|dc.description.abstract||Sphingopyxis granuli strain TFA is an ¿-Proteobacteria isolated from the Rhine river that is able to degrade the organic solvent tetralin. Until now, the Sphingopyxis genus had always been described as strict aerobic, but we have demonstrated in this work that TFA is able to respire nitrate to nitrite in anaerobic conditions, thus becoming the first facultative anaerobic Sphingopyxis strain reported.
Two putative Fnr anaerobic regulatory proteins have been found in TFA, FnrN and FixK, which have shown to be necessary for anaerobic growth, being FnrN more relevant than FixK. Transcriptomic analyses of this bacterium by dRNA-seq and RT qPCR have shown differentially regulated genes in anaerobiosis as compared to aerobiosis, which belong to different functional categories. Similar analyses using a double mutant ¿fnrN¿fixK led to identification of genes directly regulated by the Fnr regulators. A regulon of 14 operons has been defined and consensus recognition site for these regulatory proteins, the Fnr box, has also been identified. The central metabolism of this bacterium is barely affected in anaerobic conditions, with scarce exceptions. As expected, different alternative terminal oxidases have been induced in anaerobic conditions: the nitrate reductase (nar), the cytochrome bd oxidase (cyd), the cytochrome o quinol oxidase (cyo), the cbb3 cytochrome c oxidase (cco) and an NO-insensitive alternative oxidase (aox). However, only nar and cco showed Fnr sites and seemed to be regulated by Fnr. Interestingly, many genes involved in stress response and SOS repair systems have been induced in anaerobic conditions, suggesting that anaerobiosis is a hostile and mutagenic environment for TFA, probably due to nitrite accumulation as the respiration product and its partial transformation into nitric oxide. The induction of these genes is slow, as expected, as it probably needs the accumulation of toxic respiration products, such as nitric oxide, and of DNA damage, and not directly regulated by Fnr proteins. This is consistent with the fact that the nitric oxide reductase gene, norB, was highly induced, and its induction was very fast, reaching it maximum induction after 2 hours of anaerobic growth, suggesting that nitric oxide is the real inducer of this gene and is produced from the beginning. Both fnrN and fixK were also induced in anaerobiosis and an Fnr box was identified in the promoter of fixK. All the flagellar, pili and chemotaxis genes were repressed in anaerobic conditions, which is surprising taking into account that anaerobiosis is a non-favourable condition for TFA.
Moreover, EM has shown that TFA has a polar bunch of flagella and fimbriae, which is lost in anaerobic conditions. Three putative flagellar regulatory proteins have been identified in TFA, CtrA, FleQ and FliA, which are necessary for motility in this bacterium. TFA is the first Sphingopyxis strain whose flagellum has been characterised to some extent. During this characterisation, four spontaneous mutants have been isolated that swim faster than the WT strain.||es_ES