Microorganisms represent an intriguing and underexploited resource for the discovery of novel genes. Extremophiles in environments like the Namib Desert are exposed to multiple stress factors including UV radiation, desiccation, osmotic, oxidative, nutrient and temperature stress. These microorganisms are well adapted to tolerate extended exposure to stressors and rapid change in the environmental conditions. Functional metagenomics is a powerful tool for the discovery of novel genes and enzymes. As sequence based approaches are limited by sequence homologues, functional screening allows for the identification of unique genes involved in stress tolerance.
In this study, novel genes involved in stress tolerance were identified by screening a Namib Desert soil metagenomic fosmid library (>1x106 clones) in Escherichia coli EPI300 under hyperosmotic (NaCl), oxidative (H2O2), heat (46°C) and UVB radiation (280 – 315 nm) stress. Increasing the sodium chloride (NaCl) concentration in growth media to 5% resulted in the identification of 12 salt-tolerant clones. All clones, except one, showed significantly increased growth compared to the host (containing an empty vector) in liquid media augmented with 5% NaCl after 48 hours (P-value <0.05). The clones contained fosmids of approximately 42 kilobases (kb) with estimated insert sizes ranging from 30 to 34 kb. Fosmid DNA of salt-tolerant clones was sequenced using the Ion Torrent PGM platform at the University of Pretoria, generating 1.4 Gb of sequence data. Sequences were assembled using MIRA and open reading frames were predicted using the RAST server.
Phylogenetic analysis, using conserved proteins, revealed that the metagenomic DNA originated from members of the phyla Deltaproteobacteria, Fibrobacteres, Cyanobacteria, Actinobacteria and Planctomyces. Six putative salt-tolerance genes were identified through functional predictions based on conserved domains present in the encoded proteins. The proteins; an ABC transporter substrate-binding domain, a RelA-SpoT-like hypothetical protein, an HD-hydrolase domain protein, a cation export system protein, a peptidase M29 and a Na+/H+ antiporter, all shared less than 60% amino acid identity with the closest homologues. The low amino acid identity of the proteins encoded on the metagenomic DNA, to known sequences, suggests that the inserts were derived from novel taxa, highlighting the untapped microbial communities present in the Namib Desert. Over-expression of the proteins in E. coli BL21(DE3) and subsequent NaCl trials revealed that cells expressing the peptidase M29 showed significantly increased salt-tolerance at 3.5% NaCl compared to the host. Future work will entail investigating the mechanism by which the peptidase M29 confers salt-tolerance to E. coli, investigating whether any cross-protection to other abiotic stresses is conferred and determining the optimal parameters for activity and substrate specificity of the enzyme in vitro. The identification of novel stress-tolerance genes and proteins holds potential for application in industrial biotechnology, medicine and agriculture and provides insight into the mechanisms employed by extremophiles to tolerate abiotic stress.