Effect of Site-specific Changes in the Regulatory Functions of BosR
Borrelia burgdorferi, the causative agent of Lyme disease has a limited set of proteins involved in resistance to oxidative stress. Regulation of some of these proteins is mediated by Borrelia oxidative stress Regulator (BosR), a zinc-dependent DNA binding protein that recognizes motifs upstream of NapA, CoADR and SodA. We generated a bosR deficient strain in B.burgdorferi strain B31 lacking lp25(ML23) followed by restoration of minimal region of lp25 needed for infectivity using a borrelial shuttle vector (pBBE22). The bosR deficient strain was incapable of colonization of C3H/HeN mice following needle inoculation indicating that the regulatory functions of bosR is essential for infectivity. We complemented the BosR mutant with a native copy in cis, and generated site-specific substitutions in the CXXC motifs (bosRC114S-N-K-C117S; bosRC153S-N-N-C156S,) and conserved histidines (bosRH37A, bosRH 111A) present in BosR. The cis-complemented strain exhibited levels of BosR similar to the parental strain and the levels of Outer surface protein C reflected the parental phenotype. Interestingly, site-specific changes in the first CXXC motif (bosRC114S-N-K-C117S) resulted in levels of BosR similar to that of the cis complemented strain but had dramatically lower levels of OspC. Replacement of the conserved histidine at position 111 with an alanine resulted in not only a reduction in the levels of BosR but also in the levels of OspC. There was no changes in the levels of bb0646 which is co-transcribed with bosR (bb0647) as well as in the levels of a variety of other oxidatives stress response proteins such as SodA and NapA. The levels of P66 was similar in all strains serving as a control for levels of proteins loaded in each lane and as a measure of the physiological status of the spirochetes. A set of key defined site-specific mutants in B. burgdorferi has been generated and these strains will help in further analysis of the interactions of BosR with the members of its regulon. Moreover, the regulatory functions of BosR are complex and the phenotypic analysis of mutants carrying site-specific changes in bosR critical for coordinating metals such as Zn2+ would provide insights into the role of BosR in the patho-physiology of B.burgdorferi.