Transposons and plasmids combine for bacterial chromosomal transfer.

A mechanism that facilitates the horizontal transfer of large segments of chromosomal DNA has been discovered in natural isolates of the pathogenic bacterium Yersinia pseudotuberculosis.

Although horizontal gene transfer (HGT) is recognised as a major force in bacterial evolution, and many mobile genetic elements underlying genomic plasticity have been characterised, the mechanisms by which genetic exchange of chromosomal DNA occurs in natural populations have remained largely hypothetical.

In experiments analysing the mobility of a pathogenicity island (HPI) in Yersinia pseudotuberculosis, Lesic and Carniel (2005) discovered that HPI could be transferred between natural isolates in a process that occurred optimally at 4˚C. However, this process didn’t require integration/excision machinery encoded in the HPI, and also transferred adjacent sequences (>46kb). To test whether this process was specific to the HPI region, Lesic et al inserted various antibiotic resistance markers equidistantly within the Y. pseudotuberculosis chromosome. When this strain was co-incubated with a naïve recipient strain, transfer of these markers was observed in ‘transconjugants’. None of the transconjugants acquired more than one of the antibiotic resistance loci, showing that the transferred chromosomal fragments were less that 1.5Mb in size. The transfer mechanism was also capable of mediating the transmission of a non-mobilisable plasmid (pUC4K). Together these results showed the existence of a mechanism for generalised DNA transfer that functioned at low temperature (termed GDT4).

Lesic et al. found that the GDT4 performing strain contained a very high molecular weight plasmid (≥100Mb). When this plasmid was removed (‘cured’) from the strain, GDT4 was abolished. They therefore termed this plasmid pGDT4. pGDT4 was transferred during transconjugation experiments and was able to confer the ability to transfer chromosomal DNA.

Electron micrograph of aggregating Y. pseudotuberculosis. White arrows point to bridge-like structures.

Sequencing of pGDT4 showed that it contained genes involved in conjugation (the process by which a pilus acts as a conduit for transfer of DNA between cells). When part of this conjugative machinery was deleted GDT4 ability was lost. Interestingly though, pili could not be observed by electron microscopy, and strong shaking of cultures (predicted to disrupt pilus-mediated interactions) had no effect. Instead, the bacteria were seen to tightly aggregate, and seemed to be connected by ‘bridges’.

Organisation of pUC4K plasmids from transconjugants, showing novel IS insertions and IS mediated novel organisations.

pGDT4 also contains a number of Insertion Sequences (IS, short bacterial transposable elements). None of these IS were present on the bacterial chromosome or pUC4K (the non-mobilisable plasmid). After GDT4 transfer of pUC4K, it was observed the size of pUC4K was often altered. Of 10 transconjugant pUC4Ks measured, 3 were the original size, but (pGDT4 derived) IS insertions had lengthened the others. In 5 cases a single copy of ISYps1 had been acquired by pUC4K, whilst in 1 case a large section of pGDT4 had been inserted between two ISYps3 elements.

ISYps1 and ISYps3 (members of the IS6 and Tn3 families respectively) transpose by a mechanism termed replicative transposition. An intermediate stage during this mode of transposition is the formation of a ‘co-integrate’ in which the donor and target replicons (in this case pGDT4 and pUC4K) are fused by 2 copies of the transposon. The cointegrate is then resolved by homologous recombination, leaving a new copy of the transposon on the target and the original still in place on the donor.

Model describing ISYps1 transposition mediated mobilisation of pUC4K via cointegrate formation.

These observations suggested that GDT4 occurs by cointegrates of pGDT4 and other replicons (bacterial chromosome or other plasmids) being formed during IS transposition and then being transferred to recipient cells by conjugation. In further experiments, Lesic et al. confirmed that replicative transposition of ISYps1 was capable of  driving cointegrate formation between different plasmids in E.coli, that could also be transferred by conjugation.

This mode of chromosomal transfer is very similar to the ‘Hfr‘ system characterised in E. coli. In Hfr strains, transfer of chromosomal DNA occurs by conjugation because the conjugative plasmid ‘F’ becomes integrated into the chromosome. Integration of F occurs by homologous recombination at IS sequences shared between the plasmid and the bacterial chromosome. The difference in GDT4 is that no sequence identity between the two replicons is necessary. ISYps1 transposition does not seem to target any particular sequence. This makes this mode of chromosomal conjugative transfer less constrained than Hfr and potentially more powerful. It is notable that plasmids often carry a large number of IS. For instance, a plasmid in Shigella carries 93 copies of IS of 21 different types. The ability to confer chromosomal DNA transfer may be a selective advantage underlying plasmid/IS symbioses.

GDT4 was optimal at low temperature and low iron concentration. However, pGDT4 did not encode any known thermoregulators. This suggests that temperature sensitive pGDT4 conjugation is under the control host genes. GDT4 could therefore be a natural response to challenging growth conditions.

Lesic, B., Zouine, M., Ducos-Galand, M., Huon, C., Rosso, M., Prévost, M., Mazel, D., & Carniel, E. (2012). A Natural System of Chromosome Transfer in Yersinia pseudotuberculosis PLoS Genetics, 8 (3) DOI: 10.1371/journal.pgen.1002529

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