Modulation of Plasmids Conjugative Transfer by Novel Hypothetical Proteins

DIP, D.; NILSSON, J; SALTO, I. P.; ALBICORO, F.; CERVANTES, L.; BROM, S.; PISTORIO, M.; TORRES TEJERIZO, G. A.

Corboba

Congreso; XI Congreso Argentino de Microbiologia General; 2015

SAMIGE

Rhizobia are gram-negative bacteria with ability to fix atmospheric N2 in symbiotic association with the roots of legumes. The genomes of these bacteria are usually composed of a chromosome and various plasmids of sizes ranging between 40 and 2000 Kb. Plasmids have played a major role in bacterial evolution, mainly by their capacity to perform horizontal gene transfer. Plasmid pLPU83a, an accessory replicon from Rhizobium sp. LPU83, is able to transfer from its parental strain (ca. 10-5 transconjugants / donor cell), from Ensifer meliloti or from Rhizobium etli, but not from Agrobacterium tumefaciens. The mechanisms that regulate conjugative transfer (CT) of pLPU83a are not fully understood. In this work, we analysed structural and functional aspects of the CT of pLPU83a. Bioinformatics analyses were performed; this plasmid contains a complete set of transfer genes, featuring a particular organization, shared with only two other rhizobial plasmids. The elements required for CT are organized as a cluster of genes involved in formation of the mating pair (Mpf, Mating pair formation), genes involved in the processing of DNA (Dtr, DNA transfer and replication), and an oriT site, where transfer is initiated. The plasmid contains a TraR quorum-sensing (QS) transcriptional regulator, but lacks an acyl-homoserine lactone synthase gene. Furthermore, between the Dtr and the Mpf we found three genes encoding hypothetical proteins that could not be assigned to a known function. Similarly, hypothetical genes are often located in contiguous regions in bacterial genomes and, recently, Lopez-Fuentes et al. showed that the genes located among conjugation genes are involved in CT of a plasmid of Rhizobium etli CFN42, pRetCFN42a . In order to determine if the hypothetical-protein encoding genes localized in pLPU83a participate in CT, we constructed mutant derivatives and analyzed their phenotype. We obtained insertional mutations in three genes of pLPU83a (LPU83a_00145, LPU83a_00146 and LPU83a_00148) by homologous recombination and evaluated their conjugative transfer properties. The mutation of LPU83a_00145 did not modify CT (ca. 10-5 transconjugants / donor cell), the mutation of LPU83a_00146 enhanced CT (ca. 10-3 transconjugants / donor cell) while the mutation of LPU83a_00148 abolished CT (< 10-9 transconjugants / donor cell). Complementation of the mutants restored the conjugative transfer frequencies to the wild-type levels. The mode of participation of these genes in CT is still unknown. Elucidation of the mechanisms that differentially regulate plasmid CT will be helpful to understand the boundaries of plasmid exchange in bacteria.