Molecular Characterization ofBorrelia burgdorferi erpPromoter/Operator Elements

Abstract

Many Borrelia burgdorferi Erp outer surface proteins have been demonstrated to bind the host complement regulator factor H, which likely contributes to the ability of these organisms to evade the host innate immune system. B. burgdorferi controls Erp protein synthesis throughout the bacterial infectious cycle, producing the proteins during mammalian infections but repressing their synthesis during tick infections. Defining the mechanism by which B. burgdorferi regulates the expression of these virulence determinants will provide important insight into the biological and pathogenic properties of the Lyme disease spirochete. The present study demonstrates that two highly conserved DNA sequences located 5 of erp operons specifically bind bacterial proteins. Analyses with B. burgdorferi of transcriptional fusions between erp promoter/operator DNAs and the gene for green fluorescent protein indicated that the expression of these operons is regulated at the level of transcriptional initiation. These analyses also indicated significant differences in the promoter strengths of various erp operons, which likely accounts for reported variations in expression levels of different Erp proteins. Mutagenesis of promoter-gfp fusions demonstrated that at least one of the proteins which bind erp operator DNA functions as a repressor of transcription.The spirochete Borrelia burgdorferi is maintained in nature through an infectious cycle involving warm-blooded vertebrates and Ixodes sp. ticks. These bacteria regulate the expression of a number of different proteins during the infectious cycle, among which are the Erp outer surface lipoproteins. Individual bacteria encode multiple members of the Erp family, with the type strain, B31, being known to carry 17 erp genes at 10 separate loci (12,13,50). Each locus occupies an allelic position on a different plasmid of the cp32 family. Although cp32 plasmids are largely identical in their sequences, many are compatible with each other, with 12 apparent incompatibility groups identified to date (16,47). Many Erp proteins have been demonstrated to bind host factor H, a regulator of complement activation, and may thereby help protect the bacteria from the alternative pathway of complement-mediated killing (4, 5, 25-29, 34, 46). Consistent with that function, B. burgdorferi expresses Erp proteins during transmission between the tick vector and the vertebrate host, times when the bacterium is exposed to host serum and the innate immune system (22-24, 36, 37). For unknown reasons, B. burgdorferi down-regulates Erp expression during infections of ticks (22,36).A number of in vitro studies have provided insights into the regulatory mechanisms by which B. burgdorferi controls Erp synthesis. The culture temperature significantly impacts Erp expression levels, with bacteria grown at 34°C producing higher levels of Erp proteins than do bacteria cultivated at 23°C (24,44,48). These temperatures mimic those experienced by feeding and unfed ticks, respectively (40). The bacteria also respond to quorum s...

Borrelia burgdorferi complement regulator-acquiring surface proteins (BbCRASPs): Expression patterns during the mammal–tick infection cycle

“…All the erp coding sequences, including erpA, erpC, and erpP, are preceded by almost identical 5′-non-coding sequences, including binding sites for at least 3 distinct DNA-binding proteins (Babb et al, 2004. Co-regulation of erp genes on different cp32 plasmids suggests the existence of similar molecular mechanisms that could coordinate expression of genes encoding BbCRASP-3, -4, and -5 at the transcriptional level Babb et al, 2001;El-Hage and Stevenson, 2002;Babb et al, 2004Babb et al, , 2006.…”

Alternate Sigma Factor RpoS Is Required for the In Vivo-Specific Repression ofBorrelia burgdorferiPlasmid lp54-BorneospAandlp6.6Genes

“…Thus, in order to determine whether the effect of RpoS on ospA downregulation is due to decreased transcription initiation or mRNA degradation, we decided to use a transcriptional reporter construct that would enable us to discriminate between these two possibilities. The selection of green fluorescent protein (GFP) as the reporter, in conjunction with flow cytometry, has the additional advantage of enabling one to assess gene expression by individual spirochetes within a population (6,12,15,16,54). For these studies, a 189-bp region of the upstream sequence containing both the ospA promoter and a poly(T) tract reported to be involved in the regulation of ospA transcription in vitro (59) was cloned upstream of a promoterless gfp contained on our cp32-based shuttle vector pCE320 (15).…”

Dependence of the E. coli promoter strength and physical parameters upon the nucleotide sequence

“…Core prokaryotic promoters, especially from Escherichia coli for RNA polymerase complexes with the factor σ 70 usually are situated between -60 to +20 base pair from the transcription start site (+1), have two most important transcription initiation sites: at the -35 position and at -10 region (the Pribnow box). The sequences of -10 and -35 sites may affect the binding of RNA polymerase and the formation of open complexes (Babb et al, 2004).…”