The novel plasmidic type I protein secretion system RssDM of Rhizobium leguminosarum bv. viciae strain 248 exports a RTX-protein.

DOWNIE J.A.; ZORREGUIETA A.; RUSSO D.M.

San Luis

Congreso; XXIII Congreso Argentino de Microbiologia General SAMIGE 2018; 2018

Asociación Civil de Microbiología General SAMIGE

Symbiosis between Rhizobium leguminosarum (R.l.) and the legume host requires a complex interplay between the partners which induce the expression of bacterial genes encoded in the symbiotic megaplasmid (pSym). The identification of type I protein secretion systems (TISS) pSym-associated could contribute to generate genetic tools that could modulate the endosymbiont lifecycle or the symbiosis with legumes. R.l.bv.viciae 248 strain carries a pSym of about 200 kpb named pRL1JI, which confers the capability to nodulate pea and vicia plants. Our previous results confirmed that a pRL1JI-derivated cosmid, pIJ1552, contained the secretion locus of a novel plasmidic TISS in R. l. bv. viciae strain 248. This cosmid was able to restore the secretion of an extracellular protein of 49 kDa (EP49) both in a R.l. pSym-cured strain and a Tn5-pIJ1552 mutant clone that were incapable to export it. Sequence analysis of the Tn5-clone corroborated that the transposon was inserted in a putative ABC component of a TISS. In the same orientation and contiguous to the ABC transporter, genes encoding a MFP component and two ORFs of hypothetical target proteins were identified. Both ORFs belong to a family of calcium binding-proteins called RTX (Repeated in toxin) that form pores in target membranes. The plasmidic TISS was called RssDM for Rtx secretion system D (ABC) and M (MFP) components, and the putative target proteins RTX-1 and RTX-2. Based on the amino-terminal sequence of the EP49, we corroborate that RTX-1 corresponds to EP49. Three R.l.v. 248 strain deletion mutants affected in rtx-1, rtx-2 and double rtx-1 rtx-2 genes were generated by homologue recombination. The secretome analysis of extracellular proteins by SDS-PAGE and Western blot using a rabbit antiserum against EP49 confirmed that RTX-1 is absent both in rtx-1 and rtx-1 rtx-2 mutants. Coomassie Colloidal staining suggest that a 36 kDa band was absent in the extracellular medium of rtx-2 and rtx-1 rtx-2 mutants. Mass Spectrometry (MS) analysis of the triptic peptides obtained from the 36 kDa band showed that it corresponds to RTX-2. To identify possible target proteins of the RssDM system, the secretomes of R.l.v. pRL1JI (wt) and derivative 248 strains were analyzed by LC-MS. Our results confirmed that although RTX-1 is substrate, RTX-2 secretion is independent of RssDM. In order to dilucidate the role of the TISS RssDM in the bacterial lifecycle, bacteriocin activity assays were performed using R.l.v. 248 derivative strains as bacteriocin´s producers against Mesorhizobium loti sensitive strains. These results suggest that the observed Mesorhizobium´s inhibition of growth is RssDM-dependent. Finally, we corroborate that RssDM TISS secretes at least a RTX-protein, RTX-1, and although the bacteriocin activity is associated to this system further experiments are required to dilucidate its identity.