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 Tn5 mutant strain of Sinorhizobium meliloti with an insertion in tpiA (systematic identifier SMc01023), a putative triose phosphate isomerase (TPI)-encoding gene, was isolated. The tpiA mutant grew more slowly than the wild type on rhamnose and did not grow with glycerol as a sole carbon source. The genome of S. meliloti wild-type Rm1021 contains a second predicted TPI-encoding gene, tpiB (SMc01614). We have constructed mutations and confirmed that both genes encode functional TPI enzymes. tpiA appears to be constitutively expressed and provides the primary TPI activity for central metabolism. tpiB has been shown to be required for growth with erythritol. TpiB activity is induced by growth with erythritol; however, basal levels of TpiB activity present in tpiA mutants allow for growth with gluconeogenic carbon sources. Although tpiA mutants can be complemented by tpiB, tpiA cannot substitute for mutations in tpiB with respect to erythritol catabolism. Mutations in tpiA or tpiB alone do not cause symbiotic defects; however, mutations in both tpiA and tpiB caused reduced symbiotic nitrogen fixation.Triose phosphate isomerase (TPI) catalyzes the reversible interconversion of glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP). This activity makes TPI a key enzyme of central carbon metabolism, allowing it to play a role in the glycolysis (Embden-Meyerhof-Parnas [EMP]), gluconeogenesis, pentose phosphate (PP), and Entner-Doudoroff (ED) pathways. Past research on the symbiotic soil bacterium Sinorhizobium meliloti and other rhizobia has shown that hexose catabolism proceeds through the ED and PP pathways, while the EMP pathway functions at very low levels, if at all (49). Gluconeogenesis, however, is functional, and mutations in gluconeogenic enzymes have been shown to cause complex symbiotic phenotypes, including reduced or abolished nitrogen-fixing activity (6,15,16,19,24).Recent work has confirmed the roles of the ED and PP pathways as catabolic pathways in S. meliloti. Analyses of carbon flux carried out using labeled carbon compounds and gas chromatography-mass spectrometry have confirmed the absence of the EMP pathway during growth with glucose (17). Another set of experiments done using labeled carbon and nuclear magnetic resonance is in agreement, confirming that glucose is degraded primarily through the ED pathway (22, 40). The gas chromatography-mass spectrometry and nuclear magnetic resonance experiments also confirm that some of the G3P produced by the catabolism of hexoses through the ED pathway is converted back to higher-molecular-weight compounds through TPI and fructose bisphosphate aldolase (FBA) in a cyclic pathway. Because FBA requires G3P and DHAP as substrates, the interconversion of G3P and DHAP is necessary for the cyclic metabolism of hexoses as well as gluconeogenesis. An S. meliloti strain missing TPI activity would therefore be compromised metabolically.Based on gene homology, S. meliloti has two putative chromosomal genes predicted to encode TPI enzymes (18). The gene ...

In this work we have genetically defined an erythritol utilization locus in Sinorhizobium meliloti. A cosmid containing the locus was isolated by complementation of a transposon mutant and was subsequently mutagenized using Tn5 : : B20. The locus was found to consist of five transcriptional units, each of which was necessary for the utilization of erythritol. Genetic complementation experiments using genes putatively annotated as erythritol catabolic genes clearly showed that, of the 17 genes at this locus, six genes are not necessary for the utilization of erythritol as a sole carbon source. The remaining genes encode EryA, EryB, EryC and TpiB as well as an uncharacterized ABC-type transporter. Transport experiments using labelled erythritol showed that components of the ABC transporter are necessary for the uptake of erythritol. The locus also contains two regulators: EryD, a SorC class regulator, and SMc01615, a DeoR class regulator. Quantitative RT-PCR experiments showed that each of these regulators negatively regulates its own transcription. In addition, induction of the erythritol locus was dependent upon EryD and a product of erythritol catabolism. Further characterization of polar mutations revealed that in addition to erythritol, the locus contains determinants for adonitol and L-arabitol utilization. The context of the mutations suggests that the locus is important for both the transport and catabolism of adonitol and L-arabitol.