Molecular analysis of the promoter region of the Clostridium difficile toxin B gene that is functional in Escherichia coli.
Clostridium difficile is a human pathogen that produces two types of toxins, A and B, that cause a potentially lethal gastrointestinal syndrome termed pseudomembranous colitis. Virtually nothing is known about the mechanism of regulation of toxin production in this organism, and cis-regulatory regions of neither toxin have yet been identified, thus prompting this investigation. A motif homologous with the Shine-Dalgarno sequence of Escherichia coli occurs upstream from the putative initiation codon of toxin B, making this region also a candidate to contain a promoter. Therefore, a subgenomic DNA library of C. difficile in a plasmid vector was first constructed encompassing the 5'-end of the toxin B gene. A 450-bp DNA fragment was excised from the subgenomic DNA library clone and subcloned into a promoter-probe plasmid vector that contains two divergently oriented, promoterless genes to assay for promoter function. This subcloned DNA fragment directed the expression of alkaline phosphatase, a reporter gene product of the promoterless vector, thus indicating the presence of a functional promoter. To locate the promoter more precisely, a series of nested deletions of the toxin B promoter subclone was constructed with exonuclease III. The promoter that facilitates expression of the toxin B gene in E. coli was localised, based on alkaline phosphatase activity. The transcriptional initiation site of toxin B mRNA in E. coli was mapped by primer extension analysis, suggesting two closely associated tandem start sites directed by two similarly spaced promoters within this localised region.
Song
K P
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Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock 79430, USA.
Faust
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D001426:Bacterial Proteins; D001427:Bacterial Toxins / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D016360:Clostridioides difficile / Q000235:genetics* / Q000378:metabolism; D004269:DNA, Bacterial / Q000032:analysis / Q000737:chemistry / Q000502:physiology; D004926:Escherichia coli / Q000235:genetics*; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D015723:Gene Library; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004269:DNA, Bacterial; D012333:RNA, Messenger; C031815:toxB protein, Clostridium difficile