L7. S. pneumoniae Molecular Genetics 1 Flashcards
Can a compound bind to multiple PBPs at once? Why is this?
PBPs share enough homology where one compound can bind to 6 at once.
What is targeted by antibiotics for the cell wall?
Can target PBPs enzymes directly or can target the pathways more generally.
What is the building of the bacteria cell wall an example of and what can this be compared to in other gram positive bacteria?
- In S. pneumo, PBP enzymes are coordinated in time and space to build the PG-cell wall.
- Similar to the coordination seen for Staph A regulation of surface proteins and toxins
- Similar to the coordinated sporulation operons in C. Diff.
What needs to happen to the PG-cell wall during growth and division?
This needs to be enlarged for growth and division.
Describe how most bacteria grow in 3 steps?
> Bacterial cells grow and divide through alternating cycles of cell elongation and division (most bacteria do this):
- Elongate to around twice their cell volume
- Bacteria cell wall synthesis goes between elongation and division mode
- Switch to division mode, divide into two equally sized daughter cells.
What are the differences for how S. Pneumo divides compared to other bacteria
Pneumo divide like other bacteria, the only difference is how much freedom is given to the elongation complex, giving it a round shape.
What are the two main complexes controlling how most bacterial cells divide and elongate?
Elongation complex and Cell division complex.
What is the central protein in the Elongation Complex of E. coli?
MreB protein.
What protein mainly coordinates the Cell Division Complex in E. coli?
FtsZ.
How do the Elongation and Division complexes in S. pneumoniae compare to those in E. coli?
In S. pneumoniae, the Elongation complex is not centered around MreB but most other proteins are the same. Both division complexes are primarily coordinated by FtsZ.
What can be achieved by inactivating or targeting components of the cell division complex?
Dysregulation of PBP enzymes, potentially causing cell wall damage and making PBP proteins dysfunctional.
What PBPs make up the elongation complex in S. pneumo?
PBP2B and PBP1A
What PBPs make up the division complex in S. pneumo?
PBP2X and PBP2A
Which proteins are indirect targets in the Elongation complex to disrupt PBP2B and PBP1A in S. pneumoniae?
RodA and CozE.
Which proteins are indirect targets in the Division complex to disrupt PBP2X and PBP2A in S. pneumoniae?
FtsZ and DivIVA.
What was 1) forwards genetics 2) reverse genetics 3) synthetic lethality (old method) 4) Synthetic lethality and next-generation sequencing (TraDIS) used to find in S. Pneumo
- Forward genetic
>Discovery of FtsZ genes important for cell division machinery - Reverse genetics
>Characterisation of MreB genes and RodA genes, important for cell elongation machinary - Synthetic lethality used to find:
>LpoA/LpoB important cell wall regulatory factors - Synthetic lethality and next-generation sequencing (TraDIS):
>CozE and MacP
What is the general process of a forward genetic approach in bacteria?
1) Random mutagenesis using agents like chemicals, UV light, transposons, or CRISPR.
2) Observation of a phenotype, such as loss of cell wall growth.
3) Identification of the gene responsible for this phenotype.
What are common methods used for mutagenesis in the forward genetic approach?
Chemical agents, UV light, transposons, and CRISPR.
What is a fundamental problem when studying essential growth processes in bacteria?
Cells must survive to be available for study.
How is the problem of studying essential bacterial processes, where cells cannot grow, overcome?
By using conditional phenotypes.
What are the 2 conditional phenotypes essential for studying essential processes?
- Permissive: cell lives
- Non-permissive: cell dies
Explain 3 examples of permissive conditions?
- Adding Magnesium to the media:
>Magnesium stabilizes cell membranes, helping bacteria withstand more cell wall damage. This allows the cells to survive even if they have defects that would normally cause them to die. - Temperature
>Keeping the bacteria at a cooler temperature can reduce the likelihood of cell death. Some bacteria may be more viable at lower temperatures because it reduces metabolic stress or the activity of harmful enzymes. - +/- antibiotics
>Adjusting the presence or absence of antibiotics can also create conditions where bacteria with certain defects can survive. For instance, removing antibiotics can help cells that are sensitive to them to live and grow.
What is the purpose for 1) permissive 2) non-permissive conditions?
- Permissive conditions
>The key concept here is that conditional phenotypes allow researchers to study essential processes by creating environments (permissive conditions) where bacteria can survive despite having defects. This way, researchers can manipulate the bacteria and observe the outcomes. - Non-permissive conditions
>Conversely, when bacteria are placed in non-permissive conditions, the defects cause them to die, which helps in identifying and understanding the roles of specific genes or processes in bacterial survival.
What genetic approach did Jo Lukenhaus use in the 1970/80s to search for genes involved in bacterial cell division in E. coli?
A forward genetic approach.
What method did Jo Lukenhaus use to induce random mutations in E. coli?
Mutagenesis carried out by Nitrosoguanidine.
What specific phenotype did Lukenhaus screen for in his random mutants?
Cells that grew and divided at 30 degrees (permissive condition) but could not divide and died at 42 degrees (non-permissive temperature), known as filament temperature-sensitive “Fts” mutants.
What challenge did Dr. Lukenhaus face with his mutated E. coli strains?
He had strains with mutations in essential division processes but did not know which genes were responsible for the phenotype of being able to grow at 30 degrees but not 40.
What method did Dr. Lukenhaus use to identify the genes responsible for the phenotype of being able to grow at 30 degrees but not 40?
Phage complementation.
