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Cell Biology > Microbiology Pt. 2 > Flashcards

Microbiology Pt. 2 Flashcards

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1
Q

Bacterial Flagella

A
  • extracellular thin appendage

- can be polar (one direction), peritrichous (all around cell surface), loprotrichous (tuft of hair)

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2
Q

Gram Negative Bacteria Flagellum Structure

A 
Extracellular:
- cap
- filament (FliC)
- hook filament junction (FlgK/L)
- hook (FlgE)
Outer membrane:
- L ring (FlgK)
Periplasm:
- distal rod
- proximal rod
- P ring 
Plasma Membrane:
- S-M ring
- Mot Protein
- Fli Protein (motor switch) / C ring
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3
Q

Sequential Assembly of Flagellum

A
  1. MS ring assembled via secretory apparatus
  2. rod subunits assembled via secretory apparatus
  3. hook subunits
  4. flagella
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4
Q

Assembly Process

A
  1. 26 subunits of Fli integral membrane protein come together to form MS ring
  2. Flig proteins assembly under the MS ring & along with FliM/FliN make up the rotor
  3. Flagellar proteins destined to be part of the extracellular portion of the flagellum are exported by a flagellum specific export pathway and assembled at the center
  4. Mot A/Mot B form the stator. Mot B is anchored to peptidoglycan later
  5. Rod subunits move through the hollow cylinder and build up the rod in a proximal to distal fashion
  6. L/P rings are found in Gram - bacteria and penetrate the outer membrane forming a bearing for the rod
  7. rod cap dissociates and is replaced by a hook cap that guides hook protein assembly
  8. hook cap dissociates and hook filament junction proteins replace it
  9. filament cap assembles filament proteins to reach distal end > assemble in helical fashion
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5
Q

Swimming vs. Swarming Motility

A

Swimming: movement in liquid or low viscosity substances
- rotation and no coordination of population
Swarming: requires biosurfactant and flagella/higher agar concentration (semi solid)
- coordinated movement

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6
Q

Flagella Motor Energy

A
  • motor derives energy from proton gradient (higher outside the cell)
  • Protons flow through Mot A/B interface that make up the stator
  • Each stator contains 2 Mot B proteins each with an aspartic acid residue
  • Proton binding to aspartic acid residues leads to a Mot A conformational change and the first power stroke
  • Proton loss at the end of this stroke drives a second power stroke engaging the rotor
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7
Q

Directional Movement

A

Polar: counterclockwise rotation leads to forward movement and clockwise rotation leads to backwards movement
Peritrichous: counterclockwise leads for forward movement. if this switches to clockwise rotation, the bundle falls apart and the subsequent ‘tumble’ reorients the bacteria so counterclockwise rotation now drives it backwards

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8
Q

Flagella Components

A

Filament = subunits of single protein flagellum
Hook = single protein connecting filament to motor
Motor = anchored in cell wall/cytoplasmic membrane
Motor proteins = drive flagellar motions causing rotation
Fln B/FliK/Flk important in switching secretion of different substrate specificity.
ie. ordered assembly of different components

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9
Q

Chemotaxis

A

Movement dependent on environment using flagella
Experiment: capillary tube assay
- insert a tube with attractant/repellent and can graph cells in each tube over time to view chemotaxis
- Chemotaxis is controlled by TCS with a distinct mechanism

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10
Q

Pili & Fimbriae Commonalities

A
  • both commonly found on prokaryotic cell surfaces
  • many classes
  • short filamentous structures
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11
Q

Fimbriae

A
  • shorter than flagella but many more per cell
  • involved in bacterial adhesion to surfaces or other cells
  • not known to be used in motility
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12
Q

Pili

A
  • longer than fimbriae but fewer per cell
  • bridge between mating bacteria
  • receptors for certain bacteriophages
  • adherence of pathogens to host cells
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13
Q

Pili Structure

A

Type IV pili = attachment and motility

  • major subunit of T4P is type IV pilin, structurally related proteins are found as components of DNA uptake/competence systems in different species
  • Pil A protein are inserted into the plasma membrane and assemble into a filament
  • Pil C in the plasma membrane forms platform for growth
  • Pil Q is in the outer membrane allowing filament assembly
  • 2 ATPase: Pil B (ATPase assembly of filament) and Pil T (ATPase disassembly of filament)
  • peptidase cleaving end of fibrin
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14
Q

Twitching Motility

A
  • cycles of assembly and disassembly of filament
  • type IV pili
  • solid surfaces
  • retraction and coordinated population movement
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15
Q

Fimbriae Structure

A
  • helical rod with chaperone usher pathway
  • PapH terminates elongation
  • PapG is adhesion at the tip
  • Tip: filibrium proteins (Pap pili)
  • Subunits guided through the secretory pathway into the periplasm where the chaperone guides them to be secreted into the growing tip
  • driving force is the conformational change of protein
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16
Q

Fimbriae Assembly

A
  1. fimbral subunit and chaperone transported into the periplasmic space
  2. chaperone protein protects hydrophobic groove in the fimbrili (without the chaperone the protein misfolds because of this hydrophobic groove being exposed to a hydrophilic environment)
    ie. donor strand complementation between chaperone and subunit
  3. Nte extension on subunit placed in hydrophobic groove (donor strand exchange) when chaperone leaves, aiding in filament assembly
17
Q

Catabolic Depression

A
  • enzyme synthesis (primarily catabolic) is inhibited when cells are grown in an medium containing a preferred carbon source
  • ‘glucose effect’
  • catabolite repression ensure the more readily available catabolisable energy sources are used first to conserve energy
18
Q

Diauxic Growth

A
  • seen when 2 energy sources are present in medium
  • the enzyme needed to utilize one of these is under the control of enzyme repression
    eg. cell density increase until a plateau at which all glucose is used up before beginning to increase again as B-galactosidase is expressed to use lactose present
  • involves transcription control by activator protein lactose operon
  • lactose broken down into galactose and glucose
  • transcription only occurs after CAP activator protein has bound to DNA
  • CAP binding to DNA requires cAMP to bind to CAP before binding to promotor and expressing the gene
  • glucose represses cAMP and stimulates its transport out of the cell
19
Q

Metabolism in low vs high glucose conditions

A 
Low:
- cAMP high
- CAP binds to gene activator site
- enzyme synthesis
- lactose broken down
High:
- cAMP low
- CAP can't bind to lac operon binding site
- no enzyme synthesis
- no lactose synthesis
20
Q

B-galactidase expression + inducer CAP

A
  • Lac 1 acts as an active repressor binding to the operator and blocking transcription
  • Lactose is an inducer binding to Lac 1 protein to prevent this blockage
21
Q

Two Component Signal Transduction System

A
  • E. Coli alters membrane protein composition in response to environmental conditions
  • Grow bacteria in low osmolarity conditions before transfering to high osmolarity medium (effectively altering the sugar concentration in the medium)
  • Omp c and Omp z are 2 membrane proteins synthesized by E Coli
  • Omp c forms small membrane pores and is highly present in high osmolarity environments to prevent toxin passage
  • Omp f forms large membrane pores and is highly present in low osmolarity conditions to allow fast solute passage
22
Q

Env2/Omp R Two Component System

A
  • regulation mediated by sensor protein/histidine kinase Env2 localised in the cytoplasmic membrane
  • Omp R is a response regulator in the cytoplasm
  • upon stimulus sensing, kinase auto phosphorylates a specific H residue
  • the phosphate is transferred onto the response regulator with reciever domain and aspartic acid resiue
  • response reciever bound to DNA binding domain on promoter, so activation leads to gene regulation
23
Q

Low Osmolarity

A
  • limited signal sense by Env2
  • Env2 autophosphorylates and transfers PO4 to OmpR
  • OmpR-P binds to high affinity site of promotor region of OmpF
  • Promotes OmpF expression
  • larger membrane pores for better solute transport
24
Q

High Osmolarity

A
  • more signal sensed by Env2
  • increased OmpR phosphorylation
  • high concentration of Ompr-P binds to high and low affinity sites of OmpF promoter to reduce its expression
  • low affinity site overlaps RNA polymerase binding site so that it is not transcribed
  • OmpR-P binding to low affinity site also promotes Omp C expression
25
Q

Chemotaxis and TCS

A

No attractant: ultimate destination is random with movement composed of runs and tumbles
Attractant: random movements are biased towards attractant with increase attractant concentrations causing longer runs and less frequent tumbles

26
Q

Bacterial Environmental Sensing

A

Bacteria are too small to detect a concentration gradient along their body length so compare their chemical and physical state before and after a time period
Therefore there is a response to a temporal gradient as compared to a spatial one.

27
Q

Mechanism of Chemotaxis TCS

A

Movement away: increase kinase CheA phosphorylation
Movement towards: decrease kinase CheA phosphorylatoin
- MCP fully methylated no response to attractant and sensitive to repellant
- kinase connected by adaptor protein that is attracted to receptor MCPs
- 2 reciever domains Y and B
- B is a methylase that removes methyl groups from proteins
- bundled receptor means many molecules can bind to allow concentration sensing
- low level of phosphorylated kinase = movement to attractant and no phosphorylated Che-Y reciever domains
- Che-Y binds to flagellum motor causing tumbling
- At low levels = no motor interaction so carry on moving forward in counterclockwise movement for a longer run

28
Q

Chemotaxis TCS reset

A
  • Uses methyl groups
  • Che-R is methyl transferase that is constituitively active
  • when receptors are fully methylated they are no longer responding to attractors
  • Che-B demethylates receptors so they can sense again
  • Che-Z is a phosphatase that removes P group and modulates Che A-P activity
29
Q

Quorum Sensing Discovery

A
  • Discovery: light organ symbiosis of vibrio fischeri and Hawaiin squid, Euprymna scolopes
  • light organs can be mating signals or counteract shadows caused by the moon to hide fish
  • Vibrio fischeri inhabits light sensing signals
    Luciferase is essential for light generation
30
Q

Luciferase Gene Structure

A

2 regulatory genes: AI synthase, transcription activator
Acid Reductase genes
Luciferase genes (5 structural genes)

31
Q

Mechanism of Quorum Sensing

A
  • luminescent bacteria provide autoinducers that are required at high levels in culture medium for induction of gene expression and can be secreted outside the cell
  • transcriptional control exerted by LuxR is dependent on it being bound to the autoinducer
  • LuxI expression is a positive feedback cycle can transfer to other cells
32
Q

Autoinducer

A

AHL: acyl-homoserine lactone
AHL is a intra-species language
Diversity given by two R groups so structure differs between species to allow system specificity

33
Q

Quorum Sensing

A

Ability of bacteria to sense a population density and to respond by alteration of gene expression

  • high population density: high autoinducer concentration in medium > luciferase expression high
  • low population density: high autoinducer concentration in medium > luciferase expression is absent
34
Q

Quorum Sensing Functions

A
  1. cellular communication
  2. signal amplification
  3. coordinated population movement
35
Q

Examples of Quorum Sensing

A
  • P. aeroginoses pathogen uses quorum sensing as the master regulator for production of virulence factors

Cell Biology (16 decks)

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