Analysis of promoter elements involved in the transcriptional initiation of RpoS-dependent Borrelia burgdorferi genes.
Borrelia burgdorferi, the causative agent of Lyme disease, encodes an RpoS ortholog (RpoS(Bb)) that controls the temperature-inducible differential expression of at least some of the spirochete's lipoprotein genes, including ospC and dbpBA. To begin to dissect the determinants of RpoS(Bb) recognition of, and selectivity for, its dependent promoters, we linked a green fluorescent protein reporter to the promoter regions of several B. burgdorferi genes with well-characterized expression patterns. Consistent with the expression patterns of the native genes/proteins in B. burgdorferi strain 297, we found that expression of the ospC, dbpBA, and ospF reporters in the spirochete was RpoS(Bb) dependent, while the ospE and flaB reporters were RpoS(Bb) independent. To compare promoter recognition by RpoS(Bb) with that of the prototype RpoS (RpoS(Ec)), we also introduced our panel of constructs into Escherichia coli. In this surrogate, maximal expression from the ospC, dbpBA, and ospF promoters clearly required RpoS, although in the absence of RpoS(Ec) the ospF promoter was weakly recognized by another E. coli sigma factor. Furthermore, RpoS(Bb) under the control of an inducible promoter was able to complement an E. coli rpoS mutant, although RpoS(Ec) and RpoS(Bb) each initiated greater activity from their own dependent promoters than they did from those of the heterologous sigma factor. Genetic analysis of the ospC promoter demonstrated that (i) the T(-14) in the presumptive -10 region plays an important role in sigma factor recognition in both organisms but is not as critical for transcriptional initiation by RpoS(Bb) as it is for RpoS(Ec); (ii) the nucleotide at the -15 position determines RpoS or sigma(70) selectivity in E. coli but does not serve the same function in B. burgdorferi; and (iii) the 110-bp region upstream of the core promoter is not required for RpoS(Ec)- or RpoS(Bb)-dependent activity in E. coli but is required for maximal expression from this promoter in B. burgdorferi. Taken together, the results of our studies suggest that the B. burgdorferi and E. coli RpoS proteins are able to catalyze transcription from RpoS-dependent promoters of either organism, but at least some of the nucleotide elements involved in transcriptional initiation and sigma factor selection in B. burgdorferi play a different role than has been described for E. coli.
Center for Microbial Pathogenesis, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030-3710, USA. eggers@up.uchc.edu
Christian H
CH
Eggers
Melissa J
MJ
Caimano
Justin D
JD
Radolf
D000942:Antigens, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D025065:Borrelia burgdorferi / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012808:Sigma Factor / Q000378:metabolism*; D013696:Temperature; D014158:Transcription, Genetic
D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; C079616:OspC protein; D012808:Sigma Factor; C069567:sigma factor KatF protein, Bacteria