A novel pSym- borne type I protein secretion system from Rhizobium leguminosarum is responsible for the secretion of a RTX protein and a bacteriocin activity

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

Buenos Aires

Congreso; Reunion Conjunta de Sociedades de Biociencias; 2017

Diez Sociedades de Biociencias de la Argentina: SAIC, SAIB, SAI, SAA, SABiof, SABiol, SAFE, SAFIS, SAH, SAP

A NOVEL pSym-BORNE TYPE I PROTEIN SECRETION SYSTEM FROM Rhizobium leguminosarum IS RESPONSIBLE FOR THE SECRETION OF A RTX PROTEIN AND A BACTERIOCIN ACTIVITY.Effective symbiosis between Rhizobium leguminosarum (R.l.) and the legume requires the exchange of signal molecules that induce the expression of bacterial genes encoded in the symbiotic megaplasmid (pSym). The identification of secretion systems (SS) pSym-associated could contribute to generate future genetic tools for regulating symbiosis and the bacterial lifecycle. R.l.bv. viciae 248 strain carries the pRL1JI which confers the capability to nodulate pea and vicia plants. A pRL1JI-derivated cosmid library was screened using a polyclonal antiserum against an extracellular protein (EP) whose presence in rhizobial cultures was pSym-dependent. One cosmid was able to restore the secretion of the EP in R.l. pRL1JI-cured strain. By random Tn5-mutagenesis of the cosmid we isolate a clone that lost the ability to secrete the EP. Sequence analysis of the Tn5-clone corroborated that the transposon was inserted in a putative ABC component of a pSym -Type I SS (T1SS). 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; thus, we named them RTX-1 and RTX-2. Based on the amino-terminal sequence of the EP, we corroborate that the RTX-1 correspond to EP. Bacteriocin´s activity assays performed with R.l. carrying either the RTX-SS or the ABC::Tn5 derivated cosmids showed that the T1SS is required to inhibit the growth of other rhizobial strains. To dilucidate the identity of the bacteriocin activity, deletion mutants affected in rtx-1 and rtx-2 were generated. Surprisingly both mutants were able to inhibit the growth of M. strains suggesting that the RTX-proteins together or another extracellular factor secreted by the T1SS contribute with the rizobiocine activity.KEYWORDS: PROTEIN SECRETION SYSTEM, SYMBIOSIS, RHIZOBIUM