Horizontal gene transfer

Question 1

4 methods of ‘stabilising’ DNA once transferred into a new host bacterium?

Answer 1

1. Persistence as episome.
2. Homologous recombination possible between closely related strains
3. Integration into genome, by bacteriophage, integrase enzyme, or mobile genetic element (transposon?)
4. Or rarely illegitimate incorporation by DSB repair.

Question 2

Contrast transformation in gram positive and gram negative bacteria? (competent strains)

Answer 2

Gram negative bacteria transport bound and fragmented dsDNA across outer membrane by Secretin pore, into periplasmic space.

Both transport DNA in single stranded form across cytoplasmic membrane. (by competence machinery, uptake mechanisms)

Question 3

How do competent bacteria (constitutive or induced) uptake ssDNA across cytoplasmic membrane?

Answer 3

Uptake machinery related to Type II secretion system and Type IV pili.

Repeated cycles of pilus assemby and disassembly drive DNA molecule through membrane channel.

Binding energy of ssDNA binding proteins in cytoplasm provides energy for this process.

Examples PilE in N.gonorrhoea (gram neg, pilQ is secretin) and ComGC in B.subtilis.

Question 4

Compare bacterial conjugation in gram negative and gram positive bacteria:

(describe propogation genes conjugation machinery)

Answer 4

Gram positive mating pair connects via adhesins, whilst gram negatives connect using a male donor with a pilus and female recipient (exconjugant)

both form “mating pair junction” through which ssDNA is transferred (such as the F-plasmid in E-coli) and then replicated by rolling circle replication.

(transfer apparatus: Relaxosome nucleoprotein complex, Relaxase cleaves at OriT (transfer origin) and binds 5’ end on T-strand, Conjugative coupling protein connects relaxosome to Type-IVSS where it recognises relaxase protein for secretion, ssDNA hitches a ride)

Question 5

Natural plasmids like E.coli F-plasmid are modular: so which 4 types/groups of genes do they typically contain?

Answer 5

Stability: Control segregation of plasmids into daughter cells (partitioning) (e.g. one each cell with large F-plasmid)

Replication: ensures replication coordinated with host chromosome replication and controls copy number (too many plasmids –> burden on cell –> -ve selection)

Adaption genes: confer advantage for host

Propagation genes: encode for transfer machinery to transfer plasmid into recipient cell (Type IVSS, Relaxosome)

Question 6

Describe generalised and specialised transduction, and lysogenic conversion:

Answer 6

Generalised transduction occurs when bacteriophage enters lytic cycle (upon entry?) and digests host bacterial DNA. New phages are synthesised and they randomly incorporate DNA (including host DNA) into the phage head. Transducing phages released upon lysis of bacterium, infect other bacteria –> potential integration into host by recircularising plasmid or HR into host chromosome

Specialised transduction occurs with lysogenic cycle (prophage integration into host DNA). When lytic replication is induced the prophage DNA excises imprecisely taking with it adjacent host sequences, which it can then integrate into further bacteria.

Lysogenic conversion is when prophage integration into host genome confers a change in phenotype of bacterium (perhaps virulence factor, or blocking other phage entry)

Question 7

Explain how staphylococcus superantigen-carrying pathogenicity islands SaPIs (carry TSST) can be transferred between bacteria, without having their own genes for transfer:

Answer 7

SaPIs are stably integrated into host bacterial genome thanks to repression by STL transcriptional repressor.

Along comes helper lysogenic phage (excises itself in response to stress) inactivates STL repressor –> excision of SaPI and rolling circle replication

SaPI competes for uptake into helper phage’s phage heads!!