Sinorhizobium meliloti pSymB carries genes necessary for arabinose transport and catabolism

Abstract

Arabinose is a known component of plant cell walls and is found in the rhizosphere. In this work, a previously undeleted region of the megaplasmid pSymB was identified as encoding genes necessary for arabinose catabolism, by Tn5-B20 random mutagenesis and subsequent complementation. Transcription of this region was measured by b-galactosidase assays of Tn5-B20 fusions, and shown to be strongly inducible by arabinose, and moderately so by galactose and seed exudate. Accumulation of [ 3 H]arabinose in mutants and wild-type was measured, and the results suggested that this operon is necessary for arabinose transport. Although catabolite repression of the arabinose genes by succinate or glucose was not detected at the level of transcription, both glucose and galactose were found to inhibit accumulation of arabinose when present in excess. To determine if glucose was also taken up by the arabinose transport proteins, [ 14 C]glucose uptake rates were measured in wild-type and arabinose mutant strains. No differences in glucose uptake rates were detected between wild-type and arabinose catabolism mutant strains, indicating that excess glucose did not compete with arabinose for transport by the same system. Arabinose mutants were tested for the ability to form nitrogen-fixing nodules on alfalfa, and to compete with the wild-type for nodule occupancy. Strains unable to utilize arabinose did not display any symbiotic defects, and were not found to be less competitive than wild-type for nodule occupancy in co-inoculation experiments. Moreover, the results suggest that other loci are required for arabinose catabolism, including a gene encoding arabinose dehydrogenase.

A Link between Arabinose Utilization and Oxalotrophy in Bradyrhizobium japonicum

“…The bacterial strains and plasmids used in this work are listed in Table S1 in the supplemental material. Escherichia coli cells were cultivated in Luria-Bertani medium (22) at 37°C using the following concentrations of antibiotics (in g/ml): ampicillin (200), kanamycin (30), chloramphenicol (20), and tetracycline (10). Bradyrhizobium japonicum cells were routinely cultivated at 30°C on peptone-salts-yeast extract (PSY) medium (23) supplemented with 0.1% arabinose or in defined buffered Vincent's minimal medium (BVM) (24,25), referred to as minimal medium.…”

“…Previous work has shown that L-arabinose is degraded by a pathway that conceptually resembles the Entner-Doudoroff pathway (14). By analogy, L-arabinose is first oxidized to L-2-keto-3-deoxyarabonate (L-KDA) (15)(16)(17), which is then converted into ␣-ketoglurate in the case of fast-growing rhizobia (18)(19)(20) or to glycolaldehyde and pyruvate in the case of slow-growing species like B. japonicum (14). While pyruvate most likely is oxidized in the TCA cycle, the fate of glycolaldehyde has not been resolved yet.…”

l -Hydroxyproline and d -Proline Catabolism in Sinorhizobium meliloti

“…␣-KGSA dehydrogenase enzymes involved in the catabolism of L-arabinose, D-glucarate, D-galactarate, and hydroxy-L-proline have also been characterized (25,52,53). To investigate whether the reducedgrowth phenotype of the hypH mutant on trans-4-L-Hyp may be due to the presence of other ␣-KGSADH-like enzymes, we searched the S. meliloti genome for other annotated dehydrogenases and identified the most similar as AraE, which functions as an ␣-KGSADH in the catabolism of L-arabinose (18). A hypH araE double mutant was constructed, and unlike the hypH mutant, which showed a moderate growth phenotype, and the araE mutant, which grew like the wild type, the hypH araE double mutant (RmP3174) failed to grow with trans-4-L-Hyp as the carbon source (data not shown).…”

“…To construct the ⌬hypS (A) Diagram of the hydroxyproline transport and catabolic locus; (B) schematic diagram of the hydroxyproline catabolic pathway, as described by Adams and Frank (11). (A and B) Gene annotations, promoters, and enzymatic functions as deduced through this study and previous work (13,14,18). HypR, negative regulator; HypD, ⌬ 1 -pyrroline-4-hydroxy-2-carboxylate deaminase; HypT, unknown; HypS, ⌬ 1 -pyrroline-2-carboxylate reductase; HypH, ␣-ketoglutarate semialdehyde dehydrogenase; HypMNPQ, L-hydroxyproline ABC-type transport system; HypO, cis-4-hydroxy-D-proline dehydrogenase; HypRE, hydroxyproline 2-epimerase; HypX, unknown; HypY, unknown, a possible proline racemase pseudogene; HypZ, unknown.…”

Rhizobial plasmids — replication, structure and biological role

“…It also carries minCDE genes with putative function in cell division, however they can be deleted without affecting cell survival [37,52,66,67]. A large segment of this replicon is required for dulcitol, arabinose, melibiose, raffinose, P-hydroxybutyrate, acetoacetate, protocatechuate and quinate utilization [65,68]. Thus, it appears that pSymB allows the bacteria to take up and metabolize many different compounds from the environment and plays an important role in the survival of the bacterium in the soil under diverse nutritional conditions and adaptation to both saprophytic and endosymbiotic lifestyles.…”