ANALYSIS OF PROMOTERS IN BORRELIA-BURGDORFERI BY USE OF A TRANSIENTLYEXPRESSED REPORTER GENE

A transient chloramphenicol acetyltransferase (CAT) expression system was developed for Borrelia burgdorferi. An Escherichia coli vector con taining a promoterless Streptococcus agalactiae cat gene was construct ed. Promoters for ospA, ospC, and flaB were placed upstream of this ca t gene, and CAT assays were performed in E. coli from these stably mai ntained plasmids. The plasmids with putative promoters ospA and flaB w ere found to be approximately 20-fold more active than were the plasmi ds with ospC or no promoter. The level of activity correlated well wit h the resistance to chloramphenicol that each plasmid provided. Next, the nonreplicative plasmid constructs were transformed by electroporat ion into B. burgdorferi. CAT assays were performed by both thin-layer chromatography and the fluor diffusion method. Measurement of CAT acti vity demonstrated that the ospA promoter was again about 20-fold more active than the promoterless cat gene. The flaB and ospC promoters inc reased 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. burg dorferi in the absence of a well-developed genetic system.