<i>Frankia alni</i> Carbonic Anhydrase Regulates Cytoplasmic pH of Nitrogen-Fixing Vesicles

oleh: Petar Pujic, Lorena Carro, Pascale Fournier, Jean Armengaud, Guylaine Miotello, Nathalie Dumont, Caroline Bourgeois, Xavier Saupin, Patrick Jame, Gabriela Vuletin Selak, Nicole Alloisio, Philippe Normand

Format: Article
Diterbitkan: MDPI AG 2023-05-01

Deskripsi

A phyloprofile of <i>Frankia</i> genomes was carried out to identify those genes present in symbiotic strains of clusters 1, 1c, 2 and 3 and absent in non-infective strains of cluster 4. At a threshold of 50% AA identity, 108 genes were retrieved. Among these were known symbiosis-associated genes such as <i>nif</i> (nitrogenase), and genes which are not know as symbiosis-associated genes such as <i>can</i> (carbonic anhydrase, CAN). The role of CAN, which supplies carbonate ions necessary for carboxylases and acidifies the cytoplasm, was thus analyzed by staining cells with pH-responsive dyes; assaying for CO<sub>2</sub> levels in N-fixing propionate-fed cells (that require a propionate-CoA carboxylase to yield succinate-CoA), fumarate-fed cells and N-replete propionate-fed cells; conducting proteomics on N-fixing fumarate and propionate-fed cells and direct measurement of organic acids in nodules and in roots. The interiors of both in vitro and nodular vesicles were found to be at a lower pH than that of hyphae. CO<sub>2</sub> levels in N<sub>2</sub>-fixing propionate-fed cultures were lower than in N-replete ones. Proteomics of propionate-fed cells showed carbamoyl-phosphate synthase (CPS) as the most overabundant enzyme relative to fumarate-fed cells. CPS combines carbonate and ammonium in the first step of the citrulline pathway, something which would help manage acidity and NH<sub>4</sub><sup>+</sup>. Nodules were found to have sizeable amounts of pyruvate and acetate in addition to TCA intermediates. This points to CAN reducing the vesicles’ pH to prevent the escape of NH<sub>3</sub> and to control ammonium assimilation by GS and GOGAT, two enzymes that work in different ways in vesicles and hyphae. Genes with related functions (carboxylases, biotin operon and citrulline-aspartate ligase) appear to have undergone decay in non-symbiotic lineages.