Dale_Molecular Genetics of Bacteria 4th ed

Figure 6.5 Transfer of the conjugative transposon Tn916. The transposon is excised from the chromosome, using the Int and Xis enzymes and circularized. This enables the tra genes to be expressed from the promoter shown and conjugative transfer is initiated from oriT. In the recipient, the transferred DNA is circularized, the second strand is made and the transposon is integrated into the chromosome

plasmid transfer. In particular, Tn916 contains a origin of transfer (oriT) which is quite similar to that found in many plasmids. The first step in transfer is the excision of the transposon from the chromosome, using transposon-encoded enzymes (Int and Xis) which are related to those responsible for the integration and excision of bacteriophage lambda (see Chapter 4). This produces a circular molecule that resembles a plasmid in all but one vital feature – it does not have an origin of replication so is unable to be copied in the normal way. However since it does have an oriT site and carries the tra genes needed for conjugal transfer, it can be transferred to a recipient cell.

As with the plasmid transfer systems described previously, transfer of Tn916 involves single-stranded DNA synthesis initiated at oriT and transfer of the displaced strand to the recipient. The transferred single strand is then circularized and converted to a double-stranded circular form which is inserted randomly into the recipient chromosome by the action of the integrase.

One additional feature of Tn916 is worth considering. If transfer occurred without excision from the chromosome then mobilization of the chromosome would be expected to occur with incomplete transfer of the transposon. Transfer would start from oriT and would have to work right round the chromosome before reaching the rest of the transposon. This does not seem to happen. The reason is that the promoter for expression of the tra genes is found towards the left-hand end of the transposon (in Figure 6.5) and faces away from the tra genes. In the integrated linear form the tra genes will not be expressed. However, when the transposon is excised from the chromosome and circularized, this brings the promoter into the correct position and orientation for transcription of the tra genes. They will therefore be expressed from the circular intermediate, but not from the integrated form. This ensures that the transfer system will only be activated after excision has occurred.

Tn916 is the prototype of a family of related conjugative transposons that are especially widespread in Gram-positive cocci, although related elements also occur in Gram-negative bacteria (e.g. Bacteroides). For many of these elements, including Tn916, conjugative transmission is promiscuous in that they can transfer to other species or genera. It can be assumed therefore that conjugative transposons have played a significant role in the dissemination of genetic material, including antibiotic resistance genes, throughout the bacterial kingdom. In particular. many of these transposons, including Tn916, carry a tetracyclineresistance gene (tetM) which is found in a wide range of bacterial species, suggesting that they have played a role in the dispersal of this particular gene.

6.3 Transduction

Transduction is the phage-mediated transfer of genetic material. The key step in transduction is the packaging of DNA into the phage heads during lytic growth of

the phage (see Chapter 4). This process is normally highly specific for phage DNA. However, with some phages, errors can be made and fragments of bacterial DNA (produced by phage-mediated degradation of the host chromosome) are occasionally packaged by mistake leading to phage-like particles that contain a segment of bacterial genome (see Figure 6.6). These transducing particles are

Replication of DNA

Donor bacterium

Synthesis of phage particles

Lytic infection

Packaging of DNA into phage heads

Incorporation of

chromosomal DNA

Lysis

Transducing particle

Infection of recipient

Recombination with host DNA

Transducing DNA fragment

Figure 6.6 Generalized transduction

Figure 6.8 Homologous recombination: the Holliday junction. Structure iii from Figure 6.7 (iiia) is bent to an X shape (iiib) and the lower half is rotated to produce the Holliday junction (iiic). Resolution occurs by cutting the DNA at the arrowed points, producing the recombinant molecules shown (iv)

degradation is then inhibited, leaving a single-stranded tail that is able to participate in strand invasion (with the assistance of RecA). These sites are therefore hot-spots for recombination. In E. coli, this sequence is

50GCTGGTGG30 (but may be different in other bacteria). An eight-base sequence would be expected to occur within 65 kb on average if randomly distributed and most of the fragments generated during chromosome transfer by conjugation will be large enough to be likely to contain a site. However, when smaller fragments are involved the absence of a site in the DNA may limit the amount of recombination observed. This may be the case during transduction for example, and even more so during genetic manipulation experiments such as gene replacement (see Chapter 10).

Three proteins, RuvA, RuvB and RuvC, are responsible for events at the Holliday junction. RuvA binds to the Holliday junction and stabilizes the structure needed for the subsequent events, while RuvB is a helicase that unwinds the adjacent DNA, enabling the junction to migrate along the DNA (thus increasing the extent of the heteroduplex). RuvC is the nuclease responsible for cutting the DNA strands as required for resolution of the Holliday junction.

A different pathway (although still RecA-dependent) is required for repair of single-stranded gaps in the DNA, as described in Chapter 2. RecBCD is not able to participate in this system which uses instead RecF and several other proteins to prepare the single-stranded DNA for the loading of RecA which is needed for invasion of the sister strand.

Consequences of recombination

Finally, it is necessary to relate these mechanisms back to the events described earlier in this chapter. For recombination between a linear DNA fragment (introduced by transformation, transduction or conjugation) and the recipient chromosome, it is necessary for two such events to occur (see Figure 6.9) so that a portion of the linear fragment will become integrated into the circular chromosome replacing the corresponding region of the chromosome.

Figure 6.9 Recombination between a linear DNA molecule and a circular molecule. Recombination at two sites leads to replacement of a portion of the circular molecule. The reaction therefore requires at least two regions of homology

On the other hand, if both participating molecules are circular (e.g. two plasmids or a plasmid and the chromosome), then a single recombination event will suffice (see Figure 6.10) producing a fusion of the two original circles. This may be a reversible event. Recombination between the two ends of the inserted plasmid will lead to excision of the plasmid, as discussed earlier in relation to the integration of the F plasmid and also the integration and excision of phage l (Chapter 4).

In this discussion it has been assumed that two different molecules were involved, i.e. intermolecular recombination. What happens if the two homologous regions are on the same molecule, i.e. recombination is intramolecular? This will happen if there are two copies of a repetitive element such as an insertion sequence (see Chapter 7). The consequences will depend on the relative orientation of the two homologous sequences. As can be seen from Figure 6.11a, if the homologous regions are in the same orientation (direct repeats), recombination between them will result in separation into two separate circular molecules. (This can be followed by tracing the course of the DNA in the intermediate form where the two regions are paired, starting from point A and switching strands when the paired region is reached again. The course of the DNA thus misses B and C and continues straight to D and back to A). In general, one of these circular molecules will not contain a replication origin and so will be lost. The consequence is therefore a deletion of that portion of the original DNA.

On the other hand, if the homologous regions are in opposite orientations (inverted repeats), as in Figure 6.11b, recombination leads not to separation of two molecules but to inversion of the region between the inverted repeats. The circular molecule remains intact.

Figure 6.10 Recombination between two circular DNA molecules leads to integration. Recombination at a single site (a single crossover) is sufficient for integration

(a) Recombination between direct repeats leads to deletion

C B

C B

A B

Deletion of B-C

C

A D

A D

D

(b) Recombination between inverted repeats leads to inversion

B C

A B A C

C

B

A D

D D

Inversion of B-C

Figure 6.11 Intramolecular recombination between repeat sequences. (a) Recombination between two direct repeats (arrowheads) produces two separate molecules, i.e. deletion of B–C (if this structure is unable to replicate), or resolution, if B–C forms a viable replicon. (b) Recombination between two inverted repeats leads to inversion of the region (B–C) between the repeated sequences

The presence of repetitive elements can cause deletions or inversions of chromosomal regions in this way, as well as being a cause of plasmid rearrangements as described in Chapter 5. For example, if the chromosome contains two copies of an insertion sequence in the same orientation and quite close together, then recombination between the two IS elements will lead to deletion of the region of the chromosome between them. This can be a significant cause of variation between bacterial strains, as in Mycobacterium tuberculosis where much of the variation between strains arises from deletions due to recombination between insertion sequences.

6.4.2Site-specific and non-homologous (illegitimate) recombination

Recombination between DNA molecules can occur in a variety of other ways which are not dependent on the presence of extensive regions of homology nor on