Cloning and characterization of the rpoE gene encoding an RNA polymerase sigmaE factor from the deep-sea piezophilic Shewanella violacea strain DSS12.
The rpoE gene encoding an RNA polymerase sigmaE subunit was isolated from a gamma-phage library of the deep-sea piezophilic and psychrophilic bacterium Shewanella violacea strain DSS12. Structual analysis showed that the gene organization of the fragment containing S. violacearpoE was the l-aspartate oxidase-coding gene, rpoE, rseA, rseB and rseC in that order, the same as in the case of Photobacterium profundum SS9 and Escherichia coli K-12. The cloned gene, 576 bp in length, was found to encode a protein consisting of 192 amino acid residues with a molecular mass of 21,806 Da. Amino acid alignment of the RpoE protein showed that the functional domains responsible for DNA recognition, DNA melting, core binding, and RseA interaction were highly conserved. We purified hexahistidine-fused RpoE protein by constructing an overexpression plasmid. Core-binding analysis revealed that the cloned RpoE protein has the ability to bind with core RNA polymerase as a sigma factor.
Kawano
Hiroaki
H
Extremobiosphere Research Center (XBR), Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2-15 Natsushima-cho, Yokosuka, Kanagawa, 237-0061, Japan. kawanoh@jamstec.go.jp
Abe
Fumiyoshi
F
Nakasone
Kaoru
K
Kato
Chiaki
C
Yoshida
Yasuhiko
Y
Usami
Ron
R
Horikoshi
Koki
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D020592:Shewanella / Q000201:enzymology* / Q000235:genetics*; D012808:Sigma Factor / Q000737:chemistry / Q000235:genetics*; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics*
D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; D012808:Sigma Factor; D014157:Transcription Factors; C063202:sporulation-specific sigma factors