The gap gene of Rhizobium etli is required for both free life and symbiosis with common beans.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH or Gap) is a ubiquitous enzyme essential for carbon and energy metabolism in most organisms. Despite its primary role in sugar metabolism, GAPDH is recognized for its involvement in diverse cellular processes, being considered a paradigm among multifunctional/moonlighting proteins. Besides its canonical cytoplasmic location, GAPDH has been detected on cell surfaces or as a secreted protein in prokaryotes, yet little is known about its possible roles in plant symbiotic bacteria. Here we report that Rhizobium etli, a nitrogen-fixing symbiont of common beans, carries a single gap gene responsible for both GAPDH glycolytic and gluconeogenic activities. An active Gap protein is required throughout all stages of the symbiosis between R. etli and its host plant Phaseolus vulgaris. Both glycolytic and gluconeogenic Gap metabolic activities likely contribute to bacterial fitness during early and intermediate stages of the interaction, whereas GAPDH gluconeogenic activity seems critical for nodule invasion and nitrogen fixation. Although the R. etli Gap protein is secreted in a c-di-GMP related manner, no involvement of the R. etli gap gene in c-di-GMP related phenotypes, such as flocculation, biofilm formation or EPS production, was observed. Notably, the R. etli gap gene fully complemented a double gap1/gap2 mutant of Pseudomonas syringae for free life growth, albeit only partially in planta, suggesting potential specific roles for each type of Gap protein. Nevertheless, further research is required to unravel additional functions of the R. etli Gap protein beyond its essential metabolic roles.

Impact of c-di-GMP on the Extracellular Proteome of Rhizobium etli.

Extracellular matrix components of bacterial biofilms include biopolymers such as polysaccharides, nucleic acids and proteins. Similar to polysaccharides, the secretion of adhesins and other matrix proteins can be regulated by the second messenger cyclic diguanylate (cdG). We have performed quantitative proteomics to determine the extracellular protein contents of a Rhizobium etli strain expressing high cdG intracellular levels. cdG promoted the exportation of proteins that likely participate in adhesion and biofilm formation: the rhizobial adhesion protein RapA and two previously undescribed likely adhesins, along with flagellins. Unexpectedly, cdG also promoted the selective exportation of cytoplasmic proteins. Nearly 50% of these cytoplasmic proteins have been previously described as moonlighting or candidate moonlighting proteins in other organisms, often found extracellularly. Western blot assays confirmed cdG-promoted export of two of these cytoplasmic proteins, the translation elongation factor (EF-Tu) and glyceraldehyde 3-phosphate dehydrogenase (Gap). Transmission Electron Microscopy immunolabeling located the Gap protein in the cytoplasm but was also associated with cell membranes and extracellularly, indicative of an active process of exportation that would be enhanced by cdG. We also obtained evidence that cdG increases the number of extracellular Gap proteoforms, suggesting a link between cdG, the post-translational modification and the export of cytoplasmic proteins.