The analysis of plasmid rearrangements observed in the soil bacterium OR168 after the introduction of transposon Tn5 : a thesis presented in fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University

Abstract

Transposon Tn5 mutagenesis has been used extensively in Escherichia coli and various other Gram-negative bacteria to produce both random and site directed mutants. The popularity of Tn5 as a mutagen stems from its apparent random insertion into the genome, leading to non-leaky polar mutations. It also confers on many bacteria resistance to aminoglycosides, providing a strong selectable marker. The site of insertion can be mapped by Southern DNA hybridisation against a specific Tn5 probe. Tn5-containing derivatives of the Rhizobia-like soil isolate, OR168, were produced using the broad host-range suicide plasmid vector pSUP1011. After the transfer of pSUP1011 to OR168 via heterogeneric bacterial conjugation, stable OR168::Tn5 exconjugants were selectively isolated at frequencies of approximately 10-4 per recipient. None of the 53 OR168::Tn5 exconjugants screened showed the parental plasmid profile. Visible alterations to the plasmid profile were common with respect to the native plasmid profile. These events generally showed large deletions from, or additions to, the native replicons of OR168. The alterations also included a low incidence of a decrease in plasmid number. Analysis of the exconjugant population shows that the insertion of Tn5 into the genome of OR168 may not be strictly random. It was shown that 66% of OR168::Tn5 exconjugants screened contain a plasmid-borne Tn5 element, with 90% of those involving Tn5 insertion in the same episome. There is evidence that events other than classical conservative transposition have occurred after the introduction of pSUP1011 into the OR168 genome. Screening of the isolated OR168::Tn5 population for pSUP1011 vector sequences revealed the presence of the pSUP1011-derived RP4-mob fragment in 33 of 35 OR168::Tn5 exconjugants containing a plasmid-borne Tn5 element. Analysis also revealed the acquisition of Tn5 alone, presumably by conservative transposition, occurred only twice in the 35 events involving a plasmid target. This suggests that another site within the RP4 fragment can act as a surrogate transposase recognition site. Alternatively, the insertion of the RP4-mob::Tn5 sequence into a plasmid target may involve a site specific recombination process peculiar to the OR168 isolate. No mechanism was elucidated for the formation of many of the alterations in plasmid mobility. Restriction fragment lengths in the immediate vicinity of the anomalous RP4-mob::Tn5 insertion arc identical in different plasmids. This may indicate sequence duplication among the OR 168 plasmids. Such duplication may precipitate, through homologous recombination processes, the plasmid instability observed.