The lipopolysaccharide (LPS) of the Gram-negative legume symbiont Rhizobium leguminosarum biovar viciae 3841 contains several unique modifications, including the addition of a 27-hydroxyoctacosanoic acid (27OHC28 : 0), also termed the very long chain fatty acid (VLCFA), attached at the 29 position of lipid A. A transposon mutant that lacks expression of two putative 3-oxo-acyl [acyl-carrier protein] synthase II genes, fabF1 and fabF2, from the VLCFA biosynthetic cluster, was isolated and characterized. MS indicated that the lipid A of the mutant lacked the VLCFA modification, and sodium deoxycholate (DOC)-PAGE of the LPS indicated further structural alterations. The mutant was characteristically sensitive to several stresses that would be experienced in the soil environment, such as desiccation and osmotic stresses. An increase in the excretion of neutral surface polysaccharides was observed in the mutant. This mutant was also altered in its attachment to solid surfaces, and was non-motile, with most of the mutant cells lacking flagella. Despite the pleiotropic effects of the mutation, these mutants were still able to nodulate legumes and fix atmospheric nitrogen. This report emphasizes that a structurally intact VLCFAcontaining lipid A is critical to cellular traits that are important for survival in the rhizosphere.

Rhizobium leguminosarum is a soil bacterium with the ability to form nitrogen-fixing nodules on the roots of leguminous plants. Soil-dwelling, free-living R. leguminosarum often encounters desiccation stress, which impacts its survival within the soil. The mechanisms by which soil bacteria resist the effects of desiccation stress have been described. However, the role of the cell envelope in the desiccation tolerance mechanisms of rhizobia is relatively uncharacterized. Using a transposon mutagenesis approach, a mutant of R. leguminosarum bv. viciae was isolated that was highly sensitive to desiccation. The mutation is located in the ATP-binding protein of an uncharacterized ATP-binding cassette transporter operon (RL2975–RL2977). Exopolysaccharide accumulation was significantly lower in the mutant and the decrease in desiccation tolerance was attributed to the decreased accumulation of exopolysaccharide. In addition to desiccation sensitivity, the mutant was severely impaired in biofilm formation, an important adaptation used by soil bacteria for survival. This work has identified a novel transporter required for physiological traits that are important for the survival of R. leguminosarum in the rhizosphere environment.

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.

To better understand the role of proteases in Rhizobium leguminosarum biovar viciae, a gene with homology to the carboxy-terminal protease (CtpA), which belongs to a novel group of serine proteases, was studied. The ctpA gene was cloned and mutated using allelic exchange and a gusA reporter gene was used to study ctpA expression. Mutational analysis shows that ctpA is critical for the viability of R. leguminosarum when cells are grown on complex semi-solid media but is dispensable when cells are grown in complex liquid media and that this is likely due to an increase in susceptibility to desiccation on semi-solid media. The ctpA mutant also displayed an increased sensitivity to detergents, indicating an alteration in the permeability of the cell envelope. This is the first characterization of a ctpA gene within the Rhizobiaceae and the first report of a ctpA mutant that exhibits an increased sensitivity to desiccation.

The bacterial cell envelope is of critical importance to the function and survival of the cell; it acts as a barrier against harmful toxins while allowing the flow of nutrients into the cell. It also serves as a point of physical contact between a bacterial cell and its host. Hence, the cell envelope of Rhizobium leguminosarum is critical to cell survival under both free-living and symbiotic conditions. Transposon mutagenesis of R. leguminosarum strain 3841 followed by a screen to isolate mutants with defective cell envelopes led to the identification of a novel conserved operon (RL3499-RL3502) consisting of a putative moxR-like AAA ؉ ATPase, a hypothetical protein with a domain of unknown function (designated domain of unknown function 58), and two hypothetical transmembrane proteins. Mutation of genes within this operon resulted in increased sensitivity to membranedisruptive agents such as detergents, hydrophobic antibiotics, and alkaline pH. On minimal media, the mutants retain their rod shape but are roughly 3 times larger than the wild type. On media containing glycine or peptides such as yeast extract, the mutants form large, distorted spheres and are incapable of sustained growth under these culture conditions. Expression of the operon is maximal during the stationary phase of growth and is reduced in a chvG mutant, indicating a role for this sensor kinase in regulation of the operon. Our findings provide the first functional insight into these genes of unknown function, suggesting a possible role in cell envelope development in Rhizobium leguminosarum. Given the broad conservation of these genes among the Alphaproteobacteria, the results of this study may also provide insight into the physiological role of these genes in other Alphaproteobacteria, including the animal pathogen Brucella.

Two-component signal transduction systems (TCS) are a main strategy used by bacteria to sense and adapt to changes in their environment. In the legume symbiont Rhizobium leguminosarum biovar viciae VF39, mutation of chvG, a histidine kinase, caused a number of pleiotropic phenotypes. ChvG mutants are unable to grow on proline, glutamate, histidine, or arginine as the sole carbon source. The chvG mutant secreted smaller amounts of acidic and neutral surface polysaccharides and accumulated abnormally large amounts of poly-ß-hydroxybutyrate. Mutation of chvG caused symbiotic defects on peas, lentils, and vetch; nodules formed by the chvG mutant were small and white and contained only a few cells that had failed to differentiate into bacteroids. Mutation of chvG also destabilized the outer membrane of R. leguminosarum, resulting in increased sensitivity to membrane stressors. Constitutive expression of ropB, the outer membrane protein-encoding gene, restored membrane stability and rescued the sensitivity phenotypes described above. Similar phenotypes have been described for mutations in other ChvG-regulated genes encoding a conserved operon of unknown function and in the fabXL genes required for synthesis of the lipid A very-long-chain fatty acid, suggesting that ChvG is a key component of the envelope stress response in Rhizobium leguminosarum. Collectively, the results of this study demonstrate the important and unique role the ChvG/ChvI TCS plays in the physiology, metabolism, and symbiotic competency of R. leguminosarum.

Atomic force microscopy was used to investigate the surface ultrastructure, adhesive properties and biofilm formation of Rhizobium leguminosarum and a ctpA mutant strain. The surface ultrastructure of wild-type R. leguminosarum consists of tightly packed surface subunits, whereas the ctpA mutant has much larger subunits with loose lateral packing. The ctpA mutant strain is not capable of developing fully mature biofilms, consistent with its altered surface ultrastructure, greater roughness and stronger adhesion to hydrophilic surfaces. For both strains, surface roughness and adhesive forces increased as a function of calcium ion concentration, and for each, biofilms were thicker at higher calcium concentrations.

Homoserine represents a substantial component of pea root exudate that may be important for plant-microbe interactions in the rhizosphere. We identified a gene cluster on plasmid pRL8JI that is required for homoserine utilization by Rhizobium leguminosarum bv. viciae. The genes are arranged as two divergently expressed predicted operons that were induced by L-homoserine, pea root exudate, and were expressed on pea roots. A mutation in gene pRL80083 that prevented utilization of homoserine as a sole carbon and energy source affected the mutant's ability to nodulate peas and lentils competitively. The homoserine gene cluster was present in approximately 47% of natural R. leguminosarum isolates (n = 59) and was strongly correlated with homoserine utilization. Conjugation of pRL8JI to R. leguminosarum 4292 or Agrobacterium tumefaciens UBAPF2 was sufficient for homoserine utilization. The presence of L-homoserine increased conjugation efficiency of pRL8JI from R. leguminosarum to a pRL8JI-cured derivative of R. leguminosarum 1062 and to A. tumefaciens UBAPF2, and induced expression of the plasmid transfer gene trbB; however, there was no difference in conjugation efficiency or trbB expression with A. tumefaciens UBAPF2pRL8-Gm as the donor suggesting that other genes in R. leguminosarum may contribute to regulating conjugation of pRL8 in the presence of homoserine.