A novel cryptic and theta type plasmid (pHIG22) from Thermus scodotuctus sp. K6.

A cryptic plasmid pHIG22 from Thermus scotoductus sp. K6, an isolate from the Alangullu Hot Spring (Aydin, Turkey), was sequenced and characterized. The pHIG22 plasmid is a multicopy, double stranded and 2222 bp circular molecule with 62.78% GC content, which shows a characteristical nucleotide sequence without any homology to other known plasmids. Five open reading frames were predicted based on the nucleotide sequence analysis. The deduced amino acid sequence of all predicted ORFs didn't show any similarity with any known proteins. Three palindroms were detected and two promoter sequences were predicted in both strands. With electron microscopy (TEM) analysis, the replication intermediates were seen as typical Q-shaped molecules that committing pHIG22 replicates via the Theta replication mechanism. A 2012 bp region among 387 and 614 bp of pHIG22 was determined as minimal replicon which carries the elements necessary for plasmid replication and ori region. Furthermore, quantitative real-time PCR showed that the relative copy number of pHIG22 was estimated to be 148.2 ± 4.7 copies per chromosome equivalent. The new Theta type plasmid would be useful and beneficial to build vectors for cloning of thermophilic genes and in vivo protein engineering.