Diversity And Salt-tolerance Of Prokaryotes In Shaanxi Huama Salt Lake

This study explored bacterial community diversity of Huama salt lake sediments, Shaanxi, based on the high-throughput sequencing technologies. The results showed that microbial communities contained 2 Domain, 34 Phylum, 86 Class, 120 Order, 169 Family, and 242 Genus. The dominant genus of archaea was Haloarcula, Halobacterium, Halorubrum, Haloplanus, etc. and the dominant genus of bacteria was Pseudomonas, and Salinimicrobium. Our results exhibited a rich diversity of species in Huama salt lake of Shaanxi.To explore salt-tolerance mechanism halophilic microbes in Shaanxi Huama salt lake, we constructed a fosmid metagenomic library of salt lake sediments, and obtained a library containing 4126 clones. A total of 37 salt-tolerant clones were selected from the library through the 7.5% NaCl tolerance test, and the clone 2E4 was picked out for high-through sequencing to analyze its functional genes. The results showed that inserted DNA fragments in clone 2E4 could be from Pseudomonas stutzeri, and the functional genes in database COG were categorized into General function prediction, Amino acid transport and metabolism and Signal transduction mechanisms, Transcription, etc. And these genes in the database GO were classified into biological processes, Cellular component, and Molecular function. We functionally annotated 219 genes in the database SwissProt, revealing that salt-tolerant genes, encoding cation-transporting ATPase F, Na+/H+ antiporter subunit G, CDP-diacylglycerol-glycerol-3-phosphate3-phosphatidyltransferase, aspartokinase, and choline dehydrogenase, could be involved in osmotic adaptation in salt lake.And then, we made an EZ-Tn5 mutant library of the clone 2E4, obtained a mutant library containing 1670 clones, and detected the four salt-sensitive mutation clones. Gene sequencing showed that the transposon insertion destroyed the upstream promoter sequences of aminoglycoside3’-phosphotransferase(aphA). Growth tests of 2E4 and 3E7 against different ions(Na+, K+, Zn2+, Cu2+, Cd2+) showed that the growth of 3E7 was decreased in media containing Na+, K+, Zn2+ and Cu2+, comparing with that of 2E4, but growth enhancement under Cd2+ stress, indicating that up-expressed of aphA was positively correlated with ion stresses(Na+, K+, Zn2+, Cu2+) and down-regulated of aphA was positively correlated with Cd2+ stress. This demonstrated that aphA plays a key role in salt tolerance of halophilic bacteria.