The transport and targeting of a number of periplasmic proteins is carried out by the Sec‐independent Mtt (also referred to as Tat) protein translocase. Proteins using this translocase have a distinct twin‐arginine‐containing leader. We hypothesized that specific leader‐binding proteins exist to escort proteins to the translocase complex. A fusion was constructed with the twin‐arginine leader from dimethyl sulphoxide (DMSO) reductase, subunit DmsA, to the N‐terminus of glutathione‐S‐transferase. This leader fusion was bound to a glutathione affinity column through which an Escherichia coli anaerobic cell‐free extract was passed. Proteins that bound to the leader were then separated and identified by N‐terminal sequencing, which identified DnaK and a protein originating from the uncharacterized reading frame ynfI. This gene has been designated dmsD based on the findings presented in this paper. DmsD was purified as a His6 fusion and was shown to interact with preprotein forms of DmsA and TorA (trimethyl amine N‐oxide reductase). A strain carrying a dmsD knock‐out mutation showed a loss of anaerobic growth on glycerol–DMSO medium and reduced growth on glycerol–fumarate medium. This work suggests that DmsD is a twin‐arginine leader‐binding protein.

We characterized mutants of Rhizobium melloi SU47 that were unable to grow on succe as the carbon source. The mutants fell into five groups based on complmentatlon of the scchate mutato by Individual recombinant plasnulds isolated from a R. meWod done bank. Enzyme analysis showed that mutants in the following groups lacked the indicated common enzyme activities: group H, e (Eno); group m, phosphoenolpyruvate carboxykna (Pck); group IV, glyceralddhyde-3-pbosphate dehydrogena (Gap), and 3-phosphoglycerate kinase (Pgk). Mutants in groups I and V lacked C4-dicarboxylate transport (Dct-) Rhizobium meliloti form nodules which fail to fix nitrogen (i.e., ineffective nodules) (3,8,15,21,47). Ronson and co-workers (4446) have shown that in free-living cells, regulation of C4-dicarboxylate transport involves interaction of two C4-dicarboxylate-specific regulatory genes and the ntrA (rpoN) gene product, the latter of which is also required for nifH expression and nitrate assimilation.Succinate metabolism has been implicated in the regulation of a number of metabolic activities in Rhizobium species (51,52); however, unlike hexose and pentose metabolism, the pathways of succinate metabolism in Rhizobium species have been addressed in only a few reports. Mutant and enzyme analyses have shown that the Entner-Doudoroff and pentose phosphate pathways play central roles in hexose and pentose metabolism in R. meliloti L5-30 (1, 2, 11) and other fast-growing Rhizobium strains (3, 48; for a review, see reference 51). In addition, the tricarboxylic acid (TCA) cycle mutants of R. meliloti L5-30, which are deficient in aketoglutarate dehydrogenase and succinate dehydrogenase activities, have been described previously (14, 25). Glenn and co-workers (3, 37) have demonstrated that phosphoenolpyruvate carboxykinase is required for growth on gluconeogenic substrates such as succinate in R. leguminosarum.To obtain more information on succinate transport and metabolism in R. meliloti cells and, ultimately, in bacteroids, we assayed many of the enzymes that are involved in carbon metabolism in free-living cells and biochemically and genetically characterized 17 independent mutants of R. meliloti which were defective in succinate metabolism. The mutants fell into five groups (I through V), three of which were deficient in enolase, phosphoenolpyruvate carboxykinase, and glyceraldehyde-3-phosphate dehydrogenase or phos-* Corresponding author.phoglycerate kinase activities and two of which were defective in C4-dicarboxylate transport.(This work was presented in part at the 11th North American Rhizobium Conference, Quebec, Quebec, Canada, August 1987.) MATERIALS AND METHODS Bacterial strains, pla s, and mei. The bacterial strains and plasmids used in this study are listed in Table 1. Complex (LB) and defined (M9) media, antibiotic concentrations, and routine growth conditions were as described previously (18,19).Genetic techniques. Transduction, plasmid conjugation, transposon mutagenesis, and transposon replacements were done as described pre...

We report the curing of the 1,360-kb megaplasmid pRme2011a from Sinorhizobium meliloti strain Rm2011. With a positive selection strategy that utilized Tn5B12-S containing the sacB gene, we were able to cure this replicon by successive rounds of selecting for deletion formation in vivo. Subsequent Southern blot, Eckhardt gel, and pulsed-field gel electrophoresis analyses were consistent with the hypothesis that the resultant strain was indeed missing pRme2011a. The cured derivative grew as well as the wild-type strain in both complex and defined media but was unable to use a number of substrates as a sole source of carbon on defined media.

Cosmids carrying genes involved in utilization of rhamnose, sorbitol, and adonitol were isolated from a genomic library of Rhizobium leguminosarum by complementation of plasmid-cured derivatives of strain Rlt100 that were unable to grow on these carbon sources. Transposon mutagenesis was used to identify regions of each cosmid necessary for catabolism of the respective carbon source; partial DNA sequencing, as well as analysis of gene fusions created with transposon Tn5-B20, helped to determine the orientation and possible function of genes required for growth on the three substrates. Representative Tn5 insertions in the cosmids were recombined into the wild-type strain Rlt100 by gene replacement to generate isogenic strains unable to use either rhamnose, sorbitol, or adonitol. These strains were tested for their nodulation competitiveness compared with Rlt100 in co-inoculation experiments on clover plants. While sorbitol and adonitol catabolic mutants were unaltered in their competitive behavior, the nodulation competitiveness of three different rhamnose utilization mutants was significantly impaired. This result, coupled with the fact that the rhamnose catabolic genes were inducible by clover root extracts, suggests an important role for rhamnose catabolism in the early stages of the interaction of R. leguminosarum with clover plants. Hybridization studies with probes derived from the rhamnose, sorbitol, and adonitol catabolic loci demonstrated that these genes are plasmid encoded in virtually all R. leguminosarum strains, including representatives from all three biovars from a variety of different geographic locations.

Rhizobium leguminosarum bv. trifolii mutants unable to catabolize the methyl-pentose rhamnose are unable to compete effectively for nodule occupancy. In this work we show that the locus responsible for the transport and catabolism of rhamnose spans 10,959 bp. Mutations in this region were generated by transposon mutagenesis, and representative mutants were characterized. The locus contains genes coding for an ABC-type transporter, a putative dehydrogenase, a probable isomerase, and a sugar kinase necessary for the transport and subsequent catabolism of rhamnose. The regulation of these genes, which are inducible by rhamnose, is carried out in part by a DeoR-type negative regulator (RhaR) that is encoded within the same transcript as the ABC-type transporter but is separated from the structural genes encoding the transporter by a terminator-like sequence. RNA dot blot analysis demonstrated that this terminator-like sequence is correlated with transcript attenuation only under noninducing conditions. Transport assays utilizing tritiated rhamnose demonstrated that uptake of rhamnose was inducible and dependent upon the presence of the ABC transporter at this locus. Phenotypic analyses of representative mutants from this locus provide genetic evidence that the catabolism of rhamnose differs from previously described methyl-pentose catabolic pathways.Rhizobium leguminosarum bv. trifolii is a gram-negative aerobic soil bacterium that can exist as a free-living heterotropic saprophyte or can form nitrogen-fixing nodules on various species of clovers (Trifolium spp.). The interaction of rhizobia with their hosts is a sequence of events that begins with an exchange of signals in the rhizosphere, followed by the invasion of the plant through infection threads and ultimately by release of the bacteria into plant cells, where they differentiate into nitrogen-fixing bacteroids. Through this symbiotic association, the plant provides the bacteria with energy for growth, and in return the Rhizobium provides the plant with fixed nitrogen. Although a great deal has been elucidated regarding the actual steps during nodulation (27, 56) and the metabolic pathways utilized while in the bacteroid state (39), comparatively little is known about what influences the growth of the bacteria prior to or during their interaction with plant roots. The rhizosphere has been described as the region directly under the influence of secreted compounds from the plant root (12). Competition for nodule occupancy exists between strains of Rhizobium within the rhizosphere and has been well documented in the literature (16,54). A large number of biotic and abiotic factors influence competition between strains for nodule occupancy. Some of the best-characterized strain-dependent factors include production of antibiotics and bacteriocins (36,42,57) and the ability to catabolize specific compounds present in the root environment (11,13,18,22,23,35,43,49,55), as well as the synthesis or utilization of specific vitamins (38, 50).Rhamnose is a methyl-pentose s...