Exploring the diversity of arsenic resistance genes from acid mine drainage microorganisms.

The microbial communities from the Tinto River, a natural acid mine drainage environment, were explored to search for novel genes involved in arsenic resistance using a functional metagenomic approach. Seven pentavalent arsenate resistance clones were selected and analysed to find the genes responsible for this phenotype. Insights about their possible mechanisms of resistance were obtained from sequence similarities and cellular arsenic concentration. A total of 19 individual open reading frames were analysed, and each one was individually cloned and assayed for its ability to confer arsenic resistance in Escherichia coli cells. A total of 13 functionally active genes involved in arsenic resistance were identified, and they could be classified into different global processes: transport, stress response, DNA damage repair, phospholipids biosynthesis, amino acid biosynthesis and RNA-modifying enzymes. Most genes (11) encode proteins not previously related to heavy metal resistance or hypothetical or unknown proteins. On the other hand, two genes were previously related to heavy metal resistance in microorganisms. In addition, the ClpB chaperone and the RNA-modifying enzymes retrieved in this work were shown to increase the cell survival under different stress conditions (heat shock, acid pH and UV radiation). Thus, these results reveal novel insights about unidentified mechanisms of arsenic resistance.

Heterologous Expression of the Phytochelatin Synthase CaPCS2 from Chlamydomonas acidophila and Its Effect on Different Stress Factors in Escherichia coli.

Phytochelatins (PCs) are cysteine-rich small peptides, enzymatically synthesized from reduced glutathione (GSH) by cytosolic enzyme phytochelatin synthase (PCS). The open reading frame (ORF) of the phytochelatin synthase CaPCS2 gene from the microalgae Chlamydomonas acidophila was heterologously expressed in Escherichia coli strain DH5α, to analyze its role in protection against various abiotic agents that cause cellular stress. The transformed E. coli strain showed increased tolerance to exposure to different heavy metals (HMs) and arsenic (As), as well as to acidic pH and exposure to UVB, salt, or perchlorate. In addition to metal detoxification activity, new functions have also been reported for PCS and PCs. According to the results obtained in this work, the heterologous expression of CaPCS2 in E. coli provides protection against oxidative stress produced by metals and exposure to different ROS-inducing agents. However, the function of this PCS is not related to HM bioaccumulation.

Novel Genes Involved in Resistance to Both Ultraviolet Radiation and Perchlorate From the Metagenomes of Hypersaline Environments.

Microorganisms that thrive in hypersaline environments on the surface of our planet are exposed to the harmful effects of ultraviolet radiation. Therefore, for their protection, they have sunscreen pigments and highly efficient DNA repair and protection systems. The present study aimed to identify new genes involved in UV radiation resistance from these microorganisms, many of which cannot be cultured in the laboratory. Thus, a functional metagenomic approach was used and for this, small-insert libraries were constructed with DNA isolated from microorganisms of high-altitude Andean hypersaline lakes in Argentina (Diamante and Ojo Seco lakes, 4,589 and 3,200 m, respectively) and from the Es Trenc solar saltern in Spain. The libraries were hosted in a UV radiation-sensitive strain of Escherichia coli (recA mutant) and they were exposed to UVB. The resistant colonies were analyzed and as a result, four clones were identified with environmental DNA fragments containing five genes that conferred resistance to UV radiation in E. coli. One gene encoded a RecA-like protein, complementing the mutation in recA that makes the E. coli host strain more sensitive to UV radiation. Two other genes from the same DNA fragment encoded a TATA-box binding protein and an unknown protein, both responsible for UV resistance. Interestingly, two other genes from different and remote environments, the Ojo Seco Andean lake and the Es Trenc saltern, encoded two hypothetical proteins that can be considered homologous based on their significant amino acid similarity (49%). All of these genes also conferred resistance to 4-nitroquinoline 1-oxide (4-NQO), a compound that mimics the effect of UV radiation on DNA, and also to perchlorate, a powerful oxidant that can induce DNA damage. Furthermore, the hypothetical protein from the Es Trenc salterns was localized as discrete foci possibly associated with damaged sites in the DNA in cells treated with 4-NQO, so it could be involved in the repair of damaged DNA. In summary, novel genes involved in resistance to UV radiation, 4-NQO and perchlorate have been identified in this work and two of them encoding hypothetical proteins that could be involved in DNA damage repair activities not previously described.

Salt resistance genes revealed by functional metagenomics from brines and moderate-salinity rhizosphere within a hypersaline environment.

Hypersaline environments are considered one of the most extreme habitats on earth and microorganisms have developed diverse molecular mechanisms of adaptation to withstand these conditions. The present study was aimed at identifying novel genes from the microbial communities of a moderate-salinity rhizosphere and brine from the Es Trenc saltern (Mallorca, Spain), which could confer increased salt resistance to Escherichia coli. The microbial diversity assessed by pyrosequencing of 16S rRNA gene libraries revealed the presence of communities that are typical in such environments and the remarkable presence of three bacterial groups never revealed as major components of salt brines. Metagenomic libraries from brine and rhizosphere samples, were transferred to the osmosensitive strain E. coli MKH13, and screened for salt resistance. Eleven genes that conferred salt resistance were identified, some encoding for well-known proteins previously related to osmoadaptation such as a glycerol transporter and a proton pump, whereas others encoded proteins not previously related to this function in microorganisms such as DNA/RNA helicases, an endonuclease III (Nth) and hypothetical proteins of unknown function. Furthermore, four of the retrieved genes were cloned and expressed in Bacillus subtilis and they also conferred salt resistance to this bacterium, broadening the spectrum of bacterial species in which these genes can function. This is the first report of salt resistance genes recovered from metagenomes of a hypersaline environment.

Diversity of Archaea in Icelandic hot springs based on 16S rRNA and chaperonin genes.

The diversity of archaeal communities growing in four hot springs (65-90 °C, pH 6.5) was assessed with 16S rRNA gene primers specific for the domain Archaea. Overall, mainly uncultured members of the Desulfurococcales, the Thermoproteales and the Korarchaeota, were identified. Based on this diversity, a set of chaperonin heat-shock protein (Hsp60) gene sequences from different archaeal species were aligned to design two degenerate primer sets for the amplification of the chaperonin gene: Ths and Kor (which can also detect the korarchaeotal chaperonin gene from one of the samples). A phylogenetic tree was constructed using the chaperonin sequences retrieved and other sequences from cultured representatives. The Alpha and Beta paralogs of the chaperonin gene were observed within the main clades and orthologs among them. Cultivated representatives from these clades were assigned to either paralog in the chaperonin tree. Uncultured representatives observed in the 16S rRNA gene analysis were found to be related to the Desulfurococcales. The topologies of the 16S rRNA gene and chaperonin phylogenetic trees were compared, and similar phylogenetic relationships were observed. Our results suggest that the chaperonin Hsp60 gene may be used as a phylogenetic marker for the clades found in this extreme environment.

Novel nickel resistance genes from the rhizosphere metagenome of plants adapted to acid mine drainage.

Metal resistance determinants have traditionally been found in cultivated bacteria. To search for genes involved in nickel resistance, we analyzed the bacterial community of the rhizosphere of Erica andevalensis, an endemic heather which grows at the banks of the Tinto River, a naturally metal-enriched and extremely acidic environment in southwestern Spain. 16S rRNA gene sequence analysis of rhizosphere DNA revealed the presence of members of five phylogenetic groups of Bacteria and the two main groups of Archaea mostly associated with sites impacted by acid mine drainage (AMD). The diversity observed and the presence of heavy metals in the rhizosphere led us to construct and screen five different metagenomic libraries hosted in Escherichia coli for searching novel nickel resistance determinants. A total of 13 positive clones were detected and analyzed. Insights about their possible mechanisms of resistance were obtained from cellular nickel content and sequence similarities. Two clones encoded putative ABC transporter components, and a novel mechanism of metal efflux is suggested. In addition, a nickel hyperaccumulation mechanism is proposed for a clone encoding a serine O-acetyltransferase. Five clones encoded proteins similar to well-characterized proteins but not previously reported to be related to nickel resistance, and the remaining six clones encoded hypothetical or conserved hypothetical proteins of uncertain functions. This is the first report documenting nickel resistance genes recovered from the metagenome of an AMD environment.