A pSym plasmid-borne locus of Rhizobium is involved in the secretion of a RTX protein and symbiosis

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

Rosario

Congreso; L Reunión Anual de la Sociedad Argentina de Investigación Bioquímica y Biología Molecular (SAIB); 2014

Sociedad Argentina de Bioquímica y Biología Molecular

The establishment of an effective symbiosis between Rhizobium leguminosarum and the legume depends on the expression of genes encoded in the symbiotic plasmid (pSym). The profiles of secreted proteins by R. l. 8401 pSym (A34) and the pSym cured strain (A31) were compared by SDS-PAGE-Coomassie staining. The difference between both patterns was the presence of a 49 kDa-protein (EP-49) in A34, indicating that secretion of this protein is pSym dependent. A cosmid library from pSym was screened using a polyclonal antiserum against EP49. One cosmid (pIJ1552) was able to restore EP49 secretion in A31 strain. By Tn5 mutagenesis of pIJ1552 we isolated a clone that lost the ability to complement A31. Sequencing analysis revealed that the Tn5 was inserted in the gene of a putative ABC (ATP binding cassette) component of a type I secretion system (TISS). Contiguous and in the same orientation, genes encoding a MFP (membrane fusion protein) component of a TISS and EP49 (based on the amino-terminal sequence) were identified. A mutant in A34 (Z18) was obtained by recombination of Tn5-pIJ1552. By Western blot we confirmed that Z18 is impaired in EP49 secretion. Nodulation assays showed that this mutant develops white nodules in pea after 30 days of inoculation. These results suggest that the pSym harbors a novel ABC-MFP-EP49 locus of R. leguminosarum involved in the symbiotic process.