Transcriptional regulation of the ospAB and ospC promoters from Borrelia burgdorferi

Alverson, Janet; Bundle, Sharyl F.; Sohaskey, Charles D.; Lybecker, Meghan; Samuels, D. Scott

doi:10.1046/j.1365-2958.2003.03537.x

Cited by 86 publications

(92 citation statements)

References 87 publications

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“…1). These IRs are highly conserved among ospC homologs of many B. burgdorferi strains (29,55) and have been hypothesized to be involved in ospC regulation (1,55). To assess the potential role(s) of the IRs in ospC expression, we first generated complementation constructs lacking either IR 1 or both IR 1 and IR 2 (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Analysis of theospCRegulatory Element Controlled by the RpoN-RpoS Regulatory Pathway inBorrelia burgdorferi

et al. 2005

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Outer surface lipoprotein C (OspC) is a key virulence factor of Borrelia burgdorferi. ospC is differentially regulated during borrelial transmission from ticks to rodents, and such regulation is essential for maintaining the spirochete in its natural enzootic cycle. Recently, we showed that the expression of ospC in B. burgdorferi is governed by a novel alternative sigma factor regulatory network, the RpoN-RpoS pathway. However, the precise mechanism by which the RpoN-RpoS pathway controls ospC expression has been unclear. In particular, there has been uncertainty regarding whether ospC is controlled directly by RpoS ( s ) or indirectly through a transactivator (induced by RpoS). Using deletion analyses and genetic complementation in an OspC-deficient mutant of B. burgdorferi, we analyzed the cis element(s) required for the expression of ospC in its native borrelial background. Two highly conserved upstream inverted repeat elements, previously implicated in ospC regulation, were not required for ospC expression in B. burgdorferi. Using similar approaches, a minimal promoter that contained a canonical ؊35/؊10 sequence necessary and sufficient for s -dependent regulation of ospC was identified. Further, targeted mutagenesis of a C at position ؊15 within the extended ؊10 region of ospC, which is postulated to function like the strategic C residue important for E s binding in Escherichia coli, abolished ospC expression. The minimal ospC promoter also was responsive to coumermycin A 1 , further supporting its s character. The combined data constitute a body of evidence that the RpoN-RpoS regulatory network controls ospC expression by direct binding of s to a s -dependent promoter of ospC. The implication of our findings to understanding how B. burgdorferi differentially regulates ospC and other ospC-like genes via the RpoN-RpoS regulatory pathway is discussed.

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Section: Resultsmentioning

confidence: 99%

“…Relaxation of supercoiling is one of the factors that enhances promoter selectivity by E s (24). In addition, the IRs were postulated to play a role in the regulation of ospC by supercoiling (1). To examine which ospC promoter element(s) responds to DNA supercoiling, E. coli (Fig.…”

Section: Resultsmentioning

confidence: 99%

Analysis of theospCRegulatory Element Controlled by the RpoN-RpoS Regulatory Pathway inBorrelia burgdorferi

et al. 2005

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“…Expression of ospC is dependent on RpoS and is positively regulated by the Rrp2/RpoN/RpoS signaling cascade (30,67). ospA, in contrast, is believed to be transcribed via the housekeeping factor RpoD (also referred to as 70 ) (3,59) and is negatively regulated by an unknown mechanism in response to in vivo-specific signals.…”

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

Caimano¹,

Eggers²,

Gonzalez³

et al. 2005

J Bacteriol

110

155

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While numerous positively regulated loci have been characterized during the enzootic cycle of Borrelia burgdorferi, very little is known about the mechanism(s) involved in the repression of borrelial loci either during tick feeding or within the mammalian host. Here, we report that the alternative sigma factor RpoS is required for the in vivo-specific repression of at least two RpoD-dependent B. burgdorferi loci, ospA and lp6.6. The downregulation of ospA and Ip6.6 appears to require either a repressor molecule whose expression is RpoS dependent or an accessory factor which enables RpoS to directly interact with the ospA and Ip6.6 promoter elements, thereby blocking transcription by RpoD. The central role for RpoS during the earliest stages of host adaptation suggests that tick feeding imparts signals to spirochetes that trigger the RpoS-dependent repression, as well as expression, of in vivo-specific virulence factors critical for the tick-to-mammalian host transition.

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“…Electrophoresis and Southern blotting to an Immobilon-Ny ϩ membrane (Millipore) were performed essentially as described previously (1,10), except the hybridization signal was detected using a FLA3000G radioisotope imaging system (Fuji film). The probes were amplified by two rounds of PCR using oligonucleotides aadA FϩNdeI and aadA RϩAatII or oligonucleotides kanR 88F (AATGTCGGGCAATCAGGTG) and kanR 488R (TCACT CGCATCAACCAAACC), and labeled using a Prime-It II random primer labeling kit (Stratagene).…”

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aadAConfers Streptomycin Resistance inBorrelia burgdorferi

et al. 2003

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To enhance genetic manipulation of the Lyme disease spirochete Borrelia burgdorferi, we assayed the aadA gene for the ability to confer resistance to the antibiotics spectinomycin and streptomycin. Using the previously described pBSV2 as a backbone, a shuttle vector, termed pKFSS1, which carries the aadA open reading frame fused to the B. burgdorferi flgB promoter was constructed. The hybrid flgB promoter-aadA cassette confers resistance to spectinomycin and streptomycin in both B. burgdorferi and Escherichia coli. pKFSS1 has a replication origin derived from the 9-kb circular plasmid and can be comaintained in B. burgdorferi with extant shuttle vector pCE320, which has a replication origin derived from a 32-kb circular plasmid, or pBSV2, despite the fact that pKFSS1 and pBSV2 have the same replication origin. Our results demonstrate the availability of a new selectable marker and shuttle vector for genetically dissecting B. burgdorferi at the molecular level.

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Transcriptional regulation of the ospAB and ospC promoters from Borrelia burgdorferi

Cited by 86 publications

References 87 publications

Analysis of theospCRegulatory Element Controlled by the RpoN-RpoS Regulatory Pathway inBorrelia burgdorferi

Analysis of theospCRegulatory Element Controlled by the RpoN-RpoS Regulatory Pathway inBorrelia burgdorferi

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

aadAConfers Streptomycin Resistance inBorrelia burgdorferi

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