Prokaryotic Microbiology Flashcards
1
Q
Describe these different arrangements of flagella:
- Monotrichous
- Lophotrichous
- Amphitrichous
- Peritrichous
A
- a single flagellum usually at one pole
- two or more flagellum at one or both poles
- a single flagellum at each pole
- located over the entire surface
2
Q
Flagellum components and function 1
- What is the function of the ATPase complex and what proteins are involved?
- What is the function of FlhA and FlhB?
- What is the function of the C ring and what proteins are involved?
A
- present proteins involved in flagellum synthesis that are secreted through the flagellum pathway (FliH, FliI, FliJ)
- involved in secretion specificity
- used for flagellum switching (switch rotation direction) (involves FliG, FliM, FliN)
3
Q
Flagellum components and function 2
- what is the function of the MS ring and what protein is it made from?
- what is the function of the distal and proximal rods?
- what is the function of P and L rings and what protein are the made from? What type of bacteria don’t have them?
- what is the function of MotA?
- what is the function of MotB?
A
- embeds the flagellum on the inner membrane of the bacteria (FliF)
- traverses between the outer membrane and PG and PG and inner membrane respectively
- hold rotating parts of the flagellum in place (FlgI and FlhH). Gram positive.
- acts as a proton conduit to drive flagellum rotation
- attaches flagella m machinery to the peptidoglycan layer
4
Q
Flagellar rotation
- what protein makes this possible?
- how does it occur?
- how many protons are required for one rotation?
A
- MotA
- protons are pumped through MotA causing its structure to change which alters its interaction with the MS ring and C ring causing rotation
- 1000
5
Q
Model of hook-length determination by the infrequent ruler mechanism
- how long must the hook region be?
- what protein is key in this model?
- what happens if the hook is too short?
- what happen is if the hook is the correct length?
A
- 55nm
- FliK
- hook polymerisation halts, the N-terminus of FliK interacts with FlgED. FliK is then secreted and hook polymerisation continues.
- hook polymerisation halts, the C-terminus of FliK interacts with FlhB causing it to become activated and change its secretive selectivity to proteins involved in filament production
6
Q
The flagellum filament
- what protein is it composed of?
- how is it arranged?
- how large is the axial hole?
- how is it elongated?
A
- FliC
- arranged into a helical structure held together by hydrophobic interactions with polar residues lining the axial hole
- 2nm
- the filament cap (FliD) rotates and as it does so frees up space for a FliC molecule to be added to the filament chain
7
Q
Flagella filament synthesis mechanism
- in what state do FliC protein enter the flagellum?
- how are the pulled up through the flagellum?
- where does the energy for this come from?
A
- unfolded
- as the FliC protein at the FliD cap folds, it causes other molecules of FliC to be pulled up as they are attached via C-terminus to N-terminus interactions
- the folding of FliC
8
Q
Bacteria motility
- what happens when flagella rotate counter-clockwise?
- what happens when flagella rotate clockwise?
A
- flagella bundle together to create a propulsive force which causes bacteria to swim in a straight line
- flagella fly apart causing the bacteria to tumble in free space
9
Q
Basics of bacterial cell wall structure
- what cell layers do gram negative bacteria possess?
- what cell layers do gram positive bacteria possess?
A
- an inner and outer membrane with a thin layer of peptidoglycan between the two
- an inner membrane with a thick layer of peptidoglycan, no outer layer
10
Q
Peptidoglycan structure
- what are the two sugars found in peptidoglycan?
- how are these sugars arranged?
- how are the sugars joined together?
- what is the role of peptide chains?
- to which sugar are the peptide chains attached?
- which section of peptidoglycan is variable and which section is conserved?
A
- N-acetyl glucosamine (NAG) and muramic acid (NAM)
- alternate to form a glycan chain
- beta(1,4) glycosidic linkage
- covalently link to the glycan chains to form a cross-linked structure for rigidity
- NAM
- peptide chain is variable and glycan chain is conserved
11
Q
Peptidoglycan synthesis in E.coli phase 1
- where does this occur?
- what are the steps?
- why are two D-alanine residues added?
- how does this differ in gram positive bacteria?
A
- in the cytoplasm
- activated NAG assembled on bacteprenol is converted to activated NAM by MurA and MurB
MurC adds an L-alanine residue
MurD adds a D-glutamine residue
MurE adds a DAP molecule
MurF adds two D-alanine residues - to create free energy for chain incorporation
12
Q
Peptidoglycan synthesis in E.coli phase 2
- where does this occur?
- what are the steps?
A
- on the membrane
- bacteprenol-NAM complex is added to bacteprenol by MraY to form Lipid I
a NAG molecule from a bacteprenol-NAG complex is added to Lipid I by MurG to form Lipid II
Lipid II is then flipped to the periplasmic side of the membrane by FtsW
Glycosyltransferases then add Lipid II to the existing glycan chain
Transpeptidases then join the peptide side chains to form a cross-linked structure
13
Q
Gram positive cell surface
- what are its three components?
- what are the two forms of TA?
- what does each form attach to?
- what is the structure of a TA?
A
- peptidoglycan, teichoic acids (TA) and teichuronic acid (TU)
- wall teichoic acids (WTA) and lipid teichoic acids (LTA)
- WTA is covalently attached to peptidoglycan and LTA has a lipid tail embedded within the cell membrane
- polymers of glycerol and ribitol linked via a negatively charged phosphodiester bond
14
Q
Gram negative cell surface
- what are the four components?
- what is the function of the periplasm?
- what are the two forms of outer membrane proteins?
- what is the structure of LPS?
- how does variability change in LPS?
A
- inner membrane, periplasm, peptidoglycan and outer membrane proteins
- densely packed with proteins, houses peptidoglycan and isolates potentially harmful enzymes
- porins and lipopolysaccharide (LPS)
- membrane anchor Lipid A with a core polysaccharide region with a repeating oligosaccharide called the O-antigen
- LPS gets more variable the further away from the membrane
15
Q
Importance of Lipid A in gram negative cell surface
- what component of Lipid A is used in immune response?
- what two forms are there?
- give a specific immune response that Lipid A is involved in
A
- Pathogen-associated molecular patterns (PAMPS) in polysaccharides are recognised by the immune system
- soluble (manna binding proteins) and membrane bound (toll-like receptors)
- Lipid A stimulates TLR-4 to trigger an inflammatory response which can cause a bloodstream infection and endotoxinic shock
16
Q
Importance of O antigen in gram negative cell surface
- why is the variability of the O-antigen useful
A
- allows bacterial identification. Useful in vaccine development
17
Q
Gram staining
- what is the gram staining process
- what is the outcome?
- why do we add ethanol/acetone?
A
- bacteria are heat fixed, bacteria are stained with crystal violet, bacteria are fixed with Gram’s iodine, bacteria are decolourised with ethanol/acetone, bacteria are counterstained with safranin
- gram positive bacteria are stained purple, gram negative bacteria are stain ed pink
- ethanol/acetone dissolves outer membrane of gram negative bacteria causing stain to be lost, tick layer of peptidoglycan in gram positive bacteria prevents this
18
Q
S layers
- how are they assembled
- where are the found on the cell surface?
- what is its purpose?
A
- into crystalline arrays
- on the outermost layer
- forms pores with a sharp exclusion limit
19
Q
Binary fission
- what are the steps of binary fission?
- how long does each cycle usually take?
- what is the equation for exponential growth?
A
- cell elongation and chromosome replication, septum formation, cell division
- 20-30 minutes
- Nt = No x 2^n
Nt = population at time t
No = population at time 0
N = number of generations between time 0 and time t
20
Q
FtsZ protein
- what forms does it take in vitro and in vivo?
- what happens when Fts genes become mutated?
- which triose phosphate does it use?
A
- forms filaments in vitro and forms a Z ring in vitro
- mutants show a filamentous form where septum formation is disrupted so cells are elongated to become filamentous
- GTP
21
Q
Z ring formation
- what protein makes up the Z ring?
- where does the Z ring form?
- what is a divisisome?
- what is the divisisome subassembly structure?
A
- FtsZ
- at the midpoint of the cell
- proteins involved in cell division
- FtsA and ZipA attach FtsZ to inner membrane. FtsA also recruits other proteins.
FtsI is involved in peptidoglycan synthesis
FtsK is involved in chromosome separation
22
Q
How Fts proteins locate the midpoint of the cell
- what are the two mechanisms used to achieve this?
- how does nucleoid exclusion work?
- How does the cell ensure it chooses the midpoint?
A
- nucleoid exclusion and MinCDE system
- SlmA/Noc proteins prevent cell division in the vicinity of the two nucleoids. This means that the FtsZ ring can only form in the middle or at the two end points of the cell
- MinCDE system
23
Q
MinCDE system
- what is it used for?
- what are its components and what is their function?
- how does the system work?
A
- to ensure the FtsZ ring is located at the midpoint of the cell
- MinC - interacts with FtsZ to inhibit Z ring formation
MinD - is an ATPase. Associates with the membrane and MinC to form membrane bound MinCD complex
MinE - binds MinD and activates ATP hydrolysis. Forms a dynamic ring structure that oscillates from one pole of the cell to the other - MinE moves up and down the cell quickly, disrupting the MinCD complex via ATP hydrolysis causing a high concentration of Min proteins at each pole of the cell meaning the FtsZ ring cannot form there and therefore forms down the midpoint.
24
Q
Bacterial cell division - separation
- how does the FtsZ ring split the cells?
- what happens to FtsZ after this?
A
- it constricts
- it depolymerises via FtsZ hydrolysing GTP
25
Q
Spores
- what is the toughest form of spore?
- What type of bacteria can make spores?
- what is the model system for sporulation?
A
- Endospore
- gram positive
- Bacillus
26
Q
Sporulation process overview
- describe the process of sporulation
A
- DNA is duplicated. Unequal division of the cytoplasm by septum formin at one pole of cell. Smaller compartment is engulfed by larger compartment forming a double membrane. Peptidoglycan deposited between two membranes forming the cortex. Protein deposited on outside of spore to form coat layers. Cell lyases and spore is released
27
Q
Spore anatomy
- describe the anatomy of a spore
A
- core surrounded by an inner membrane, surrounded by the cortex, surrounded by the coat layer, surrounded by expos porins in some cases
28
Q
Spore anatomy - formation of cortex
- what is the spore made out of? How is it modified?
- how is cross-linking different in the cortex and why?
- when does cortex formation occur?
A
- modified peptidoglycan, no teichoic acids
- cross-linking is light, allows cortex to be elasticated - can withstand shrinking and expanding
- during dehydration of cortex
29
Q
Spore anatomy - accumulation of spore specific contents
- what two compounds are found accumulated in spores?
- how are these two compounds found?
- what does this cause?
- why does it do this?
A
- calcium and DPA
- in complex with each other
- dehydration of the core and therefore formation of cortex
- dehydration reduces metabolic process in the spore
30
Q
Spore anatomy - protection of DNA
- what molecules protect DNA in spores?
- what forms is it found in? Which one is more important for survival?
- what does it provide resistance to?
- how does it do this?
A
- small acid soluble proteins (SASPs)
- alpha and beta. Alpha
- UV
- binds to DNA and prevents pyrimadine dimers which are caused by UV radiation
31
Q
Sporulation in pathogens
- what bacteria causes tetanus?
- what bacteria causes antibiotic associated diarrhoea?
- what bacteria is weaponised as anthrax?
- what bacteria causes botulism poisoning
A
- Clostridium tetani
- Clostridium difficile
- Bacillus anthracis
- Clostridium botulinum
32
Q
Sporulation initiation
- what receptors recognise stimulants for sporulation?
- what do they trigger? Describe this process
A
- histadine kinases A-E
- phosphorylation cascade. Histadine kinase phosphorylates Spo0F. Spo0F phosphorylates Spo0B. Spo0B phosphorylates Spo0A. Spo0A activates key genes involved in sporulation
33
Q
Sporulation initiation - histadine kinases
- what doe histadine kinases A and B do?
- what does KinC do?
- what does KinD do?
A
- trigger the phosphorylation cascade by phosphorylating Spo0F
- directly phosphorylates Spo0A
- acts against Spo0A
34
Q
Sporulation initiation - KinA
- what are PAS domains?
A
- domains on KinA that recognise stimulants that trigger sporulation
35
Q
Sporulation initiation - Spo0A
- what domain does it posses?
- how many genes does it directly regulate?
A
- DNA-binding domain
- 121
36
Q
Gene regulation during sporulation
- what factor leads to asymmetric division of the septum?
- what factors are expressed in the larger compartment and smaller compartment?
A
- sigma factor H
- sigma factor E and sigma factor F
37
Q
Spore germination
- describe the process
- what triggers this?
A
- Ca-DPA complex is released causing hydration of the cortex which causes the cortex to hydrolyse. The core begins to expand, SASPs begin to degrade until the core outgrows the coat and becomes freed
- initiated by nutrients such as sugars, amino acids and purines
38
Q
VanS/VanR
- what is vancomycin and how does it affect bacteria?
- what type of bacteria does it affect and why?
- explain the two component sensory system resulting in bacterial resistance to vancomycin
A
- it is a glycopeptide antibiotic that binds to D-Ala-D-Ala on the terminus of Lipid II preventing crosslinking of peptide chains in peptidoglycan causing a weaker cell
- gram positive bacteria as they have no outer membrane which blocks vancomycin from entering
- VanS responds to vancomycin and auto phosphorylates histidine residues on itself, the phosphate group is passed on to aspartame residues on VanR which promotes the transcription of more VanS and VanR alongside vanHAX genes causing D-Ala-D-Ala to be swapped for D-Ala-D-Lac preventing vancomycin from binding
39
Q
EnvZ/OmpR in E.coli
- what does it respond to?
- explain the system
- what is the difference between the two outcomes?
A
- osmotic changes
- unknown osmotic signals are recognised by EnvZ which auto phosphorylates itself on histadine residues, this phosphate group is passed on to OmpR. With low levels of activation OmpR expressed ompF and in high levels of activation OmpR expressed ompC.
- ompF creates larger pores that ompC so more molecules can be absorbed when there is low osmolarity
40
Q
Salmonella PhoQ/PhoP
- what is it in response to?
- explain the system
A
- cationic antimicrobial peptides from the host
- PhoQ recognises cationic antimicrobial peptides from the host and auto phosphorylates itself on histidine residues, the phosphate groups is passed on to PhoP which causes the expression of pag and RPG genes which cause the structural remodelling of the salmonella outer membrane making it more resistant to the hosts immune system
41
Q
Quorum sensing
- what is quorum sensing?
A
- a mechanism to asses population density and respond accordingly
42
Q
Quorum sensing in gram positive bacteria
- name an example
- what is competence
- what type of sensing molecules are used by gram positive bacteria?
- what is the signal molecule?
- explain the mechanism for quorum sensing for competence
A
- competence in streptococcus pneumoniae
- uptake of free DNA in the environment and expression of its phenotype
- protinaceous compounds
- competence sensing protein
- ComD interacts with CSP causing transphosphorylation of its histidine residues. The phosphate group is passed on to ComE which causes the expression of more ComD and ComE, ComX which is a sigma factor for genes associated with competence and ComC which produces more CSP which is released into the environment.
43
Q
Quorum sensing in gram negative bacteria - overview
- explain the basic mechanism
A
- AHL freely diffuses into the cell and interacts with activator proteins which interact with the chromosome which produced quorum specific proteins and AHL synthase which releases more AHL into the environment
44
Q
Quorum sensing in gram negative bacteria - Vibrio fischeri
- what does quorum sensing in V.fischeri produce?
- where is the bacteria found?
- what is the signal molecule?
- explain the mechanism
A
- bioluminescence
- in the light organ of the Hawaiian bobtail squid
- N-acyl homoserine lactone (AHL)
- AHL freely diffuses into the cell and is recognised by LuxR which interacts with the Lux box on the genome causing the expression of LuxICDABE. LuxI is an AHL synthase that produces more AHL into the environment. LxABCDE are all involved in the production of luciferase which causes bioluminescence
45
Q
Quorum sensing in gram negative bacteria - AHL structures
- what is the AHL in V.fischeri and what is its function
- in P.aeruginosa ?
- in A.tumefaciens?
A
- LuxI bioluminescence
- LasI - virulence factors
- TraI - transfer of Ti plasmids
46
Q
Quorum sensing in gram negative bacteria - Vibrio harveyi
- what are the two sensing systems and how are they different?
- what is the signal molecule in AI-2 signalling system?
- what happens at low cell density?
- what happens at high cell density?
- what is the function of luxR?
A
- AI-1 species specific, AI-2 general
- AI-2 signal
- AI-2 signal is recognised by LuxQ and kinase activity is activated so a phosphate group is passed onto LuxU and then LuxO which causes the expression of 5 sRNAs that destabilise luxR so no luxR protein is produced
- phosphatase activity is activated so the phosphate group is passed from LuxO to LuxU to LuxQ so luxR is not longer destabilised and can produce luxR protein
- translational activator controlling up to 50 genes
47
Q
Integral Outer Membrane Proteins - functions
- what are the four functions of OMPs?
A
- transport, catalysis, structural and adhesion
48
Q
OMP basic structure
- what secondary structure do OMPs form?
- what type of residues are found on the other surface and why?
- where would you find the N and C terminus of an OMP and why?
A
- a beta barrel
- hydrophobic residues to embed the OMP inside the lipid membrane
- around the same position as it forms a closed barrel
49
Q
OMPs- Porins
- what type of transport do they do
- what type of molecules do they transport?
- how many beta strands do they have?
- give an example of a porin in E.coli
- what two additional structures does it have and what is their function?
A
- passive transport
- small molecular weight solutes e.g. salts, monosaccharides
- 16 and 18
- OmpF
- a loop region which regulates the size of solute that can pass through the barrel and an eyelet region with charged amino acids which regulates what charges can pass through the barrel
50
Q
OMPs - TonB-Coupled Receptors
- what type of transport are they involved in
- what types of molecules does it transport?
- = how many beta strands does it have
- what additional domain does it have and what is its function
- where does the energy come from?
- explain how it opens
A
- active transport
- iron, heme and vitamin B
- 22
- a plug domain which sits in the lumen of the barrel
- a proton motile force across the inner membrane through TonB
- TonB binds to the plug domain and causes it to dissociate from the barrel allowing molecules to pass through
51
Q
OMPs - FhaC
- what is its function
- how many beta strands does it have?
- what additional structure is found in the lumen?
- what additional structure is found on the intracellular side of the membrane?
- explain the process of secretion
- where does the energy come from?
A
- mediates the secretion of filamentous haemagluttinin (FHA)
- 16
- an alpha helix and a large extracellular loop called L6
- two potra domains
- unfolded FHA binds to the POTRA domains which causes L6 to become displaced from the lumen of the barrel. The POTRA domains then feed FHA through the barrel where it becomes folded on the extracellular side
- the folding of FHA
52
Q
OMPs - Secretins
- how are these different to other OMPs?
- what do they transport?
A
- they do not form beta barrels
- folded proteins
53
Q
Biogenesis of OMPs
- what complex promotes this?
- explain the process
- explain the structure of DegP
A
- Bam complex
- unfolded OMPs are transported into the periplasmic through the Sec translocon. Chaperones such as SurA and DegP stabilise the OMP in its unfolded state and transport it to the Bam complex. The Bam complex the folds the OMP and BamA inserts it into the OM
- DegP has a cage like structure that traps the unfolded OMP inside
