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

The rpoA gene encoding the alpha subunit of RNA polymerase from the deep-se a piezophilic bacterium Shewanella violacea DSS12 was cloned and sequenced. The rpoA gene was found to encode a polypeptide consisting of 329 amino ac ids with a molecular mass of 36 238 Da. S. violacea alpha protein was expre ssed in a ts Escherichia coli mutant. to confirm whether the rpoA gene is f unctional. It complemented this mutation, indicating a chimeric RNA polymer ase is assembled at the non-permissive temperature. Recombinant alpha prote in was overexpressed using an expression plasmid harboring the rpoA gene an d purified to near homogeneity. Primer extension analysis revealed that two transcriptional initiation sites are recognized by sigma (70) RNA polymera se. It also indicated that pressure response (piezoresponse) in the alpha o peron occurred at the transcriptional level. suggesting some positive regul ators may interact with the transcriptional apparatus and regulate the expr ession of the operon at different pressure conditions. (C) 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. A ll rights reserved.