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Succinate transport is not essential for symbiotic nitrogen fixation by Sinorhizobium meliloti nor Rhizobium leguminosarum

ABSTRACT: Symbiotic nitrogen fixation (SNF) is an energetically expensive process performed by bacteria known as rhizobia during endosymbiotic relationships with leguminous plants. The bacteria require the plant to provide a carbon source for generation of the reductant to power SNF. Although it is well known that C4-dicarboxylates (succinate, fumarate, malate) function as the primary, if not sole, carbon source provided to the rhizobia, the relative contribution of each C4-dicarboxylate is not known. Here, we employ genetic and systems-level analyses to address this issue. Expression of a malate specific transporter (MaeP) in Sinorhizobium meliloti Rm1021 dct mutants unable to transport C4-dicarboxylates resulted in malate import rates up to ~ 30% that of wild type S. meliloti. This was sufficient to support SNF with Medicago sativa, with acetylene reduction rates up to ~ 50% those of plants inoculated with wild type S. meliloti. Rhizobium leguminosarum bv. viciae 3841 dct mutants unable to transport C4-dicarboxylates but expressing the maeP transporter had strong symbiotic properties, with Pisum sativum plants inoculated with these strains appearing similar to plants inoculated with wild type R. leguminosarum. This was despite malate transport rates by the mutant bacteroids being < 10% those of the wild type. A transcriptomics analysis of the combined plant/bacterium nodule transcriptome was performed using RNA-sequencing to identify any systems-level adaptations in response to the inability of the bacteria to import succinate or fumarate. Few transcriptional changes, with no obvious pattern, were revealed by this analysis. Overall, these data illustrated that succinate and fumarate are not essential for SNF, and that at least in specific symbioses, L-malate is likely to naturally serve as the primary C4-dicarboxylate provided to the bacterium.

ORGANISM(S): Pisum sativum

PROVIDER: GSE101430 | GEO | 2017/09/16

SECONDARY ACCESSION(S): PRJNA394202

REPOSITORIES: GEO

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Project description:Malic enzymes decarboxylate the tricarboxylic acid (TCA) cycle intermediate malate to the glycolytic end-product pyruvate and are well positioned to regulate metabolic flux in central carbon metabolism. The bacterium Sinorhizobium meliloti has a NAD(P)-malic enzyme (DME) and a NADP-malic enzyme (TME) and DME is required for symbiotic N2-fixation. To help understand the role of these enzymes, we examined growth, metabolic and transcriptional consequences resulting from the deletion of these enzymes. Few effects were observed upon growth with glucose, whereas growth with the gluconeogenic substrate, succinate, resulted in transcriptional and metabolic effects particularly in the dme mutant strains. When grown with succinate, DME mutant cells accumulated hexose sugar phosphates and trehalose, while TME mutants accumulated putrescine. Succinate-grown DME mutant cells also showed increased transcription of genes for gluconeogenesis and for pathways such as amino acid and fatty acid synthesis that divert metabolites away from the TCA cycle. These data suggested that, DME is required to regulate the levels of TCA cycle intermediates and that the activity of TME is insufficient to prevent the accumulation of TCA cycle intermediates in cells utilizing succinate as carbon source. Consistent with this, in short-1-3 hour incubations with succinate, dme mutant cells excreted large amounts of malate whereas little malate was excreted from tme or wild-type cells. These results support the suggestion that DME is required for N2-fixation in alfalfa because it is required for synthesis of pyruvate and acetyl-CoA and the rapid metabolism of C4-dicarboxylates supplied by the plant. RNA expression was measured for S. meliloti in exponential phase grown in MOPS (morpholinpropanesulfonic acid) buffered minimal media under 2 growth conditions: 1) 15 mM succinate, 2) 15 mM glucose; using wild type cells as well as dme and tme mutant strains (6 experiments, 2 replicates: total 12 samples)

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Succinate transport is not essential for symbiotic nitrogen fixation by Sinorhizobium meliloti nor Rhizobium leguminosarum

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