Verification and dissection of the ospC operator by using flaB promoter as a reporter in Borrelia burgdorferi.

The Lyme disease spirochete Borrelia burgdorferi must repress expression of outer surface protein C (OspC) to effectively evade specific humoral immunity and to establish persistent infection. This ability largely relies upon a regulatory element, the only operator that has been reported in spirochetal bacteria. Immediately upstream of the ospC promoter, two sets of inverted repeats (IRs) constitute small and large palindromes, in which the right IR of the large palindrome contains the left IR of the small one, and may collectively function as the ospC operator. In the study, the large palindrome with or without the small IR was fused with an flaB promoter, which was used to drive expression of a promoterless ospC copy as a reporter gene, and introduced into OspC-deficient B. burgdorferi. The presence of the large palindrome alone significantly reduced ospC expression driven by the fused flaB promoter in the joint tissue of severe combined immunodeficiency (SCID) mice, and rescued spirochetes from elimination by passively transferred OspC antibody in infected SCID mice and specific immune responses elicited in immunocompetent mice, confirming a function of the IRs as an operator. Inclusion of the small IR further enhanced the ability of the large palindrome to reduce the activity of the fused flaB promoter, indicating that the small IR is a part of the operator. Taken together, the study led to successful verification and dissection of the ospC operator.

Affinities of mAbs to Tet repressor complexed with operator or tetracycline suggest conformational changes associated with induction.

We isolated five monoclonal antibodies (mAbs) made against tetracycline repressor (TetR), one against the TetR tetracycline complex (Tc) and two against the TetR-tet operator (tetO) complex. The epitopes of the anti-TetR mAbs are localized in the alpha-helix-turn-alpha-helix motif (HTH), at different sites near the Tc binding pocket and at the dimerization interface. The anti-TetR-Tc and one of the anti-TetR-tetO mAbs recognize epitopes near the Tc binding pocket. The other anti-TetR-tetO mAb binds to an epitope within the HTH. Quantitative immunoprecipitation and competitive ELISA employing TetR, TetR-Tc, or TetR-tetO revealed different affinities of the mAbs for TetR in these functional states. Binding of the two mAbs to epitopes in the HTH was identical for TetR and TetR-Tc indicating the same conformation in both forms. The epitope located in the dimerization interface is bound more strongly in TetR compared to TetR-Tc, supporting the idea of different conformations of that epitope in these forms of TetR. The greatest affinity differences were found for epitopes around the Tc binding pocket. Two anti-TetR mAbs have the highest affinities for free TetR, somewhat reduced affinity for TetR-tetO and the lowest affinities for TetR-Tc. The anti-TetR-Tc mAb has a discontinuous epitope, formed in TetR-Tc, which is less well bound in TetR and not bound in the TetR-tetO complex. One anti-TetR-tetO mAb does not recognize TetR-Tc. Since the epitopes do not overlap with the respective ligand binding sites on TetR, these results are interpreted as conformational differences of the epitopes in these forms of TetR.