Metagenomics and culture-dependent insights into the diversity of halophiles microorganisms indigenous to the Karak salt mine, Pakistan. Metagenomic study of diversity of microorganisms in the salt mine of Pakistan

Hypersaline regions are terrestrial analogs of the Earth’s primitive ecosystem and extraterrestrial environment. The salt range in Pakistan is considered among few of the ancient salt deposits in the subcontinent. Karak salt mine is situated at the Northwest end in Pakistan. Despite the fact that halophiles initiated the formation of terrestrial ecosystems, their products and identities remain hidden. Some preliminary studies limited to culture dependent isolations have been reported. Understanding the microbiome that spans over centuries of ecosystem development is crucial, given its role in shaping landscape succession and biogeochemical cycles. Here, we used metagenomics and culturing techniques to explore the diversity of Karak salt mine and to screen the extreme halophiles for the presence of unique genes for halophilic adaptations. We used DGGE and Illumina sequencing to study the halophiles entrapped in Karak mine. The results were interpreted using Basespace, QIIME & Cytoscape. Cultures were isolated at 16-25% salinity. With the help of bioinformatics tools, genes of interest were located, identified and characterized. Metagenomics data was consistent with culturing, indicating remarkable species to strain level diversity of unique halophiles. A total of 53,947 (brine) and 85,418 (salt) reads were obtained. 16S rRNA based sequencing revealed a microbiome with bacteria (66% brine & 72% salt) dominated by Bacteroidetes, Proteobacteria, and Firmicutes with a strikingly high abundance of Archaea (18% brine & 13% salt). Alpha diversity has higher values in salt than in the brine. Genes responsible for the production of ectoine, betain, halozymes, bacteriorhodopsin and gas vesicle proteins were characterized. The study of the halophiles in the Karak salt mine gives clue for the maintenance of the ecosystem. Genes producing bioactive compounds were located in the isolates, establishing their potential in industrial applications. This is the first report of metagenomics studies of any hypersaline region of Pakistan.