Lecture 6: Archaea

Archaea
 Relationship with eukaryotes
o Eukaryotes likely arose when an early archaea engulfed a bacteria
 Hypothesis explains important similarities between eukaryotes and
archaea including:
 Similar transcriptional and translational machinery
 DNA binding proteins
 Precense of introns
 Informational structure is similar
 Use histone homologs to wind DNA (in contrast to bacteria)
 Relationship with bacteria
o Similarities between bacteria & archaea:
 Circular chromosomes
 Genes organized into operons
 High gene density
o Same size as bacteria
 Unique traits of archaea:
o Either-linked, isoprene based lipids
 Make isoprene subunits using a pathway that is similar to the one
used by humans and different from that of bacteria
o Non-phosphorylated intermediates during sugar metabolism
o Unique nucleotides in tRNA
 Such as archeosine
o Methanogenesis is unique to archaea, though not all archaea can perform
this process
 Produce methane from hydrogen and oxidized carbon sources
such as carbon dioxide, methanol (and related methyl donors) or
acetate / pyruvate
 Different electron acceptors and donors for methanogenesis
 Proton motor force leads to ATP production
 Some methanogens contain a cell wall structure similar to the
peptidoglycan or murein found in bacteria
 Archaeal pseudomurein:
o Resistant to lysozyme because it is built from different
sugars that are bonded to one another in a different
fashion than the bonds in peptidoglycan
 B1-3 rather than B1-4
o Resistant to beta-lactam antibiotic, such as penicillin,
because of differences in the enzyme that links the
peptide side chains of pseudomurein
 Usually not found in highly acid or saline environments
 Can be found in thermophilic environments
  The current record high temperature growth is held by
Methanpyrus (122 C)
 Has important implications for human activities such as wastewater
treatment and agriculture
 Also impacted by human activities:
 As the permafrost has begun to melt, increased
methanogenesis in these highly-anaerobic, high-organic
matter soils is resulting in increased emission of this
important greenhouse has
o Archaea do not do:
 None are pathogenic
 None can perform photosynthesis
 Though some (notably haloarchaea) do have primitive light-
driven pumps that help provide them with proton motive
force for ATP synthesis, ion transport, & phototaxis
 Archaeal diversity:
o Active research topic and dozens of new uncultured phyla were recently
identified from whole genomes reconstructed from metagenomic data
o Three main phyla of cultured archaea have been characterized
 Thaumarchaeota
 Chemoautotrophs that oxidize ammonia (nitrifiers)
 Widespread in many environments
 Important in oligotrophic environments since they are able to
grow:
o At ammonia concentrations a hundred times lower
than bacterial nitrifiers
o In acidic soils
 Euryarcheota
 Have more metabolic diversity
 Found in surprisingly high number of environments
o Include:
 Methanogens
 Halophiles
 Acidophiles
 Alkalinophiles
 Haloarchaea – a class of euryarcheota
o Accumulate high levels of K+ in order to deal with
their saline environments
o Many of their cytoplasmic and cell membrane proteins
are highly acidic and contain fewer hydrophobic or
basic amino acid residues
o Are obligate aerobic heterotrophs
 But can supplement their energy production
with proton gradients generated by bacterial
rhodopsin
  Other rhodopsins are involved in
sensing light or pumping sodium ions or
amino acids
 Ferroplasma & Thermoplasma
o Acidophily – Can grow at pH below 1
o Lack cell wall
o Maintain cell membrane integrity by producing
tetraether glycolipids
 Work great at low pH
 Fall apart at neutral pH, limiting growth of these
organisms to pH’s below 4
 Other adaptations to low pH include the
production of very basic DNA-binding proteins
that are similar to bacterial proteins, but serve
the same function as histone-like proteins
found in other archaea and eukaryotes
o Ferroplasma
 Commonly found in acid mine drainage
 Chemolithotroph
o Thermoplasma
 Commonly found in self-heating coal mines
 Heterotroph
 Crenarcheota
 More ecologically diverse
 Found in surprisingly high number of environments
o Can be found in the most extreme temperatures – low
and high
 Genomes
o Hyperthermophiles use positive supercoils
 Seems to be critical for their ability to grow in high temperature
o Most DNA is for coding (not junk DNA)
o Both archaea and bacteria tend to organize their genes based off of
function
 Different Cell Walls: Peptidoglycan vs. pseudoglycan vs. S layer
o Length of peptidoglycan is predictable based off of type of organism
 Flavor can differ, but chemistry is similar
o Sugars are linked differently
o Organisms are insensitive to lysozyme
o Both have page-like appearance that gives resistance to osmotic stress
and lets them be resilient to different environmental conditions
 Cell wall diversity
o No uniform archaeal cell wall (lots of diversity)
o Cell structure allows to grow at high temperatures and very low pH’s
o Different proteins and lipids contribute to cell wall
 Some even appear to not have a membrane
 o Many archaea have variation to lipid bilayer
 Branched nature of lipids give stability to thermophilic conditions
 Ether lipids
o Different chemical motifs for archaea
o Can get good idea of type of archaea just by analyzing lipids

Lecture 7: Bacteria

Bacteria
 Relationship with eukaryotes
o Use of ester-linked lipids
o Ability of some species to perform photosynthesis
o Inability to perform methanogenesis
 Relationship with archaea
o Same:
 Coupled transcription and translation
 Circular chromosomes
 Genes organized into operons
 Metabolic diversity
 Use of electron donors and acceptors for energy generation
 Do:
 N2 fixation
 Denitrification
 Lithotrophy
 Respiration
 Fermentation
o Different:
 Bacteria do not do methanogenesis
 Important facts about bacteria
o They are essential to ecosystem function
o They are sensitive to antibiotics and have similar machinery of life
o Volume range: 1-100 micrometers cubed
o Bacteria can be critical in producing biofuels – further fermentation of
ethanol
o As a group, bacteria share common transcriptional and translational
apparatus
o Many, not all, rely on a cell wall made of peptidoglycan to protect them
from osmotic stress in dynamic environments
o Although more than 80-phylum level clades were previously recognized
from 16S sequences amplified from environmental bacteria, only 29 phyla
have cultured representatives, or so called, “type strains”
 o Our understanding of the diversity of uncultured bacteria has been greatly
expanded in the last 2 years thanks to reconstruction of nearly complete
genomes from metagenomic sequences
 Nearly 40 new candidate phyla were added early in 2016
 Some estimates suggest that there may be as many as 1400 more
bacterial phyla waiting to be characterized
 The “Candidate Phyla Radiation” Project published this year more
than doubled the amount of known bacterial phyla
 Gram Stain
o Not useful outside of a clinical setting
o Diversity in peptidoglycan thickness can make it difficult to make
phylogenetic conclusions about natural bacterial samples using the gram
stain
o Outside of the main 5 phyla, gram stain is of limited value since it does not
correlate well with either phylogeny or the presence of an outer membrane
in many cases
o LPS outer layer is usually a toxin
 Disk Diffusion
o Can determine the effect of an antibiotic based on diffusion diameter on a
lawn plate
 The main bacterial phyla are (from most diverse to least diverse):
o Proteobacteria
 Mono layer of peptidoglycan and an outer membrane
 Outer membrane contains lipopolysaccharides (LPS aka
endotoxin) that are very distinctive and recognized by the
immune systems of numerous eukaryotes
 Gram Negative
 Most metabolically diverse, include:
 Photoautotrophic bacteria
 Chemoautotrophic bacteria
 Chemoheterotrophs
 Even closely related proteobacteria can have very different
metabolic strategies and live in different niches
 Divided into 6 phylum-level clades
 Alphaproteobacteria
o Rickettsia
 Intracellular pathogens that are related to
eukaryotic mitochondria
o Rhizobia
 Plant symbionts that fix nitrogen
o Rhodobacter
 Metabolically versatile organisms
 Can grow via either photoautotrophy,
lithoautotrophy, or chemoheterotrophy
 Betaproteobacteria
 o Nitrosomonas
 Lithotroph
 Oxidize ammonia
o Neisseria gonorrhoaea
 Pathogen that causes STD
 Gammaproteobacteria
o Pseudomonas
 Remarkable for their ability to degrade
manmade pollutants
 Can also be opportunistic pathogens
 Kill people with cystic fibrosis
 Can sense the population size of
competitors (quorum sensing) and can
become pathogenic upon sufficiently
increasing their population size
o Enterobacteria
 E.coli
 Can be pathogenic but are more often
harmless gut commensals
 Salmonella
 Usually pathogenic
 Deltaproteobacteria
o Myxococcus
 Resembles tissue developments in eukaryotes
o Bdellovibrio
 Inserts itself into other bacteria and devours
 Epsilonproteobacteria
o Helicobacter pylori
 Causes stomach ulcers but also beneficially
modulates the host immune system
 On its way to extinction, but, the presence of
too few “training” bacteria such as these can
cause immune weakness and lead to
autoimmune diseases
o Bacteroidetes
 Mono layer of peptidoglycan and an outer membrane
 Outer membrane contains lipopolysaccharides (LPS aka
endotoxin) that are very distinctive and recognized by the
immune systems of numerous eukaryotes
 Gram Negative
 Obligate fermenters that specialize in degrading complex
polysaccharides and are among the most abundant bacteria in the
mammalian gut
  Also one of the few bacteria that produce sphingolipids, which are
important immune modulating compounds produced by their
mammalian hosts
o Actinobacteria
 Thick layer of peptidoglycan
 Gram Positive
 High GC content
 Many have rather large genomes (>8000 Kb)
 Some have linear, rather than circular chromosomes
 Well represented in culture collections and have made important
contributions to the human pharmacopeia
 Genus Streptomyces
o Well known for ability to produce antibiotics
o Often found in coils where they produce the geosmin
that gives many soils its distinctive smell
o Also found on decaying vegetation and some are
plant pathogens
o Well known for ability to form multinucleated hyphae
structures that septate to yield spores known as
conidia
 Genus Mycobacterium
o Do NOT sporulate
o Best known for their waxy outer cell wall
 Even though they have a thick layer of
peptidoglycan, gram stains cannot penetrate
these mycolipids, and neither can safranin
o Include pathogens that cause tuberculosis & leprosy
o Include non-pathogenic species, too
 M. spegmatis
 Harmless human commensal bacteria
o Firmicutes
 Known to form endospores
 Thick layer of peptidoglycan
 Gram Positive
 Low GC content
 Important orders that form endospores:
 Endospores are heat resistant
 Related to Epulopiscium whose method of reproduction
resembles the early stages of spore production, but results
in live offspring and is a rare exception to rule of binary
fission followed by most bacteria
o Bacilliales
 Well known for their ability to form heat and
desiccation resistant endospores
  Typically rods and include important pathogens
such as:
 Bacillus anthracis
o Causes anthrax
 Bacillus thuringensis
o Makes a crystalline protein that is
toxic to insects and has been
cloned into crops, such Bt Corn
 Harmless bacilli are also common and include:
 B. subtilis
o Used as a model for
understanding the biology of
gram positive bacteria
o Clostridiales
 Lack a respiratory chain
 Obligate fermenters
 Clostridia difficile - pathogen
 Can be difficult to treat
 The current best treatment for C. difficile
is fecal transplant
 “Clean” is not the best defense against a
disease – a balanced bacterial ecology
is
o Some endospore-forming bacteria can even survive
autoclaving (C. botulinum)
 Important orders that do NOT form endospores:
 Listeria
o Food-borne pathogen
 Probiotic lactic acid bacteria
o Important in the production of fermented foods
 Resident human microbiota
 Opportunistic pathogens
o Streptococcus
o Staphylococcus
o Tenericutes
 Closely related to Firmicutes
 Represented by a single class: Mollicutes
 Includes genus Mycoplasma
o Intracellular pathogens that completely lack a cell wall
o Use sterols to reinforce their cell membrane and
many have lipoglycans that resemble the
lipopolysaccharides of gram negative bacteria
o Many have undergone genome reduction and have
small genomes that range from 750-1500 Kb
o Cyanobacteria
 o Planctomycetes
 Have an outer membrane, but no peptidoglycan at all
 Inner membrane is highly invaginated which led some to
conclude that it actually had a nuclear membrane
o We know now that this is not the case
 Gram negative

Lecture 8: Microbial Growth and Nutrition

Energy
 Living organisms must acquire nutrients and energy in order to be able to
perform the anabolic reactions of biosynthesis and make the material needed for
growth
o Essential elements:
 C, O, N, H, P, S, Se
 Usually need to be accompanied by:
 Cations:
o Mg+2, Na+, K+, Ca+2
 Trace metals:
o Fe, Mo, Co, Cu, Ni, Zn
o In practice, rarely need to be intentionally added to
media because it is nearly impossible to completely
exclude them from even the purest water
o Cells require energy to transform these elements into complex
biomolecules (in order of most to least abundant):
 Protein
 RNA
 Lipids
 Polysaccharides
 DNA
o The energy can come from several sources:
 Heterotrophy
 Oxidation of already reduced organic material
 Autotrophs
 Must fix their carbon by reducing CO2
 Chemolithotrophy
 Oxidation of reduced mineral
 Chemoorganotrophs (chemoheterotrophs) and
chemolithotrophs do not compete over sources of carbon
o Require sulfur, nitrogen, phosphorus, and oxygen
 o Nutritional Variance: The identity of carbohydrates
forms the competition between the bacteria
 Phototrophy
 Process garnering energy from the photolysis of water or
reduced compounds (organic or inorganic)
 Direct pumping of ions such as protons or sodium ions
 Nutritional diversity confers ecological advantages
o Driven by this shift: Balanced community  Dysbiosis (explosion of small
bacteria population due to an increase in advantageous nutrients)
 Diet change, genetic predispositions, illness / infection can cause
this

Media
 In the lab, bacteria are usually grown in liquid media or on solid agar surfaces
that supply all of their nutritional requirements
 Types of media:
o You can determine the catabolism patterns from the medium
o Minimal
 Have defined amounts of pure compounds
 Prototrophs grow on minimal media (type of autotroph)
 Can simply use a defined source of carbon and nitrogen to
produce EVERYTHING they need to grow
 Oxytrophs need a mix of nutrients and amino acids
o Complex
 Typically made from enzymatically digest or chemically hydrolyzed
polymers proteins or even whole cell extracts) whose exact make
up is not fully known
 If you do not know what conditions your isolate prefers, start with
dilute complex media to ensure growth, then design appropriate
defined medium
o Selective
 Ingredients can be added to media to make them favor one or more
species or phenotypes at the expense of others
 Often used to enrich (increase the number) for bacteria performing
a specific metabolic function or inhibit the growth of others
 Example: ammonium free medium is used to select for
bacteria capable of fixing nitrogen
o Nitrogen fixation is an energetically expensive
process, so cells that do it are at a disadvantage if
ammonium is available from another source
o They are not very abundant in most environmental
samples, but would increase in number during the
enrichment process only if no other nitrogen source
was available
o Differential
  Include an indicator that does not select for or against organisms,
but rather helps to identify those performing a specific task
 Common differential ingredient is a pH indicator that will change
color when specific bacteria ferment a sugar or other substance
and lower the pH
 Enrichment
o Driving the growth of one organism at the expense of others to make it
identifiable
 Ex: With antibiotics, differential growth media, dyes, infection
o Enrichment process is biased in that it selects for the organism that grows
fastest under the defined conditions and not necessarily for the organism
that is most abundant in the original microbial community
 Big problem when trying to isolate poorly characterized, slow
growing bacteria
 Case when scientists first tried to isolate Pelagibacter
o To overcome this obstacle, they took advantage of
this organism’s numerical abundance and just diluted
away other organisms until they only had single cells
of the ones they wanted
o This relieved the Pelagibacter of competition and
eventually allowed scientists to isolate and grow this
bacterium that was previously only known from the
sequences of 16S genes amplified from the
environment
o Pelagibacter (proteobacterium)
 The most abundant chemoorganotroph in the
ocean
 We could only isolate it a couple of years ago
 Oligotrophic – Likes very low nutrient
concentration
 Grow on simply filtered water (no added
nutrients)
 Highlights the problem of the “great plate count anomaly”
o Typically less than 1% of the cells seen under the
microscope can be cultured
o Single cell genomics is a powerful tool that shows
promise in helping us learn more about and
eventually isolate many currently uncultured bacteria
o FISH
 Fluorescent In-Situ Hybridization
 Attempts to circumlocute this by isolating
fluorescent cells with flow cytometry
Growth of Bacteria
 Careful aseptic (sterile) technique is required to keep cultures of isolated bacteria
pure and free of contamination
o Growth in a flask or a test tube is the most common method, usually called
batch culture, but results in changing conditions as bacteria consume
nutrients and produce waste products
 Results in a growth curve
 1) Lag phase
o No apparent change in number
o Cells are first assembling the resources and
machinery for division
 2) Exponential phase / Log phase
o Double at a uniform rate
o Growth slow as resources are depleted
 3) Stationary phase
o Growth stops
 4) Death phase
o Many of the cells die due to the accumulation of toxic
waste products
 The turbidity (OD) of the solution follows the bacterial growth curve,
making OD a good indicator of bacterial growth. Error comes from
the fact that dead cells also absorb light, but the general shapes of
the curves are similar.
o When a more precise measure of microbial growth is required, scientists
will use a chemostat
 Fresh medium dilutes out spent medium at a precisely controlled
rate
 Resembles natural systems like the human intestine or the bank of
a stream
o Change in bacterial number in either batch culture or chemostats:
 Can be determined using:
 Direct microscopic examination
o Observations using dyes reveal the true number of
live/dead cells in a culture
 Dilution into liquid growth medium (MPN) or onto plates
 Using a spectrophotometer to measure changes in the
transmission of light through the increasingly turbid medium
o Measure of optical density
o Increase in cell density gradually decreases accuracy
of measurements
 Data are used to calculate:
 Generation time / Doubling time
o Time it takes for bacteria to double in number
o Dependent on the medium and other environmental
factors like pH and dissolved gas concentration
o Dt=T/n
 Growth rate
o Number of generations or doublings per unit of time
 o 1/doubling time
 Cells grow by binary fission
o Selection: Plate target colony on selective plate (select target CHEST
colony and use selective medium to grow)
o Isolation: Isolate target colony from a pure culture (for other tests to streak
pure culture)
 Growth of bacteria in the environment is often very different from either a batch
culture or a chemostat
o Dominated by cells that are attached to surfaces since this allows them to
stay put as new resources flow by
 Biofilm = surface attached bacteria
 Experience very different environmental conditions than
those that are found in the bulk medium flowing past them
 Typically require the production of either:
o Extracellular polymeric substances (EPS)
o Adhesive proteins and extracellular structures
 Curli
 Pilli
 Fimbriae
 Bacteria in a biofilm are usually in a different physiological
state than those in the bulk liquid around them
o In many bacteria this process is regulated by an
intracellular metabolite called cyclic diGMP that is
derived from the nucleotide GTP
 More resistant to antibiotics
 Grow more slowly
 Often engaged in process called “Quorum Sensing”
o They sense the accumulation of diffusible
metabolites, such as homoserine lactones, that help
them communicate with one another and coordinate
the regulation of specific behaviors, including motility
and virulence
 This is common to gram negative bacteria
 Gram positive bacteria usually use
polypeptides to achieve quorum sensing
 The ability to form biofilms is not uniform
 Many members of the same species show very different
biofilm forming capabilities, though most bacterial cells in
natural systems reside in biofilms

Lecture 9: Bacterial Cell Structure & Envelope

Understanding of the Bacterial Cell

  Our understanding of the bacterial cell has come in large measure from a
reductionist approach that led scientists to look at pieces of the whole in an effort
to find out how they worked together
 This was made possible by advances in separation sciences, including
centrifugation, that were critical for enabling the isolation of individual
components of the cell and thereby permitting biochemical and ultimately
structural analyses
 This reductionist approach was complemented by advances in bacterial genetics
that enabled microbiologists to understand the importance of individual
components in a cellular context and by advances in microscopy that let
scientists see how the pieces fit together

Cell Membrane
 Arguably one of the most quintessential components of life
 A membrane is a permeability barrier, a protein anchor, a structural
reinforcement, and a facilitator of energy conservation
 Characteristics are critical to bacteria that largely depends on their membrane’s
selective permeability for absorptive nutrition and maintenance of chemical
gradients that are critical for energy generation
 Important membrane constituents:
o Lipopolysaccharides (LPS=endotoxins) on membranes contribute to our
understanding of bacterial phylogeny because of their immense diversity
o Arrangement of peptidoglycan
o Lipoteichoic acid arrangement
o S-layers: crystalline array of proteins outside of membrane (usually in
archaea)
o Endospores
 The small size of most bacteria means that they have a high surface area to
volume ratio, making absorptive nutrition a reliable way of garnering resources
needed for growth.
o The normal is a large SAV: small volume, large surface area
o Some bacteria defy the size norm and appear to have developed
specialized strategies to overcome the reduction in their surface area to
volume ratio that accompanies their large size
o These strategies include:
 Membrane invaginations
 Overexpression of nutrient transporters
 Extreme polyploidy
 Many copies of their chromosomes
 Selective Permeability
o Hallmark of membrane integrity and essential for life
o Most charged and hydrophilic chemicals cannot diffuse across the
membrane without the help of specialized transporters
o Many neutral hydrophobic compounds can pass through the membrane
unaided
o In addition to being a permeability barrier and a means of conserving
energy through ion gradients, the membrane is also an anchoring place
for the many proteins that a cell employs to help to acquire nutrients an
sense its environment
 Bacteria have active strategies for controlling the composition of
their membranes so that these important functions remain intact in
the face of changing environmental conditions.
 Bacteria can change:
o The length and degree of saturation of the lipid
component of the phospholipid membrane
o Nature of the polar head group
 Changes in polar head groups of the
phospholipids can determine membrane fluidity
and structure
 For example, development of cyclopropane
rings can add rigidity to a membrane, giving
heat tolerance
 Another example is changing the charge of the
surface of a bacterium
 This can cause antibiotic resistance
against charged antibiotics (eg.
Daptomycin)
o Some can produce sterols
 Like hopanes that reinforce the membrane and
pack the lipids more tightly
o Protein transporters are the main way that selective membrane
permeability is achieved
 Simple transport do not require any other energy source
 Porins
o Allow molecules to move down the concentration
gradient
o Aquaporins are important for allowing almost all
bacteria to deal with osmotic stress
o Gram negative bacteria also deploy specialized porins
to their outer membranes to aid in sugar uptake
 Active transporters require energy
 Uniport
o One molecule goes through unidirectionally (usually
with the concentration gradients)
 Symport
o Two molecules transported together in one direction
 Antiport
o Molecules move in opposite directions
 ABC transport
o ATP-Binding Cassette
  Group translocation systems
o Chemically modifies glucose upon entry into the cell,
tricking the concentration gradient

Cell Wall
 Cell wall is layer of peptidoglycan that is present in most bacteria
o Large heteropolymer of n-acetylglucosamine and n-acetylmuramic acid
that is cross linked by short peptide chains to give it rigidity
o The nature of the peptides is different for gram positive and gram negative
organisms, sometimes being species specific
 Gram Positive
o Thick peptidoglycan layer
 Reinforced by sugar-peptide heteropolymers called techoic acids
 In some bacteria like Mycobacteria, can be coated in large amounts
of glycolipids
 Gram Negative
o Thin layer of peptidoglycan is connected to the outer membrane by
lipoproteins
 Outer membrane is also home to lipopolysaccharides which contain
2 lipids connected to phosphorylated n-acetylglucosamine dimer
(lipid A)
 Next comes the core polysaccharide composed of a
relatively invariant mixture sugar polymers including
ketodoxyglucose, which is the base upon which the O-
specific polysaccharide sits
o It is the O antigen that gives lipopolysaccharide (LPS)
its highly immunogenic nature
o The O polysaccharide is not only species, but is often
strain specific
o Can undergo changes as pathogens seek to evade
host immune surveillance
 Gram positive & negative
o Some gram positive & gram negative bacteria can produce large capsule
layers
 Consist of a mixture of compounds that is species specific but can
include
 Polysaccharides
 Proteins
 Nucleic acids in some cases
 Capsule is often referred to as EPS (extracellular polymeric
substances)
 Contributes to biofilm development
o Other surface structures:
 Pilli
 Fimbriae
  Curli
 Stalks
o Flagella

Intracellular Structures
 Many bacteria can also make specialized intracellular structures
 Depending on the species, can include:
o Magnetosomes
o Carbonate mineral deposits
o Reduced sulfur droplets
o Carbohydrates
 Polyhydroxyalkanoates
 Glycogen
 Polyphosphate
 Most autotrophic (litho and photo) bacteria have icosahedral carboxysomes that
help to concentrate CO2 and keep oxygen away from RUBISCO
o RUBISCO is the enzyme responsible for the majority of the carbon fixation
on earth
o Phototrophic bacteria have specialized structures such as:
 Highly invaginated membranes that house their light harvesting
complexes called thylakoids
 Some free swimming (aka planktonic) aquatic photosynthetic
bacteria also have gas vesicles them maintain their position in the
water column
Lecture 10:

Syntrophy
 One reason some cells aren’t easy to culture in the lab is because they have
adapted to growing with another organism that either:
o Supplies them with something they need
o Removes a waste product whose accumulation would inhibit their growth
 Syntrophy is an important example of this phenomenon
o Allows bacteria to grow even when the Gibbs free energy calculations
would seem to make a reaction non-spontaneous
o Growth of fermentative microorganisms in close association with
methanogens
 Hydrogen gas usually accumulates during fermentation, making
some reactions energetically unfavorable
 By consuming the hydrogen, methanogens or sulfate reducing bacteria
can make possible certain fermentation reactions that wouldn’t otherwise
occur under standard (1 Molar) concentrations.

Redox Chemistry
 Electron donor = Oxidized
 Electron acceptor = Reduced
  Reduction potential (E’) is inversely related to Gibbs free energy (-∆G, the energy
available to do work under standard conditions)
o So the more thermodynamically favorable a reaction is (-∆G), the larger
(and more positive) it’s reduction potential (E’) will be (-∆G=nF(∆E’), where
n=moles of electrons and F=Faraday’s constant)
o Gibbs free energy of a reaction is a pretty good predictor of how much
biomass cells can produce when using that reaction

Breaking Down Sugar

 During fermentation, an organic compound serves as both the electron acceptor
and the electron donor
o Example: Glycolysis -- (the Embden Meyerhoff Parnas Pathway)
 Glucose is oxidatively split into two molecules of pyruvate, yielding
2 ATP and reducing NAD+ to NADH
 There are other ways of breaking down sugars including:
o Entner Doudoroff pathway for sugar acids
o Pentose phosphate pathway for the metabolism of 5-carbon sugars like
ribulose
o These pathways differ with respect to the amount of ATP they generate,
whether they produce NADH or NADPH, and the nature of the oxidized
products
 The formation of NADPH and intermediate sugars or organic acids is very
important for biosynthesis.
 Glycolysis and these other pathways would stop if there wasn’t a way to
regenerate the NAD+
o In the absence of oxidative phosphorylation, glycolysis is completed by
fermentation: pyruvate is reduced while NADH is oxidized back to NAD +
o The ATPs made during fermentation get their phosphate from an organic
phosphate donor, not from inorganic phosphate
 “Substrate level phosphorylation”
 The fate of pyruvate depends on the enzyme performing the reaction
o Some bacteria can convert pyruvate to acetaldehyde or ethanol, while
others produce lactate
o Acetyl-CoA can also be made by some organisms and is a major building
block for many other eventual products, such as solvents like butanol
o The enzymes responsible for performing these reactions are typically
dehydrogenases or decarboxylases
 With respect to food preservation, many fermentation end products help to limit
the growth of spoilage microorganisms and food pathogens, while preserving
much of the nutritional value of foods
o Some fermentative microorganisms like Lactobacillus may also play an
import role in regulating immune reactions in the gut mucosa and are
considered to be “probiotics” that can promote good colonic health
 In the absence of oxidative phosphorylation and an electron transport chain
o Much of the pyruvate produced during glycolysis will be reduced in order
to oxidize NADH back to NAD+
o Some of it will undergo decarboxylation by the pyruvate dehydrogenase
complex to yield NADH and ATP (the later via substrate level
phosphorylation) and acetyl CoA
o Acetyl CoA is then free to enter the citric acid cycle where it will eventually
be fully oxidized to two molecules of CO2, generating 2 ATP, 3 NADH, and
1 FADH2 in the process
o The citric acid cycle actually shares important similarities with glycolysis in
that all ATP is made via substrate level phosphorylation
o Also, as with fermentation, the highly reduced byproducts (in this case
both NADH and FADH2) need to be reoxidized if further metabolism is to
occur
 Fermentation can be used to reoxidize these reduced cofactors in
the same way that the NADH from glycolysis was reoxidized, by
reducing pyruvate
 This helps explain why even many obligate anaerobes have
complete citric acid cycles even though they don’t perform oxidative
phosphorylation
 In part addition to energy via substrate level phosphorylation, the
citric acid cycle also supplies important biosynthetic precursors.