BIMM 120 (Microbiology)

F-type ATPase

goal: generating ATP with proton gradient

F0: A (embedded in membrane); B (Connects F1 + F0); C (c-ring, 12 subunits, rotor rotation)

F1: 2 hydrophilic subunits (y & e); rotating rod; free in outside membrane

Components of bacterial flagellum

basal body: motor, anchors substrate to envelope

• extends into cytoplasm with rings containing switch proteins

• responsible for direction of rotation (dependent on chemotactic signal transduction)

• tail: components added to tail after traveling through hollow tube that is capped by HAP2

  • transports export substrates

  • HAP2 stops growth of tail

filament: cell lengths extend from body to propel bacterium

hook: attaches body to filament

• ~ 10 flagella

Bacterial flagella rotation

Driven by PMF or sodium motive force

Direction driven by chemotactic signal transduction (based on environment)

Flagellar motor (5 proteins)

MotA/B = proton channels

Proton movement causes conformational change to MotA (power stroke) → turns on switch and motor turns

T3SS

needed for assembly of components outside of the membrane

Flagellar rings and their functions

M ring: plasma membrane → rotation of flagella

S ring: periplasma → works with M ring for stability

C ring: cytoplasm → interacts with motor proteins for rotation (charged rig)

P ring: peptidoglycan → stablilized flagellum

L ring: outer structure (LPS membrane) → maintains structure

FliC vs. FljB switching in salmonella “Flagellar phase variation”

goal: to invade host immune system and avoid immune detection

• mediated by Hin invertase

• FljB and FljA are repressed

• FliC not is expressed, making Phase 1 flagellin to avoid aB

  • FliC good for invading and colonizing tissue/tumbling

  • FljB good for straight movement

“Diverse Ways That Prokaryotes Move” paper

• Genome Economics: needing a lot of structures but having a small genome → express a lot of one thing that is capable of aggregating easily so you can make various structures

• swimming, swarming, twitching, gliding → movements are mediated with T4P, flagella, and nanomotors

  • swarming example: numerous flagella in cell and it moves

flagella assembly

basal body → hook → filament

critical checkpoint that is mediated by FliK (internal ruler)

power source for bacterial flagellum (MotAB, PomAB, etc)

MotA/B driven by H+ in salmonella

PomA/B driven by NA+ in Vibrio

MotP/S driven by NA+ in Bacillus

Archaeal Flagella

• thinner, faster than bacteria

• similar in structure (rotating structures with filament and hook)

• lack peptidoglycan

• contain signal peptides

• shares homology with T4P but lacks hollow interior → general secretory system exports subunits

• assemble at the base, not tip

• driven by ATP hydrolysis

  • J at base

  • I ATPase

  • H binding protein that interacts with I (forms motor)

Swimming without flagella

• helical cells lack cell wall and cannot maintain morphology with internal cytoskeletal filaments

  • contractile cytoskeletons function as linear motor

• moving kink made from differential length changes of filament building blocks so liquid can move past itself

Moving over surfaces without flagella

• dependent on T4P and twitching motility

  • T4P utilizes pilus extension attachment and retraction to surface

Pilus fibre

• made of many copies of PilA (protein)

  • rapid assembly made by recycling this protein

• ATP hydrolysis powers extension (PilB) and retraction (PilT)

  • Gram (-) bacteria: PihQ forms pore to outside of membrane to allow passage of pilus

  • Gram (+) bacteria T4P: secretion not needed bc outer membrane lacking

Pili

adheres to surfaces at tips (involves PilA or PilY1)

extension/retraction: atp hydrolysis

• used for bacterial conjugation, adhesion, twitching and gliding

• found as archaeal flagella

• assembled by inserting subunits into base

Gliding and lateral movement with surface adhesions

• cells attach at one pole and rotate

• twitching = ATP hydrolysis

• gliding = PMF

• 669 kDA SprB required for movement over agar (mobile component)

M. Xanthus (polysaccharides) and E. Coli Tol

• when they run out of nutrients, they form spores by aggregating multicellular fruiting bodies

• homologues of E. Coli tol required for A-motility

• Tol proteins involved in active transport of molecules across outer membrane

Types of Motility

S Type: T4P mediated motility (twitching)

A Type: Pilus independent; generates propulsion and leaves behind snail trail

mycoplasma

mycoplasma: tiny bacteria with simple genomes; asymmetrical and move with head

mobile: large surface Gli proteins localized at neck region (becomes its legs)

• centipede movement, wave length

pneumoniae: gliding motility in direction of terminal organelle

• inch worm movement; dynamic motor - extension and contraction

Passive vs Parasitic

Passive: utilize gas-vesicle for vertical movements

Parasitic: dependent on host cell actin for movement (Listeria and Shigella)

bacterial locomotion

• similar to bacterial organelle; conserved function and structure across species

• basal body: bidirectional rotary motor

  • CCW = coherent swimming

  • CW = tumbling

• hook: universal joint

• filament: helical propeller

How to build flagellum

bottom up (basal → filament)

• MS ring (FliF): weird symmetry 34x FliF; different copies of the same protein with same sequence have multiple conformations

C-ring

• C ring assembles onto M ring (FliG/M/N)

  • force generation (rotor)

  • number of complexes dependent on direction

    • less in CW compared to CCW

• switches to CW when CheYP binds to c-ring

  • MFXF motif (reverse gear of motor)

P ring and L ring

• c26 symmetry

• FlgI (p) FlgA assembles around rod with FlgA

• water tight (1-2 molecules)

• rod centered during rotation due to charges

Rod (flagellar driveshaft)

Proximal: FliE6 + FlgB5 + FlgC6 + FlgF5

Distal: FlgG24

• tight association with MS ring

Hook assembly

FlgE; cannot twist, only bend

• flagella can bunch up and form bundles

Filament assembly

FliC or FljB

• 15uM or 1-3uM for bacteria

• part that bundles when multiple motors turn CCW

Previous model of bundling

CCW: Left handed super coiling → bundling → normal filament

CW: Right handed super coiling → semicoiled → curly filament

• overturned by recent Cryo-EM results

Hook-filament junction

FlgK11, FlgL11

• prevents leakage from flagellin and provides mechanical intermediate

Cap structures

filament cap (FliD)

• c5 symmetry, prevents leaking

hook cap (FlgD)

rod cap (FlgJ)

• pierces cell wall to allow distal rod assembly

Flagellar type 3 secretion system

• homologous to injectisomes of pathogenic bacteria

• components in C,M,S rings

  • C rings: ATPase (reverse of ATP synthesis)

  • M rings: Transmembrane export gate complex

    • chemiosmotically powered (H+ or NA+ antiport → protein export)

    • substrate specific chaperones required to export working flagellin/components

MotA5/B2

• key parts: cytoplasmic domain, TM H+ channel, Linker, Peptidoglycan binding

• binds and dissociates from rotor during rotation

  • high loads = 10

  • low loads = 3

  • sensitivity mediated by PGB

3 promoter classes (gene regulation)

Class 1: flhDC (transcription factors)

• controlled by global regulators (master operon)

• required to express subsequent promoters

Class 2: hook basal body (HBB)

• FliA (turns on class 3 genes); FlgM (turns off class 3 genes until hook is ready)

  • hook completion → FlgM secretion → active FliA → Class 3 promoters

Class 3: filament but also regulatory proteins

Gliding motility

SprB adhesin

• organelle that grabs onto something and goes through conformational change

• movement occurs during retraction conformational stage

Nanowires

• Nanowires = broad class of conductive structures with diameters on the order of nanometers (10^-9)

• microbes can produce biocompatible nanowires safely and efficiently

  • peptide based

  • low energy requirements

  • renewable feedstocks

Types of microbial nanowires

1. Pilin-based nanowrires/electrically conductive pili (e-pili)

   • based on type 4 pili of archaeal homologs

   • conductive in absence of native metal cofactors or added metals

   • function as conduits for long range e-transport to extracellular terminal e-acceptors or surface sensors

   • high abundance and close packing of aromatic rings (higher conductivity)

   • modification: C-terminal exposed peptide ligands enhances sensory function

2. Curli fibers

   • CsgA monomers self assemble outside of cell

   • serve to adhere bacteria to surfaces, promoting biofilm formation

   • not conductive

   • modification: exposed metal-binding peptides or more closely packed aromatic residues

3. Protein wires from cable bacteria

   • cable bacteria in aquatic sediments form long chains of cells that shuttle excess e- from sulfide oxidation to oxygen rich areas

   • difficult to purify nanowires and can lose conduction

   • extraction of filaments done by chemically dissolving other cell components

carbon nanotubes

nanowires and carbon nanotubes are important class of electronic materials

• enable miniaturization and high density integration of components and construction of flexible electronics

Sensors

• one device is highly responsive to ammonia gas in wide range of concentrations

• another is continuously monitoring skin hydration or breathing rate

• can attach aB to increase analyte specificity/binding

Electricity production

exposure of upper layer or nanowire film to atmospheric humidity = vertical moisture gradient within film

• constant output voltage = 0.5 volts

Memristors (memory+resistor)

• device designed to function as analog of biological neurons

• mimics history-dependent recording of inputs by brain

• conductivity increases with increased duration/frequency of electrical input

The Force Awakens Paper

• surface sensing and attachment gives resistance to fluid flow and enables injection of virulence factors

Adhesion to surface

• helps with antibody resistance by enhancing stability of plasma membrane

• adhesion = initial stage of biofilm development

• sensing of surface attachment → alteration in protein expression/modulation of gene expression

biofilms

protection against chemical and biological agents

non-pathogenic mechanosensing

attachment sensing, biofilm forming

Flagella impedance

• physical cues form environmental recognition

• for pathogenic bacteria, sensing physical interactions is vital for survival

• flagella plays major role in surface sensing, adhesion, and biofilm forming

• surface sensing mediated by interference in rotation of bacteria flagella by proximal surface

type 4 pili

membrane bound filamentous structures that extend via polymerization by assembly ATPase

(retraction = depolymerization by ATPase)

completes twitch motility

type 4 pili attach → signal generated when organelle start to retract → Pil Chp chemosensory system → cAMP → express virulence gene

Psudomonas aerginosa

opportunity pathogen with acute or chronic effected

• acute = cytotoxic, systematic infection common with burn infections

• chronic effect = noncytotoxic; causes cystic fibrosis

• has polar flagella that normally attaches to surface temporarily then detaches

• activation of virulence require the physically rigid surface

quorum sensing

sensing of cell population density which can regulate gene expression

positive feedback loop

attachment is the path to the dark side

• leads to Vfr activity → immobility → biofilm formation → attachment → more virulence

escherichia cell

commensal bacteria

common health concern

• regulatues virulence through mechanosensors

• uses T3SS

• e coli senses changes in physical force and alters expression of virulence factors in response

tests in soft agar

increased motility observed in pathogenic strains

commensal bacterial shower normal motility speed

flagellar mechanosensing

aids in successful host colonization by increasing expression of flagella in early stages of infection when motility is useful in promoting contact with epithelial cells

Vibrio Cholerae

marine bacteria that can exist in free-living planktonic state

• inhibition of flagellar rotation through surface contact disrupts ion flux through flagellar motor = biofilm cannot form

• defective motility showed enhanced expression of virulence factors (toxins)

Vibrio parahaemolyticus

• liquid media they swim with single polar flagellum

• solid surfaces = forms filamentous flagellated cells

• accurate sensing of environmental conditions vital to allowing pathogenic and non-pathogenic bacteria to produce appropriate behavioral response to surroundings

Proteus mirabilis

UTI infections

Swimmers → longer swarmer cells → express a lot of flagella

• flagella = mechanosensory organelle

  • can detect proximity to surface

Swimmer Vs Swarmer

swimmer = discrete rods, 10 flagella

swarmer = filamentous cell, thousands of flagella

triggered by flagellar impedance

Common themes

• positive detection of stress →

• reduction in motility

• expression of adhesions (biofilm formation)

• expression of virulence genes (toxin delivery)

• small molecule secondary messengers to ensure reliable signaling

• positive feedback loops resulting in robust transitions to virulent states

Inter-Kingdom signaling paper

previous understanding of quorum sensing: signals used for bacteria-bacteria communication

recent updates: quorum sensing signals affect mammalian host cells and their signals affect bacterial behaviors too

which hormonal signals can cross kingdom barriers?

• gasotransmitters (NO, H2S, CO) can freely diffuse through membrane

• hydrophilic small molecules cannot (noradrenaline)

• oligopeptides: noncanonical bonding to insert directly into membrane

• lipids: diffuse or dissolve in membrane (acyl-homoserine lactones)

• proteins = too bulky to cross membrane

how do cells detect these signals?

• Intracellular receptors (for molecules that can cross the membrane)

• cell surface receptors (for molecules that cannot)

Noradrenaline

• release in human correlated with stress

  • pathogens will pick this up and turn on virulence factors in response

    • ex: flagella, T3ss, toxins, adhesins

  • bacterial counterpart = AI-3

AHLs

• produced by bacteria

• can respond to and degrade through QS jamming

• induce pro-inflammatory expression patterns in mammalian cells

• pathogens can become more virulent in response

• induce apoptosis through Ca2+ influx

• immunogenic in plants

why are mammalian cell-cell signaling pathways found in bacteria but not invertebrates?

arose from horizontal gene transfer from bacteria

non-pathogenic communication

• root nodule formation

• legumes secrete hormones for bacterial receptors → NodD activates nod factors → plant hosts initiate nodulation → bacteria fix nitrogen. plants fix carbon

• alfalfa secretes QS inhibitors like L-canavanine (suppressing QS signal production in symbiotes)

Flagella direction of motion

CCW → run (bindle flagella)

• faster, smoother directed motion

CW → tumble (random)/reverse (polar); (scatter flagella)

• less organized motion

Chemotaxis

attractants attract, repellents repel

• hydrophilic substances attract sugars and amino acids

• hydrophobic substances repel membrane dissolving oils

bacterial chemotaxis system

dramatis personae:

• MCP (receptor) - methylation reduces sensitivity

• CheR: methylase (always active)

• CheY response regulator (inactive CCW) → active is CheYP

why do bacteria need methylation

methylation/demethylation = slower = better

• activation (fast) vs. adaptation (slow)

  • bacteria detects concentration differences over time

  • expands measurable range of chemoattractant/repellent concentrations

no methylation for bacteria

constantly run in high attractant areas and tumble/reverse in low attractant areas = bacteria stuck in unfavorable areas

Multiple Functions of Flagellar Motility paper

• multiple functions for flagella even in same species

• polar flagella allow run reverse or run tumble (random reverse)

why is adaptation for methylation/demethylation necessary

bacteria can detect gradients

• constant high → desensitization (easier to tumble)

• constant low → sensitization (easier to run)

without adaptation, run/tumble based on concentration, not gradient

• cannot stay in high attractant areas, but can stay in high repellent ones

trade-offs

higher sensitivity → faster localization

• more frequent overshoots

why power up flagella when nutrients are scarce?

search strategy: benefits of sampling more space to find nutrients outweigh metabolic costs

growth strategy: risks of sampling more space to find even more nutrients outweigh the benefits of staying around

• use natural selection to reason about how things happened/developed

100% search - 0% growth

• everyone swims to edge, center empty

• abrupt starvation waves as nutrients deplete

0% search - 100% growth

• everyone stays in center, edges unexploited

• abrupt starvation waves as nutrients deplete

what other roles do flagella and chemotaxis play?

secreted attractant/quorum sensing modulator → autoaggreggation → biofilm formaton

flagella - potential initial adhesin

above a certain density, swirls develop in cell swimming patterns → why does chemotaxis become less effective round here?

below threshold: neighbor motility helps smooth chemotactic motion

above threshold: neighbor motility becomes self-reinforcing and interferes with chemotaxis

contrast flagella

subunits inserted under FliD at tip

spirochetes

periplasmic flagella

• enable swimming in viscous fluids

Capsules

• adhesion (sticky polysaccharide-rich slime)

• immune evasion (slippery/polymorphic polysaccharides)

storage structures

• carbon storage structures (induced by carbon starvation)

• PHB/PHV granules: TCA intermediates shunted to generate cytoplasmic granules

• glycogen granules (starch)

Magetosomes

single domain magnetite crystals encased in membrane

single domain = largest size with clear direction of magnetization

Gas vesicles

• protein bound, permeable to gas only (buoyancy)

  • carboxysomes: protein bound organelles for carbon fixation in cyanobacteria

  • enterosomes: toxic metabolite containment chambers in enterobacteria

nuceloid

not nucleus, but contains DNA

not membrane bound but can fast growing bacteria have multiple

DNA packing challenges

negative charge → coulomb’s law (like repels like) → counterions neutralize charge

• Mg2+ ions or histone-like proteins

stiffness → histone like proteins

total size in general → supercoiling to reduce “wasted” space

• adding supercoil = gyrase (topoisonmerase 2): atp dependent

• reducing supercoil = topoisomerase 1; atp independent

origin of chromosomal replication

• AT rich (weaker base pairs on average) → easier to open → easier to replicate

• DnaA binding sites: prepares strands for separation by helicase

  • recruits B (inactive helicase)

  • recruits C (generates active helicase)

goal of respiration

oxidize carbon source (remove electrons) to prevent metabolism from stalling

• easier with strong oxidizer

geobacter

give excess electrons to FE3+

• side benefit: solubilizes iron

cellulosomes

• degrade cellulose

• key components:

  • anchoring protein: bind scaffold to cell

  • scaffoldin: hold enzymes out at useful distance

  • CBM: bind carbohydrates

  • catalytic enzymes: cut cellulose

carboxysomes (fix co2)

• Rubisco (very inefficient but very abundant enzyme)

• Carbonic anhydrase → fastest known enzyme

• protein shell keeps CO2 gas out

anammoxosomes

oxidize NH3 using NO2 to produce dinitrogen and water anaerobically

acidocalisomes

found in bacteria and eukaryotes

only shared organelle

protein secretion and membrane insertion paper

• transport of smell molecules = channels and carriers

• cytoplasmic protein domains or subunits superimpose upon integral membrane channel and carrier proteins to allow coupling of chemical energy/PMF

inner membrane secretory systems

export across/insertion into both membranes or inner membranes in gram negative bacteria

outer membrane secretory system

export across/insertion into outer membrane in gram negative bacteria

type 1 ATP-binding cassette (ABC) transporters

• ATP dependent systems

• 2 integral membrane domains and 2 cytoplasmic energizer domains to hydrolyze atp

• limitations = size and ease of unfolding of substrate protein

• ABC transporters exhibit substrate specificities

what are membrane fusion proteins and outer membrane factors and what do they do?

allow transport across both membranes of gram-negative bacterial envelope in single step

• OMF = provides transperiplasmic channel

• MFP = interlinks inner and outer membrane transport pathways

TolC in E. Coli

OMF that functions with several types of transporters.

• connects inner membrane permease to outer membrane pore

• beta barrel pore structure

• alpha helical conduit

Type 3 flagellar and pathogenicity related systems

• found in gram (-) bacteria, allow secretion of proteins across both membranes of the cell envelope

• responsible for descretion of virulence factors

• proteins encoded by pathogenicity islands

• shares notable homology with flagellar proteins

• secretes proteins directly into host cell cytoplasm without getting exposed to extracellular environment

type 4 conjugation and virulence related systems

• multiple subunits that span the 2 membranes and peptidoglycan wall of gram (-) bacteria

• also spans the single membrane pilus of gram (+) bacterial celll envelope

• export proteins and DNA Protein complexes of cell into cytoplasm