Analysis of promoters in Borrelia burgdorferi by use of a transiently expressed reporter gene.
A transient chloramphenicol acetyltransferase (CAT) expression system was developed for Borrelia burgdorferi. An Escherichia coli vector containing a promoterless Streptococcus agalactiae cat gene was constructed. Promoters for ospA, ospC, and flaB were placed upstream of this cat gene, and CAT assays were performed in E. coli from these stably maintained plasmids. The plasmids with putative promoters ospA and flaB were found to be approximately 20-fold more active than were the plasmids with ospC or no promoter. The level of activity correlated well with the resistance to chloramphenicol that each plasmid provided. Next, the nonreplicative plasmid constructs were transformed by electroporation into B. burgdorferi. CAT assays were performed by both thin-layer chromatography and the fluor diffusion method. Measurement of CAT activity demonstrated that the ospA promoter was again about 20-fold more active than the promoterless cat gene. The flaB and ospC promoters increased the activity seven- and threefold, respectively, over that with the promoterless construct. This simple transient-expression assay was shown to be an effective method to study promoter function in B. burgdorferi in the absence of a well-developed genetic system.
Sohaskey
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Department of Microbiology & Molecular Genetics, University of California Irvine, 92697-4025, USA.
Arnold
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C
Barbour
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D000942:Antigens, Bacterial; D000954:Antigens, Surface / Q000235:genetics; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics; D001428:Bacterial Vaccines; D015748:Borrelia burgdorferi Group / Q000235:genetics*; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics; D018274:Electroporation; D004926:Escherichia coli / Q000235:genetics; D005408:Flagellin / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D017930:Genes, Reporter; D008074:Lipoproteins; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014162:Transfection
D000942:Antigens, Bacterial; D000954:Antigens, Surface; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D001428:Bacterial Vaccines; D008074:Lipoproteins; C072045:OspA protein; C079616:OspC protein; D011993:Recombinant Fusion Proteins; D005408:Flagellin; C416532:flaB flagellin; D015500:Chloramphenicol O-Acetyltransferase