Isolation of a novel paraquat-inducible (pqi) gene regulated by the soxRS locus in Escherichia coli.
We have isolated promoters inducible by paraquat, a superoxide radical-generating agent, from Escherichia coli, using promoter-probing plasmid pJAC4 (Y.S. Koh and J.H. Roe, Korean J. Microbiol. 31:267-273, 1993). One promoter clone pqi-5 (pqi denotes paraquat-inducible gene) was mapped at 21.8 min on the E. coli chromosome by using the Kohara phage library. We constructed an operon fusion of the lacZ gene with the pqi-5 promoter to monitor the expression of the gene in the single-copy state. LacZ expression was induced about 7- to 13-fold by 77 to 780 microM paraquat. Other known superoxide generators such as menadione, phenazine methosulfate, and plumbagin also induced the expression of beta-galactosidase in this fusion strain. On the other hand, no significant induction was observed with treatment with hydrogen peroxide, ethanol, and heat shock. Induction of beta-galactosidase was significantly reduced by introducing a delta sox-8::cat or soxS3::Tn10 mutation into the fusion strain, indicating that pqi-5 is a member of the soxRS regulon. A DNA fragment containing the pqi-5 promoter was cloned and sequenced from the Kohara phage E2E5. We identified two pqi-5 open reading frames (ORFs); ORF-A encodes a predicted protein of 342 amino acids, and ORF-B is truncated at the cloning site. The transcription start site from the pqi-5 promoter was determined by primer extension and S1 nuclease protection analyses. Northern (RNA) and S1 analyses indicated that there are two kinds of pqi-5 transcript; one covers ORF-A only and the other covers ORF-A and possibly also ORF-B.
Koh
Y S
YS
Department of Microbiology, College of Natural Sciences, Seoul National University, Korea.
Roe
J H
JH
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial / Q000235:genetics*; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016366:Open Reading Frames / Q000235:genetics; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000235:genetics; D010269:Paraquat / Q000494:pharmacology*; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D018085:Regulon / Q000235:genetics; D012097:Repressor Proteins; D017422:Sequence Analysis, DNA; D015534:Trans-Activators; D014157:Transcription Factors / Q000235:genetics
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D015534:Trans-Activators; D014157:Transcription Factors; C499299:oxyR protein, E coli; C093588:pqi5 protein, E coli; C070724:SoxR protein, Bacteria; C070725:SoxS protein, E coli; D010269:Paraquat