill or inhibit pathogen while damaging host as
little as possible
o Therapeutic dose drug level required for clinical treatment
o Toxic dose drug level at which drug becomes too toxic for patient (i.e., produces
side effects)
o Therapeutic index ratio of toxic dose to therapeutic dose
Very high if drug has low or no effect on host, eg penicillin as humans cells
dont have cell walls
Low if the drug has toxic side effects
Antibiotic production
o naturally produced
o Synthetic (e.g. sulfonamides)
o semisynthetic antibiotics are natural antibiotics that have been chemically modified
to make them less susceptible to inactivation by pathogen
e.g., ampicillin and amoxycillin are semisynthetic whereas penicillin G and
penicillin V are naturally produced
 Determining the Level of Antimicrobial Activity
o Effectiveness expressed in two ways
minimal inhibitory concentration (MIC)
lowest concentration of drug that inhibits growth of pathogen
minimal lethal concentration (MLC)
lowest concentration of drug that kills pathogen
o Two techniques are routinely used to determine MIC and MLC
Static inhibits growth of bacteria
Cidil kills bacteria
Broad spectrum kill both gram negative and gram positive
Determining the Level of Antimicrobial Activity
o Dilution susceptibility tests for MIC
o Disk diffusion tests Kirby Bauer
Dilution Susceptibility Tests
o Involves inoculating media containing different concentrations of drug
broth or agar with lowest concentration showing no growth is MIC
if broth used, tubes showing no growth can be subcultured into drug-free
medium
broth from which microbe cant be recovered is MLC
Dilute multiple tubes with the antibiotic (serial dilutions). Then add the
same amount of test microbe to each tube. The first tube which shows no
growth is the MIC (growth is inhibited by may not be dead). You then take
the broth of that tube and add to a tube with no drugs. If there is no growth
then that is MLC.
Can also do this with agar
Disk Diffusion Tests
o Disks impregnated with specific drugs are placed on agar plates inoculated with test
microbe
o Drug diffuses from disk into agar, establishing concentration gradient
o Observe clear zones (no growth) around disks
o Use the table to see which zone diameter corresponds to effectiveness
o Larger the zone, the more susceptible, but you cant compare directly between
antibiotics, as each antibiotic has different properties which vary.
o So you have work out MIC plotted against inhibition zone diameter for each
antibiotic to design table. On the graph, higher parts of the line are resistant. Lower
parts are where they are susceptible
Kirby-Bauer Method
o Standardized method for disk diffusion test
o Sensitivity/resistance determined using tables relating zone diameter with microbial
resistance
o Table values plotted, used to determine if effective concentration of drug in body
can be reached
Antibiotic Misuse and Drug Resistance
o An increasing problem
 once resistance originates in a population it can be transmitted to other
bacteria
a particular type of resistance mechanism is not confined to a single class of
drugs
o Microbes in abscesses or biofilms may be growing slowly and not be susceptible
o Resistance mutants arise spontaneously and are then selected
The Origin and Transmission of Drug Resistance
o Resistance genes can be found on
Chromosome
Plasmids
Transposons
o When found on mobile genetic elements they can be freely exchanged between
bacteria
Antibiotic misuse and drug resistance
o Tons of antibiotics valued at billions of dollars are produced. Approx 40-50% of these
are added to livestock feed. A resistant pathogen in animals can develop and then
transfer the genes to a human pathogen.
o Neisseria gonorrhoea infections
o a methicillin-resistant Staphylococcus aureus (MRSA) that developed resistance to
vancomycin
o these drug resistant organisms are a serious threat to human health
o Over 50% of the antibiotics prescriptions are given without clear evidence of
infection
o Antibiotics given to patients with colds, influenza, viral pneumonia and other viral
diseases
o Antibiotics are prescribed without culturing or without determining bacterial
sensitivity to the drug
o Patients not completing their course of medication: drug-resistant mutants may
survive
o Freely available in many countries
o Use of antibiotics in animal feed: drug-resistant bacteria may develop in animal GI
tract
12 steps to prevent antimicrobial resistance
o Prevent Infection
1. Immunise to prevent common infections
2. Avoid unnecessary introduction of parenteral devices, such as catheters
o Diagnose and Treat Infection
3. Target the pathogen
4. Access the experts do antibiotic susceptibility tests to find correct
treatment
o Use Antimicrobial Agents wisely
5. Practice antimicrobial control
6. Use local data - like what resistance there is and what infections there are
7. Treat infection, not contamination
8. Treat infection, not colonisation
 9. Treat with the least exotic antimicrobial agent that will eliminate the
pathogen ie not broad spectrum first
10. Stop antimicrobial treatment when unnecessary, ie when sore throat is
caused by virus
o Prevent pathogen transmission
11. Isolate the pathogen, isolate the infected and their body fluids to
prevent spread of the infection
12. Break the chain of contagion
o Prevention is always better than curing
The Search for New Antimicrobial drugs
o New analogs of existing antimicrobial compounds
Make existing drugs better, eg ampicillin is a synthetically made from
penicillin
o Computer drug design
Once you identify targets in the bacterial cell, then you can use computers
to design a drug to bind to the target site.
o Natural products as antibiotics
Need methods to detect them cause sometimes they are not produced at
detectable concentrations
o Drug combination
Want to retain efficacy of a drug its a good idea to have combination
therapy.
Penicillin/ampicillin have beta-lactam ring which is degraded by beta-
lactamase in resistant microbes. So give ampicillin in combo with beta-
lactamase inhibitor.
o Bacteriophages
Approved by FDA as a controlling agent used to control E. coli and
Salmonella on foods. However has same problem as may get resistance,
mutations in the receptor phages bind to could mutate so phage cant bind.
To avoid this use a cocktail of phages, so if resistance to one phage is
detected, then the others will still kill it.
Food Microbiology
Why study? Microbes like to grow on food as it is a food source. Microbes are also used to
change food and make food. Beer wine, chocolate and coffee are all products of microbial
activity. Also food can act as a vehicle for disease transmission.
3 kinds, the good, the bad (spoil the food) and the ugly (cause disease)
The Good Microorganisms
o Naturally found on foods in the environment
Mechanism of preservation in the absence of refrigeration
Microbial modifications - texture, flavour etc
o Starter cultures in a variety of fermented foods
a large proportion of our daily diet is composed of fermented foods (beer,
wine, chocolate etc)
fermented food industry in Australia (cheese, beer and wine) Food
o Microbiologist today - improving and expanding fermented foods
 The Bad, and the Ugly Microorganisms
o Food-borne Illness and Spoilage
o Food Microbiologists today - try to increase the understanding of
The environmental source of these MO
How they persist in foods
Improving control and detection of these organisms
o The economic costs of food-borne illness are substantial.
Interesting Facts
o Numbers of meals eaten here every year - 20.8 billion
o Percentage of meals which cause food-borne illness - 0.02%
o 1 in 5 cases arises from incorrect food handling at home
o 60-80% arises from the Food Service Industry
Microbial Growth and Food Spoilage
o Results from growth of microbes in food
alters food visibly and in other ways, rendering it unsuitable for
consumption
o Different foods undergo different types of spoilage processes
o Toxins are sometimes produced
o Microbial growth is controlled by
o intrinsic factors
factors related to the food itself
o extrinsic factor
environment where food stored

Intrinsic Factors
o Food composition - carbohydrates
o mold predominates
o degrades food by hydrolysis
o little odour
o aflatoxin produced by some microbes
o ergotism
hallucinogenic alkaloids released by Claviceps purpurea
may cause death
o Food composition proteins or fats
o bacterial growth predominates
o Putrefaction proteolysis and anaerobic breakdown of proteins; foul smelling amine
compounds such as cadverine and putrescine
o Unpasteurized milk spoilage
 Lactobacillus lowers the pH of the milk and then mould grows and proteins
degrade the milk proteins.
o Butter short-chained fatty acid production; rancid butter

o pH
impacts make up of microbial community and therefore types of chemical
reactions that occur when microbes grow in food
e.g., low pH favors yeast and mold
o Presence and availability of water
in general, lower water activity inhibits microbial growth
o Oxidation-reduction potential
altered by cooking
lower redox more bacteria and anaerobes
o Physical structure
grinding and mixing distribute microbes; promotes microbial growth mincing
increases the surface area of the meat and allow pathogens to distribute all over
outer skin of vegetables and fruits slows microbial growth
o Antimicrobial Substances
Coumarins fruits and vegetables
Lysozyme found in egg and cows milk, can break down the cell wall of gram
positive bacteria
Aldehydic and phenolic compounds herbs and spices
Allicin garlic
Polyphenols green and black teas
o Extrinsic Factors
Temperature lower temperatures retard microbial growth by decreasing
metabolic activity stopping replication
Relative humidity higher levels promote microbial growth vacuum sealed
food protects it
Atmosphere oxygen promotes growth
Controlling Food Spoilage
o Methods of preservation
Primary goal is to eliminate or reduce the populations of spoilage and disease
causing microbes while maintaining food quality
Filtration
o Water, wine, beer, juices, soft drinks, and other liquids usually by filtration
o May better preserve flavour and aroma
 Low temperature
o Refrigeration at 5C retards but does not stop microbial growth
microorganisms can still cause spoilage with extended storage
growth at temperatures below 10C has been observed
fruit juice concentrates
ice cream
some fruits
High Temperature
o Food heated in special containers to 115C for 25 to 100 minutes
o Kills spoilage microbes, but not necessarily all microbes in food
o Spoilage of canned foods
spoilage prior to canning
underprocessing
leakage of contaminated water into cans during cooling process
Cans are a very good anaerobic environment, so if there are microbes in it, they
will produce gas and the shape of the can will change.
Pasteurization
o Kills pathogens and substantially reduces number of spoilage organisms
o Different pasteurization procedures heat for different lengths of time
shorter heating times result in improved flavour
Water Availability
o Dehydration
e.g., lyophilization to produce freeze-dried foods is commonly used to eliminate
bacterial growth
food preservation occurs as a result of free water loss and an increase in solute
concentration (add salt or sugar)
Chemical-Based Preservation
o GRAS
chemical agents generally recognized as safe
agents include organic acids, sulfite, ethylene oxide gas, ethyl formate
sodium nitrite inhibits spore formation in meats, forms nitrosamines
nitric acid binds to haem in the meat and gives red colour
o pH of food impacts effectiveness of chemical preservative
High Hydrostatic Pressure (HHP)
o Fairly new, so no standards yet
o Alternate way to preserve food
o Applies pressures from 100-800 milliPascals (MPs) without significant changes in
temperature highly detrimental to cell membranes, so the bacterial cell, especially
gram positive bacteria dies
o No industry standards for HHP conditions (yet)
Radiation
o use of ionizing radiation (gamma radiation) to extend shelf life or sterilize meat,
seafoods, fruits, and vegetables
o excellent penetrating power food not rendered radioactive
 o kills microbes in moist foods by producing peroxides from water
peroxides oxidize cellular constituents
Microbial Product-Based Inhibition
o Bacteriocins
bactericidal proteins active against related species
some form pores in plasma membranes, so leakage of intracellular material and
the cell lyses
some inhibit protein or RNA synthesis
e.g., nisin from Lactococcus lactis used in low-acid foods to inactivate
Clostridium botulinum (gram positive) during canning process
e.g., bacteriophages that kill Listeria monocytogenes sprayed onto ready-to-
eat meats prior to packaging
Packaging
o Modified atmosphere packaging (MAP)
gases in stored food affect microbial growth
shrink wrap materials and vacuum technology control atmosphere
impermeable to gases
high CO2 content packaging can be used to prevent fungal growth
high O2 content packaging produces superoxide radicals that inhibit
microbial growth
MAP is used for products: deli meats and cheeses, pizza, grated cheese,
coffee etc
Types of Food-Borne Disease
o Pathogens Noroviruses, Campylobacter jejuni, Salmonella are major causes
o E. coli and Listeria are also important pathogens
o Two primary types
food-borne infections pathogen on the food gets into the body and makes you
sick
food intoxications food gets contaminated by the bacteria, and the bacteria
produce toxins on the food. So dont need the bacteria, just the toxin to make
you sick
in some cases its not a clear cut boundary between the two
o Transmission
breakdown in hygiene causes
faecal-oral route key main route, so need to control it
fomites also important taps, chopping board, door handles also play a role in
the faecal oral route.
Food-Borne Infection
o Ingestion of pathogen, followed by growth, tissue invasion, and/or release of toxins
o Raw foods (e.g., sprouts, raspberries, and seafood) are important sources
o Staph aureus from meats, cold cuts and salads and cream-filled bakery goods
o Bacillus cereus intoxication and infection from reheated fried rice, sprouts and
cucumber, short incubation time as only need the toxin to make you sick
o Clostridium botulinum intoxication
o Infections usually have a longer incubation periods
 o Salmonellosis gastroenteritis from ingestion of contaminated meats, poultry or eggs
o Campylobacteriosis transmitted by uncooked or poorly cooked poultry products, or
raw milk
o Listeriosis pregnant women, the young and old and immunocompromised individuals
most vulnerable at risk people should not eat soft cheeses, refrigerated smoked meats,
deli meats and undercooked hot dogs
Campylobacteriosis
o Causative agent - Campylobacter jejuni - most common bacterial cause of gastroenteritis
in humans
o Campylobacter - are helically curved, motile rods, Gram-negative, microaerophilic
o Environmental Reservoirs:
50-100% of domestic animals (chickens, turkeys, cattle)
Also found in dogs and cats - typically a harmless inhabitant
Can also be isolated in large numbers from water
o Foods associated:
meat, poultry, raw milk, mushrooms, contaminated water, processed foods
presumably contaminated by an infected worker (not common),
o Person to person spread is unusual
o Growth characteristics:
Fastidious organism - does not thrive and multiply in foods well - even under
optimal conditions, grows slowly
Requires low concentration of oxygen
Growth temp best is 30-470C but survives well in refrigeration temps
o Control of C. jejuni in foods:
Sensitive to freezing, heat, presence of oxygen
measures to limit cross contamination should be observed
o Characteristics of campylobacteriosis:
First identified as a human pathogen in 1973
Low infectious dose (approx 100 cells)
2-10 days incubation period
o Symptoms:
ranges from mild, brief enteritis to grossly bloody stools
diarrhoea, abdominal pain, malaise, fever, nausea, vomiting.
Symptoms may persist from 1 day to a few weeks
o Mechanism of pathogenesis:
Only 100 cells required
adhesion and colonization of the small intestine, they colonize the epithelium of
the small intestine
enterotoxigenic - production of enterotoxin which is responsible for causing
diarrhoea
o Invasion:
production of enterotoxins and cytotoxins (associated with bloody diarrhoea)
penetration of the epithelial cells, intracellular multiplication, and systemic
dissemination
o Long term effects:
 immune reaction to the infection results in arthritic joints but no invasion of the
joint by the bacteria
Guillain Barre Syndrome autoimmune disorder of the peripheral nervous
system (serotype O:19) paralysis. The serotype has a specific LPS structure
which has resemblance to components found on peripheral nervous, so the
immune system attacks the nerves instead of the bacteria, which leads to
paralysis.
Listeriosis
o Gram-positive coccobacilli often arranged in pairs
o Facultative anaerobe
o Motile and capable of growth at 4 degrees C and in high-salt concentrations
o Epidemiology:
isolated in soil, water, and vegetation and from a variety of animals
disease associated with consumption of contaminated food products (soft
cheese, milk, turkey, raw vegetables (transplacental spread from mother to
neonate)
The young, elderly, and pregnant women (the bacteria can cross the placenta
barrier), as well as patients with defects in cellular immunity, are at increased
risk
Largest meat recall in the U.S. in 2002 (27.4 m pounds of deli meats) death,
illness, bankruptcy and changes to food safety inspection policy
o Mechanism of pathogenesis:
Noninvasive listeriosis (listerial gastroenteritis)
Symptoms: fever, diarrhoea, muscle aches, nausea, vomiting and fatigue
(incubation period: usually 1 day)
Invasive listeriosis (blood, CNS, uterus of pregnant women)
Symptoms: : typically an influenza-like illness with or without
gastroenteritis In non-pregnant adults: meningitis (incubation period: 3
days to 3 months) as can get into the brain by crossing the blood brain
barrier
Virulence:
Invasion (intracellular) Listeriolysin O breaks down the vacuole wall
Live in cytoplasm of the cell, take its nutrients and multiply, lyse the cell
and then enter other cells
Cross the blood-brain and placental barriers
Vibrio parahaemolyticus
o Curved Gram-negative rods, Facultative anaerobe and requires salt for growth
o Epidemiology: found in marine environment worldwide associated with consumption of
contaminated seafood and shellfish, get from seafood salads where food is not cooked
o Pathogenesis: produces haemolysin
o Symptoms: gastroenteritis - generally self-limited with an explosive onset of watery
diarrhoea and nausea, vomiting, abdominal cramps, headache and low-grade fever
Food-borne infections
o Escherichia coli diarrhoea caused by enteropathogenic, enteroinvasive (able to invade
host cells) and enterotoxigenic (produce toxins) types
 o E. coli 0157:H7 is thought to have acquired enterohaemorrhagic genes from Shigella,
including genes for the Shigalike toxin
o other organisms
viruses Rotavirus, common in kids and day care centres
protozoan pathogens Giardia, amoeba, dysentery, cryptosporidium
o other concerns
foods that are transported and consumed in uncooked state
sprouts
shellfish and finfish
raspberries
Food-Borne Intoxications
o Ingestion of toxins in foods in which microbes have grown
o Produce symptoms shortly after the food is consumed because growth of the disease
causing microorganism is not required
o Include staphylococcal food poisoning, botulism, and Bacillus cereus food poisoning
Detection of Food-Borne Pathogens
o A challenge
o Must be rapid, sensitive (as generally low levels of contamination) and simple and for
prevention
o Methods include: culture techniques immunological techniques molecular
techniques (much faster)
o Pulse Net
established by Centers for Disease Control
uses pulsed-field gel electrophoresis to determine distinctive DNA pattern of
each bacterial pathogen
Take a bacterial strain, prepare it for gnomic DNA and digest it with a
restrictive enzyme. Then you get a pattern you can compare to a
database. It separates large bits of DNA to create a pattern
enables public health officials to link pathogens associated with disease
outbreaks in different parts of the world to a specific food source
o In addition to PFGE, PCR and RFLP (restriction fragment length polymorphism) are
increasingly developed and used to create a genomic finger print to compare it to the
database.
amplify small quantities
species specific
o microarray techniques directly from food or water used to identify genes which are
differentially expressed in different environments. Look at the difference in mRNA of
samples
o whole genome sequencing
Microbiology of Fermented Foods
o Fermented Foods
o traditionally, fermentation has been a major way of preserving food
o raw food (naturally or artificially inoculated)
results in a food that is very different from the original product in texture,
flavour and aroma
 can be stored for extended periods
o Produced from fruits, vegetables, beans and related substrates
o Major fermented milk products
Yoghurt, Butter, Sour cream, Cottage cheese, Buttermilk, Other cheese
Fermented Milks
o Majority of fermented milk products rely on lactic acid bacteria (LAB) in the genera
Lactobacillus, Lactococcus, Leuconostoc, and Streptococcus
o Gram-positives that tolerate acidic conditions, non-spore forming, aerotolerant, strictly
fermentative
Cheese production
o Approximately 2,000 distinct varieties representing 20 general types
o Classified based on texture, hardness (soft, semi-soft, hard, very hard)
o All from lactic acid fermentation molds may further enhance flavour (blue cheese and
Camembert cheese)
Microorganisms as Food Amendments
o Probiotics (Lactobacillus and Bifidobacterium)
microbial dietary adjuvants good bacteria which help maintain good health
microbes added to diet in order to provide health benefits beyond basic
nutritive value
Possible Benefits of Probiotics
o Immunomodulation
o Control of diarrhoea
o Possible modulation of Crohns Disease
o Lactobacillus acidophilus and Bifidobacterium help minimize lactose intolerance
improve general intestinal health and balance may lower serum cholesterol may
have anti-tumor activity
Probiotic Microbes in Farm Animals
o Probiotics Lactobacillus acidophilus in beef cattle
decrease E. coli O157:H7 (cattle are reservoir for this strain) hamburger
disease
o Bacillus strain in poultry
limit colonization of gut by the process of competitive exclusion
reduces Salmonella and Campylobacter
Prebiotics ones which promote growth of good bacteria in the gut
o Oligosaccharide polymers (sources: soybeans, Jerusalem artichoke, chicory root, raw
oats, unrefined wheat and barley, onion, garlic, bananas, leek etc) that are not
processed until they enter large intestine
o Symbiotic system
combination of prebiotics and probiotics
results in production of certain acids that may be responsible for possible
beneficial effects of probiotics
Viruses
o Acellular infectious agents
Early Attempts to Prevent Viral Disease
 o Lady Wortley Montagu (early 1700s) proponent of inoculation of children with
material from smallpox lesions observed this practice among Turkish women, so that
children would get a mild case of the disease and develop life-long resistance
o Edward Jenner (1798) published case reports of successful attempts to prevent
smallpox by exposure to cowpox
Viruses versus cellular organisms
o Viruses
simple organization
Usually DNA or RNA but not both (almost all viruses)
unable to reproduce outside of living cells
obligate intracellular parasites
o Cellular organisms
Complex organization
Both DNA and RNA
Carry out cell division
Some are obligate intracellular parasites
Viruses Infect All Cell Types
o Bacterial viruses called bacteriophages (phages)
o Few archaeal viruses
o Most are eukaryotic viruses plants, animals, protists, and fungi
o Classified into families based on genome structure, life cycle, morphology, genetic
relatedness
Structure of viruses
o Virion size range is ~10400 nm in diameter and most viruses must be viewed with
electron microscope
o All virions contain a nucleocapsid which is composed of nucleic acid (DNA or RNA) and a
protein coat (capsid) some viruses
consist only of a nucleocapsid, others
have additional components
o Envelopes
Capsids
o Large macromolecular structures which
serve as protein coat of virus
o Protect viral genetic material and aids in
its transfer between host cells
o Made of protein subunits called
protomers
o Capsids are helical, icosahedral, or
complex
Helical Capsids
o Shaped like hollow tubes with
protein walls
o Protomers self assemble
o Tobacco Mosiac Virus structure
 Influenza Virus an Enveloped Virus with a Helical Nucleocapsid
Icosahedral Capsids
o Maximises the use of space, able to fit the most amount of
DNA inside the capsid
o An icosahedron is a regular polyhedron with 20 equilateral
faces and 12 vertices
Capsids of Complex Symmetry
o Some viruses do not fit into the category of having helical or
icosahedral capsids
o poxviruses largest animal virus
o large bacteriophages
T4 phage of E. coli
o Binal symmetry: head resembles icosahedral,
tail is helical
Viral Envelopes and Enzymes
o Arise from host cell membrane or nuclear
membrane
Viral Envelope Proteins
o Envelope proteins, which are viral encoded,
may project from the envelope surface as
spikes or peplomers
o involved in viral attachment to host cell
e.g., haemagglutinin of influenza virus
o used for identification of virus
o may have enzymatic or other activity
e.g., neuraminidase (helps the virus release from the cell, it facilitates release
from host cell) of influenza virus
o may play a role in nucleic acid replication
RNA dependent polymerase required but may not be present in host cell, so
code for it
Viral Genome
o Most animal viruses contain all 4 types
o Most plant viruses contain single stranded DNA
o Bacterial viruses have double stranded DNA
o
o
 o
o Positive strand RNA have same sequence for
mRNA, as soon as a virus infects the cell the
RNA gets in and starts transcribing.
o Negative strand RNA requires mRNA to be
produced via RNA dependent polymerase.
Viral Multiplication
o Mechanism used depends on viral structure and
genome
o Steps are similar
attachment to host cell
entry and uncoating of genome
synthesis
assembly
release
Attachment (Adsorption)
o Specific receptor attachment
o No receptors for plants, so there has to be
mechanical damage done to the plant for the
virus to get in. Damage caused by insects eating
the plants
o Lambda can only infect E. coli has e. coli only
has the particular receptor.
o Some viruses use LPS, flagella, pilli as receptors
o Receptor determines host preference for animal
viruses
 may be specific tissue (tropism) eg polio viruses, due to receptors only present
in specific organs and tissues
may be more than one host (rabies virus)
may be more than one receptor (HIV CD4 and CCR5)
may be in lipid rafts providing entry of virus
Viral Entry and Uncoating
o Entire genome or nucleocapsid
o Varies between naked or enveloped virus
o Three methods used
fusion of the viral envelope with host membrane; nucleocapsid enters by
receptor mediated endocytosis
endocytosis in vesicle; endosome aids in viral uncoating
injection of nucleic acid
Animal Virus entry see slides
Synthesis stage
o Genome dictates the events
o ds DNA typical flow
o RNA viruses
virus must carry in or synthesize the proteins necessary to complete synthesis
o Stages may occur, e.g., early and late
Assembly
o Late proteins are important in assembly
o Assembly is complicated but varies
o Head proteins are produced together and form the head capsid in one assembly line. In
another assembly line for the tube and sheath and then another assembly line for the
attachment of tail fiber proteins.
Virion Release
o Nonenveloped viruses lyse the host cell
viral proteins may attack peptidoglycan or membrane
Holin makes the hole and Lysin goes through the hole and cleaves the
peptidoglycan.
o Enveloped viruses use budding
in this case the cell is not lysed and will keep producing viruses for a while
viral proteins are placed into host membrane
nucleocapsid may bind to viral proteins
envelope derived from host cell membrane, but may be Golgi, ER, or other IM
virus may use host actin tails to propel through host membrane (Vaccinia)
Bacteriophage Lambda: A Temperate Bacteriophage
o Phage lambda () can enter either the lytic or lysogenic cycle upon infection of E. coli
o lysogenic dsDNA becomes prophage - integrated into hosts chromosome
o upon induction, viral genome is excised and lytic cycle begins
lysogenic conversion when there is an advantage to the host by having a prophage, ie phage
bringing toxin gene into a non-virulent bacteria, making them virulent.
Advantages of lysogeny survival strategy for the phage. If conditions are not right for bacteria
to multiple then the lytic phage cant multiply effectively. But if its a lysogenic phage, then it
 can survive until the conditions are right, and so it can enter the lytic cycle when conditions are
right
Lambda Phage
o Linear ds DNA genome with cohesive ends
o circularizes upon injection into host cytoplasm
o 40 genes, genes clustered together by function
o transcription from different promoters determine if lytic cycle or lysogeny occurs, cell
has to decide which cycle to enter based of external cues. Expression at different stages
of the cycle determines which cycle is taken
Regulatory Proteins Determine Lysogeny or the Lytic Cycle
o Function as repressors, activators, or both regulate transcription, termination, and
antisense RNA molecules
o cII activator plays pivotal role in determining if will establish lysogeny or the lytic cycle
o cII levels high early in infection lysogeny
o cII levels not high early in infection lytic cycle
o cI is the main repressor, cI is the only protein expressed during the lysogenic cycle, so all
other genes are repressed
o when cII is produced, cIII is produced which protects cII from being cleaved by an
enzyme. Cro represses the left side of the genes and the cI gene and causes expression
of Q protein which causes expression of main genes. When no cI, no lysogenic cycle.
Phage and High cII Levels
o Increases int gene transcription integrase catalyzes integration of into host genome
lysogeny is established
o Increases transcription of cI gene ( repressor) repressor represses all transcription
except its own binds to PRM promoter and activates transcription of cI therefore,
lysogeny is maintained
Phage and Low cII Levels
o cII is quickly degraded by a host enzyme, HflB, unless it is protected by viral cIII
o If cII not protected, protein Cro increases Cro is repressor inhibits transcription of cIII
and cI genes this further decreases cII and repressor Cro is activator increases
transcription of itself, Cro increases transcription of regulatory protein, Q
o Q activates genes needed for the lytic cycle
If Lambda Repressor Wins Race with the Cro Protein
o Lysogeny is established
o Lambda genome is integrated into the host genome in a reaction catalyzed by the
enzyme, integrase
How Does Induction Reverse Lysogeny?
o Triggered by drop in repressor levels
due to UV light, mutagenic chemical
DNA damage alters host cell RecA protein interacting with repressor,
causing repressor to cleave itself
cI transcription decreased, repressor levels reduced further
o Transcription increases
xis gene, excisionase increases and binds integrase
 reverse integration; phage freed from host chromosome
Cro protein levels increase
synthesis of repressor blocked
protein Q increases, lytic cycle proceeds
The cultivation of viruses
o Requires inoculation of appropriate living host
Hosts for bacterial and archaeal viruses
o Usually cultivated in broth or agar cultures of suitable, young, actively growing bacteria
o Broth cultures lose turbidity as viruses reproduce, as the cells will be lysing, so its also an
indication that viruses are growing in the broth culture
o Plaques observed on agar cultures make the lawn of E. coli and look for plaques which
is where the viruses will be multiplying. Can use this to work out plaque forming units
Hosts for animal viruses
o Tissue (cell) cultures
cells are infected with virus (phage)
viral plaques
localized area of cellular destruction and lysis
Cytopathic effects (CPEs) microscopic or macroscopic degenerative
changes or abnormalities in host cells and tissues dying, shrinkage
Have a monolayer of animal cells eg monkey kidney cells and see
plaques forming on them similar to bacterial. However not all animal
viruses form plaques so also look for CPEs.
o Embryonated eggs
Disinfect the egg shell, then make a small hole and inject the viruses into the
embryos within the eggs at different sites. Then reseal the whole.
See POCK within the embryonated egg lesions within the embryo which
contain the viruses
The right inoculation site is determined by which viruses as different viruses
grow better at different sites.
Hosts for plant viruses
o Plant tissue cultures
o Plant protoplast culture prepare cells without cell walls so virus can gain entry
o Suitable whole plants
may cause localized necrotic lesions or generalized symptoms of infection
plants have no receptors, so need to have mechanical damage done to the plant
for the virus to enter eg rub plant leaves together
Virus Assays
o used to determine quantity of viruses in a sample
o two types of approaches
direct
count particles
indirect
measurement of an observable effect of the virus
Particle counts
 o Direct counts made with an electron microscope, however very time consuming and
need the right concentration, also need to know the shape of what you are looking for
to count the correct stuff. Also might not be accurate as need to count many fields. To
help with this use latex beads with known concentration to know if your count is
accurate.
o Virus number can be quantified by qPCR
o Indirect counts eg hemaglutination assay
determines highest dilution of virus that causes red blood cells to clump
together
make a dilution, and look for the highest dilution which causes glutination of
RBC. Basically determine the relative quantity of virus in each sample not the
actual number, so if a sample has glutination at 10-8 dilution then that sample
has more viruses than a plate that only glutinated at 10-6 dilution.
Measuring Biological Effects
o Infectious dose and lethal dose assays
determine smallest amount of virus needed to cause infection (ID) or death (LD)
of 50% of exposed host cells or organisms (ID50 or LD50)
make dilutions and look for the dilution that will cause 50% of cells to die, or
when 50% are infected.
Measuring Concentration of Infectious Units
o Plaque assays
dilutions of virus preparation made and plated on lawn of host cells
number of plaques counted
results expressed as plaque-forming units (PFU) - plaque forming units (PFU)
PFU/ml = number of plaques/sample dilution
Final exam
o 120 minutes
o 12 questions 10 minutes per questions, write half a page to a page, can also do dot
points
9 from Naresh
3 from Ulles section
o All questions will be short answer questions.
o All content covered
o Example questions on wattle
o Tutorial on Wednesday 2nd Nov at 12-1 in Phys T.