Identification of rpoBC genes encoding for βand β′ subunits of RNA polymerase in a deep-sea piezophilic bacterium, Shewanella violacea strain DSS12

Abstract

RNA polymerase from cells of the deep-sea bacterium Shewanella violacea DSS12 was purified using three chromatographic steps. An in vitro transcription assay indicated that the purified enzyme was σ70 containing RNA polymerase. The enzyme activity was inhibited in the presence of rifampicin when the sensitive domain was targeted. The rpoBC genes encoding for the β and β′ subunits of RNA polymerase were cloned and their nucleotide sequences determined. Expression plasmids, designated pQSVB and pQSVC, to overproduce these proteins were constructed, and the proteins were purified using a Ni2+ affinity column. In vitro reconstitution using all proteins for the holoenzyme (α, β, β′, σ70) was carried out and the activity of the recombinant RNA polymerase was detected.