Modulation of Plasmids Conjugative Transfer by Novel Hypothetical Proteins

DIANA DIP; JULIET NILSON; ILEANA SALTO; FRANCISCO ALBICORO; LAURA CERVANTES; SUSANA BROM; MARIANO PISTORIO; GONZALO A. TORRES TEJERIZO

Cordoba

Congreso; XI Congreso Argentino de Microbiologia General; 2015

Rhizobia are gram-negative bacteria withability to fix atmospheric N2 in symbiotic association with the rootsof legumes. The genomes of these bacteria are usually composed of a chromosomeand various plasmids of sizes ranging between 40 and 2000 Kb. Plasmids haveplayed a major role in bacterial evolution, mainly by their capacity to performhorizontal gene transfer. Plasmid pLPU83a, anaccessory replicon from Rhizobium sp. LPU83, is able to transfer fromits parental strain (ca. 10-5 transconjugants / donor cell), from Ensifermeliloti or from Rhizobium etli, but not from Agrobacteriumtumefaciens. The mechanisms that regulate conjugative transfer (CT) of pLPU83aare not fully understood. In this work, we analysedstructural and functional aspects of the CT of pLPU83a. Bioinformatics analyseswere performed; this plasmid contains a complete set of transfer genes,featuring a particular organization, shared with only two other rhizobialplasmids. The elements required forCT are organized as a cluster of genes involved in formation of the mating pair(Mpf, Mating pair formation), genes involved in theprocessing of DNA (Dtr, DNA transfer and replication), andan oriT site, where transfer isinitiated. Theplasmid contains a TraR quorum-sensing (QS) transcriptional regulator, but lacksan acyl-homoserine lactone synthase gene. Furthermore, between the Dtr and the Mpf we found three genes encoding hypothetical proteins that could not beassigned to a known function. Similarly, hypothetical genes are often locatedin contiguous regions in bacterial genomes and, recently, Lopez-Fuentes et al. [1] showed that the genes located among conjugation genes are involved in CTof a plasmid of Rhizobium etli CFN42,pRetCFN42a .In order to determine if the hypothetical-proteinencoding genes localized in pLPU83a participate in CT, we constructed mutantderivatives and analyzed their phenotype. We obtained insertionalmutations in three genes of pLPU83a (LPU83a_00145,LPU83a_00146 and LPU83a_00148) by homologous recombination and evaluated theirconjugative transfer properties. The mutation of LPU83a_00145 did notmodify CT (ca. 10-5transconjugants / 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 tothe wild-type levels. The mode of participation of these genes in CT is still unknown. Elucidation of the mechanisms thatdifferentially regulate plasmid CT will be helpful to understand the boundariesof plasmid exchange in bacteria. 1.            Lopez-Fuentes, E., et al., Genes encoding conservedhypothetical proteins localized in the conjugative transfer region of plasmidpRet42a from Rhizobium etli CFN42 participate in modulating transfer and affectconjugation from different donors. Front Microbiol, 2015. 5: p. 793.