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

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Abstract
Outer surface lipoprotein C (OspC) is a key virulence factor ofBorrelia burgdorferi. ospCis 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 ofospCinB. burgdorferiis governed by a novel alternative sigma factor regulatory network, the RpoN-RpoS pathway. However, the precise mechanism by which the RpoN-RpoS pathway controlsospCexpression has been unclear. In particular, there has been uncertainty regarding whetherospCis controlled directly by RpoS (σs) or indirectly through a transactivator (induced by RpoS). Using deletion analyses and genetic complementation in an OspC-deficient mutant ofB. burgdorferi, we analyzed theciselement(s) required for the expression ofospCin its native borrelial background. Two highly conserved upstream inverted repeat elements, previously implicated inospCregulation, were not required forospCexpression inB. burgdorferi. Using similar approaches, a minimal promoter that contained a canonical −35/−10 sequence necessary and sufficient for σs-dependent regulation ofospCwas identified. Further, targeted mutagenesis of a C at position −15 within the extended −10 region ofospC, which is postulated to function like the strategic C residue important for Eσsbinding inEscherichia coli, abolishedospCexpression. The minimalospCpromoter also was responsive to coumermycin A1, further supporting its σscharacter. The combined data constitute a body of evidence that the RpoN-RpoS regulatory network controlsospCexpression by direct binding of σsto a σs-dependent promoter ofospC. The implication of our findings to understanding howB. burgdorferidifferentially regulatesospCand otherospC-like genes via the RpoN-RpoS regulatory pathway is discussed.