A Genetic Locus Necessary for Rhamnose Uptake and Catabolism in Rhizobium leguminosarum bv. trifolii

Abstract

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...

Identification of an Indigenous Atrazine Herbicide Tolerant Microbial Consortium in Beans (Phaseolus vulgaris L.) as a Potential Soil Bioremediator

“…(Table 3). Similar results were reported by Acosta and Martinez [27] and Richardson et al [28]. In these reports, Rhizobium strains were isolated from nodules of legumes growing in pesticide contaminated soils.…”

Sinorhizobium meliloti pSymB carries genes necessary for arabinose transport and catabolism

“…The bacterial strains and plasmids used and generated in this work are listed in Table 1. S. meliloti strains were routinely grown at 30 uC on complex LuriaBertani (LB) medium or defined Vincent's minimal medium (VMM), as previously described (Sambrook et al, 1989;Vincent, 1970;Richardson et al, 2004). Carbon sources used in defined media were filter-sterilized and used at a final concentration of (Mulligan & Long, 1985).…”

“…Overnight cultures at mid-exponential phase were then subcultured into fresh medium of the same type, and grown to OD 600~0 .3, before being assayed as described by Miller (1972). Richardson et al (2004), with the following modifications. Cells were grown in defined media containing the appropriate carbon source(s), washed twice, and then resuspended in defined media with the original carbon source(s) (omitting arabinose or glucose as necessary) to OD 600 0.1-0.3.…”

“…However, glucose has also been shown to repress the expression of catabolic genes in R. leguminosarum (Oresnik et al, 1998;Richardson et al, 2004). To determine if either succinate or glucose catabolite repression plays a role in arabinose catabolism, strain SRmA240 was grown with arabinose or galactose in combination with succinate, glycerol or glucose, and gene expression was measured by b-galactosidase activity (Table 3).…”

Rhizobial plasmids — replication, structure and biological role

“…Furthermore, it was found that the Rlt nodule strains belonging to defined genetic groups and varying in the plasmid profile differ also in metabolic capabilities and symbiotic effectiveness with clover [95]. It was also demonstrated that Rlt mutants in plasmid-located rhamnose utilization genes were significantly impaired in nodulation competitiveness [96,97]. The rhamnose catabolic genes were inducible by clover root extracts, suggesting their important role for early stages of the interaction of Rlt with the host plant.…”