Analysis of promoters in Borrelia burgdorferi by use of a transiently expressed reporter gene

Abstract

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.

The extended promoters for two outer membrane lipoprotein genes of Borrelia spp. uniquely include a T‐rich region

“…In a previous study, we developed a transient transfection system with a chloramphenicol acetyltransferase (CAT) gene as a reporter to compare the function of three B. burgdorferi promoters, ospAB,¯aB and ospC, in B. burgdorferi, as well as in Escherichia coli (Sohaskey et al, 1997). [OspC is an outer membrane lipoprotein that is encoded by a gene on a circular plasmid (Sadziene et al, 1993).]…”

lacZ Reporter System for Use in Borrelia burgdorferi

“…S1 of the supplemental material. The flaB promoter was chosen for initial experiments because it is constitutively expressed in B. burgdorferi and E. coli and has been used to drive expression of antibiotic resistance markers in B. burgdorferi (5,13,33). We transformed all constructs into electrocompetent B. burgdorferi B31-A34 and a ⌬lac E. coli strain and assessed ␤-galactosidase activity in liquid and solid media.…”

“…A chloramphenicol acetyltransferase (CAT) gene was the first reporter gene that was fused to B. burgdorferi promoters for analysis of promoter strength (33). The development of luciferase (4) and multiple fluorescent proteins (9,11,30) as reporter systems in B. burgdorferi followed.…”

“…The lacZ gene is one of the most widely used reporter genes, and B. burgdorferi promoters have previously been fused to lacZ, but ␤-galactosidase activity was assessed in E. coli, not B. burgdorferi (8,32). However, since many studies have detected significant differences in the expression profiles of B. burgdorferi and E. coli (1,12,33,34), using lacZ as a reporter gene directly in B. burgdorferi will allow a more accurate analysis of native promoter activity. Additionally, there are many substrates for ␤-galactosidase, and this allows lacZ to be used in multiple applications, including simple in vitro assays to assess promoter activity and rapid screening for transformants on solid media.…”

Characterization of secA-sod operon in Borrellia burgdorferi.

“…The transcription included both alternative monocistronic and polycistronic messages based on PE experiments identifying promoters for bmpA, bmpC, and bmpD, but not for bmpB (Dobrikova et al, 2001). seven-and three-fold, respectively (Sohaskey et al, 1997).…”