MCB C112 Final Study Guide

Lecture 22-More Regulation

- Operon vs regulon
o Operon-set of genes next to each other and transcribed on same mRNA
 Transcriptional regulators affect expression
o Regulon-entire set of genes regulated by a transcriptional regulatory protein
 Genes in regulon can be all over the chromosome
 Can include many single genes or whole operons
- Lac operon
o Regulation simultaneously detects lactose presence and glucose absence
o When glucose is present, uptake of other sugars is inhibited
o Low glucose, high cAMP
- PTS system
o Involved in linking carbon utilization gene transcription with metabolism
o Glucose absent:
 Slow entry of glucose
 EnzIIa-P binds to adenylate cylase and stimulates cAMP production
 High phosphorylation of PTS proteins
 High PEP:pyruvate ratio
o Glucose present:
 Rapid entry of glucose
 EnzIIa inhibits uptake of alternative sugars by binding to their transporters
 Low PEP:pyruvate ratio
 Low phosphorylation of PTS proteins
- Heat shock recovery
o How do cells recover?
 During heat shock, proteins denature
 Cells make chaperones that refold proteins and proteases that remove
damaged proteins
 Heat shock-induced genes have promoter sequences that bind σ32
o σ32 RpoH
 transcription in normal conditions:
 After transcription of heat-shock gene: insufficient translation due to
stem-loop at 5’ end of the mRNA
 Some σ32 is translated, but it is bound to DnaK, so it’s inactive
 DnaK sends σ32 to FtsH protease, which degrades it
 Transcription during heat shock:
 Heat melts stem loop on mRNA, so more σ32 is made
 DnaK binds to denatured proteins instead of σ32 and promotes
refolding or degradation
o If σ32 is bound to DnaK, it is not inactivated or degraded
  σ32 interacts with RNA Pol core to promote transcription of heat-
shock induced genes
 Recovery from heat shock
 one of the genes activated by σ32 is dnak itself, creating a homeostatic
mechanism that returns the cell to its normal state
 over time, denatured proteins are refolded, DnaK levels increase, and
there is enough DnaK to bind σ32
- SOS response to DNA damage
o 1. RecA binds to damaged ssDNA
o 2. RecA-ssDNA stimulates LexA auto-cleavage
o LexA
 in normal conditions, LexA represses its own transcription in a negative
feedback loop
 kept in a steady-state level in cell
 when LexA is present, transcription of it stops
 when LexA levels drop, transcription is allowed
 partially represses recA expression
o LexA represses around 40 genes by binding to DNA sites called SOS boxes
o RecA-ssDNA complex stimulates LexA auto-cleavage
o LexA-repressed genes are turned on as LexA is depleted
 sulA- SulA inhibits cell division
 umuCD- UmuCD performs translesion DNA synthesis
- How cells reset after DNA damage is fixed
o SulA is degraded by Lon protease
 Once sulA is repressed by newly made LexA, SulA is depleted and cell division
can begin again
o UmuCD is degraded by ClpXP protease
 When transcription of UmuCD stops, the protein will be degraded, so no more
mutagenic replication occurs
- Two-component signal transduction
o 1. Histidine kinases in cytoplasmic membrane
o 2. External signal dimerization, cross-phosphorylation
o Phosphoryl group is transferred to an Asp residue on a response regulator, which
increases or decreases its activity
o What do response regulators do?
 Activate or repress transcription
 Output domains can be enzymes turned on or off by phosphorylation of the
receiver domain
 Sometimes a receiver domain acts alone by binding to a downstream protein
only in one state
- nif regulon genes for N2 fixation
o Genes for N2 are only expressed when needed (no NH4+ present) and when they can
function properly (low O2)
o nif genes
  NifA positively regulates genes needed to make nitrogenase complex
 Expression controlled by NtrC and activity controlled by NifL
o NifL- negative regulator of NifA, binds to NifA when O2 is
present
 Presence of O2 or NH4+ blocks nif transcription
o In Klebsiella pneumoniae
 NtrB-histidine kindase activated by low NH4+
 NtrC-DNA-binding response regulator, works with σ54
 σ54- positive regulator of N-scavenging and N2-fixing genes
- Regulation of N2-fixing genes in S. meliloti during symbiosis with plants
o NtrC not present in root nodules
o O2 regulates nif and fix transcription via FixL histidine kinase
o FixL-histidine kinase with heme in sensing domain
 Does not autophosphorylate when O2 is bound to heme
o FixJ-DNA-binding response regulator
o NifA and FixK-transcription factors that activate nif and fix gene expression
o Other nif and fix genes encode nitrogenase subunits and enzymes that synthesize FeMo
—co and assemble it into the protein

Lecture 23-Microbial growth and antibiotics

- Growth control
o Physical methods
 Temperature, autoclaving, UV and other radiations, filters
o Chemical methods
 Disinfectants, antiseptics, antibiotics
o Sterilization-killing or removal of all living cells, spores, and viruses on an object
o Disinfection-killing or removal of microorganisms, but not necessarily spores
 Used on inanimate surface
 Used in medical/lab settings
o Sanitizing-reducing microbial numbers on surfaces less strong than disinfectants
 Used in food industry
o Antisepsis-removal of pathogens from surface of living issue
- Autoclaving
o Used for heat+pressure sterilization of liquids and dry objects
o Temperature around 121C
- Pasteurization
o Precisely controlled heat reduces microbial populations
o Prevents spread of pathogens
o Results in reduced spoilage and longer shelf-life of products
o Kills all pathogens we know of that can be transmitted in infected milk
 Does not kill all bacteria!
o Flash pasteurization
 Most common method
  Liquids flowing through heat exchanger raised to 71C for 15 seconds, then
cooled rapidly
o Ultra-high temperature pasteurization (UHT)
 Kills endospores
 Liquid heated to 135C for 1-2 seconds
- Antimicrobial drugs/antibiotics
o Chemicals used in humans that kill or inhibit growth of microorganisms
o Can be naturally produced or synthetic
o Must have selective toxicity-the ability to inhibit or kill a pathogenic microbe at some
concentration without hurting the host
o 3 different outcomes of antibiotic treatment
 Bacteriostatic- growth is inhibited, but bacteria are not killed
 If antibiotic is removed, growth will resume
 Bactericidal- bacteria are killed, but not lysed
 If cells are removed from antibiotic, they will not resume growth
 Bacteriolytic- bacteria are killed and lysed
 Turbid culture becomes translucent
- Measuring antimicrobial activity
o Minimal inhibitory concentration (MIC)- lowest concentration of an agent that
completely inhibits growth
 Make series of cultures with equal cell numbers and different concentrations of
antibiotic
 Grow overnight
- Naturally occurring antibiotics
o Streptomyces produce over 500
o Antibiotic production often triggered by competition for resources
- Spectrum of action
o Isoniazid has narrow spectrum, only harms mycobacteria
o Tetracycline has broad spectrum
- Mechanisms of action
o Target: cell wall synthesis
 Penicillin
 Structure: B-lactam ring
 MOA: suicide inhibitor of PBPs that perform transpeptidation in PG
synthesis
 Bacteriolytic
 Spectrum: G+ and some G-
 Semisynthetics have altered R groups that make them more acid-stable
o Target: DNA supercoiling/replication/integrity
 Ciprofloxacin
 Structure: quinolone
 MOA: quinolone interacts with DNA gyrase
o Stabilize covalent intermediate between the enzyme and DNA
 o Gyrase-DNA complex blocks replication, causing broken DNA
ends
 Irreversible damage
 Bactericidal
 Spectrum: G+ and G-
o Target: Metabolism
 Sulfanilamide
 Structure: PABA analog
 MOA: growth factor analog that resembles PABA and blocks synthesis
of folic acid
 Bacteriostatic
 Spectrum: G+ and G-
 Acts as a competitive inhibitor of folate biosynthesis
o Target: Protein Synthesis
 Tetracycline
 Structure: napthacene ring system
 MOA: binds reversibly to 30S subunit of the ribosome at the A site and
prevents charged tRNA from entering
o Blocks protein elongation
 Bacteriostatic
 Spectrum: G+ and G-
 Substitutions of R groups on molecule create different analogs with
different spectra of activity
- Antibiotic resistance mechanisms
o Slightly alter target so drug no longer binds
 ex. streptomycin resistance can result from point mutation in rpsL, encoding SR
 critical that target protein can still function with this AA substitution
o modify antibiotic structure so that it no longer binds
 ex. enzyme nptII phosphorylates kanamycin, preventing it from binding to the
ribosome
 ex. Beta lactamases cleave Beta lactam ring
 ex. enzyme cat acetylates chloramphenicol, so it no longer can bind the
ribosome
o Pump drug out of cell
 tetA is a membrane protein that catalyzes the exchange of tetracycline (out) for
a protein (in)
- Discovering MOA of an antibiotic
o 1. Measure MIC of the compound toward a specific bacterium
o 2. Plate bacteria on plates containing the compound at a concentration greater than the
MIC
o 3. Select mutants resistant to compound
o 4. Use complementation and/or whole genome sequencing to ID the mutated gene
responsible for the resistance
  Since you can’t predict whether this gene is dominant or recessive,
 Make genomic DNA library from resistant mutant in replicating plasmid
o Transform population of WT cells and select for growth on
medium containing the compound
 Works if drug resistance is dominant
 Make a genomic DNA library from WT and transform on population of
resistant mutant.
o Plate these mutant colonies on medium without antibiotic, then
replica plate onto medium with antibiotic.
 Colonies that die on antibiotic have WT gene conferring
sensitivity
 Works if gene is dominant

Lecture 24-Early Earth History and Microbial Evolution

- Hydrothermal mounds
o May have provided first compartments where nutrients could be produced and
accumulated by abiotic reactions
 Nitrogen bases and nucleotides would be among the precursors made in these
compartments
o Must hypothesize that favorable reactions were somehow encoded in a molecule within
a compartment so that a compartment had a record of its abilities that it could replicate
and transmit
- RNA
o Thought to connect abiotic reactions with cellular life since they have coding and
catalytic ability
- Living stromatolites
o Sedimentary layers of limestone accreted by the growth of microbial mats over years to
centuries
o Cycles produce ascending layers
- Fossil stromatolites
o Layered structures resembling living stromatolites
o However, some layered formations once called stromatolites have been shown to be
formed by abiotic processes
 As a result, having layered structures in a rock does not indicate that living
organisms were present when it was formed
- Comparing microfossils with modern species
o Minerals have precipitated and filled in the forms of ancient microbial cells
 Dated by the age of the rock formation in which they were found
 Need to show regular 3D patterns that can’t be explained by abiotic processes
 Requires subjective interpretation
o Hard to prove that a fossil of a unicellular microbe could not have been formed by an
abiotic process
- Isotope ratios as evidence of life
o Abiotic processes that we know of use 13C and 12C equally
 o many enzymes favor 12C
 more 12C than 13C in organic product
o CO2 fixed into living cells is later converted to CaCO2 in sedimentary rock
o Rocks formed from fossilization of living organisms have higher C12 to C13 ratio
o Isotope ratios tell nothing about the form of early life
- Likely properties of early microbial life
o Anaerobic (prior to atmospheric O2)
o Thermophilic or psychrophilic
o Underwater
 Due to no protection from UV on land
o Possible energy sources
 Light-driven ion pumps- use light energy to pump protons or ions across
membrane
 Extant bacteriorhodopsins do this
 Redox reactions-involve reduced substances rising from ocean vents (H2S) and
oxidized substances falling from the UV-driven reactions in the atmosphere
(nitrate, etc)
 Methanogenesis-reaction is consistent with models of early earth atmosphere
 Primitive hydrogenase-oxidizes H2 coupled to reduction of an acceptor
- Phylogenetic trees
o Relationship diagrams that show how living organisms are related to each other through
time
o Ancestors of microbes are nearly all unknown, but we use shared characteristics of
extant organisms to infer relationships
o Different trees can be made for a group of species depending on characteristics
o Good model of relationships when:
 DNA used in tree is passed vertically
 Genes change due to point mutations
o Limitations
 More than one tree can be made with a set of data
 Similar sequences can result from convergent evolution instead of inheritance
 DNA can be acquired through sources other than parents (horizontal gene
transfer)
 Tree assumes all genes are inherited vertically from parents
- Difficulties in determining phylogenetic relationships among bacteria
o Relative lack of complex structures
o Little fossil record
o Metabolic characteristics may change according to how a strain is cultured
o Phenotypes often result in conflicting trees
o Bacteria cannot be defined using the traditional definition of a species
- Molecular clocks
o Could determine polygenetic relationships between bacteria
o Assumes
  Mutations in DNA accumulate randomly due to errors in replication
 Mutations can cause phenotypic changes subject to natural selection, or they
can be neutral
 Random mutations with neutral effects accumulate at a steady rate
 More sequence differences between two organisms means they diverged from
each other further in the past
o Uses 16S rRNA
- 16S rRNA
o Used to build phylogenetic tree of all living organisms
o Has highly conserved regions that make it easy to align sequences from all organisms
o Has less conserved regions (V1-V9) where sequence variability occurs
 Used to measure evolutionary distance
o Basic method for comparing 16S rRNA sequences of 2+ organisms
 1. Isolate genomic DNA from organisms to be compared
 2. Amplify 16S rRNA genes
 3. Run PCR
 4. Obtain sequences
 5. Align each pair of sequences and compare
 6. Make tree
o Distance=sequence differences/total nucleotides
- What did researchers expect from first phylogeny based on SSU rRNA?
o Two groups, prokaryotes and eukaryotes
 Eukaryotes evolved from prokaryotes
- What did they actually find?
o Three groups!
 Bacteria, archaea, eukarya
o New group-archaea
 Formerly grouped with prokaryotes
o Support for endosymbiotic hypothesis
- Species definition in microbio uses 16S rRNA and ANI
o ANI-average nucleotide identity
 To compute, algorithm breaks one genome into 1000 bp chunks and compares
this sequence to the orthologous region of the other genome

Lecture 25-Ecology

- Enrichment cultures
o Select growth of one bacterium in a mixed culture
o Must repeat process several times before isolating single colonies
o Goal is to isolate individual microbes or communities with particular metabolic activities
o Begins with inoculum from specific habitat
o Enrichment conditions promote growth of specific microbes
o Positive result
 Isolation of colonies on selective plates
  Indicates that organism with selected property was present
 Doesn’t indicate any info on abundance
o Negative result
 Inconclusive

- Enrichment bias
o Most rapidly growing microbes that can do the selected metabolism dominate the
culture, even if they are not the most abundant microbes in the environment capable
of doing it
o There could be more of a different microbe doing the desired metabolism
o We select for both particular metabolism and fastest-growing bacteria
o How to avoid enrichment bias?
 Dilute initial inoculum and grow multiple independent enrichment cultures
 Ensures that weed species do not take over every culture
 Cell sorting to separate individual cells of inoculum into 96 well plates
 Microbes no longer in competition with each other
o Can be used to culture previously unculturable microbes
- Single gene analysis of the diversity of microbes in an environment sample
o using 16S-based techniques, ecologists always find sequences that are different from all
known species
o most abundant organisms are the ones that have not been cultured
o rare biosphere-very rare 16S sequence types in environmental samples
- Single gene analysis vs metagenomics
o With metagenomics, can answer who is doing a process in an environment
- Metatranscriptomics
o Collect total RNA from population
o Reverse transcribe RNA into DNA to revel what genes in environment are being
transcribed
o Allows you to determine which microbes in environment where transcribing which
genes at the time of collection
- Other techniques to find out what microbes are doing
o Chemical assays of major metabolic reactions
 give rates of reactions occurring in environmental samples
 however don’t tell you what organisms are present or which ones are doing the
reaction
o microelectrodes
 measure chemical species, such as pH, O2, CO2
 measure changes in chemical concentrations over extremely small distances
 can be used directly in environment
 reveal what reactions are occurring in environment
 however don’t reveal what organisms are doing them
 o Stable isotope probing
 Method to associate reactions with organism

Lecture 26-Biofilms

- Bacterial habitats in the environment

o Bacteria directly experience microenvironments
o Places where microbes live can have very large changes over small distances
o Access to nutrients is often limited by diffusion
- Growth rates in natural environments and in the lab can be very different
o Microbes often experience intermittent exposure to nutrients
 Creates feast-or-famine lifestyle
- Biofilms
o Predominant mode of bacterial growth in nature
o Group of bacteria enclosed in an adhesive, self-made matrix made of
exopolysaccharides (EPS), proteins, and nucleic acids
o May adhere to abiotic or living surfaces
o May also exist as free-floating communities (flocs) in aquatic environments
o Bacteria living in biofilms are in physiologically different states than the same species
living in a free, planktonic lifestyle
 Can have different growth rates
 different transcriptional profiles
 enhanced antibiotic tolerance
 enhanced interactions with other microbes
o multispecies communities
- Biofilms are highly relevant to human health and industry
o grow on catheters, IV lines, artificial joints
o cause fouling of water treatment systems and fuel storage tanks
o cause corrosion of water pipes, contribute to water-borne disease outbreaks
o help corrode submerged objects
o found in teeth, promote tooth decay
- Cells in biofilm are embedded in an extracellular matrix that they make
o Bacteria colonize surface sparsely at first
o Attached cells preproduce and excrete polysaccharides and proteins to form matrix
o Biofilm grows by cell division and by attracting additional cells from the liquid
- Biofilm components
o EPS
 Hydrophilic, protects biofilm cells from desiccation
o as mobilized bacteria grow in the matrix, they form a habitat which is more favorable for
these processes:
 localized gradients
 sorption
 enzyme retention
 cooperation
  competition
 tolerance
 resistance
- Physiological activities of immobilized cells create gradients and chemical heterogeneity within
biofilms
o Can also be gradients of secondary metabolites (antibiotics) made by microbes in the
biofilm
 These gradients all intersect and lead to spatial heterogeneity in the biofilm

- Biofilms and antibiotics

o Bacteria in biofilms often tolerate higher levels of antibiotics than their planktonic
counterparts
o Antibiotics and toxins diffuse into biofilm, but the matrix leads to lower levels of them
entering
o Some bacteria become tolerant because they grow slower than planktonic cells
 Antibiotics usually affect active growth processes
o Co-existence in biofilm enhances opportunities for resistance genes on plasmids to
spread via horizontal gene transfer
- Crystal violet assay for biofilm formation
o Used to test organisms, mutants, surfaces, and anti-biofilm chemicals and drugs
o Amount of crystal violet remaining indicates adherent cells and extracellular biofilm
matrix
- Stages of biofilm formation and dissolution
o Attachment-adhesion of some motile cells to surface
o Colonization-intercellular communication, growth, polysaccharide formation
o Development-more growth and polysaccharide formation
o Active dispersal-triggered by environmental factors like nutrient availability
- Mechanisms important for biofilm formation
o Flagellar motility is important for reaching the surface
 Flagella and pili help with initial attachment to surface
o Surfaces are sensed by:
 Outer membrane proteins
 The hindrance of flagellar rotation
 Increased tension on retracting type IV pili
o Surface sensing results in changes in gene expression that prepare cell for biofilm
lifestyle
 EPS synthesis
 Stop motility or switch to surface motility
 Express adhesins
 Secrete enzymes for nutrient digestion
- How do extracellular signals cause internal changes that lead to biofilm formation?
o Switch from planktonic to biofilm lifestyle is often triggered by increase in cellular
concentration of cyclic di-GMP
  Synthesized by diguanylate cyclase (DGC) enzymes and hydrolyzed by
phosphodiesterase (PDE) enzymes
 Activity of these proteins is regulated by chemical and physical signals
 Sensed by specific receptor proteins
 Different c-di-GMP binding receptors cause different cellular outputs
 Binding allosterically modulates activities of receptor proteins, leading to
changes in gene expression of other processes
- Wsp system
o Found in Pseudomonas aeruginosa
o Elaborate two-component system
o Surface binding sensed by WspA
 WspA bound in cell to linker WspB and histidine kindase WspE
o Surface sensing causes increased WspE phosphorylation and increased phosphotransfer
to WspR
o Phosphorlyation of WspR leads to activation of its DGC, increasing cyclic-di-GMP levels
o Happens minutes to hours after surface interaction
- Rapid surface-sensing in Pseudomonas
o Occurs when type IV pilus retracts under tension and stimulates a different two-
component system
o PilJ-receptor
o PilI-CheW linker
o ChpA-histidine kinase
o PilG- interact with CyaB when phosphorylated by ChpA
o CyaB=adenylate cyclase stimulated by PilG binding
o Vfr- cAMP-binding transcriptional activator of virulence genes
o Works in seconds to minutes after surface interaction
 Increases cAMP, not c-di-GMP

Lecture 27-Human Microbiome (global, skin, oral cavity)

- The human body consists of sterile and non-sterile sites

o Non-sterile: anything exposed to environment
 Skin, gut, UT, respiratory tract, vagina
o Sterile: unexposed to environment
 Blood, heart, liver, kidney
 If microbes found here, indicates disease
- Human microbiome project conclusions
o No reference microbiome for healthy adult humans
 No single “healthy” type of microbiome
 Probably many
 Major variability at body sites among people
 Different microbial communities at body sites
o One body site on an individual is likely to be more similar to the same site on another
individual than to a different site on the same person
 o No virulent pathogens, but opportunistic pathogens present
o At a given site on different individuals, may be different communities of microbes
 However, no matter who the organism are, roughly same metabolic pathways
are seen in metagenome of community
 Microbial communities perform similar functions
 Hypothesis: different bacteria groups can provide the same metabolites to host
- Distribution of microbes in skin habitats
o Different microbes occupy sweat glands, sebaceous glands, hair follicles, and the
epidermal surface
 Physical and chemical properties of these areas are different

o Acne
 P. acnes lives in sebaceous glands, where it digests triglycerides in skin oils
 Products of this metabolism include inflammatory free fatty acids, which
attract immune cells
 Inflammation and keratins from epidermis can clog pores and create acne
- Oral Cavity microbiome and dental caries
o Above the gum line, bacteria must withstand antimicrobial factors in saliva
 Ex. lysozyme
o Streptococci ferment sugars into acids
 When pH drops below 5, enamel dissolution occurs
 When sugars are exhausted, salivary production raises pH and enamel
dissolution stops
o Poor oral hygiene and highly prolonged sugar intake cause dental caries
 Accompanied by shift in community structure toward S. mutans and lactobacilli
 These bacteria produce acid and are very acid-tolerant
 Continue to grow at low pH, while other less destructive bacteria fail to
thrive
o States
 State 1 and 2: biofilm contains commensal bacteria
 State 3 and 4: damaging states, contain acid-tolerant fermenters that damage
enamel
 Cleaning teeth brings stages back to 1 or 2 and prevents continuing acid
damage to enamel
- Babies are sterile until they acquire microbes during birth
o Birthing method has major impact on microbiome of baby
o Microbiomes of C-section babies are different from vaginal-born babies
 C-section babies lack Bifidobacterium
 Babies born by C-section had greater incidence of asthma and allergies
o Early exposure to microbes can influence immune system
 Maternal-child microbial seeding-inoculation of C-section baby with mother’s
vaginal and perineal microbiomes

Lecture 28-The Human Microbiome-GI Tract and Immune System

- Human gut microbiome
o Each person has around 1000 species
o Chemical properties of different areas of GI tract promote growth of different species
o Human populations differ in GI microbiota due to combined effects of different inputs
 Host genes
 Birth procedure
 Early feeding
 Diet
 Microbial exposure
 Antibiotic perturbation

- GI microbes have a complicated relationship with our immune system

o We have a large microbial population for optimal health, but we still have to maintain
an immune response against pathogens
o Changes in GI microbiome have been associated with several immune system disorders,
even ones not based in GI tract
o There are correlations between GI microbes and neurological diagnoses
- Germ-free (gnotobiotic) mice
o Mice that lack microbiome
o Can add a specific microbial species or community to mice while keeping all other
conditions constant
o Experiments where the microbiome is the independent variable are critical for
concluding that the microbiome causes particular health/disease outcome
- Microbiota are necessary for proper development of the immune system
o Ex. germ-free mice accumulate a large number of one kind of T-cell (iNKT) in the GI
mucosa
 causes greater susceptibility to chemicals that cause IBD and asthma
 if germ-free mice are treated with antibody to block iNKT cells, they have
reduced sickness
 mice are protected from T-cell imbalance if they are colonized with bacteria
neonatally, but if they are colonized after growing into adults
 suggests there is developmental window where normal microbiota is needed
for proper, balanced development of cells in immune system
- Balance in GI microbial community gives immune cell balance
o Segmented filamentous bacteria (SFB) promote growth of TH17 cells that respond to
infection
 SFB and Th17 cells are pro-inflammatory
o Bacteroides fragilis and clostridiae promote develop of Treg cells, which reduce
inflammation
o High TH17 to Treg cell ratio leads to increased chances of inflammatory diseases
 Imbalance can occur due to overgrowth of SFB or depletion of B. fragilis and
clostridiae
- Diet can influence host body composition and composition of GI microbes
o Species diversity is reduced in western-fed mice compared to CHO-fed mice
  CHO diet: complex plant carbs
 Western diet: high fats, high sugars
 Each diet gives mice different microbial community
 Western-fed mice had more fat
o Microbiome can cause change in body composition, even if other variables don’t
change
 Microbiomes from western-fed and CHO-fed mice transplanted into donor mice,
given same diet
 Donor mice with western-fed mice transplant had more fat
o Microbes from obese co-twins leads to greater fat gain than microbes from lean co-
twins
- How diet and microbiota affect health and disease
o Ex. dietary fiber break down
 Dietary fiber cant be broken down by host enzymes
 In colon, bacteria break down dietary fiber and release short-chain fatty acids
(SCFA) as waste product
 SCFA is used by host cells, leads to beneficial effects
o PYY and GLP-1 are released
 PYY reduces appetite
 GLP-1 lowers blood glucose levels
- Perturbation of GI microbiome by antibiotics
o Antibiotics reduce species diversity
o Clostridium difficile
 Found in GI tracts of healthy people
 If it survives antibiotics, it can proliferate and become dominant species in GI
tract
 Causes diarrhea
 Acquired after other bacteria that keep its growth in check are killed by
antibiotics
 Treatment: Fecal microbial transplants (FMT)
 Put in stool sample of healthy relative
o Sample contains other healthy gut bacteria
- Probiotics
o Health-inducing GI bacteria
o “good bacteria”
o Found in yogurt, freeze-dried capsules
- Prebiotics
o Foods that contain plant polysaccharides indigestible by humans
o Enter colon and provide source of food for bacteria that make SCFAs
 Diet high in prebiotics promotes growth of these bacteria
- Different interventions exist or are being developed to modulate GI microbiome
o Untargeted
 ex. exercise, nutrition, pre/probiotics, fecal transplant
 general improvement in microbial composition and function
 o Targeted
 Ex. phage therapy, CRISPR-Cas9 therapy, drugs
 Specific modification in metabolism-related gut microbiota
Lecture 29-Chemotaxis

- Chemotaxis
o Movement toward preferred chemicals (attractants) or away from toxic chemicals
(repellants)
o Accomplished by regulating the direction of flagellar rotation
 Counterclockwise-multiple flagella form a bundle and work together to propel
cell forward
 run
 Clockwise-individual filaments are pushed out of the bundle and stop working
together
 Tumble
 Tumbling reorients cell randomly, so next run may be in different
direction
- Bacteria with single flagellum use different mechanisms to change direction
o Reorientation is also random
- Chemotaxis assay
o Soft agar+ compound that attracts bacteria and can be metabolized
o Cells inoculated at center metabolize compound and swim out in circle, up gradient
they’ve made
o Must distinguish nonmotile mutants from nonchemotactic mutants by observing
motility under microscope
- Screen for chemotaxis mutants
o Plate mutants on hard agar, transfer to soft agar
o ID mutations that cause small swarms
- Chemotaxis is a biased 3D random walk
o If cell is swimming toward greater concentration of attractant, flagellum keeps on
rotating counterclockwise more
- Amazing properties of chemotaxis
o Chemotaxis signaling is very sensitive
 Can respond to very small changes in attractant concentration
o Signaling is effective over a wide range of attractant concentrations
o These features allow bacteria to sense a slightly better environment over a wide range
of background conditions
o System sense between new and old levels of chemical, not exact amounts
- Chemotaxis system senses changes in level of chemical and sends this info to flagellum
o Sensor proteins are methyl-accepting chemotaxis proteins (MCPs)
o Periplasmic sensing domain binds attractant
o HAMP domain transmits info of ligand binding to rest of protein
o Methylation region functions in adaptation
 o Signaling region binds CheW and CheA, which relay information to downstream proteins
o MCPs influence activity of two-component signaling proteins: CheA and CheY
 CheA
 Increased attractant binding, decreased CheA-P
 CheY-response regulator
 CheY dephosphorylated by CheZ
 CheB methylesterase-response regulator
 CheR methyltransferase-always active
o Basal level of attractant binds to MCPsbasal CheA activity, making CheA-Pbasal
level of CheY-Pbasal level of CheY-P binding to FliM on flagellumCheZ deactivates
CheY
 Baseline level of CheY-P binds to FliM
 Flagellar reversals occur every 2-4 seconds
o Increased attractant binding to MCPsdecreased CheA activity, so less CheA-P
madeless CheY-Pless binding of CheY-P to FliMCheZ deactivates CheY
 Less clockwise rotation
 Fewer tumbles, longer runs
o Decreased attractant binding to MCPsincreased CheA activity, so more CheA-P
mademore CheY-P mademore binding of CheY-P to FliMCheZ deactivates CheY
 More clockwise rotations
 More tumbles, shorter runs
- How does bacterium know if it’s swimming up a gradient of attractant?
o Temporal sensing mechanism
 Is there more signal now than a few seconds ago?
 Bacteria reset their signaling system rapidly in order to quickly detect
concentration changes
- Adaptation
o Resetting the signaling baseline to reflect current conditions
o CheR methylates MCPCheA phosphorylates CheBCheB demethylates MCP
o Increased attractant binding to MCP: CheR methylates MCPCheA phosphorylates
CheB lessCheB demethylates MCP less
o Decreased attractant binding to MCP: CheR methylates MCPCheA phosphorylates
CheB moreCheB demethylates MCP more
o Less attractant binding: More methyl groups on MCPmore ability to stimulate CheA
autophosphorylation
o More attractant binding: less methyl groups on MCPless ability to stimulate CheA
autophosphorylation