MCB C112 Midterm 2 study guide

Lecture 11-Nitrogen Fixation

- The Nitrogen Cycle

o Biogeochemical cycle that depends on microbial metabolism
o Nitrogen fixation: N2NH3
- Nitrogen fixation
o Because N2 is chemically stable, it must be reduced into NH3 before it can be put into
biomolecules
o Performed by some bacteria and archaea
- Industrial vs biological nitrogen fixation
o Conversion of N2 and H2 into NH3 is thermodynamically favorable
 However, triple bond of N2 is hard to break, and N2 is an almost inert gas
 Lots of energy required for rxn to happen
o Industrial nitrogen fixation- energy supplied via high temperature and high pressure,
iron catalyst
o Biological nitrogen fixation- energy supplied via ATP, occurs at lower temperature,
catalyst is nitrogenase
 Electrons provided by reduced ferredoxin or flavodoxin
- Nitrogenase complex
o 2 separate enzymes
 Dinitrogenase reductase=Fe protein
 Dinitrogenase=FeMo protein
o 1. Fe protein accepts electrons from reduced flavodoxin and binds ATP
o 2. Fe protein passes electrons to FeMo and hydrolyzes ATP
o 3. FeMo uses electrons to reduce bound N2 to NH3
o Co-rxn centers are very susceptible to O2
- Nitrogenase can reduce N2 to ammonia or acetylene to ethylene
- Demonstrating nitrogenase activity
o Definitively shown by growing organisms in sealed container with stable N2 isotope
o Cells are digested to release NH3 from biomolecules
 NH3 shown to contain N2 isotope
o Acetylene reduction to ethylene is an alternative nitrogenase assay that doesn’t require
isotope labeling
- Enrichment cultures to isolate nitrogen-fixing bacteria from environmental samples
o Growth medium with carbohydrates and other nutrients, but N2 gas is only source of
nitrogen
 Incubate with and without NH4+
 If NH4+ is always supplied, nitrogen fixers will not be enriched since
they are not forced to perform nitrogen fixation
- How do aerobes protect nitrogenase from O@?
o Cyanobacteria
 O2 is produced by phototrophy
  O2 irreversibly inhibits nitrogenases
 Solutions
 Temporal separation: photosynthesis happens during day, nitrogen
fixation happens at night
 Spatial separation: photosynthesis happens in vegetative cells, nitrogen
fixation happens in heterocysts
o In heterocysts, PSII is dismantled and oxidases are produced to
keep O2 concentration low
o Obligate aerobes
 Ex. Azotobacter
 Produce slime layer that limits O2 influx into cell
 High respiratory rate removes O2 quickly
 Protective protein that binds to nitrogenase and protects it from O2 damage
o Symbionts
 S. meliloti
 Invade alfalfa root tissues to form root nodules
o Develop nitrogen fixing ability
 In the root nodule, leghemoglobin made by alfalfa binds O2, reducing
free O2 that could damage nitrogenase

Lecture 12-Microscopy

- Bright-field microscopy
o Magnification is theoretically infinite, but resolution is limited by:
 Wavelength of light use to capture image
 Light-gathering ability of lens
o Total magnification
 Product of magnification by the objective lens (10-100x) and the oculars (10x)
o Resolution (resolving power)
 Smallest distance between two objects that allows them to be seen as distinct
objects
 R=λ/2NA, λ=wavelength, NA=numerical aperture
 Smaller the R, higher the resolution
o Tell things apart from shorter distances
 Magnification not involved
o To see an object, we need contrast between it and the background
 Bacteria can be visualized via staining
- Staining
o Gram stain- differential stain that can be used to differentiate between two classes of
bacteria in the same specimen
 Crystal violet stain
 Gram-positive cells are purple
 Gram-negative cells are colorless
o Turn pink/red when safranin is added
 o Contain outer lipid membrane that is destroyed by alcohol, so
they can’t hold the purple dye
- Creating contrast optically
o Able to visualize live cells
o Phase contrast, differential interference contrast (DIC)
o Use polarized lights, prisms, specialized lens systems
- Transmission electron microscopy (TEM)
o Uses electrons as radiation and electromagnets as lenses
o λ=1.23/V1/2
 typically, λ=0.0055 nm
o very small NA since they can only gather refracted electrons over a very small angle
 however, decrease in wavelength more than compensates for decrease in NA,
so resolution in TEM is better than in light microscopy
o can visualize features in bacteria not visible in light microscopy
- Visualizing cell proteins
o Immunofluorescence microscopy (IF)
 An antibody that recognizes a specific protein is attached to a fluor
 Fluor- molecules that absorb light at one wavelength (high energy) and
emit light at another wavelength (low energy)
 Fixed, permeablized cells are treated with the antibody and viewed under
microscope
 Can determine location of protein, not structure
o Green fluorescent protein (GFP)
 Contains 3 AA side chains that react covalently to form fluor that absorbs blue,
emits green
 GFP is transcribed from a gene, unlike chemical fluors
 Gene for GFP can be fused with protein of interest via recombinant DNA
technology
 can visualize proteins in live bacteria
o Comparing results
 Typically take two pictures of same bacteria, one using IF or GFP and the other
using phase or DIC
 Compare photos by overlaying them or putting them side-by-side
 How do we know if IF or GFP gives correct location of protein?
 Gold standard: IF and GFP give you same results
 Other experiments to increase confidence:
o IF: compare fluorescence signal in WT bacteria to genetically
altered strain without target protein
o GFP: determine that GFP fusion protein is functional by making
it complement a deletion of the target protein

Lecture 13-Genetics I

- Genetic logic
 o What causes this trait or phenotype?
 Forward genetics
- How do you isolate the mutants you want and identify the phenotype?
o Depends on phenotype
o Selectable mutations- confer distinct growth advantage on the mutant strain under
some condition
 Ex. antibiotic resistance, bacteriophage resistance
o Nonselectable mutations- may result in very clear changes, but do not confer a growth
advantage
 Ex. mutant cells can’t make their own histidine
- Selection vs screen
o Selection- establish where only mutants you want will grow
 Parent strain and all unrelated mutants die
 Very efficient, can select desired mutations from large populations of unrelated
ones
 However, not always possible
 Can’t select a disadvantage
o Screen- after mutagenesis, grow all survivors under permissive conditions, and then
under nonpermissive conditions
 Patching and replica plating are two techniques
 Strains that die only under nonpermissive conditions are the desired
ones
 All survivors of mutagenesis must be grown first as independent colonies before
they can be tested
 Less powerful than selection
- Essential genes
o Genes whose function is required under all known growth conditions
 Ex. DNA replication, cell division, DNA translation, chromosome segregation
o Can only isolate mutations in essential genes by screening for conditional loss-of-
function mutations
 Most common technique: screen for temperature-sensitive (ts) mutants
 Mutations only active at nonpermissive temperature
 Find mutants with specific phenotype at nonpermissive temperature
- Mutations
o Mutation rate- probability that a given gene will acquire a mutation in one generation
o Spontaneous mutation- arise from errors in DNA replication
o Mutagens- can increase mutation rate by 10-1000x
o Microlesions- base pair substitutions, small insertions/deletions
o Macrolesions- large deletions, insertions, duplications, or inversions
 Transposons
- Mutagenesis
o Expose bacteria to mutagens (chemicals, UV radiation, transposons)
o Chemical mutagens
 Ex. nucleotide base analogs
  Can create single-base substitutions during subsequent rounds of replication
o UV radiation
 Bases strongly absorb UV light
 Pyrimidine dimers form, which can’t be repaired by normal polymerases
 RecA- detects UV damage, induces SOS response
 During repair of lesions, polymerase adds random bases across from the
lesion, creating mutations
o Transposons (Tn)
 Move from place to place in the genome
 Can be used to make random mutations
 Cause loss-of-function mutations
 Transposase- recognizes inverted repeat sequences (IR) of transposon
 Cuts at ends of transposon, cuts target DNA
 Ligates transposon into new site
 Each transposase is specific for its own IR sequence
 Engineered so that they hop only once in the genome
 Transposase encoded outside of the transposon
 Antibiotic resistance gene encoded in transposon
 Plasmid can’t be replicated in host strain to be mutagenized
 Transposase catalyzes transposon insertion into random site on chromosome in
host
 Select or screen for mutants with desired phenotype
 Cannot test essential genes

Lecture 14-Genetics II

- Genetic exchange
o 3 types of gene transfer
 Transformation
 Transduction
 Conjugation
- Transformation
o Griffith experiment-demonstrated transformation
o Natural transformation by free DNA
 DNA acquire is integrated into the host by homologous recombination
 Integrated DNA replaces similar genes/sequences already in
chromosome
o Incoming DNA must be similar to sequence in genome
 RecA- initiates strand exchange
o Homologous recombination
 Physical exchange between two highly similar DNA molecules
 Requires base pairing of donor and recipient DNA strands over 30-1000 bases
 Can’t be used to integrate completely foreign DNA into chromosome
 No homology to initiate strand invasion
 Mediated by RecA, RecBCD, RuvABC, SSB
  Not involved in transposition
o Competence
 Ability of a bacteria to take up free DNA
 Can be artificially induced, since not all bacteria are capable of natural
transformation
 Incubate cells with high concentrations of Ca2+
 Electroporation
 Non-homologous replicating plasmids can be introduced by artificial
competence
 Plasmids do not need to be integrated into the cell to be replicated
o Plasmids
 Circular genetic element that replicates independently of chromosome
 Replicated by normal machinery
 Low, medium, high copy plasmids exist
 Have specific host ranges- range of bacteria in which they can replicate
- Conjugation
o DNA transfer involving cell-to-cell contact
o Medicated by F plasmid, which encodes proteins that catalyze its own transfer
 F pilus made by F+ cell (already has plasmid) binds to surface of F- cell and
retracts, bringing the two cells together
 Cell surface proteins make and stabilize conjugation bridge where DNA will
cross
 one strand of F plasmid moves across bridge into recipient cell
 F plasmid is replicated in recipient cell, so now both cells have the F plasmid
 Certain F plasmid proteins in the donor and certain outer membrane
proteins in the recipient are required to form the conjugation bridge
o F plasmid
 tra region contains genes needed for plasmid transfer
 makes plasmid self-transmissible
 transferred last into host cell
 oriT- origin of transfer, where plasmid mobilization starts
 oriV- allows plasmid replication in the host cell without transfer so that
daughter cells inherit plasmid
 If plasmid can replicate outside of the chromosome, it can be used for:
 Complementation
 Housing a transcriptional reporter
 Expressing GFP protein
 If plasmid can’t replicate outside of chromosome, it can be used for:
 Delivering an engineered transposon to mutagenize the recipient strain
 Homologous recombination to insert some part of the new plasmid into
the host chromosome to make a gene knockout
- Generalized Transduction
o Phage-mediated transfer of genes between bacterial chromosomes
 1. Phage attacks bacterium and injects its DNA
  2. Host chromosome is cut into pieces
 3. New phage proteins and DNA are made, DNA is packed into phage particles
 4. Rare packaging of host chromosome, rather than phage DNA, creates a
transducing particle
o When transducing particle injects its DNA, a fragment of the donor chromosome is
transferred instead of phage DNA
 Genes on this fragment can be incorporated into host chromosome via
homologous recombination
o Not all phages perform generalized transduction because some package DNA with a
specific packing sequence
o Must select for desired piece of DNA that you hope to transfer from one cell to another
 Usually select for transfer of DNA that confers antibiotic resistance
o After a transducing particle infects a new cell, donor chromosome fragment can be
recombined into recipient chromosome to yield a transductant
o DNA acquired by transduction replaces part of recipient chromosome
o Can be used to investigate double mutations

Lecture 15-Genetics III

- Complementation
o Transform mutant with genomic DNA library from WT parent
o Select mutant cells that have received a plasmid
o Select or screen for colonies that have WT phenotype restored
o “rescue” specific genes
- Random mutagenesis
o When is it used?
 When you don’t know at all what genes are involved in your process of interest
or if you have no sequence info on the organism
 Tools: chemical mutagens, UV, transposons
- Targeted mutagenesis
o When is it used?
 When you can predict what genes are involved in the process of interest
 Tools- must have sequence information about gees you are targeting, or whole
genome sequence of organism
- Targeted gene disruption
o Creates knockout mutations
o PCR orfA and surrounding region, clone into plasmid
o Cut orf with restriction enzymes
o Replace with antibiotic resistance gene
o Transform competent bacteria with KO plasmid
o Recombination needs to occur to obtain antibiotic resistant cells
- When doesn’t gene knockout work?
o When you are trying to knockout an essential gene
o Screen for ts mutation
 o or use inducible promoter to make depletion strain
 gradually deprive bacterium of essential protein
- Change gene regulation with inducible promoter
o Some proteins are not expressed at all times
 Ex. xyl proteins
o Express xyl promoter in front of any gene
 Gene only transcribed in presence of xylose
- Making a depletion strain to learn null phenotype of essential gene
o Create plasmid with essential gene regulated by inducible promoter
o Transform plasmid into host strain, keep gene on with inducer
o Knockout chromosomal copy of gene with targeted gene disruption
o Turn off plasmid gene by removing inducer
o Wait for existing protein to be depleted, observe cells
- Use reporter to determine when gene expression is on/off
o On a phenotypic level, regulation using repressor + inducer looks similar to regulation
with activator + coactivator
o Ex. studying utilization of raffinose
 Enzyme RafA breaks down raffinose
 RafA is only made when raffinose is present
 Make a plasmid where rafA promoter drives expression of GFP
 WT strain with this plasmid only makes GFP when raffinose is present
 Mutagenize reporter with transposon
 If colonies grown in raffinose don’t fluoresce, activator was knocked out
 If colonies grown without raffinose fluoresce, repressor was knocked
out

Lecture 16-Bacterial Cell Walls

- Two types
o Gram-negative
 Outer membrane + peptidoglycan cell wall
 Covering layers called the cell envelope
 PG wall is 1-3 layers thick
 OM contains lipopolysaccharide (LPS)
 LPS determines how cell interacts with world
 OM also contains porins for permeability based on size
 Lipoproteins in OM are covalently bound to PG for integrity of envelope layers
o Gram-positive
 Only peptidoglycan cell wall
 Can be >20 layers thick
 Teichoic acids polymers impart negative charge to cell surface and bind
Ca2+ and Mg2+ for eventual reuptake
o Represent around 50% of cell wall
 Lipoteichoic acids- embedded in membrane and extend through PG
 o Important virulence factors
o Stimulate innate immune response in animals
o Type of wall is important determinant of antibiotic resistance/susceptibility
- LPS structure
o Lipid A- fatty acids linked to sugar phosphates
 Cause immune response
 In outer leaf of OM
 Phosphates and sugars bind divalent cations to make a barrier that excludes
many hydrophobic compounds
o Ketodeoxyoctanate (KDO)
 Found in core polysaccharide
o O-specific polysaccharide (O-antigen)
 Involved in binding to host tissues and evading immune response
o Hydrophilic polysaccharides
 Protect cell surface from bible salts, hydrophobic antibiotics
 Complement activation
- Peptidoglycan
o PG sacculus is one large macromolecule
o Glycan chains around circumference
o Peptide crosslinks connect adjacent glycan strands
o Protects against lysis in hypotonic environments
o Not static structure
 Over 50% of PG is recycled during each generation
- E. coli peptidoglycan
o Monomer= disaccharide pentapeptide
o NAG and NAM are connected in glycan strands/chains
o DAP helps form peptide cross links in gram-negative PG
o Bacteria have enzymes to make, break, or remodel these linkages
- PG synthetic and remodeling enzymes
o PG monomers are synthesized in the cytoplasm in the Mur Pathway
o During last few steps of pathway, monomer is attached to bactoprenol in the
cytoplasmic membrane
o Finished monomer lipid II is flipped by flippase so that it faces the periplasm
o Monomer is added to glycan strand during transglycosylation
o Bactoprenol is recycled
o Crosslinks form between peptide chains attached to NAM molecules during
transpeptidation
- Proteins that synthesize or modify PG
o Penicillin-binding proteins (PBPs)
 High-MW PBPs
 PBP1- performs both transglycosylation and transpeptidation
 PBP2, 3- perform only transpeptidation
o PBP2 is in lateral walls
 o PBP3 is at division site
 Low-MW PBPs- make alternative peptide crosslinks, remove AAs, break peptide
crosslinks
o Lytic transglycosylases
 break B(1,4) linkage between NAM and NAG
 ex. lysozyme

o SEDS proteins
 Have glycosylation activity, can replace PBPs
 Ex. RodA, FtsW
- Penicillin
o Blocks transpeptidation step of PG synthesis
 1. Ser residue on PBP transpeptidase domain forms covalent intermediate with
penultimate D-ala, releasing the terminal D-ala residue
 2. PBP-ala intermediate is attacked by DAP on neighboring peptide to form
crosslink and release PEP enzyme
o Penicillin is a B-lactam antibiotic
 Mimics D-ala-D-ala residues at end of PG peptides
 Attacked by PBPs and substrate
 B-lactam ring is broken during attack, forming irreversible intermediate
with PBP
 Transglycosylation happens, but peptide links cannot form between glycan
strands, weakening PG
 Cell eventually lyses
- B-lactamases
o Confer resistance to penicillin
o Break B-lactam ring on penicillin, inactivating it
- Cell shape
o Different bacteria have different shapes
o Shape of cell is determined by PG sacculus
o PBPs, LTGs, and SEDS proteins influence cell shape by working at certain positions at
certain times
 PBP2, RodA synthesize lateral cell walls
 PBP3 and FtsW synthesize septa
o Protein localization corresponds with function
 PBP3 is at division septum
 PBP1 is at lateral walls and septa
 PBP2 is at lateral walls

Lecture 17-Cell Shape and Division I

- Cytoskeletal proteins
o Create distinct shapes and required for mobility
o All bacterial cytoskeletal proteins are homologs of eukaryotic ones
 MreB=actin
  FtsZ=tubulin
 CreS=intermediate filaments
- MreB
o Polymerizes in an ATP-dependent manner into filaments
o In living cells, GFP-MreB assembles into short filaments adjacent to the cytoplasmic
membrane and is associated with PBPs in large complexes
o MreBCD
 forms complex that helps position and regulate PG synthesis machinery along
lateral cell walls
 complexes perform transglycosylation, transpeptidation, and synthesis
of PG monomers
o enzymes perform each task
 PG monomer synthesis enzymes- MurG, MraY
 without MreBCD, PBP2 is mislocalized and misregulated, so lateral cell walls are
not made normally
 cell lyses
- Elongasome
o Includes MreB
o Moves around circumference of cell as it synthesizes new PG
o Movement is dependent on active PG synthesis
o MreB associates with elongasome via lipid-linked PG monomers, made by MurG
 Without precursors, MreB stops moving and dissociates from the membrane,
elongasome disassembles
- Crescentin (CreS)
o Responsible for curved shape of caulobacteria and other bacteria
o Polymerizes into filaments in vitro without added nucleotide
o Located in cytoplasm at inner curvature of cell
o Requires MreB to induce curvature
 CreS modulates activity of elongasome
- CrvA
o Found in vibrio cholerae
o Similar to CreS
 However, found in periplasm instead of cytoplasm
o Forms complex with CrvB
o Can generate curvature even without MreB
o CrvAB causes asymmetric addition of new PG, with more at outer face than inner face
- Why is curvature important?
o Allows bacteria to swim through viscous environments

Lecture 18-Cell Shape and Division II

- FtsZ
o forms ring at site of cell division between the two replicated chromosomes
o polymerizes into short protofilaments with GTP in vitro
 o constriction of FtsZ ring drives cell division
 Z-ring
o Assembly site for the divisome- large protein complex that spans cytoplasmic
membrane and synthesizes septal cell wall
 FtsA and ZipA tether FtsZ to membrane
 FtsW brings PBP3 to the septum
 FtsK helps with chromosome segregation
 FtsQLP complex triggers Z-ring and cell constriction
- Septal PG is synthesized in concentric rings moving forward with time
o Ftsz treadmilling drives motion of PBPs a septum
 Drugs that block PBP do not stop motion of FtsZ, but drugs that block FtsZ
GTPase activity do
- Division site
o E. coli divides at center of cell with remarkable precision and fidelity
o Minicells- normal amounts of RNA but no DNA
 Cells divided at wrong site
 Had mutation in MinB
 Deletion inactivating minC, minD, minE
o Abesence of MinC, MinD, or both
 Minicell phenotype
 MinC inhibits z-ring formation
o Absence of MinE
 Filamentous cells
o MinE restricts action of MinCD so that division only occurs at midcell
 Involves pole-to-pole oscillations of MinCD
 MinE forms ring at medial edge of MinCD polar zone
 E-ring moves toward MinCD
 As E-ring moves, MinCD is released
 MinCD move to other end of cell, making new zone, MinE follows
 Biochemical explanation
 When MinC is bound to MinD-ATP, it inhibits binding of FtsZ filaments
 MinE binds to MinD-ATP, converting it to MinD-ADP
o MinC, MinD-ADP are released cytoplasmic membrane
 MinE rebinds to membrane
 In cytoplasm, MinD-ADP becomes MinD-ATP, then MinCD binds to
membrane again
 MinE follows MinCD
 Net result is that MinC spends most time at poles of cell, not in middle
 FtsZ will build Z-ring at midcell
- Nucleoid occlusion
o Prevents division from occurring at DNA sites
o Nucleoid gives off signal preventing Z-ring from forming nearby
o SlmA
  Binds to 20 bp sequence (SBS) in E. coli genome
 SBS are not present near terminus, which is located at the center of the cell near
the division plane
 SlmA+SBS inhibits FtsZ polymerization
 SlmA by itself does not

Lecture 18-Chromosome Replication and Segregation

- Replication
o Starts at a specific site called the origin (oriC),
 Place where initiation proteins bind and start process
o Bidirectional, happens at two forks at same time
o Cells tightly regulate initiation of replication because unfinished replication is lethal
o DNA Pol III
 Adds nucleotides 5’ to 3’
o Tau holds Pol III proteins together in single replisome
o Sliding clamp DnaN holds polymerase on the DNA
o DNA primase-adds primer to start replication
o DNA gyrase- removes supercoils by unwinding DNA strands
- Protein-DNA interactions at the origin of replication oriC
o Properties of initiation proteins
 DnaA-catalyzes unwinding of AT-rich repeats
 Must be ATP-DnaA, not ADP!
 IHF- binds and bends DNA to facilitate DnaA binding
 DiaA helps DnaA bind to low-affinity sites
- Steps in initiation of chromosome replication at oriC
o DNA binds DnaA, DnaA and IHF make open complex, AT-rich region is unwound
o DnaC loads helicase DnaB onto SSR, DiaA and DnaC leave oriC
o DnaB helicase expands region of ssDNA, primase DnaG binds to helicase and makes first
primers
o DnaN (sliding clamp) and Pol III are loaded onto DNA, DNA synthesis begins
- Replication regulation
o Dam methylase-methylates entire chromosome on A residues within sequence 5’ GATC
3’
 DnaA can bind to fully methylated DNA
o Immediately after initiation, DNA becomes hemimethylated
 SeqA binds to hemimethylated GATC sites within oriC and prevents ATP-DnaA
from rebinding
o SeqA and Dam methylase compete for binding to GATC sites and remethylate them
 At that point, SeqA can no longer bind, and DnaA can rebind to initiate more
replication
- RIDA- replicative inactivation of DnaA
o Hda- hydrolyzes ATP-DnaA into ADP-DnaA, inactivating it
 Another round of replication can’t begin
 - How are plasmids inherited?
o No segregation system- plasmid is eventually lost if selection is not maintained
o Dedicated segregation system- plasmid is maintained in population even without
slection
o Plasmid R1 segregation
 ParR binds to DNA sites called parC
 ParM binds ParR and polymerizes to push two plasmids apart
 ParM stabilized by binding to ParR-parC
- Chromosome segregation
o Bacterial chromosome is a nucleoid
 Not surrounded by membrane
o ParABS system
 parS
 parC analog
 where ParB binds
 ParB
 ParR analog
 ParB-parS interacts with ParA-ATP dimers on DNA and stimulates ATP
hydrolysis by ParA
 ParA
 ParM analog
 Binds ATP and dimerizes
 Interacts with polar anchoring proteins
 Facilitates movement of ParB-parS complex to opposite pole
 Doesn’t push two ParB-parS complexes apart

Lecture 19-Membrane Transport Systems

- Lipid bilayer construction

o Made of phospholipids
 Hydrophilic head determines identity
 Bacteria have different amounts of different phospholipids
o Rigid planar molecules intercalate into membrane and make it less fluid
 Bacteria have hopanoids
o Many different proteins in the membrane
 Proteins can diffuse in the plane of the membrane
- Cytoplasmic membrane function
o Membrane is permeable to H2O, CO2, NH3, short/medium chain fatty acids, and small
hydrophobic molecules
o Processes that occur
 Lipid biosynthesis
 Some PG and LPS biosynthesis
 Sensing external conditions
 Importing nutrients
 o ETCs and ATPase are in cytoplasmic membrane
 Enzymes of ETCs create a proton gradient, with H+ higher outside and lower
inside
 PMF can power
o ATP synthesis
o Transport reactions
o Flagellar rotation
- Protein-mediated transport
o Properties
 Transport rate is saturable-a finite number of transport sites exist per cell
 Mutants that do not transport individual substances can be isolated
 Inactivation of individual protein can block transport
o Active transport systems
 Use ATP or energy of ion gradient
 Allow accumulation of solutes in cell to concentrations higher than in
surrounding fluid
- Glycerol
o Enters/exits cell by facilitated diffusion through GlpF transporter
o If glycerol concentration is higher outside than inside, net entry of glycerol into cell will
occur until concentrations are equal
- Energy-dependent transporters
o Simple transport
 Driven by energy in PMF or other ion gradients
 Symport- two solutes enter cell
 Antiport- one solute enters, other one leaves
o Group transport
 Facilitated diffusion coupled with substrate phosphorylation
 Modification of imported sugar changes its identity, so internal
concentration of unmodified sugar stays low
 Reverse transport can’t happen
 Energy for directionality of transport comes from hydrolysis of
phosphoanhydride bond in PEP
 Common elements are El and HPr
 Every sugar has its own IIA, IIB, IIC
o ATP-binding cassette (ABC) transport
 Energy of ATP hydrolysis powers transport
 Some function in reverse to expel substrates
 Binding proteins determine substrate specificity
o TonB-dependent transport (TBDT)
 Some substrates are too big to cross OM via nonspecific proteins
 TonB/ExB/ExbD complex uses PMF across IM to power uptake of substrates
across OM via TBDT
 ABC transporter helps bring substrate to IM from periplasm
 Ex. Fe3+-siderophore complex for iron uptake
 - Protein secretion across cytoplasmic membrane
o Sec system
 SecY, SecE, SecG form protein-translocating channel in membrane
 Two paths lead to this channel
 1. SecB
o Binds signal sequence of protein, keeps it unfolded
o SecA ATPase brings protein to Sec channel
o Protein folds in periplasm after being transported
o Used for OM proteins, lipoproteins, periplasmic proteins
 2. Signal recognition particle (SRP)
o Binds to signal sequences as protein comes out of ribosome
 Translation pauses
o Entire complex binds to SRP receptor on membrane
o Growing protein chain moved to Sec channel, translation
continues via GTP hydrolysis
 Protein is pushed through channel across membrane
o Used for IM proteins because they are too hydrophobic for SecB
to maintain in conformation that can be exported
o Tat pathway
 Used to transport proteins that can’t fold in the periplasm
 Proteins first folded in the cytoplasm
 PMF provides energy for transport
 Problem- permeability barrier is hard to maintain
 Since Tat channel has big opening, small molecules can diffuse through,
which is bad
 Signal peptide binds to protein
 TatABC
 Complex for Tat system
 TatA dispersed in membrane when not in use
 PMF and Tat signal binding required for TatABC assembly
 Protein crosses TatABC channel
o After crossing, signal peptide is removed
 TatA dissociates from complex, shutting channel
o Since TatA can’t maintain permeability barrier unless engaged
with a substrate

Lecture 21-Appendages and secretion systems

- Capsules
o Polysaccharide+protein
o Hydrophilic polymer
o Repels viruses, hydrophobic anti-microbial compounds
o Attaches to host tissues
o Protects against phagocytosis desiccation
 o Involved in biofilm formation
- Flagella
o Enables cell to swim through liquid
o Anchored in cell membrane by rotary motor
o Flagellar rotation is like a boat propeller
o Motor driven by proton gradient across cytoplasmic membrane
o Structure
 MS ring- anchor in cytoplasmic membrane
 P ring-anchor in peptidoglycan
 L ring- anchor in outer membrane
 C ring- proteins on cytoplasmic surface
 Involved in export of flagellar proteins
 Can change direction of flagellar rotation
 MotA, MotB
 Stators of flagellar rotation
 Help rotate flagella
o Protons move through MotA, causing changes that push FliG of
the rotor in the C-ring
 Stay still as rest of flagella turns
 Bound to PG
 FliM, FliN
 Determine which direction flagella rotates
o Hook-basal body complex (HBB)
 Assembled from inside to outside
 Early proteins are secreted into membrane or periplasm via SecYEG pathway
 Later flagellar components secreted through channel in axis of
rod/hook/filament
 Long axis- channel through which protein components are secreted
o Transcription
 Transcribed in a transcriptional cascade
 Early (class II) genes encode proximal components of flagellum
 MS ring, C ring, rod, P ring, L ring, hook
 Two class II genes are a transcription factor that promotes expression of
class III genes (sigma 28) and a protein that binds to sigma 28 and blocks
its action (FlgM)
 When HBB complex is complete, FlgM is exported through flagellar channel, so
sigma28 can activate class III genes
- Pili (fimbriae)
o Type IV
 Extend and retract
 Used for surface motility
 Pilus proteins exported into cytoplasmic membrane or periplasm via Sec system
 PilG- assembly site
 PilF ATPase- powers assembly
  PilT ATPase- powers retraction
 Assembled pilus travels through OM in large channel made of PilQ subunits
o Type I
 Used for attachment
 Subunits move to periplasm via Sec pathway
 PapA- main subunit
o Type II
 Related to Type IV pili
 Transfers proteins
 Substrates sent to periplasm via Sec system
 Secrete proteins into extracellular space
o Type III
 Related to flagellum
 Transfers proteins
 Secretes proteins into host cell cytoplasm
 Sec system not involved
o Type VI
 Related to contractile phage tails
 Transfers proteins
 Secretes substrates into cytoplasm of host cell
 Sec system not involved