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

Abstract

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.

Borrelia burgdorferi Uniquely Regulates Its Motility Genes and Has an Intricate Flagellar Hook-Basal Body Structure

“…Recently, using microarray and transcriptional analysis, Caimano et al have shown that whereas flagellum biosynthetic genes are controlled by 70 both in vitro and in vivo, the methyl-accepting chemotaxis genes mcp1, mcp4, and mcp5 and the chemotaxis gene cheW2 are upregulated by S (RpoS) (11) during growth in vivo. For B. burgdorferi, evidence suggests that S is directly controlled by 54 (61,66). 54 directly participates in the transcription of motility gene expression in other bacteria, such as Caulobacter crescentus, Vibrio species, Pseudomonas aeruginosa, and Helicobacter species (4,16,44,50).…”

“…In addition, all of the motility promoters identified so far are 70 -like (19,22,38). However, discrimination between S and 70 promoters in B. burgdorferi based on promoter consensus sequences is problematic (11,66). Recently, using microarray and transcriptional analysis, Caimano et al have shown that whereas flagellum biosynthetic genes are controlled by 70 both in vitro and in vivo, the methyl-accepting chemotaxis genes mcp1, mcp4, and mcp5 and the chemotaxis gene cheW2 are upregulated by S (RpoS) (11) during growth in vivo.…”

Transcriptional interplay among the regulators Rrp2, RpoN and RpoS in Borrelia burgdorferi

“…pJD55 is a derivative of pJD44 in which the aph[39]-IIIa marker is replaced with PflgB-Kan of pBSV2 (Stewart et al, 2001). BbJSB18-B2 was transformed with the resulting construct, designated pJSB208B, as described previously by Yang et al (2005), and transformants were selected using kanamycin at a concentration of 160 mg ml 21 . Clones were confirmed by plasmid recovery, as described by Blevins et al (2007).…”

“…This was achieved by removing the 4.2 kb region from pXY240 by digestion with KpnI and XbaI and ligating it into pJD44 digested with the same enzymes. The resulting construct, designated pJSB259, was transformed into BbJSB19-A7B, as described previously (Yang et al, 2005).…”

lacZ Reporter System for Use in Borrelia burgdorferi

“…1B). The intergenic region between guaA and ospC, which includes the ospC promoter and regulatory operator sequences up to the ospC start codon (12,20,21,(37)(38)(39), was amplified from B. burgdorferi genomic DNA with primers 13 and 14 (Table 2), cloned into pCR2.1, sequenced, and digested with BamHI and XhoI for ligation into pBH-lacZBb, creating pBHospCp-lacZBb. Constructs were transformed into electrocompetent B. burgdorferi (27) and ⌬lac E. coli cells.…”