Isolation of a novel, cryptic low molecular-weight, multicopy plasmid, pHIG22, from Thermus sp. K6 and its partial characterization

In this study, thermophilic bacteria of both water and mud samples were isolated from Alangullu (Aydın) hot spring. Two isolates of those were determined belongs to Thermus genus and it was examined whether they harboured plasmid or not.Plasmid content of two isolates, named Thermus sp. K6 and Thermus sp. M5, respectively, were examined. A small, novel and multicopy cryptic plasmid, named pHIG22, was isolated from Thermus sp. K6. The nucleotide sequence of pHIG22 revealed that plasmid was 2222 bp long, with a total G + C content of 63%.According to results of BLAST searches, the sequence didn?t show any similarities to any other plasmids; also the amino acid sequences of five putative open reading frames did not show similarities to those in the databases. ORF-1 426 bp long, ORF-2 741 bp long in leading strand; ORF-3 696 bp long, ORF-4 387 bp long and ORF-5 741 bp long in complementary strand were determined. Two promoter regions were predicted in complementary strand according to promotor analyses tools.E.coli-Thermus shuttle vector were constructed by using pHIG22, pUC18 cloning vector and kanamycin cassette. Desired regions from genome of pHIG22 were amplified with PCR and cloned to pET28a+ vector. Constructed recombinant plasmids were tried to transform to thermophilic bacteria.