Aid

Question 1

List the functions of the capsul:

Answer 1

Carbon storage, protection against desiccation, capture of nutrients (acquisition of ions) , attach to surface, exclude phage, anti microbial sand disinfectants, pathogens often capsular and resist phagocytosis.

Question 2

What retains pigment for gram staining +ve ?

Answer 2

Peptidoglycan layer

Question 3

Compare + and - ve:

Answer 3

+ ; thicker, 25 sheets of peptidoglycan , teichoic acid
- : only 10% peptidoglycan in cell wall, between inner and outer membranes.

Question 4

Compare bacteria to archaea cell wall:

Answer 4

Bacteria: peptidoglycan, n-acetylmuramic acid, 1,4 beta linking , degraded by lysozymes or penicillin , d-amino in linkers

Archaea : pseudomerein , N- acetylosaminuronic acid, 1,3 beta linking, not effected by lysozymes or penicillin, no d-amino acids in linkers

Question 5

Different types of flagella:

Answer 5

Monotrichous, amphitrichous, lophotrichous, peritrichous

Question 6

List the different tactic responses:
(Flagella L-6)

Answer 6

Aero taxis , chemotaxis , magneto taxis , photo taxis

Question 7

What are MCP and for what tactic response are they involved in? (Flagella L-6)

Answer 7

Methyl - accepting chemotaxis proteins + involved in the direction and the ‘run and tumble” movement

Question 8

Large complex molecules containing lipid and carbohydrate
Lipid A
Core polysaccharide
O side chain
Best described is from Salmonella
E. coli LPS is nearly identical and also well studied
Also called endotoxin when free in host
Can induce massive immune response (septic shock)
Not a function of LPS but an effect of its interaction with innate immune system

Question 9

what can cause septic shock ?

Answer 9

endotoxin aka Lipopolysaccharide free in host

Question 10

describe the Lipid A on the lipopolysaccharide

Answer 10

it is Made of two glucosamine residues linked to fatty acids and phosphates
it is integrated into the outer membrane
most immunogenic as endotoxin

Question 11

describe the Core polysaccharide “ R-antigen or R-polysaccharide”

Answer 11

In Salmonella, 10 (mostly) unusual sugar residues
Glucose; Galactose; Heptulose (x 2);2-keto-3-deoxyoctonate (x 3)
Side chains of NAG, phosphate and ethanolamine

Question 12

describe the O side chain

Answer 12

• Variable region, responsible for antigenic make-up of bacteria
  Different O serotypes linked to disease
  Species specific attachment to specific receptors
  Extends outwards from the cell
  Lipid A and core polysaccharide are straight and O side chain is flexible and bent
  Highly variable composition (at least 20 different sugars)
  Rough (R)/Smooth (S) variants depend on side chain length

Question 13

Functions of LPS

Answer 13

Lipid A stabilises outer membrane structure
Core polysaccharide charged
Contributes to negative charge on surface
Charged, hydrophilic external layer reduces permeability of hydrophobic substances
e.g. preventing entry of bile salts, antibiotics
Protects against host defences
Rough variants more susceptible to phagocytosis
loss of O-antigen in E. coli and Salmonella leads to reduced virulence
O-antigen very variable, a key diagnostic tool
e.g. E. coli O157:H7; E. coli O104:H4

Question 14

what are endotoxins?

Answer 14

immune responce
Released during cell division or by lysis of bacterial cells
Can act to prime immune system against a pathogen
If in the blood LPS can cause septic shock syndrome
No direct treatment
Immunogenic even in absence of living cells
LPS of some non-pathogens can also be endotoxin
Test for endotoxin
Rabbit pyrogen test
Limulus amoebocyte lysate (LAL) assay

Question 15

properties of endotoxins

Answer 15

Heat stable
Toxic in nanogram amounts
Interacts with innate immune system cells
Can trigger the release of cytokines in a cascade
Activates transcription factors such as interferon-β and tumour necrosis factor (TNF)
Can result in
Inflammation, fever, vasodilation, thrombosis, acute disseminated intravascular coagulation, depletion of platelets/clotting factors leading to internal bleeding (haemorrhage), shock, sometimes death

Question 16

why and what are Limulus polymephus blood cells used for?

Answer 16

Limulus Amaebocyte Lysate – LAL test
- asey for endotoxins / LPS
–Amaebocytes are the blood cells of Limulus polymephus
Contains a clotting factor that is released if coming into contact with bacterial endotoxin
Natural immune mechanism that contains bacterial infection

Question 17

why is the outer membrane of gram - bacteria more permeable then the cytoplasmic membrane

Answer 17

porins –> protein channels that permit passage of small molecules up to around 600 Da
and: carriers & active transport systems → larger molecules attached to, NOT through porins

Question 18

are all porins non selective? give examples

Answer 18

most= non selective

some:
E. coli LamB, a maltose and maltodextrin selective porin
First identified as the receptor for bacteriophage Lambda

Question 19

describe the periplasmic space of bacteria cells

Answer 19

= space between outer membrane and cytoplasmic membrane

Ranges in size from 1 to 70 nm
Can be up to 40% of cell volume
Gel like consistency due to abundant proteins

Question 20

what are the functions of the enzymes in the periplasmic space

Answer 20

*Nutrient acquisition
*Hydrolytic enzymes such as alkaline phosphatase
*Energy conservation
*e.g. electron transport proteins
*Some peptidoglycan synthesis enzymes are periplasmic
*Periplasmic binding proteins
*Deliver specific compounds to ABC transporters in cytoplasmic membrane
*Chemoreceptors
Involved in chemotaxis

Question 21

Protein export into periplasm

Answer 21

Sec pathway
Exports nascent polypeptide through cytoplasmic membrane using a translocase
Folding of protein occurs after translocation
Proteins have an N-terminal signal peptide
TAT pathway (twin arginine translocase)
Exports fully folded enzymes across cytoplasmic membrane
Proteins have a twin arginine in N terminal region

Question 22

describe the flagella

Answer 22

Complex ring structures anchored into the membrane and cell wall/outer membrane
Rings and hook are rigid and attached
Flagellum shaft is easily removed by vigorous shaking
Different antigenic properties of shaft, tip of shaft and hook
The motor is driven due to transfer of protons through the ring structure (from periplasm to cytoplasm)

Question 23

describe flagella synthesis

Answer 23

1. MS and C rings in cytoplasmic membrane
2. Motor proteins
3. P and L ring, hook and cap
4. Flow of flagellin through hook, growth of filament

Question 24

how does the motor work

Answer 24

Rotary motion provided by the basal structures
Mechanism not completely understood
Powered by proton motive force
Protons move across the cytoplasmic membrane through the mot complex
One rotation requires translocation of 1000 protons
Mutational analysis suggests dependence on an Asp residue in MotB
Hypothesis: Changes in charge lead to conformational change of MotA, causing movement relative to MotB
Second conformational change upon loss of charge may provide another step of the motor

Question 25

are the flagella of gram positive bacteria different to gram positive bacteria?

Answer 25

In Gram +ve, no L and P rings
Anchor in the membrane layer is more complex
Mot proteins surround inner ring and movement of these relative to each other provides the force

Question 26

Flagellum antigenic structure

Answer 26

aka - H antigen
Escherichia and Salmonella serotypes defined in part by flagella structure
E. coli O157:H7 best known
Strain causing food-borne illness including hemorrhagic diarrhoea and kidney failure
E. coli O104:H4 caused the 2011 Germany outbreak
Many fewer H serotypes than O serotypes (LPS O polysaccharide) and therefore of limited diagnostic value

Question 27

To identify the proteins in a sample, the typical proteomics experiment breaks proteins into peptides, usually with the protease trypsin.
Tryptic digestion creates many more peptides than proteins, so we need to fractionate the mixture, usually by liquid chromatography (LC), to give the MS time to do MS/MS on as many peptides as possible.
A typical LC system for MS uses a C18 reverse phase column to separate peptides by hydrophobicity before electrospray ionisation (ESI) into MS.
Potential peptide ions are measured (MS1) and selected for fragmentation in a MS2 scan.
Peptide fragmentation patterns are matched to predicted protein sequences. Modifications can be identified by mass additions.
Protein identities are inferred from peptide sequences.