bacterial genetics screencasts - transduction and phage therapy

what is transduction?

the mobilisation of genes by bacteriophages

what does mobilisation refer to in transduction?

the transfer of genes from one bacterial cell to another

what is a bacteriophage?

a virus that infects bacteria

how do phage replicate?

by infecting a host bacterial cell

what do viruses consist of in the most basic terms?

genetic material contained in a protective protein coat (the capsid)

what two types of bacteriophage exist?

virulent and temperate phage

what does infection by virulent phage usually lead to?

phage multiplication and death of the host

describe what happens to phage in the lytic cycle (5 steps)

• 1) the phage is absorbed by the host cell

• 2) entry or injection of the genome

• 3) synthesis of phage proteins and replication of its genome (degradation of the host genome)

• 4) assembly of phage in the host cell

• 5) lysis of the cell and release of the free phage

what is a T-even phage?

a bacteriophage with a double stranded DNA genome

how long does the lytic cycle of T-even phage take?

20-30 minutes

what is the purpose of degrading the host genome in the lytic cycle?

ensuring that the cell is devoted to expressing phage, not chromosomal genes

what are generalised transducing phage?

types of virulent phage (not T-even) that can ‘accidentally’ package bacterial DNA into capsids during the lytic cycle

how are generalised transducing phage detected experimentally?

by the frequency that they package bacterial DNA into capsids

why are they called GENERALISED transducing phage?

because they can carry any part of the chromosome

give 2 examples of generalised transducing phage

P1 and P22

what are transducing phage often used for in the lab?

moving chromosomal DNA (often containing a mutation) between strains

what opportunity can transfer of chromosomal DNA from HFR strains by transducing phage set up?

opportunities for recombination

is recombination reciprocal?

no, one cell is the donor, one is the recipient and is changed by the DNA received from the donor

give and example of a temperate phage

the lambda phage

what can temperate phage establish by undergoing the lytic cycle?

lysogeny

what is lysogeny?

the blocking of the lytic cycle via synthesis of a repressor molecule and integration of phage genome into the chromosome

what are the cells resulting from lysogeny called?

lysogenic bacteria/ lysogens

what is a prophage?

the genome of a temperate phage incorporated into the host genome

what can happen to the prophage under conditions of stress?

it can excise from the host genome and enter the lytic cycle

how does the lambda genome integrate into the host genome?

• via a single crossover

• mediated by site-specific recombination

• is highly efficient

how is integration of the lambda genome similar to integration of the F plasmid?

both integrate via a single crossover

how is integration of the lambda genome different to integration of the F plasmid?

• the f plasmid integrates via homologous recombination, not site-specific recombination

• it integrates at a low frequency, whereas the lambda genome is highly efficient

why is homologous recombination not efficient?

the regions of homology are too short to be efficient

what how does lambda recognise specific sequences flanking the core?

it has dedicated recombinases

how does the excision of lambda normally happen?

• is normally a reversal of integration

• using the recombination sites flanking the prophage

what occasionally happens when lambda excises and what does this result in?

• occasionally, it excises with a site beyond the boundaries of the prophage

• this results in the acquisition of of a segment of chromosomal DNA

what is rare abnormal outlooping?

when a phage excises using sites beyond the prophage, picking up a segment of chromosomal DNA

from which directions can DNA be acquired from when excising?

from the left or right

what process is rare abnormal outlooping analogous to?

the formation of F’ plasmids

why is lambda a ‘specialised’ transducing phage?

it can only carry regions from the left or right point of integration (normally called attlambda)

is lambda unique in being a specialised transducing phage?

no, there is evidence that other lysogenic phage can integrate/ excise by homologous recombination

what is the evidence to suggest phages other than lambda can integrate. excise via homologous recombination?

several coliphage (E. coli phage) genomes contain IS elements

what has genome sequencing revealed many phage to have in their genomes?

several genes that are not for their replication

what does phage containing genes not for their replication suggest?

like conjugative plasmids, they are acquiring genes originally from bacterial genomes

why is phage having bacterial genes clinically relevant?

• these could be resistance genes

• sequencing of E. coli 0157:H7 showed it encodes 1632 proteins that are not found in the E. coli lab strain K12

where are the extra genes of E. coli 0157:H7 found and give 1 example?

in lots of clusters, for example the Shiga-like toxin

what are lots of the extra E. coli 0157:H7 genes associated with?

24 different prophages and prophage-like elements

what is an EHEC?

enterohemorrhagic E. coli

why has phage played a significant role in the evolution of EHECs?

they have provided the extra genes needed to encode proteins that cause the enterohemorrhagic diarrhoea

2 examples of promiscuous phages

P1 and Mu

what species can P1 infect?

E. coli and other enteric species

some phages have been found to infect both E. coli and P. aeruginosa, why is this important?

• they are both gram negatives

• but P. aeruginosa is not an enteric species, showing a wider host range

what is phage therapy?

the therapeutic use of lytic phage to treat bacterial infections

why has there been renewed interest in phage therapy?

lots of antibiotics are becoming less useful because of antibiotic resistance

what is phage therapy a potential alternative to?

antibiotics

what is the idea behind the phage therapy process?

• phage have definable host ranges

• so we could use specific phage to infect and kill specific bacteria without any interaction with surrounding human tissue or other harmless bacteria

why is it hoped that only a small single dose of phage would be needed for phage therapy?

phage replicate quickly

why might phage therapy not work?

previous resistance suggests that bacteria may quickly acquire resistance to the phage e.g. by modifying the molecule used by the phage to absorb into its surface

how could phage combat bacterial resistance to them?

phage also evolve at a fast rate so could evolve to recognise the surface molecule on the bacteria again or recognise a new surface molecule

what did the US drug administration approve in 2006 relating to phage?

they approved spraying ready to eat meat with a cocktail of phage as an antimicrobial agent against listeria

what have phage been used for mediacally?

developing therapies to treat bacterial infections e.g. in someone a few years ago with a seemingly untreatable bacterial infection