Rhizobia are an agronomically valuable group of bacteria capable of entering into endosymbiotic relationships with leguminous plants, during which they fix atmospheric nitrogen using energy derived from the metabolism of plant-provided dicarboxylic acids. It is generally assumed that the gluconeogenic catabolism of dicarboxylic acids proceeds via the Embden-Meyerhoff-Parnas pathway in rhizobia. However, rhizobia are classically thought to lack the phosphofructokinase enzyme required for conversion of fructose-1,6-bisphosphate to fructore-6-phosphate as part of this pathway. Here, we demonstrate that a model rhizobium, Sinorhizobium meliloti, encodes a phosphofructokinase, completing the Embden-Meyerhoff-Parnas pathway of this organism. Biochemical characterization of the S. meliloti phosphofructokinase demonstrates that it can catalyze the reversible phosphorylation of fructose-6-phosphate under in vitro conditions in a pyrophosphate-dependent, rather than ATP-dependent, manner. We further show that S. meliloti also encodes a distinct fructose-1,6-bisphosphatase that can phenotypically complement the loss of the phosphofructokinase enzyme. Loss of both enzymes results in a block of the gluconeogenic pathway in S. meliloti and results in S. meliloti being unable to fix nitrogen in symbiosis with alfalfa (Medicago sativa). Phylogenetic analyses and complementation studies demonstrate that PPi-dependent phosphofructokinases are broadly distributed across the phylum Pseudomonadota (syn. Proteobacteria), including most rhizobial species of the class Alphaproteobacteria, suggesting both that PPi-dependent phosphofructokinases are likely more broadly distributed than is generally recognized, and that the catabolism of dicarboxylic acids in most rhizobia proceeds via a PPi-dependent phosphofructokinase.
Payton, R. D. J., Kaur, S., diCenzo, G. C., Oresnik, I. J.
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