Analysis of a periplasmic thiol oxidoreductase in Rhizobium leguminosarum

Abstract

In this work the expression and cellular localization of a predicted periplasmic thiol oxidoreductase encoded by Rhizobium leguminosarum 3841, ORF RL1083, were analysed. Based on the homology of the encoded protein with DsbA proteins from other bacteria we named it dsbA. The genetic organization of dsbA region showed that it is a monocystronic gene. A putative σ70 promoter was predicted upstream dsbA gene. The promoter region fused to gusA reporter gene revealed that dsbA is expressed in free-living conditions in different media and also, although at a lower level, in pea bacteroids. R. leguminosarum DsbA contains a potential Tat-dependent signal peptide. To localize this protein in different cellular fractions the protein was labelled by means of a C-terminal Strep tag. The DsbA-Strep protein was localized in the periplasmic fraction. At present three type of experiments are in progress: first, the study of DsbA Tat-dependence by using a tat mutant strain harbouring dsbA-Strep; second, the construction of a dsbA mutant and third the evaluation of periplasmic disulfide oxidoreductase activity of different strains: wild-type, tat mutant and dsbA mutant.