Deciphering the Role of Multiple Thioredoxin Fold Proteins of Leptospirillum sp. in Oxidative Stress Tolerance

Abstract

Thioredoxin fold proteins (TFPs) form a family of diverse proteins involved in thiol/disulfide exchange in cells from all domains of life. Leptospirillum spp. are bioleaching bacteria naturally exposed to extreme conditions like acidic pH and high concentrations of metals that can contribute to the generation of reactive oxygen species (ROS) and consequently the induction of thiol oxidative damage. Bioinformatic studies have predicted 13 genes that encode for TFP proteins in Leptospirillum spp. We analyzed the participation of individual tfp genes from Leptospirillum sp. CF-1 in the response to oxidative conditions. Genomic context analysis predicted the involvement of these genes in the general thiol-reducing system, cofactor biosynthesis, carbon fixation, cytochrome c biogenesis, signal transduction, and pilus and fimbria assembly. All tfp genes identified were transcriptionally active, although they responded differentially to ferric sulfate and diamide stress. Some of these genes confer oxidative protection to a thioredoxin-deficient Escherichia coli strain by restoring the wild-type phenotype under oxidative stress conditions. These findings contribute to our understanding of the diversity and complexity of thiol/disulfide systems, and of adaptations that emerge in acidophilic microorganisms that allow them to thrive in highly oxidative environments. These findings also give new insights into the physiology of these microorganisms during industrial bioleaching operations.

Keywords: Leptospirillum sp. CF-1; bioleaching; oxidative stress; thioredoxin; thioredoxin fold proteins.

Figure 1

Predicted genetic context of TFP-encoding genes from Leptospirillum sp. CF-1. A.1. The tfp13 gene codes for a: thioredoxin reductase (TrxB); hyp1: hypothetical protein 1; hyp2: hypothetical protein 2; tfp11: thioredoxin domain-containing protein (YyaL); ypfH: predicted esterase. A.2. rpl28: ribosomal protein L28; end: endonuclease; hyp1: hypothetical protein 1; tfp1: thioredoxin-like protein; hyp2: hypothetical protein 2. A.3. groES: co-chaperone GroES; groEL: chaperone GroEL; trp: TRP-containing small protein; tfp2: thioredoxin-like protein (chaperedoxin); recN: DNA repair protein RecN. A.4. tfp6: thioredoxin-like protein; psa: puromycin- sensitive aminopeptidase. B.1. tfp3: thiol disulfide oxidoreductase; yjbQ: thiamine synthase YjbQ; prmA: ribosomal protein L11 methytransferase PrmA; pyrF: Orotidine 5’-phosphate decarboxylase PyrF. B.2. tfp4: thioredoxin-like protein; nadE: NAD(+) synthetase. C.1. acnA: aconitase A; ccl: citryl-CoA lyase; hyp1: fdrA: fumarate reductase subunit A; frB: fumarate reductase subunit B; sucC: succinyl-CoA synthetase alpha subunit; sucD: succinyl-CoA synthetase beta subunit; tfp10: 2-Cys peroxiredoxin (Prx). D.1. ccsB: cytochrome c-type biogenesis protein CcsB; ccsA: cytochrome c-type biogenesis protein CcsA; tfp5: cytochrome c-type biogenesis protein CcsX; tfp7: thioredoxin-like protein; ccdA: cytochrome c-type biogenesis protein CcdA; ggr: geranylgeranyl reductase. E.1. tfp8: thiol disulfide oxidoreductase; dgc: GGDEF diguanilate cyclase; hyp: hypothetical small membrane protein. E.2. agc: adenylate/guanylate cyclase; trp: tetratricopeptide repeat (TRP)-containing protein; tfp9: thiol disulfide oxidoreductase. F.1. tfp12: cytoplasmic thiol disulfide oxidoreductase; pkk2: polyphosphate kinase 2; hyp: hypothetical protein; pulO: type IV prepilin peptidase.

Figure 2

Relative expression of tfp genes from Leptospirillum sp. CF-1. Cells were stressed with (Fe₂(SO₄)₃) (A) or diamide (B) for 1 h as indicated in Material and Methods. The RNA samples were analyzed in triplicate and ∆Ct results compared to the control 16S RNA gene. Bars represent the average of three independent experiments ± standard deviation. (*): p < 0.05.

Figure 3

Growth of Escherichia coli JEM-136 complemented with tfp genes under oxidative stress culture conditions. Expression induction of the tfp gene in E. coli carrying pBADTopo-tfp was carried out with l-arabinose, as described in Materials and Methods. The complemented strain was grown on minimum tryptone yeast extract salts (TYES) medium under stress conditions with 0.5 mM (Fe₂(SO₄)₃) (A) for 15 min, then transferred to a fresh medium, and grown for another 13 h. For diamide stress, the cells were exposed to 0.4 mM diamide and then grown for 20 h (B).