Lecture 4 - the nucleoid and cell division Flashcards
What is notable about E. coli O157:H7?
Produces Shiga toxin –> bloody diarrhea and hemolytic uremic syndrome in humans.
Where does E. coli O157:H7 come from?
Bovine rectum
How did E. coli O157:H7 acquire the ability to produce shiga toxin
Uptake of a prophage gene that encoded the toxin
E. coli K-12 genome vs E. coli O157:H7
K-12 (lab strain) doesn’t have the same virulence or host interaction factors. O157 (virulent strain) genome is 20% larger due to insertion of virulent factors.
Variance of bacterial genome within a species
Two members of the same species can vary by up to 40% of their genome
Where is mycoplasma genitalium found?
Only found in the human genital urinary tract; sexually transmitted disease
Why is the genome of mycoplasma genitalium so small?
Loss of traits including peptidoglycan production because it only lives in an iso-osmotic fluid in the urinary tract. PG isn’t needed to protect against osmotic pressure.
What makes the genes in plasmids (episomal) different than chromosomal genes?
Generally not critical for life, but can give bacteria advantageous traits. Can be gained and lost depending on the circumstance.
E. coli vs human genome
Density: E. coli genome has a much higher concentration of coding sequences than human (more introns and repeats, only 1% of genome is actually coding)
Length: human genome is 1000x larger than E. coli (but only 5x more genes)
What defines a gene as “essential?”
The definition of “essential” is very conditional.
- Some genes are necessary in any circumstance (ex: ileS)
- Some gene deficiencies can be compensated for by the addition of the gene product (ex: dapB)
- Some genes are only necessary in certain conditions (ex: dnaK only helps above 37 C)
Function of dapB gene
Encodes diaminopimelic acid (peptide involved in PG crosslinking)
Eukaryotic vs prokaryotic storage of DNA
- Eukaryotes have a membrane-bound nucleus
- Prokaryotes have a nucleoid region extending through the cytoplasm
Purpose of macrodomains in bacterial chromosomes
Certain regions of the genome need to be kept in a specific 3D orientation relative to one another. Macrodomains need to be spatially organized
Mechanism of studying relative localization of genome macrodomains in bacteria?
Fluorescence labeling with sequence-specific fusion proteins. Place the sequence that the protein binds to anywhere in the chromosome.
What are ParB (binds parS), LacI (binds lacO), TetR (binds tetO) used for?
Can fluorescence tag these proteins to mark regions of the DNA for study of 3D orientation of certain sequences in the bacterial chromosome in relation to one another.
Limits of fluorescence labeling in determining relative position of two parts of the bacterial chromosome?
If the dots are below 200 nm apart (below the Abbe diffraction limit), we can’t resolve the dots as separate. We can only differentiate things that are over 200 nm apart to prove that two sequences are kept apart in the cell
What helps neutralize the charge of DNA for close-packing in bacteria?
Polyamines (putricine and spermine) and magnesium; positive charge neutralizes negative DNA
What is H-NS?
Histone-like nucleoid structuring protein that aids in DNA packing in bacteria
Toroidal vs plectonemic coiling
- Toroidal creates crossovers between nearby regions of DNA
- Plectonemic creates crossovers between far apart regions of DNA
What is SeqA?
DNA binding protein that regulates DNA synthesis and therefore bacterial division. Binding sites excluded from the Ter macrodomain
What is SlmA?
DNA binding protein involved in positioning chromosomes for cell division. Binding sites are located in the Ori and NS-right and NS-left macrodomains. Involved in nucleoid occlusion.
What is MatP?
“Macrodomain ter protein”
Binding sites are only found in the Ter domain of the chromosome
Process of DNA replication in prokaryotes
- Replication begins at the origin (oriC) site
- Two replication forks are generated as two replisomes (groups of DNA polymerase molecules and other proteins) travel in opposite directions from the origin
- Replication is terminated by ter sequences and the Tus (ter utilization substance) protein which acts as a roadblock to stop replication.
- Result: two linked daughter chromosome that must be separated by topoisomerase IV or Xer resolvases
How fast can DNA polymerase make DNA?
1000 base pairs per second, therefore 2000 base pairs are made per second during chromosome replication
How does DNA replication keep up with growth rate of fast-growing bacteria?
In good conditions, bacteria will begin a second round of DNA replication before the first is complete. Daughter cells will have already begun the process of DNA replication before full separation.
Relationship between gene prevalence in bacteria and location on the chromosome
Genes closer to the oriC are usually seen in higher levels due to constant gene replication. Up to 4 copies of a gene could be be present at a given time.
Differences between prokaryotic and eukaryotic cell division
Prokaryotic: Cells mostly undergo binary fission, involving cell wall expansion and septation into daughter cells. Continue synthesizing RNA and proteins during DNA replication
Eukaryotic: Mitosis, stops protein and RNA synthesis during DNA replication
What is the septum? (cell division)
Dividing partition that grows inward and constricts to seal off the two daughter cells
What is septation triggered by?
Replication of the termination site
MreB (cytoskeletal protein)
“Mecillinam resistance”
- structurally related to actin, may form similar filaments for DNA organization
- ESSENTIAL for rod-shaped bacteria, may coordinate PG synthesis
FtsZ (cytoskeletal protein)
“Filamentous temperature sensitive”
- tubulin homolog that forms a circumferential ring at the division septum
- UNIVERSALLY ESSENTIAL FOR BACTERIA
What happens if bacteria lose FtsZ?
Bacteral morphology becomes long and filamentous; able to grow, but unable to divide
CreS (cytoskeletal protein)
aka crescentin
- forms polymer along the inner side of crescent-shaped bacteria (ex: caulobacter crescentus)
What is the divisome?
Multi-protein complex required to form a contractile ring
Important proteins involved in the divisome
- FtsZ, FtsW, FtsI, FtsK-C
- MinCD
Function of FtsW in the divisome
Acts as a PG synthase only during division
Function of FtsI in the divisome
Makes peptide crosslinks in the new PG; inhibited by penicillin
Function of FtsK-C in the divisome
Binds DNA that is still at the mid-cell and pumps it over to the proper side to avoid DNA cleavage during septation
Function of MinCD in the divisome
System that regulates where the divisome is in the cell; keeps the divisome at the mid-cell
How does the Min system control localization of the divisome?
Min C and D oscillate between the ends of the dividing cell to form patches that inhibit polymerization of FtsZ. Therefore, FtsZ can only polymerize and form the division septum in the middle of the cell
What happens if you inhibit Min C and D?
Septation can occur at the ends of the cell, leading to a budding action and defective dvision due to division at the poles
What is nucleoid occlusion?
Mechanism to prevent division septa from closing if the bacterial chromosomes haven’t been separated to the daughter cells properly.
How does nucleoid occlusion work?
SlmA (synthetic lethal with min) binds DNA near the division site. If FtsZ comes into contact with SlmA, it depolymerizes and prevents the cell from guillotining the chromosome.
What is a baeocyte?
Offspring cell of certain cyanobacteria that undergo multiple rounds of chromosome replication before multiple rounds of cell division
Bdellovibrio method of cell division
Obligate predator. Invades the periplasm of Gram - cells and degrades prey cell material. Cell division doesn’t occur until it has grown in a prey cell.
What makes pedomicrobium and plantomyces different in terms of cell division?
No FtsZ
Vivipary
“Live birth”
- cell division involving daughter cells maturing and developing inside the mother cell
Example of vivipary
Epulopiscium: daughter cells divide from the cell poles and develop in the mother cell. Mother deteriorates to release daughter cells
