A locus necessary for the transport and catabolism of erythritol in Sinorhizobium meliloti

Abstract

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.

Carbohydrate Kinase (RhaK)-Dependent ABC Transport of Rhamnose in Rhizobium leguminosarum Demonstrates Genetic Separation of Kinase and Transport Activities

“…All the alleles that were generated were screened for their ability to complement the rhaK mutant strain Rlt144 for growth on rhamnose. In addition, since the buildup of a phosphorylated intermediate has also been correlated with the inability to complement (40), the alleles were also screened on medium containing rhamnose and glycerol ( Table 2). The results show that 25 of the insertions were not affected in their ability to complement Rlt144 (Table 2), and thus these were not further examined.…”

Inability To Catabolize Galactose Leads to Increased Ability To Compete for Nodule Occupancy in Sinorhizobium meliloti

“…SRmD144 (dgoK1), SRmD298 (galD), SRmD312 (SMc00883), and SRmD313 (ilvD1) were able to grow in the presence of both sugars, whereas SRmD311 (dgoA) did not grow (data not shown). This type of toxic phenotype was previously suggested to be the result of the buildup of a phosphorylated intermediate (1,24,51,55). The inability of a dgoA mutant to grow on this medium is consistent with the placement of the aldolase reaction in the De Ley-Doudoroff pathway following phosphorylation, presumably carried out by DgoK1.…”

“…The induction of galD was modest (4-fold) compared to inductions that were previously observed for other catabolic genes using qRT-PCR (24). A significant induction of galD by arabinose or seed exudate was not observed for Rm1021.…”

“…pCO37 is a derivative of broad-host-range plasmid pRK7813, which contains attB sites (31). This facilitated the recombination of araA and dgoK1 from an S. meliloti ORFeome entry vector into pCO37 as previously described (24). The identities of the resulting plasmids were confirmed by sequencing of the inserts.…”

“…Cells were harvested by centrifugation and resuspended in TE (10 mM Tris, 1 mM EDTA [pH 8]) buffer with lysozyme (0.4 mg/ml). RNA was then isolated by using the Qiagen RNeasy kit as previously described (24). Approximately 200 ng of cDNA sample was used as a template for quantitative reverse transcription-PCR (qRT-PCR).…”