Microbiology Pt. 2 Flashcards
1
Q
Bacterial Flagella
A
- extracellular thin appendage
- can be polar (one direction), peritrichous (all around cell surface), loprotrichous (tuft of hair)
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
3
Q
Sequential Assembly of Flagellum
A
- MS ring assembled via secretory apparatus
- rod subunits assembled via secretory apparatus
- hook subunits
- flagella
4
Q
Assembly Process
A
- 26 subunits of Fli integral membrane protein come together to form MS ring
- Flig proteins assembly under the MS ring & along with FliM/FliN make up the rotor
- 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
- Mot A/Mot B form the stator. Mot B is anchored to peptidoglycan later
- Rod subunits move through the hollow cylinder and build up the rod in a proximal to distal fashion
- L/P rings are found in Gram - bacteria and penetrate the outer membrane forming a bearing for the rod
- rod cap dissociates and is replaced by a hook cap that guides hook protein assembly
- hook cap dissociates and hook filament junction proteins replace it
- filament cap assembles filament proteins to reach distal end > assemble in helical fashion
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
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
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
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
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
10
Q
Pili & Fimbriae Commonalities
A
- both commonly found on prokaryotic cell surfaces
- many classes
- short filamentous structures
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
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
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
14
Q
Twitching Motility
A
- cycles of assembly and disassembly of filament
- type IV pili
- solid surfaces
- retraction and coordinated population movement
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
16
Q
Fimbriae Assembly
A
- fimbral subunit and chaperone transported into the periplasmic space
- 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 - 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
Chemotaxis and TCS
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
Bacterial Environmental Sensing
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
Mechanism of Chemotaxis TCS
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
Chemotaxis TCS reset
- 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
Quorum Sensing Discovery
- 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
Luciferase Gene Structure
2 regulatory genes: AI synthase, transcription activator
Acid Reductase genes
Luciferase genes (5 structural genes)
31
Mechanism of Quorum Sensing
- 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
Autoinducer
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
Quorum Sensing
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
Quorum Sensing Functions
1. cellular communication
2. signal amplification
3. coordinated population movement
35
Examples of Quorum Sensing
- P. aeroginoses pathogen uses quorum sensing as the master regulator for production of virulence factors
