IS26-mediated loss of the translocatable unit from Tn4352B requires the presence of the recA1 allele.

The pseudo-compound transposon Tn4352B is unusual in that the translocatable unit (TU) consisting of one of the bounding IS26 copies and the central portion containing the aphA1a gene has been found to be readily lost in the Escherichia coli strains used as host. Rapid loss required the presence of an additional 2 G residues adjacent to the internal end of one of the IS26 that flank the central portion and an active Tnp26 transposase. However, Tn4352B was found to be stable in wild-type Klebsiella pneumoniae strains. Though it was concluded that the difference may be due to the species background, the E. coli strains used were recombination-deficient. Here, we have further investigated the requirements for TU loss in E. coli and found that Tn4352B was stable in recombination-proficient strains. Among several recombination-deficient strains examined, rapid loss occurred only in strains that carry the recA1 allele but not in strains carrying different recA alleles, recA13 and a novel recA allele identified here, that also render the strain deficient in homologous recombination. Hence, it appears that a specific property of the RecA1 protein underlies the observed TU loss from Tn4352B.

The RuvABC Holliday Junction Processing System Is Not Required for IS26-Mediated Targeted Conservative Cointegrate Formation.

The insertion sequence IS26 plays a key role in the spread of antibiotic resistance genes in Gram-negative bacteria. IS26 and members of the IS26 family are able to use two distinct mechanisms to form cointegrates made up of two DNA molecules linked via directly oriented copies of the IS. The well-known copy-in (formerly replicative) reaction occurs at very low frequency, and the more recently discovered targeted conservative reaction, which joins two molecules that already include an IS, is substantially more efficient. Experimental evidence has indicated that, in the targeted conservative mode, the action of Tnp26, the IS26 transposase, is required only at one end. How the Holliday junction (HJ) intermediate generated by the Tnp26-catalyzed single-strand transfer is processed to form the cointegrate is not known. We recently proposed that branch migration and resolution via the RuvABC system may be needed to process the HJ; here, we have tested this hypothesis. In reactions between a wild-type and a mutant IS26, the presence of mismatched bases near one IS end impeded the use of that end. In addition, evidence of gene conversion, potentially consistent with branch migration, was detected in some of the cointegrates formed. However, the targeted conservative reaction occurred in strains that lacked the recG, ruvA, or ruvC genes. As the RuvC HJ resolvase is not required for targeted conservative cointegrate formation, the HJ intermediate formed by the action of Tnp26 must be resolved by an alternate route. IMPORTANCE In Gram-negative bacteria, the contribution of IS26 to the spread of antibiotic resistance and other genes that provide cells with an advantage under specific conditions far exceeds that of any other known insertion sequence. This is likely due to the unique mechanistic features of IS26 action, particularly its propensity to cause deletions of adjacent DNA segments and the ability of IS26 to use two distinct reaction modes for cointegrate formation. The high frequency of the unique targeted conservative reaction mode that occurs when both participating molecules include an IS26 is also key. Insights into the detailed mechanism of this reaction will help to shed light on how IS26 contributes to the diversification of the bacterial and plasmid genomes it is found in. These insights will apply more broadly to other members of the IS26 family found in Gram-positive as well as Gram-negative pathogens.