Characterization of the anaerobic metabolism of the tetralin degrader Sphingopyxis granuli TFA strain

Abstract

Sphingopyxis granuli TFA strain is an alphaproteobacteria isolated from the Rhine river which is able to degrade the pollutant compound tetralin. We have shown recently that TFA is able to grow anaerobically using nitrate as final electron acceptor for respiration and transforming it to nitrite. In fact, a cluster of genes (narUGHIJ) coding for proteins involved in nitrate respiration has been found in its genome. To date, no anaerobic growth had been described in Sphingopyxis representatives. We are interested in studying the regulation mechanisms of anaerobic respiration in TFA. The genome sequence of TFA revealed the presence of three regulatory genes encoding for homologs of FixLJ proteins and two fnr-like regulatory genes (SGRAN_2447 and SGRAN_3861). FixLJ is a two component regulatory system able to detect low oxygen concentration and activate the expression of the transcription factor FixK, which in turn regulates the expression of many micro-aerobic genes. Fnr proteins are transcriptional regulators able to detect oxygen levels by themselves and directly regulate the expression of a wide range of genes in response to changes in oxygen. In Escherichia coli, Fnr protein shows four highlhhy conserved cysteine residues involved in the co-ordination of a redox sensitive [2Fe-2S] cluster. In other bacteria as Rhodobacter sphaeroides the third Cys residue is displaced two positions downstream (FnrN group). TFA SGRAN_2447 and SGRAN_3861 exhibit the redox-responsive cysteine motif with the third cysteine displaced as in the FnrN group. SGRAN_2447 shares a 24.6% identity with E. coli Fnr and a 38.2% with R. sphaeroides FnrL while SGRAN_3861 shows a 27.0% and a 31.3% identity respectively. In order to study their role in regulation we have constructed TFA mutant derivatives lacking either SGRAN_2447 or SGRAN_3861 genes and analyzed their growth at low oxygen concentrations. In addition, we have performed functional complementation assays in some E. coli fnr mutants to examine both the ability to restore the anaerobic growth and activate expression from a FNR-dependent promoter fused to lacZ. Our results are presented here.