Isolation and piezoresponse of the rpoA gene encoding the RNA polymerase alpha subunit from the deep-sea piezophilic bacterium Shewanella violacea.

Abstract

The rpoA gene encoding the alpha subunit of RNA polymerase from the deep-sea piezophilic bacterium Shewanella violacea DSS12 was cloned and sequenced. The rpoA gene was found to encode a polypeptide consisting of 329 amino acids with a molecular mass of 36238 Da. S. violacea alpha protein was expressed in a ts Escherichia coli mutant, to confirm whether the rpoA gene is functional. It complemented this mutation, indicating a chimeric RNA polymerase is assembled at the non-permissive temperature. Recombinant alpha protein was overexpressed using an expression plasmid harboring the rpoA gene and purified to near homogeneity. Primer extension analysis revealed that two transcriptional initiation sites are recognized by sigma(70) RNA polymerase. It also indicated that pressure response (piezoresponse) in the alpha operon occurred at the transcriptional level, suggesting some positive regulators may interact with the transcriptional apparatus and regulate the expression of the operon at different pressure conditions.