Infection-Blocking Genes of a Symbiotic Rhizobium leguminosarum Strain That Are Involved in Temperature-Dependent Protein Secretion

Abstract

Rhizobium leguminosarum strain RBL5523 is able to form nodules on pea, but these nodules are ineffective for nitrogen fixation. The impairment in nitrogen fixation appears to be caused by a defective infection of the host plant and is host specific for pea. A Tn5 mutant of this strain, RBL5787, is able to form effective nodules on pea. We have sequenced a 33-kb region around the phage-transductable Tn5 insertion. The Tn5 insertion was localized to the 10th gene of a putative operon of 14 genes that was called the imp (impaired in nitrogen fixation) locus. Several highly similar gene clusters of unknown function are present in Pseudomonas aeruginosa, Vibrio cholerae, Edwardsiella ictaluri, and several other animal pathogens. Homology studies indicate that several genes of the imp locus are involved in protein phosphorylation, either as a kinase or dephosphorylase, or contain a phosphoprotein-binding module called a forkhead-associated domain. Other proteins show similarity to proteins involved in type III protein secretion. Two dimensional gel electrophoretic analysis of the secreted proteins in the supernatant fluid of cultures of RBL5523 and RBL5787 showed the absence in the mutant strain of at least four proteins with molecular masses of approximately 27 kDa and pIs between 5.5 and 6.5. The production of these proteins in the wild-type strain is temperature dependent. Sequencing of two of these proteins revealed that their first 20 amino acids are identical. This sequence showed homology to that of secreted ribose binding proteins (RbsB) from Bacilus subtilis and V. cholerae. Based on this protein sequence, the corresponding gene encoding a close homologue of RbsB was cloned that contains a N-terminal signal sequence that is recognized by type I secretion systems. Inoculation of RBL5787 on pea plants in the presence of supernatant of RBL5523 caused a reduced ability of RBL5787 to nodulate pea and fix nitrogen. Boiling of this supernatant before inoculation restored the formation of effective nodules to the original values, indicating that secreted proteins are indeed responsible for the impaired phenotype. These data suggest that the imp locus is involved in the secretion to the environment of proteins, including periplasmic RbsB protein, that cause blocking of infection specifically in pea plants.