Abstrakt

We report the identification and characterization of the single-stranded DNA binding protein (SSB) from extreme psychrophilic bacterium Psychromonas ingrahamii 37 (PinSSB) that grows expotentially at -12°C and may well grow at even lower temperatures. PinSSB is one of the largest known bacterial SSB protein consisting 222 amino acid residues with a calculated molecular mass of 25.1 kDa. The analysis by gel filtration chromatography revealed single peak with a molecular mass of about 77,5 kDa that indicates PinSSB is functional as homotetramer containing one single-stranded DNA binding domain (OB-fold) in each monomer. Psychromonas SSB possesses a high sequence homology to Escherichia coli SSB (45% identity and 58% similarity). Fluorescence spectroscopy assays indicated that PinSSB binds single-stranded DNA with a binding site size of 8 - 10 nucleotides only, depending on the salt concentration. Bacterial SSBs proteins indentified to date have at least twice as big ssDNA-binding site. It indicates that very probably PinSSB as huge homotetramer can binds ssDNA in special manner. The half-lives of PinSSB was 5 min at 60°C and 1 min at 70°C. These results showed that PinSSB as the first characterized SSB from strictly psychrophilic microorganizm is partly thermostable SSB protein with unique very short ssDNA-binding site, offering an attractive alternative for other SSB proteins in their applications for molecular biology techniques.

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