Lecture 1 Intro 8-11-2018

Micro organisms are small organisms consisting of one cell or a cluster of cells
● diverse in form/function
● inhabit every environment supporting life
● live in microbial communities
● oldest form of life
● major fraction of Earth’s biomass
● surround plants and animals
● affect human life (health & infectious diseases, food and water, fuel)

Energy sources
Chemoorganotroph → Oxidation organic compounds
Chemolithotroph → Oxidation inorganic compounds
Only in prokaryotes
Phototroph
Light = energy source (photopigments)
Oxygenic photosynthesis: O2 production
(cyanobacteria, algae)
Anoxygenic photosynthesis: no O2 production
(purple and green bacteria)

Autotroph
C-source = CO2
Most chemolithotrophs and phototrophs
“Primairy producers”

Heterotroph
C-source = one or more organic compounds
Feed on autotrophs or (waste) products of autotrophs
Lecture 2
Transmission electron microscopy (TEM)
● Much greater resolving power (0.2 nm) than light microscope
● Enables visualization of structures at the molecular level
● Specimen must be very thin (60 nm) and stained to improve contrast
● Sees through the cell, the inside

Scanning electron microscopy (SEM)

● Specimen is coated with a thin film of heavy metal (e.g. gold)
● An electron beam scans the object
● Scattered electrons are collected and projected to produce an image
● Even very large specimens can be observed
● magnification range of 15–100,000x
● Only surface visualized

Pure cultures: cells from only a single type of microorganism

Enrichment culture techniques: isolate microbes having particular metabolic characteristics
from nature, then making a culture with about an abundance of them, but not exclusively.

Louis Pasteur
● discovered that living organisms discriminate between optical
isomers
● discovered that alcoholic fermentation was a biologically (not
just chemically) mediated process
● developed vaccines for anthrax, fowl cholera, and rabies
● Using the swan-necked Pasteur flask, he disproved theory of
spontaneous generation = led to sterilization methods and
food preservation

Robert Koch
● identified causative agents of anthrax, tuberculosis, and cholera
● developed solid media for obtaining pure cultures of microbes
● observed that masses of cells (colonies) have different shapes, colors, sizes
● experimentally demonstrated the link between microbes and infectious diseases
(germ theory of
infectious disease)
● Koch's postulates
● Developed enrichment
culture technique
Sergei Winogradsky
● proposed concept of chemolithotrophy: oxidation of inorganic compounds to yield
energy
● demonstrated chemolithotrophs use carbon from CO2 (autotrophs)
● first to demonstrate nitrogen fixation (Clostridium pasteurianum) and nitrification
● demonstrated that specific bacteria are linked to specific biogeochemical
transformations (e.g., N and S cycles)

Carl Woese
● discovered rRNA from methanogens distinct from Bacteria and Eukarya
● named new group Archaea
● found relationships can be deduced by comparing genetic information in the different
specimens
Bacteria
● prokaryotes Eukarya
● usually undifferentiated single cells ● plants, animals, fungi
1–10 μm long but vary widely ● first were unicellular, may have
● 30 major phylogenetic lineages, appeared two billion years ago
mostly diverse species with diverse ● at least six kingdoms
physiological and ecological ● vary dramatically in size, shape,
strategies physiology

Archaea
● prokaryotes
● less morphological diversity than
Bacteria
● mostly undifferentiated cells 1–10
μm long
● five well-described phyla
● historically associated with
extreme environments, but not all
extremophiles
● lack known parasites or pathogens
of plants and animals
Viruses
● obligate parasites that only
replicate within host cell
● not cells & do not carry out
metabolism - take over other
metabolic systems to replicate
● have small genomes of
double-stranded or single-stranded
DNA or RNA
● very diverse & classified based on
structure, genome composition,
and host specificity
Bacteria and Archaea
Morphology: cell shape
● Major morphologies of prokaryotic cells: coccus (pl. cocci), rod, spirillum
When clustered: Streptococcus, Sarcina, Staphylococcus
● Cells with unusual shapes: spirochetes (tightly coiled), appendaged bacteria,
filamentous bacteria
● Many variations on basic morphological types known

● Morphology typically does not predict physiology, ecology, phylogeny, or other

properties of a prokaryotic cell.
Some selective forces involved in setting morphology:
○ optimization for nutrient uptake
○ swimming motility in viscous environments or near surfaces
○ gliding motility

Sizes
Size range for prokaryotes: 0.2 μm to >700 μm in diameter
Most cultured rod-shaped bacteria: 0.5-4.0 μm wide & <15 μm long
Size range for eukaryotic cells: 2 to >600 μm in diameter

Advantages to being small

● more surface area (S) relative to cell volume (V) than large cells: higher S/V ratio
● support greater nutrient and waste product exchange per unit cell volume
● tend to grow faster than larger cells
● Mutations lead to faster evolution, Eukaryotic cells adapt slower

Cellular organisms <0.15 μm in diameter are unlikely, they need volume to house proteins,
nucleic acids, ribosomes, and so on

The Cytoplasmic Membrane

● Surrounds cytoplasm
● Separates it from environment
● Main function: selective permeability, nutrients transported in and waste products out
Bacterial cytoplasmic membrane
● 8–10 nm wide
● phospholipid bilayer:
● fatty acids (hydrophobic, point inward)
● ester linkages in phospholipids
● glycerol + phosphate + functional group (e.g., sugars, ethanolamine, choline)
(hydrophilic, point outward)
● some species hopanoids (sterol-like)
● embedded proteins: integral membrane proteins or peripheral membrane proteins

Archaeal membranes
● ether linkages in phospholipids (Bacteria and Eukarya have ester linkages)
● lipids have isoprenes instead of fatty acids
major lipids:
○ phosphoglycerol diethers with phytanyl C20 side chains
○ diphospho glycerol tetraethers with diphytanoyl C40 side chains (lipid
monolayer)
○ Crenarchaeol (Thaumarchaeota) (lipid monolayer)

Cytoplasmic membrane function

● permeability barrier: polar and charged molecules must be transported, transport
proteins accumulate solutes against the concentration gradient
● protein anchor: holds transport proteins in place
● energy conservation and consumption: generation of proton motive force

Bacterial Cell Walls

● Bacteria separated into two groups based on Gram stain
● Gram-positives and gram-negatives have different cell wall structures
○ gram-negative cell wall: peptidoglycan & outer membrane
○ gram-positive cell wall: thick peptidoglycan layer

Structure of Peptidoglycan
● rigid layer that provides strength
● can be destroyed by lysozyme (cleaves glycosidic bond between sugars) - found in
human secretions, major defense against bacterial infection
● cross-linked differently in gram-negative bacteria and gram-positive bacteria (often
“interbridges”)
Gram-positive cell wall
● up to 90 percent peptidoglycan
● common to have teichoic acids covalently bound to
peptidoglycan
● bind divalent metal ions (e.g., Ca2+ and Mg2+)
● lipoteichoic acids: teichoic acids covalently bound to
membrane lipids

[Few prokaryotes lack cell walls]

Cell Nutrition
Nutrients → supply of monomers (or precursors of) required
by cells for growth
● Macronutrients
○ nutrients required in large amounts
● Micronutrients
○ nutrients required in small amounts
○ trace metals and growth factors

H-C-N-O-P-S (and Se) : required by ALL

cells
Carbon (C): major element in ALL classes
of macromolecules
● Typical bacterial cell is ~50%
carbon (by dry weight)
● Most microbes (heterotrophs) use
organic carbon
● Autotrophs use carbon dioxide
(CO2)
Nitrogen (N): proteins, nucleic acids, and
many more cell constituents
● Bulk of nitrogen in nature is
ammonia (NH3), nitrate (NO3-), or
nitrogen gas (N2)
● Nearly all microbes can use NH3
Oxygen (O) and hydrogen (H): from water
Phosphorus (P)
● nucleic acids and phospholipids
● Sulfur (S)
● sulfur-containing amino acids
(cysteine and methionine)
● vitamins (e.g., thiamine, biotin,
lipoic acid)
Potassium (K)
● required by enzymes for activity
Magnesium (Mg)
● stabilizes ribosomes, membranes,
and nucleic acids
● also required by many enzymes
Calcium (Ca) and sodium chloride (NaCl)
● required by some microbes (e.g.,
marine microbes)

Transporting Nutrients into the Cell

Active transport: how cells accumulate solutes against concentration gradient
Transporters: energy-driven (proton motive force, ATP, energy-rich compound)
Three classes:
● simple transport
● group translocation
● ABC system
Simple transport
● driven by proton motive force
● either symport: solute and H+ cotransported in one
direction
● or antiport: solute and H+ transported in opposite
directions

Group translocation
● Substance transported is chemically modified
● Energy-rich organic compound (not pmf) drives transport
● Phosphotransferase system in E. coli

ABC (ATP-binding cassette) systems

● 200+ different systems identified in prokaryotes for
organic and inorganic compounds
● high substrate affinity
● ATP drives uptake
● requires transmembrane and ATP-hydrolyzing proteins
plus:
○ Gram- employ periplasmic binding proteins
○ Gram+ & Archaea employ substrate-binding
proteins on external surface of cytoplasmic
membrane
Lecture 3
The outer membrane
Gram - bacteria have an outer membrane
● Barrier against antibiotics and other harmful agents
● LPS = core polysaccharide, O-polysaccharide, and lipid A (endotoxin)
● Porins: transmembrane protein channels for entrance and exit of solutes

Periplasm: space located between cytoplasmic and outer membrane

● houses many extracellular proteins
● thin layer of peptidoglycan
● ~15 nm wide

Archaeal Cell Walls

No peptidoglycan
Pseudomurein
● found in cell walls of certain methanogenic Archaea
● polysaccharide similar to peptidoglycan
● composed of N-acetylglucosamine (in peptidoglycan) and N-acetyltalo-saminuronic
acid (different)
● β-1,3 glycosidic bonds instead of β-1,4
● amino acids all L-stereoisomer
● cannot be destroyed by lysozyme and penicillin

Cell walls of some Archaea lack pseudomurein - contain other polysaccharide polymers
Surface protein (S-) Layers
● most common cell wall type
● consist of protein or glycoprotein
● paracrystalline structure
● in many organisms, S-layers present in addition to other cell wall
components
● always outermost layer
Cell Surface Structures
Capsules and slime layers
● not considered part of cell wall because these do not confer
significant structural strength
● polysaccharide layers – may be thick or thin, rigid or flexible
● capsule: if tightly attached, tight matrix; visible if treated with India
ink
● slime layer: loosely attached, easily deformed (e.g., Leuconostoc)

● assist in attachment to surfaces

● role in development and maintenance of biofilms
● virulence factors: protect against phagocytosis
● prevent dehydration

Fimbriae and pili

● filamentous protein structures ~2–10 nm wide
● fimbriae enable organisms to stick to surfaces or form pellicles (thin sheets
of cells on a liquid surface)
● pili are typically longer, and fewer (1 or a few) found per cell
than fimbriae
○ Conjugative pili facilitate genetic exchange between
cells (conjugation)
○ Type IV pili adhere to host tissues and support
twitching motility
Hamus/hami
● Archaeal “grappling hooks” assist in surface attachment, forming biofilms
● structurally resemble type IV pili

Cell Inclusions
● Inclusions function as energy reserves, carbon reservoirs, and/or have
special functions
● Enclosed by thin membrane
● Reduces osmotic stress

Carbon storage polymers

● poly-β-hydroxybutyric acid (PHB): lipid polymer produced upon excess of
carbon used as C-/E- source under tough conditions
● glycogen: glucose polymer

Other
● Polyphosphate granules: inorganic phosphate
● Sulfur globules: elemental sulfur found in periplasm, oxidized to sulfate
(SO42–)
● Carbonate minerals: biomineralization of barium, strontium, and magnesium
● Magnetosomes: magnetic iron oxides; allow cell to undergo magnetotaxis: migration
along magnetic field lines
Gas Vesicles
● Confer buoyancy in planktonic cells
● Conical-shaped, gas-filled structures made of protein
● Gas = from external environment
● Impermeable to water and solutes
● Molecular structure:
○ composed of two proteins: GvpA and GvpC
○ function by decreasing cell density, increasing
buoyancy
● Benefit to phototrophic MO: adjust cells according to light
intensity

Endospores
● Formed during (endo)sporulation
● Highly differentiated cells resistant to heat, harsh
chemicals, radiation
● Survival structures to endure unfavorable growth
conditions
● “Dormant” stage of bacterial life cycle
● Ideal for dispersal via wind, water, or animal gut

Formation and germination

● vegetative cell converted to nongrowing,
heat-resistant, light-refractive structure
● only occurs when growth ceases due to lack of
essential nutrient such as carbon or nitrogen
It can remain dormant for years but converts rapidly back to being vegetative in three steps:
● activation: heated for several minutes at elevated but sublethal temperature
● germination: rapid (minutes), loss of refractility and loss of resistance to heat and
chemicals
● outgrowth: swelling from water uptake and synthesis of RNA, proteins, and DNA

Structure and features

● many layers: exosporium (outermost), spore coats, cortex, core enriched
in Ca2+
● contains dipicolinic acid (DPA): binds free water (with calcium -
dehydration) and stabilizes DNA
● core contains small acid-soluble spore proteins (SASP), which bind and
protect DNA and function as carbon and energy source for outgrowth
Cell Locomotion
Flagella
● structure that assists in swimming in
Bacteria
● different arrangements: polar (incl.
lophotrichous, amphitrichous), peritrichous
● increase or decrease rotational speed
relative to strength of proton motive force

Flagellar structure and activity

● helical in shape
● consists of several components
● Filament composed of flagellin
● reversible rotating machine
● movement at expense of pmf via Mot proteins

Flagellar synthesis
● Several genes are required
● Filament grows from tip
● MS ring is made first
● Other proteins and hook are made next

Archaella
● half the diameter of bacterial flagella
● move by rotation
● composed of several different filament proteins with little homology to
bacterial flagellin
● speeds vary from 0.1–10x Escherichia coli
● structurally similar to type IV pili

Gliding Motility
● Bacteria only; no Archaea
● Slower and smoother than swimming
● Movement typically occurs away from colony
● Requires surface contact
● Mechanisms:
○ excretion of polysaccharide slime
○ type IV pili/twitching motility
○ gliding-specific proteins (adhesion complexes or other specialized proteins)

Taxis: directed movement in response to chemical or physical gradients

● chemotaxis: response to chemicals
● phototaxis: response to light
● aerotaxis: response to oxygen
● osmotaxis: response to ionic strength
● hydrotaxis: response to water
Chemotaxis
● “run and tumble” behavior: run: smooth forward motion,
flagellar motor rotates CCW tumble: stops and jiggles,
flagellar motor rotates CW, flagella disperse
● Bacteria respond to temporal, not spatial, difference in
chemical concentration
● monitor/sample environment with chemoreceptors that
sense attractants and repellents

Chemotaxis in polarly flagellated Bacteria

● similar but not identical to peritrichous cells
● Many (e.g., Pseudomonas) can fully reverse flagellar
rotation, avoiding tumbling and reversing direction
● Some (e.g., Rhodobacter) stop and are affected by
Brownian motion

Measuring chemotaxis
● measured by inserting a capillary tube containing an
attractant or a repellent in a medium of motile bacteria
● can also be seen under a microscope

Phototaxis and Scotophobotaxis

● Phototaxis using photoreceptors allows phototrophic organisms to optimize position
for light harvest
● Scotophobotaxis: entering darkness causes cell to tumble, reverse direction, head
back to light
Lecture 4
Growth: increase in cell number
Binary fission: cell division following enlargement of a cell
to twice its minimum size
● Septum: partition between dividing cells
● each daughter cell receives a chromosome and
sufficient copies of all other cell constituents to exist
as an independent cell

Generation time: time required for microbial cells to double

in number
● depends on nutritional and genetic factors and
temperature

Budding division: unequal cell growth - forms new

daughter cell

Types of growth
Planktonic growth: growth as suspension
Sessile growth:
● attached to surface
● can develop into biofilms - polysaccharide
matrix containing bacteria
Biofilms form in stages:
● Planktonic cells attach
● Sticky matrix forms

● Biofilms: protection against harmful

chemicals (e.g., antibiotics), protists,
washing away of cells
● Biofilms affect human health, water
distribution systems, and fuel storage
● Microbial mats: multilayered sheets with different organisms in each layer

Exponential growth: growth of a microbial population in which cell numbers double within a
specific time interval

Calculation
Relationship between the initial number of cells present in a culture and the number present
after a period of exponential growth:
Generation time (g) of the exponentially growing population:

Specific growth rate (k) expresses growth rate at any instant

Initial increase is slow but rapidly increases, resulting in huge increase in cell numbers
(e.g. lactic acid bacteria spoiling milk)

The Microbial Growth Cycle

Batch culture​: a closed-system microbial culture of fixed volume. Typical growth curve for
population of cells grown in a closed system is characterized by four phases
● lag phase
● exponential phase
● stationary phase
● death phase

Lag phase
● Interval between
inoculation of a culture
and beginning of growth
● Biosynthesis of new
enzymes, production of
required metabolites

Exponential phase
● cell numbers double within a specific time interval
● cells in this phase are typically in the healthiest state

Stationary phase
● growth rate of population is zero
● either an essential nutrient is used up or waste products accumulate
● metabolism continues at greatly reduced rate
● some cells grow while others die, balancing each other

Death phase
● if incubation continues after cells reach stationary phase, the cells will eventually die
● exponential rate but typically much slower than exponential growth
● viable cells remain for months or years
Continuous culture:​ an open system microbial culture of fixed volume
Chemostat: most common type of continuous culture device

Both growth rate and population density (yield) of culture can be

controlled independently and simultaneously, depending on:
● dilution rate (D)= F/V (F = flow rate of adding fresh medium
and removing spent medium, V = culture volume)
● concentration of a limiting nutrient
Steady state: cell density and substrate concentration do not change
over time

Experimental Uses
● can maintain exponential growth phase for weeks/months
● used to study physiology, microbial ecology and evolution, enrichment and isolation
of bacteria from nature
● growth rate controlled by dilution rate
● growth yield controlled by (limiting) nutrient concentration

Growth Media and Laboratory Culture

Culture media
● nutrient solutions used to grow
microbes in the laboratory
● typically sterilized in an autoclave
Two broad classes:
● defined media: exact chemical
composition known
● complex media: composed of
digests of microbial, animal, or
plant products
Enriched media
● contain complex media plus highly
nutritious materials (e.g. serum or
blood)
● used to culture fastidious
(nutritionally demanding) microbes
Selective media
● contain compounds that selectively
inhibit growth of some microbes
but not others

Differential media
● contain an indicator, usually a dye,
that detects particular metabolic
reactions during growth

For successful cultivation of a microbe, it is important to know the nutritional requirements

and supply them in proper form and proportions in a culture medium.
Cells can be grown in liquid or solid culture media
● Solid media are prepared by addition of the gelling agent agar to liquid media
● When grown on solid media, cells form isolated masses (colonies)

Microbes are everywhere

● Sterilization of media is critical
● To prevent contamination, aseptic technique should be followed

Total cell count

● microscopic cell count: observing and enumerating cells present
● dried on slides or on liquid samples
● counting chambers with squares etched on a slide for liquid samples

Limitations of microscopic cell counts

● cannot distinguish between live and dead cells without special stains
● Precision is difficult to achieve
● Small cells can be overlooked
● phase-contrast microscope required if a stain is not used
● cell suspensions of low density (< 106 cells/ml) hard to count
● Motile cells need to immobilized
● Debris in sample can be mistaken for cells

Microscopic cell counts in microbial ecology

● often used on natural samples
● use stains to visualize and provide phylogenetic information or metabolic properties
● use DAPI to stain DNA

Viable (plate) counts: measurement of living, reproducing population

● count colonies on plates with 30–300 colonies
● to obtain the appropriate colony number, the sample should always be diluted
Sources of error in plate counting
● depends on inoculum size,
viability, culture medium,
incubation conditions
● mixed cultures grow at different
rates
● plating inconsistencies
● reporting in colony-forming
units instead of # viable cells
accounts for clumps

Applications
● quick and easy
● used in food, dairy, medical and aquatic microbiology, and water analyses
● high sensitivity
● can target particular species in mixed samples

“The great plate count anomaly”: Direct microscopic counts of natural samples reveal far
more organisms than those recoverable on plates. Why is this?
● microscopic methods count dead cells, whereas viable methods do not
● different organisms may have vastly different requirements for growth

Turbidimetric Measures
● cell suspensions are turbid (cloudy) because cells scatter light
● turbidity can be measured with a spectrophotometer: optical density (OD) at specified
wavelength
● for unicellular organisms, OD is proportional to cell number within limits
● to relate a direct cell count to a turbidity value, a standard curve must first be
established

Advantages
● quick and easy to perform
● typically do not require destruction or significant disturbance of sample
● Same sample can be checked repeatedly

Disadvantages
● sometimes problematic (e.g., microbes that form clumps or biofilms in liquid medium)

Effects of temperature
● Temperature is a major environmental factor controlling microbial growth
● Cardinal temperatures: the minimum, optimum, and maximum temperatures at which
an organism grows
● Range is typically < 40°C
Microorganisms can be classified into
groups by their growth temperature
optima.

Extremophiles: grow under very hot or

very cold conditions

Psychrophiles
● optimal growth temperature ≤ 15°C, maximum ≤ 20°C, minimum ≤ 0°C
● inhabit constantly cold environments
Psychrotolerant
● grow at 0°C but have optima of 20°C to 40°C
● More widely distributed in nature than psychrophiles
● isolated from soils and water in temperate climates and food at 4°C
Molecular adaptations to life in the cold
● cold shock proteins (chaperones)
● cryoprotectants (antifreeze proteins, certain solutes) prevent ice crystal formation
● exopolysaccharide cell surface slime
● cytoplasmic membranes remain functional at low temperatures
○ high unsaturated and shorter-chain fatty acid content
○ some polyunsaturated fatty acids, which remain flexible at very low
temperatures
● production of enzymes that function optimally in the cold
○ more α-helices than β-sheets → greater flexibility for catalysis at cold T
○ more polar and fewer hydrophobic amino acids
○ fewer weak bonds (e.g. hydrogen and ionic bonds)
Thermophiles
● growth temperature optima between 45°C and 80°C
Hyperthermophiles
● optima greater than 80°C inhabit hot environments, including boiling hot springs and
seafloor hydrothermal vents, that can experience temperatures in excess of 100°C

Above 65°C, only prokaryotic life forms thrive, but extensive diversity present

Protein and membrane stability at high temperatures

Modifications in cytoplasmic membranes to ensure heat stability:
● Bacteria: lipids rich in long-chain and saturated fatty acids, fewer unsaturated fatty
acids
● Archaea: C40 hydrocarbons made of repeating isoprene units bonded to glycerol
phosphate, lipid monolayers
Enzymes and proteins function optimally at high temperatures, features that provide thermal
stability:
● Critical amino acid substitutions in a few locations provide more heat-tolerant folds
● Increased number of ionic bonds between basic and acidic amino acids resists
unfolding in the aqueous cytoplasm
● highly hydrophobic interiors
● Production of solutes helps stabilize proteins

Enzymes commercially useful

● prolong shelf life (e.g. Taq polymerase for polymerase chain reaction)

Effects of pH
● pH: acidity / alkalinity of a solution
● acidic pH < 7, alkaline pH > 7
● each microbe has a pH range ~2–3 pH units within which growth is possible
● most natural environments: pH 3–9
● the intracellular pH must stay relatively close to neutral (pH
5–9) even if the external pH is highly acidic or basic
● microbial culture media typically contain buffers to maintain
constant pH

Neutrophiles:​ organisms that grown optimally at pH 5.5–7.9

Acidophiles:​ organisms that grow best at low pH (< 5.5)
● stability of cytoplasmic membrane critical
● Some are obligate acidophiles membranes destroyed at
neutral pH

Alkaliphiles​: organisms that grow best at high pH (≥ 8)

● found in soda lakes and high carbonate soils
● used commercially (e.g. secreted proteases and lipases that
are added to laundry detergents)
● Some have sodium (Na+) motive force rather than proton
motive force

Water activity (aw): water availability

● defined as ratio of: [vapor pressure of air in equilibrium with
a substance/solution] to the [vapor pressure of pure water]
● varies from zero (no free water) to one (pure water)

Osmolarity
● Osmosis: water diffuses from high to low concentrations
● Typically: [solute]cytoplasm > [solute]environment =
tendency for water to move into the cell (positive water
balance)
● When a cell is in an environment with a higher external solute concentration, water
will flow out unless the cell has a mechanism to prevent this
Halotolerant​ : can tolerate some additional dissolved solutes
but generally grow
best without
Halophiles:​ grow best at aw = 0.98 (seawater); have specific
NaCl requirement
Extreme halophiles: require very high NaCl levels (15-30
percent) often unable to grow at lower conc.
Osmophiles​: live in environments high in sugar
Xerophiles:​ able to grow in very dry environments

Compatible solutes​: used by cell to maintain positive water

balance
● pumping solutes from environment into cell
● synthesizing cytoplasmic solutes
● highly water-soluble (e.g. sugars, alcohols, glycine betaine, KCl)

Oxygen
● Aerobes: require oxygen (respiration) and grow at full oxygen tension (~21%)
● Microaerophiles: can use oxygen only when it is present at levels reduced from that
in air due to limited respiration or oxygen sensitivity
● Facultative organisms: can live with or without oxygen
● Anaerobes: cannot respire oxygen
● Aerotolerant anaerobes: tolerate oxygen and grow in its presence even though they
cannot respire
● Obligate anaerobes: inhibited or killed by oxygen

Special techniques are needed to grow aerobic and anaerobic microorganisms

● Aerobes need extensive aeration (e.g., shaking, bubbling)
● Anaerobes need oxygen excluded
● reducing agents: chemicals that may be added to culture media to reduce oxygen (to
H2O)
● complex medium that separates microbes based on oxygen requirements
● Oxygen can penetrate only the top of the tube
● Microbes grow at different heights based on oxygen exposure
● can flush or consume oxygen (e.g. glove box)

Why is oxygen toxic?

● Molecular oxygen (O2) is not toxic
● Exposure to oxygen yields toxic byproducts
○ superoxide anion (O2-)
○ hydrogen peroxide (H2O2)
○ hydroxyl radical (OH·)
Enzymes are present to neutralize most of these toxic oxygen species
Growth Control by Heat
● Decontamination: the treatment of an object to make it safe to handle
● Disinfection: the removal of all pathogens, not necessarily all microorganisms

Heat sterilization​: most widely used method of controlling microbial growth

● amount of time required to reduce viability tenfold = decimal reduction time (D)
● exponential relationship
● heat killing faster as temperature rises
● moist heat works better than dry heat
● thermal death time: time to kill all cells at a given temperature; affected by population
size
● endospores can survive heat that would rapidly kill vegetative cells
Autoclave​: sealed device that uses steam under pressure
● allows temperature of water to get above 100°C
● kills endospores
● not the pressure but the high temperature kills the microbes
Pasteurization:​ process of using precisely controlled heat to reduce the microbial load in
heat-sensitive liquids
● does not kill all organisms, so it is different from sterilization

Radiation and Filtration

Ultraviolet radiation​: (between 220-300 nm) causes modifications and breaks in DNA
● UV useful for decontaminating surfaces
● cannot penetrate solid, opaque, or light-absorbing surfaces

Ionizing radiation
● electromagnetic radiation, produces ions & other reactive molecules upon collision
● amount of energy required to reduce viability tenfold (D10) is analogous to D value
● some microorganisms more resistant to radiation than others
● used for diverse items including surgical supplies, plastic labware, drugs, fresh
produce, meat

Sources of radiation​: cathode ray tubes, X-rays, and radioactive nuclides

● Radiation is used for sterilization in the medical field and food industry
● approved by the WHO and used in USA for decontaminating foods particularly
susceptible to microbial contamination
○ hamburger, chicken, and spices

Filtration​: avoids the use of heat on sensitive liquids and gases

● pores of filter (0.45 and 0.2 μm) are too small for living organisms to pass through but
do not trap most viruses
● pores allow liquid or gas to pass through
● Depth filters made of overlapping paper or glass fibers (HEPA filters)
● Membrane filters function more like a sieve
● Nucleopore filters for scanning electron microscopy
Lecture 5
Antimicrobial agents​ are chemicals that kill or inhibit growth
● -cidal kills microorganisms (e.g., bactericidal, fungicidal, viricidal) Bactericidal agents
bind tightly and kill the cell
● -static inhibits growth (e.g., bacteriostatic, fungistatic, viristatic) Bacteriostatic agents
inhibit biochemical processes such as protein synthesis and bind weakly
● Bacteriolytic agents kill by lysis (e.g. detergents)

Minimum inhibitory concentration (MIC)​: smallest amount of an agent needed to inhibit

growth of a microorganism
● Disc diffusion assay uses solid media
○ Antimicrobial agent added to filter paper disc,
diffuses into agar
○ MIC is reached at some distance
○ Zone of inhibition: area of no growth around disc

Sterilants, disinfectants, sanitizers, and antiseptics are used to

prevent growth on inanimate surfaces and external body surfaces
● Sterilants: destroy all microorganisms, including endospores
● Disinfectants: used on surfaces to kill microorganisms but not necessarily
endospores
● Sanitizers: reduce microbial numbers but do not sterilize
● Antiseptics (germicides): kill or inhibit microbial growth but are nontoxic enough to be
applied to living tissues

Visualizing Molecular Growth

Super-resolution microscopy​ is a powerful new form of light microscopy using fluorescent
molecules
● can resolve structures as small as 10–50 nm in living cells
● observes dynamic behaviors in real time
Fluorescent Tagging
● Reporter genes encode proteins easy to detect or assay and are fused to genes of
interest, enabling visualization of proteins and monitoring gene expression (e.g.
green fluorescent protein [GFP])

Super-resolution techniques
● can reveal and quantify single molecules in living cells
● employ photoactivatable probes that switch from bright to dark emission states
depending on light wavelength
● Photoactivated localization microscopy maps movement of individual molecules
Regulation of chromosome replication initiation: DnaA
Several proteins initiate and inhibit chromosome replication in E. coli
DnaA binding to specific sequences within oriC region leads to DNA
unwinding and loading of replisome
● most active when linked to ATP (DnaA-ATP)
After initiation, DnaA-ATP needs to be inactivated:
● competition for oriC binding
● repression of dnaA expression
● titration of DnaA-ATP away from oriC
● inactivation of DnaA-ATP

Blocking of OriC regions after replication initiation: SeqA

● Before replication initiation: both DNA strands methylated (adenine
residues)
● After replication initiation, only parental strand is methylated =
hemimethylated DNA
● SeqA strongly binds hemimethylated OriC = blocks DnaA-ATP
● Appr. 10 min after replication initiation: newly synthesized daughter
strand is methylated by DNA adenine methylase
● Binding of SeqA also represses dnaA expression
● dnaA expression also autoregulated by DnaA binding to DnaA
boxes in its promoter region
● ATPase HdaA targets and hydrolyzes DnaA-ATP to
DnaA-ADP

Genome replication in fast-growing cells

● circular genome replication bidirectional from origin (on
both strands)
● E. coli’s genome replication takes 40 minutes but is
independent of generation time (20 minutes) = problem?

Generation time < genome replication time:

● multiple DNA replication forks present in each cell: new
round starts before previous round completed
● some genes present in multiple copies

Chromosome segregation
● Required so daughter cell gets copy of genome and for safe septum formation
● In many bacteria, Par (partitioning) system distributes chromosomes and plasmids
equally ​[]
Plasmids replicated similarly
● Replication at poles - transfer to daughter cells
● partitioning systems
The Divisome
● several essential proteins called Fts proteins
● FtsZ protein crucial in binary fission (related to tubulin - eukaryotic cell division
protein)
● Fts proteins interact to form the divisome (cell division
apparatus)
○ In rod-shaped cells, formation begins with
attachment of FtsZ molecules around cell center in a
ring that becomes cell-division plane
○ Ring attracts other divisome proteins including FtsA
and ZipA
○ ZipA: anchor that connects FtsZ ring to cytoplasmic
membrane
○ FtsA: related to actin; recruits FtsZ and other
divisome proteins and helps connect FtsZ ring to
membrane
● divisome forms about 3⁄4 of the way into cell division
● also contains Fts proteins needed for peptidoglycan synthesis
FtsI: penicillin-binding protein (activity inhibited by penicillin antibiotic)
● orchestrates synthesis of new cytoplasmic membrane and cell wall material (division
septum), then cell divides

Min proteins and cell division

● before nucleoids segregate, they block formation of FtsZ ring
(nucleoid occlusion)
● MinC and MinD (poles), and MinE (center) guide FtsZ to cell
midpoint instead of poles
● FtsK and other proteins mediate separation of chromosomes
to daughter cells
● FtsZ depolymerizes, triggering inward growth of wall
materials to form septum
● FtsZ also hydrolyzes GTP to provide energy for
polymerization and depolymerization of FtsZ ring

MreB and Cell Morphology

Prokaryotes contain a dynamic cell cytoskeleton
Rod-cell-shape and MreB
● major shape-determining factor in Bacteria and a few Archaea
● forms simple cytoskeleton with patchlike filaments around inside of cell just below
cytoplasmic membrane
● recruits other proteins for cell wall growth to group into a specific pattern
● Filaments move from one side to another, localizing synthesis of peptidoglycan and
allowing new cell wall to form at several points
● inactivation causes cells to become cocci
● most coccoid bacteria lack MreB
Crescentin
● shape-determining protein found in vibrio-shaped Caulobacter
● organizes into filaments ~10 nm wide that localize on concave face of the curved
cells
● thought to impart curved morphology
● similar proteins found in other curved cells (e.g., Helicobacter pylori)
[Evolution of cell division and cell shape]

Peptidoglycan Biosynthesis
● Preexisting peptidoglycan needs to be temporarily severed to allow newly
synthesized peptidoglycan to form
● In cocci, cell walls grow in opposite directions outward from the FtsZ ring
● In rod-shaped cells, cell wall growth occurs at several points along cell length
● In cells that grow by budding, cell wall growth localized
● wall band: junction between new and old peptidoglycan on surface of gram-positive
bacteria
● Must synthesize new peptidoglycan and export it
outside the cytoplasmic membrane

Insertion of new peptidoglycan

● requires controlled cutting of existing peptidoglycan and
simultaneous insertion of precursors
● Bactoprenol​: precursor insertion
○ hydrophobic C55 alcohol
○ binds N-acetylglucosamine/N-acetylmuramic acid/pentapeptide PG precursor
○ transports precursors across membrane
● Outside cell, bactoprenol complex interacts with transglycosylases that insert
peptidoglycan precursors into growing points and catalyze glycosidic bond formation.

Insertion of new peptidoglycan

● Autolysins cut small gaps by hydrolyzing M-G bonds
● Bactoprenol transports precursor over membrane
● Transglycolases insert precursor via glycosidic bonds
● Transpeptidation
○ peptide cross-links between muramic acid residues in adjacent glycan chains
○ In gram-negative bacteria, cross-links between DAP and D-alanine
○ Removal of extra D-alanine from precursor is exergonic and drives
transpeptidation
○ In E. coli, FtsI is a transpeptidase
○ inhibited by antibiotic penicillin - continued autolysin activity weakens PG –
cell lysis
Biofilm formation
Biofilms are attached polysaccharide matrices containing embedded bacterial cells
Steps to biofilm formation: attachment, colonization, development, dispersal
● Random collision accounts for initial attachment - facilitated by flagella and pili or cell
surface proteins
● Attachment = signal for expression of biofilm-specific
genes (proteins that produce intercellular signaling
molecules and extracellular polysaccharides)
● Once committed to biofilm formation, cell loses
flagella and becomes nonmotile
● Cells can be released through active dispersal

Signals guide bacteria in transitioning from planktonic growth

to life in matrix
● triggered by accumulation of regulatory molecule cyclic di-guanosine monophosphate
(c-di-GMP)
● c-di-GMP widely distributed only in Bacteria
● Synthesis and degradation depend on environmental and cellular cues
● Synthesis triggers physiological events: c-di-GMP binds proteins that reduce activity
of flagellar motor, regulates attachment proteins, and mediates biosynthesis of
extracellular matrix polysaccharides

Pseudomonas aeruginosa and biofilms

● forms a tenacious biofilm containing polysaccharides that increase pathogenicity and
prevent antibiotic penetration
● classic opportunistic pathogen
● primary reservoir soil
● infects blood, lungs, urinary tract, ears, skin, other tissues
● cystic fibrosis symptoms caused by thick biofilms in lungs
● significant nosocomial (hospital-acquired) pathogen

Pseudomonas aeruginosa and biofilms

● intercellular communication: accumulation of acyl homoserine lactones (AHLs)
- signal to adjacent cells that population is growing
● also triggers genes for extracellular polysaccharide and c-di-GMP synthesis
● Elevated c-di-GMP initiates extracellular polysaccharide production and leads to
decreased flagellar function
Vibrio cholerae and biofilms
● uses both inter- and intracellular signaling to control biofilm formation
● Signaling by c-di-GMP activates genes for formation
● Intercellular communication acts opposite compared with P. aeruginosa:
accumulation of signaling molecules represses biofilm formation genes and activated
flagellar and virulence genes
● biofilm formation triggered by low densities, repressed by high cell densities
● more likely to occur when V. cholerae is found in natural environment compared with
intestinal cells where nutrients are more plentiful
● allows cell to attach to marine surfaces (e.g., plankton, crustaceans, sediments) for
better access to nutrients and protection

Mechanisms of action of major antibacterial agents

Antibiotics are antimicrobials naturally produced by microbes
● kill or inhibit bacterial growth
● target essential molecular processes

Antibiotic resistance
Four classes of resistance mechanisms:
○ metabolic bypasses
○ enzymatic inactivation
○ removal via efflux pumps
○ modification of drug target
● Metabolic bypass: produce more of the target (enzyme) than the AB can bind
● Enzymes that inactivate antibiotic: e.g. β-lactamase
● Resistance genes can exist on plasmids and be transferred by horizontal transfer
● Random chromosomal mutations can lead to resistance Example: Spontaneous
mutants resistant to antibiotic rifampin can be obtained by exposing a large
population
Efflux pumps transport various molecules, including antibiotics, out of the cell
● lowers intracellular concentration, allowing cell to survive at higher external
concentrations
● Many act promiscuously and transport different classes outside cell
● contribute to multidrug resistance
● AcrAB-TolC of E. coli is one of the best characterized and pumps out rifampicin,
chloramphenicol, fluoroquinolones
Biofilm growth leads to increased resistance
● makes infections difficult to treat
● AcrAB-TolC efflux pump genes upregulated in biofilm growth mode
● P. aeruginosa several multidrug efflux pumps that are more active in biofilm

Antibiotic target is no longer essential

Example: MRSA Methicillin-resistant Staphylococcus aureus
● Methicillin is a β-lactam AB resistant to β-lactamase cleavage
● MRSA strains contain a DNA island called Staphylococcus chromosomal cassette for
methicillin resistance (SCCmec) that encodes MecA, an alternative penicillin-binding
protein that is not recognized by β-lactams
● MRSA synthesize MecA only in the presence of β-lactams due to repressor MecI and
β-lactam sensor MecR1

Persistence: population of antibiotic-sensitive

bacteria produces rare cells that are transiently
tolerant to multiple antibiotics

Persisters are genetically identical but dormant

(viable but do not grow)
● Dormancy prevents antibiotic from killing cell
● When treatment is stopped, cells emerge
from dormancy and grow
● believed to be cause of recurring
Mycobacterium tuberculosis and
Pseudomonas aeruginosa infections