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

Abstract

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.

Mutation of a Broadly Conserved Operon (RL3499-RL3502) from Rhizobium leguminosarum Biovar viciae Causes Defects in Cell Morphology and Envelope Integrity

“…(8,17). The expression data for the RL3499::gusA fusion in the chvG mutant background suggests that ChvG does play a role in RL399-RL3502 operon expression; however, another factor is required for the induction of the RL3499-RL3502 operon in the presence of peptides.…”

“…We previously reported that the sensor kinase ChvG regulates expression of the outer membrane protein gene ropB in the closely related strain R. leguminosarum VF39 (17). ChvG is also important in the expression of the homologous ropB in S. meliloti (8).…”

“…Many of the phenotypes described for the operon mutants are similar to those described for a mutant strain no longer expressing the outer membrane protein gene ropB in R. leguminosarum bv. viciae VF39 (17), a strain that is closely related to R. leguminosarum 3841 (17,63). We used pDF4, a ropB::gusA transcriptional fusion, to determine whether the sensitivity of these mutants to outer membrane stressors could be, in part, due to diminished expression of ropB.…”

“…Homologs of the ChvI/ChvG two-component signal transduction system which regulates several key components of the cell envelope in rhizobia, are required for growth of R. leguminosarum, S. meliloti, and Agrobacterium spp. on complex media (4,17,32). A mutation in a periplasmic protease gene, ctpA, which has been implicated in cell envelope development results in an inability of R. leguminosarum to grow on standard complex media, and the mutant forms large, spherical cells on solid TY after incubation for 24 h (22).…”

Mutation of the Sensor Kinase chvG in Rhizobium leguminosarum Negatively Impacts Cellular Metabolism, Outer Membrane Stability, and Symbiosis

“…Recently, we reported isolation of a chvG mutant in Rhizobium leguminosarum VF39 and described its role in regulating the ropB outer membrane protein-encoding gene and an operon required for cell envelope integrity and control of cell morphology (RL3499-RL3502) (10,38). We have also found that ChvG is important for induction of the fabXL genes required for biosynthesis of the lipid A very-long-chain fatty acid during growth under membrane stress conditions (E. M. Vanderlinde and C. K. Yost, unpublished data).…”