title: Influence of transcriptional and translational control sequences on the expression of foreign genes in Caulobacter crescentus.
abstract: The influence of expression control sequences (ECSs; promoters and ribosome-binding sites [RBSs]), transcriptional terminators, and gene orientation on the expression of the Escherichia coli lacZ gene in the gram-negative microorganisms Caulobacter crescentus and E. coli was investigated. A series of broad-host-range expression vectors, based on the RK2 plasmid derivative pRK248, were constructed. The ECSs included the tac promoter, the promoter for the surface layer protein of C. crescentus, and promoters from a number of gram-positive bacteria together with their associated RBSs. In addition, synthetic ECSs were constructed by using different combinations of promoters and RBSs. lacZ expression was found to be dependent on the nature of the promoter and RBS and, to a lesser extent, on the presence of a transcriptional terminator and the orientation of the promoter-lacZ construct in pRK248. The relative efficiencies of the various ECSs in driving lacZ expression differed markedly in C. crescentus and E. coli. In C. crescentus, the ECS ptac1 (tac promoter and consensus RBS for C. crescentus mRNAs) appeared to be the most efficient, producing 12-fold-higher activity than did pSL (promoter for the surface layer protein of C. crescentus and its putative RBS). pSL was not transcribed in E. coli, whereas various promoters from gram-positive microorganisms were transcribed in both C. crescentus and E. coli. A number of ECSs were also used to drive mosquitocidal toxin gene expression in C. crescentus, and a correlation between toxin expression and lacZ expression was observed.
authors.affiliation: Institute of Molecular and Cell Biology, National University of Singapore.
authors.forename: W H
authors.initials: WH
authors.lastname: Yap
authors.forename: T
authors.initials: T
authors.lastname: Thanabalu
authors.forename: A G
authors.initials: AG
authors.lastname: Porter
mesh_terms: D001407:Bacillus / Q000235:genetics; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D001427:Bacterial Toxins; D001483:Base Sequence; D016935:Caulobacter crescentus / Q000235:genetics*; D004731:Endotoxins / Q000096:biosynthesis / Q000235:genetics; D004790:Enzyme Induction / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D015870:Gene Expression; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors / Q000235:genetics*; D006460:Hemolysin Proteins; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D056968:Lac Repressors; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics; D014176:Protein Biosynthesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D012097:Repressor Proteins / Q000378:metabolism; D013728:Terminator Regions, Genetic / Q000235:genetics; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins; D056968:Lac Repressors; C539064:LacI protein, E coli; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D007544:Isopropyl Thiogalactoside; D001616:beta-Galactosidase