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Abstract
A moderately thermophilic Gram-negative bacterium isolated from the Polok hot spring, Sikkim, India, was identified as a strain (PL17) of Tepidimonas fonticaldi by 16S rDNA sequencing. T. fonticaldi PL17 produces a Type IIP restriction endonuclease; named TfoI. Restriction mapping, run-off sequencing of TfoI-digests of dsDNA fragments, and end compatibility of TfoI with NdeI confirmed that the enzyme recognizes and cleaves the sequence 5'-T^TAA-3', and is thus an isoschizomer of MseI. The TfoI restriction-modification genes in the T. fonticaldi PL17 genome were identified, and the annotated TfoI protein encodes a protein of 181 amino acid residues that shares 47.2% sequence identity with MseI. The native enzyme was purified using a four-column chromatography protocol, and its functional homogeneity was confirmed by standard quality control tests. The ESI-MS measured molecular weight of purified TfoI (20.696kDa) is in agreement with that of the calculated monomeric molecular weight of the predicted TfoI protein sequence (20.694kDa). TfoI exhibits optimal activity in the temperature range of 55-70 °C with Mg+2 or Co+2 as cofactor. Similar to its isoschizomers, TfoI can be used as the frequent cutter for genome analysis.
Published Version
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