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.

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Leptospirillum ferriphilum is an acidophilic iron-oxidizing bacterium that is relevant for chemical leaching of sulfide ores. In the extremely acidic conditions found in bioleaching operations, this microorganism deals with an abundant supply of soluble iron and other metals that might induce oxidative damage to biomolecules through the generation of reactive oxygen species (ROS). We evaluated the role of Dyp-type peroxidase in the protection against oxidative stress in L. ferriphilum DSM14647. The genetic region encoding dypA was cloned and sequenced. The predicted DypA enzyme is 295 amino acids long with an estimated molecular mass of 32.9 kDa containing a highly conserved peroxide reduction motif. Genetic complementation of catalases/peroxidases-deficient Escherichia coli cells indicated that expression of dypA from L. ferriphilum restored the resistance to hydrogen proxide to levels exhibited by the wild type strain. Exposure of L. ferriphilum to hydrogen peroxide leads to a significant transcriptional activation of dypA suggesting its involvement in the response to oxidative stress in this bacterium. This is the first Dyp-type peroxidase characterized from an acidophilic microorganism, making it a potential candidate for research in basic and applied biology.

Fe-S clusters are versatile and essential cofactors that participate in multiple and fundamental biological processes. In Escherichia coli , the biogenesis of these cofactors requires either the housekeeping Isc pathway, or the stress-induced Suf pathway which plays a general role under conditions of oxidative stress or iron limitation. In the present work, the Fe-S cluster assembly Isc and Suf systems of acidophilic Bacteria and Archaea, which thrive in highly oxidative environments, were studied. This analysis revealed that acidophilic microorganisms have a complete set of genes encoding for a single system (either Suf or Isc). In acidophilic Proteobacteria and Nitrospirae, a complete set of isc genes ( iscRSUAX-hscBA-fdx ), but not genes coding for the Suf system, was detected. The activity of the Isc system was studied in Leptospirillum sp. CF-1 (Nitrospirae). RT-PCR experiments showed that eight candidate genes were co-transcribed and conform the isc operon in this strain. Additionally, RT-qPCR assays showed that the expression of the iscS gene was significantly up-regulated in cells exposed to oxidative stress imposed by 260 mM Fe 2 (SO 4 ) 3 for 1 h or iron starvation for 3 h. The activity of cysteine desulfurase (IscS) in CF-1 cell extracts was also up-regulated under such conditions. Thus, the Isc system from Leptospirillum sp. CF-1 seems to play an active role in stressful environments. These results contribute to a better understanding of the distribution and role of Fe-S cluster protein biogenesis systems in organisms that thrive in extreme environmental conditions.