Identification and characterization of the Escherichia coli gene dsbB, whose product is involved in the formation of disulfide bonds in vivo.
We have identified and characterized the Escherichia coli gene dsbB, whose product is required for disulfide bond formation of periplasmic proteins, by using two different approaches: (i) screening of a multicopy plasmid library for clones which protect E. coli from the lethal effects of dithiothreitol (DTT), and (ii) screening of insertion libraries of E. coli for DTT-sensitive mutants. Mapping and characterization of mutations conferring a DTT-sensitive phenotype also identified the dsbA, trxA, and trxB genes, whose products are involved in different oxidation-reduction pathways. Null mutations in dsbB conferred pleiotropic phenotypes such as sensitivity to benzylpenicillin and inability to support plaque formation of filamentous phages, and they were shown to severely affect disulfide bond oxidation of secreted proteins such as OmpA and beta-lactamase. These phenotypes resemble the phenotype of bacteria carrying either a null mutation in the dsbA gene or the double mutation dsbA dsbB. Sequencing and expression of the dsbB gene revealed that it encodes a 20-kDa protein predicted to possess an "exchangeable" disulfide bond in -Cys-Val-Leu-Cys-. The dsbB gene maps at 26.5 min on the genetic map of the E. coli chromosome, and its transcription is directed from two promoters, neither of which resembles the canonical E sigma 70-recognized promoter.
Missiakas
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Department of Cellular, Viral and Molecular Biology, University of Utah School of Medicine, Salt Lake City 84132.
Georgopoulos
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C
Raina
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D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D002874:Chromosome Mapping; D002876:Chromosomes, Bacterial / Q000648:ultrastructure; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004220:Disulfides; D004229:Dithiothreitol / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D012329:RNA, Bacterial / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D001618:beta-Lactamases / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004220:Disulfides; D009838:Oligodeoxyribonucleotides; D012329:RNA, Bacterial; D012333:RNA, Messenger; D001618:beta-Lactamases; D004229:Dithiothreitol