Isolation of gene conferring salt tolerance from halophilic bacteria of Lunsu, Himachal Pradesh, India.

BACKGROUND: Halophiles offer an attractive source of genes conferring salt tolerance. Halobacillus trueperi SS1 strain of Lunsu, Himachal Pradesh, India, a strict halophile, was exploited to isolate and clone the genes for salt tolerance. The genomic library of BamH1 digest of H. trueperi SS1 was constructed in pUC19, and recombinants were screened for salt tolerance on an LB medium containing ampicillin (100 µg/ml) and NaCl (0 to 1.5 M). RESULTS: One recombinant clone named as salt-tolerant clone (STC) conferred salt tolerance to host Escherichia coli/DH5α, which showed growth in the LB medium supplemented with ampicillin and 1.2 M NaCl. Restriction digestion and PCR analysis revealed the presence of an insert of approximately 2000 bp in the STC. DNA sequencing of the 2-kb insert on both strands yielded a sequence of 2301 nucleotides. Protein BLAST analysis of 2301-bp sequence of H. trueperi SS1 present in STC showed 97% identity to multidrug transport ATP binding/permease protein of Halobacillus karajensis. The insert contained in STC was subcloned into pGEX4T2 vector, and the recombinant clone STC/pGEX4T2 conferred salt tolerance to the bacterial host E. coli. CONCLUSIONS: The present study led to the isolation of salt tolerance gene encoding a putative multidrug transport ATP binding/permease protein from H. trueperi SS1. The salt tolerance gene can be subcloned for transferring salt tolerance traits into agricultural crop plants for cultivation in saline and coastal lands.

A diverse group of halophilic bacteria exist in Lunsu, a natural salt water body of Himachal Pradesh, India.

Five halophilic bacterial isolates namely SS1, SS2, SS3, SS5 and SS8 were isolated from soil sediments of Lunsu, a salty water body. All the bacterial isolates showed growth in LB medium containing up to 8.7% NaCl, pH 7-8 and at temperature range of 30-37°C. The bacterial isolates SS1 and SS3 require at least 3.8% NaCl for their growth, indicating their strict halophilic nature. Interestingly, bacterial isolates SS2, SS5 and SS8 but not SS1 and SS3 exhibited growth in medium supplemented with KCl. Accordingly, Na(+) and K(+) ions were detected at 1.39 and 0.0035%, respectively in Lunsu water. All the bacterial isolates were analyzed by random amplification of polymorphic DNA (RAPD) using four different random primers and produced PCR fragments ranging from 0.1 to 5 kb in size. Phylogenetic tree based on RAPD finger prints showed that SS1 and SS3 formed one group, while SS2 and SS5 formed the second group, whereas SS8 was out group. Sequence analysis of 16S rDNA identified SS1 and SS3 as Halobacillus trueperi, SS2 as Shewanella algae, SS5 as Halomonas venusta, and SS8 as Marinomonas sp. were deposited in GenBank with accession numbers of KM260166, KF751761, KF751760, KF751762 and KF751763, respectively. This is the first report on the presence of diverse halophilic bacteria in the foot hills of Himalayas.