INSIGHTS INTO THE ROLE OF A PLASMID-BORNE TYPE I SECRETION SYSTEM (RssDM) OF Rhizobium leguminosarum

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

Virtual

Congreso; Reunión Conjunta on line de SAIB-SAMIGE 2020. LVI Reunión Anual de la Sociedad Argentina de Investigación Bioquímica y Biología Molecular (SAIB). XV Congreso Argentino de Microbiología General (SAMIGE 2020); 2020

Sociedad Argentina de Investigación Bioquímica y Biología Molecular (SAIB) y Asociación Civil de Microbiología General (SAMIGE)

INSIGHTS INTO THE ROLE OF A PLASMID-BORNE TYPE I SECRETION SYSTEM (RssDM) OF Rhizobium leguminosarum Russo DM (1), Downie JA (2), Zorreguieta A (1)(1) IIBBA-CONICET, Fundación Instituto Leloir, Argentina. (2) John Innes Centre, United Kingdom. E-mail: drusso@leloir.org.ar The effective symbiosis between Rhizobium leguminosarum and legume plants requires the exchange of signal molecules which induce the expression of bacterial genes encoded in the symbiotic megaplasmid (pSym). Protein secretion plays an important role in modulating the interactions between bacteria and their environments. Hence, the study of protein secretion systems associated with the pSym plasmid may provide new insights into the mechanisms that contribute to rhizobial ecology and symbiosis. Using a genetic approach, we have identified a novel type I protein secretion system (T1SS) encoded in the pRL1JI symbiotic plasmid of Rhizobium leguminosarum bv. viciae 248 strain (R.l.v 248). The ABC and MFP components of this system are encoded by the rssD and rssM genes, which are upstream of two ORFs encoding putative calcium-binding proteins that belong to the RTX-family (Repeated in ToXin) called RTX-1 and RTX-2. Comparison of the secretomes of a T1SS rssD mutant and the parental R.l.v 248 strain by SDS-PAGE analysis showed the absence in the mutant protein profile of a 49 kDa band, which corresponds to RTX-1. The secretion of this protein was restored by the rssDM genes cloned into the pBBR1MCS vector, indicating that the RssDM system is responsible for RTX-1 secretion. LC-MS/MS (LFQ labelled free quantification) analyses suggest that RTX-1 is the unique RssDM substrate. Furthermore, RTX-2´s secretion was independent of this T1SS, as well as other pRL1JI encoded RTX-proteins such as the Rhizobiocin 248 (Rzb248) and the pore-forming protein NodO. The analysis of the RTX-1 and RTX-2 predicted proteins indicate the presence of six acidic nonapeptide repeats (L/I/F-X-GG-X-G-N/D-D-X) and predominant beta-strand secondary structures (Phyre-2). BLASTP search of homologues resulted in several uncharacterized calcium-binding proteins present in members of the Rhizobiaceae family, including bacteriocins. Bacteriocin plate assays indicated that an RssDM-dependent bacteriocin activity against Mesorhizobium loti is produced by R.l.v 248. However, the mutational analysis indicated that this activity is independent of RTX-1 secretion or both RTX-1 and RTX-2. Nodulation experiments showed that although the rssD mutant is able to nodulate legume plants, it is outcompeted by the wild-type strain in co-inoculation experiments, suggesting that the RssDM system could be involved in competitiveness in the rhizosphere environment. Taken together, these results show that the pSym plasmid that defines the symbiosis between Rhizobium and the legume harbours a T1SS, whose substrate could have a role in competition for the rhizosphere niche or for nodulation.