Characterization of a Gene Family of Outer Membrane Proteins (ropB) in Rhizobium Leguminosarum Bv. Viciae VF39SM and the Role of the Sensor Kinase ChvG in Their Regulation

The outer membrane of Gram-negative bacteria represents the interface between the bacterium and its external environment. It has a critical role as a protective barrier against harmful substances and is also important in host-bacteria interactions representing the initial physical point of contact between the host cell and bacterial cell. RopB is a previously identified outer membrane protein from Rhizobium leguminosarum bv. viciae that is present in free-living cells but absent in bacteroids (H. P. Roest, I. H. Mulders, C. A. Wijffelman, and B. J. Lugtenberg, Mol. Plant Microbe Interact. 8:576-583, 1995). The functions of RopB and the molecular mechanisms of ropB gene regulation have remained unknown. We identified and cloned ropB and two homologs (ropB2 and ropB3) from the R. leguminosarum VF39SM genome. Reporter gene fusions indicated that the expression of ropB was 8-fold higher when cells were grown in complex media than when they were grown in minimal media, while ropB3 expression was constitutively expressed at low levels in both complex and minimal media. Expression of ropB2 was negligible under all conditions tested. The use of minimal media supplemented with various sources of peptides resulted in a 5-fold increase in ropB expression. An increase in ropB expression in the presence of peptides was not observed in a chvG mutant background, indicating a role for the sensor kinase in regulating ropB expression. Each member of the ropB gene family was mutated using insertional mutagenesis, and the mutants were assayed for susceptibility to antimicrobial agents and symbiotic phenotypes. All mutants formed effective nodules on pea plants, and gene expression for each rop gene in bacteroids was negligible. The functions of ropB2 and ropB3 remain cryptic, while the ropB mutant had an increased sensitivity to detergents, hydrophobic antibiotics, and weak organic acids, suggesting a role for RopB in outer membrane stability.