Succinate Transport Is Not Essential for Symbiotic Nitrogen Fixation by Sinorhizobium meliloti or Rhizobium leguminosarum

Abstract

Symbiotic nitrogen fixation (SNF) is an energetically expensive process performed by bacteria during endosymbiotic relationships with plants. The bacteria require the plant to provide a carbon source for the generation of reductant to power SNF. While C₄-dicarboxylates (succinate, fumarate, and malate) appear to be the primary, if not sole, carbon source provided to the bacteria, the contribution of each C₄-dicarboxylate is not known. We address this issue using genetic and systems-level analyses. Expression of a malate-specific transporter (MaeP) in Sinorhizobium meliloti Rm1021 dct mutants unable to transport C₄-dicarboxylates resulted in malate import rates of up to 30% that of the wild type. This was sufficient to support SNF with Medicago sativa, with acetylene reduction rates of up to 50% those of plants inoculated with wild-type S. melilotiRhizobium 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. An RNA-sequencing analysis of the combined P. sativum-R. leguminosarum nodule transcriptome was performed to identify systems-level adaptations in response to the inability of the bacteria to import succinate or fumarate. Few transcriptional changes, with no obvious pattern, were detected. Overall, these data illustrated that succinate and fumarate are not essential for SNF and that, at least in specific symbioses, l-malate is likely the primary C₄-dicarboxylate provided to the bacterium.IMPORTANCE Symbiotic nitrogen fixation (SNF) is an economically and ecologically important biological process that allows plants to grow in nitrogen-poor soils without the need to apply nitrogen-based fertilizers. Much research has been dedicated to this topic to understand this process and to eventually manipulate it for agricultural gains. The work presented in this article provides new insights into the metabolic integration of the plant and bacterial partners. It is shown that malate is the only carbon source that needs to be available to the bacterium to support SNF and that, at least in some symbioses, malate, and not other C₄-dicarboxylates, is likely the primary carbon provided to the bacterium. This work extends our knowledge of the minimal metabolic capabilities the bacterium requires to successfully perform SNF and may be useful in further studies aiming to optimize this process through synthetic biology approaches. The work describes an engineering approach to investigate a metabolic process that occurs between a eukaryotic host and its prokaryotic endosymbiont.

Keywords: Sinorhizobium; cross-kingdom interactions; dicarboxylate; endosymbionts; malic acid; metabolic engineering; metabolism; nitrogen fixation; nutrient transport; transcriptome.

FIG 1

Nodule carbon metabolism. A schematic representation of the primary nodule carbon metabolic pathways is shown. Sucrose in the plant cytosol is split into glucose and fructose, which are then catabolized via glycolysis to phosphoenolpyruvate (PEP). Carbon from PEP and carbonic acid is diverted to oxaloacetate (OAA) and l-malate via PEP-carboxylase and the nodule-enhanced malate dehydrogenase, and then possibly to fumarate, and succinate. These metabolites then cross both the peribacteroid and bacteroid membranes, which requires DctA, where their metabolism provides the ATP and reductant required to power the nitrogenase reaction. TCA, tricarboxylic acid.

FIG 2

Alfalfa plants inoculated with S. meliloti. Alfalfa plants were imaged 53 days postinoculation with S. meliloti. Strains: dctA⁺, wild-type S. meliloti; dctA, dctA14::Tn5; dctA maeP⁺, dctA14::Tn5, multicopy P_dctA::maeP.

FIG 3

Symbiotic phenotypes of pea plants inoculated with R. leguminosarum. Pea plants were harvested 25 days postinoculation with R. leguminosarum. (A) Images of the shoots of the pea plants. (B to G) Images of nodule sections visualized with bright-field microscopy following iodine staining. (H to M) Confocal microscopy images of nodule sections stained with the nucleic acid binding dye Syto9. Strains: dctA⁺, wild-type R. leguminosarum bv. viciae; dctA maeP⁺, dctA::ΩSp^r, multicopy P_dctA::maeP; dctA, dctA::ΩSp^r.

FIG 4

RNA-sequencing sample distance analysis. RNA-sequencing count tables were statistically analyzed with DESeq2 (57), and the Euclidean distances were calculated between each sample. Samples were clustered using hierarchical clustering analysis, and the dendrograms represent the clustering results. The heatmap illustrates the pairwise distances between the indicated samples, with the colors indicating the distances as shown in the key in the bottom left; i.e., the more blue the square, the more similar the samples.