Cell Envelope Biogenesis Flashcards
The major biosynthetic processes involved (23 cards)
Why is cell wall biogenesis challenging to bacteria
- Needs to be made accurately at the highest possible pace
- Built from the inside out so susceptible to lysis
What enzymes make the cell wall
PBP1A and PBP1B
- Class A have TP and TG activity
- Class B have only TP
What 2 complexes allow cell division and growth
Division complex
Elongation complex
What did forward genetics help classify
FtsZ
What method was used to classify FtsZ
Filament temperature sensitive method
- Mutants growing well at 30, become filamentous at 42 as their division complex (not elongation) has MUTATED
- Filter out those that cannot form filaments at 42
- Complement with phages using E.coli DNA that is not temperature sensititve
- Those that can grow at 42 will have had mutations in their division complex
How were FtsZ inhibitor drugs discovered?
Tubulin homologue aspect means cancer drugs targeting tubulin can be modified
- Destabilise polymerisation
- Shown little cytotoxicity in our cells
What did reverse genetics help classify?
MreB
What method was used to classify MreB
Conditional expression system
- MreB was added to an inducible promotor (Xylose)
- (-) Xylose will inhibit MreB expression
- Saw cells could not control cell width and inflated then lysed
- Used immunofluorescence microscopy to show it localised in patches of filament around the cell wall
What is the function of MreB
Finds points of high cell curvature and promotes cell wall synthesis to smooth elongation
- Works alongside MreCD complex
What drugs target MreB
Drugs like A22 target the nucleoid binding pocket = where it binds ATP
MreB is an ATPase, so A22 competing with it will decrease function and lyse
Why does genetic redundancy affect our genetic methods?
Makes forward and reverse genetics difficult as will mean one gene knockout will not cause phenotype changes
Give 3 examples of forward genetics identifying PBP modulator proteins
- Synthetic lethal screen for LpoA/B (E.coli)
Subsequent synthetic lethal screen specific to S.P: CozE and MacP
Describe the synthetic lethal screen for LpoA/B and the synthetic lethal pair used
PBP1B/1A pair
4 aspects:
- Strains without LacZYA and PBP1B made = PBP1A becomes essential
- Random mutagenesis on the strain
- Unstable plasmids were inserted that contain LacZYA and PBP1B
- If a gene essential for PBP1A function is knocked out then the plasmid will have to become essential
- This is seen with a blue phenotype
LpoA identified from this, then LpoB
30,000 mutants made, only 16 colonies showed lethality and of those 2 attributed to LpoA
What synthetic lethal pair is in S.P.
PBP1A and PBP2A
How did the insertions of CozE vary from w.t to mutant?
Insertions were not found in w.t but were found in mutant
- Shows it loses essentiality when PBP1A is lost = only role is regulating it
What is the role of CozE
Coordinator of Zonal Elongation
- Downregulates activity of PBP1A
- Keeps cell wall biosynthesis only at the sites of cell DIVISION and where new cell wall is NEEDED
Why would CozE inhibitors counteract penicillin
CozE inhibitors would increase cell wall synthesis
Penicillin works to stop cell wall synthesis
How did the insertions of MacP vary from w.t. to mutant
Insertions are in w.t but not in mutant
- Becomes essential upon PBP1A deletion as PBP2A is essential
- Its role is dependant on PBP2A
What is the role of MacP
Localises at division site and ACTIVATES PBP2A to function
-StkP phosphorylates MacP which allows PBP2A activation
-Binds Lpo factors as well (not in S.P)
How was Next Gen Sequencing used in synthetic lethal studies?
Tn-Seq
- Used to create pools of transposon mutants (up to 50,000 in one pool)
- Maps the locations of insertions onto one genome
Describe the process of Tn-Seq
- Many unique transposon mutants created (insert transposons using phages)
- Typically MARINER transposon
- Sequence DNA flanking the transposon insertions
- Amplify with PCR
- Massively parallel sequencing sequences them
- Create an insertion library one one genome
Give pros and cons of Tn-Seq
PROS
- widely used
- less laborious than making mutants traditionally
- Can detect absence of data with high accuracy
CONS
- Need a wide genetic variety of insertions: 10^5 unique ones
- Genes involved in pathogenesis typically only consist of 10^3 cells
- Bottlenecking therefore a big problem
Why is the cell wall a good antibiotic:
The cell wall prevents cell lysis, as its tensile forces counteract the osmotic forces of the environment
