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 (bosR C114S-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.
|Commitee:||guentzel, neal, wormley, floyd|
|School:||The University of Texas at San Antonio|
|School Location:||United States -- Texas|
|Source:||MAI 49/05M, Masters Abstracts International|
|Keywords:||Borrelia burgdorferi, BosR, Lyme disease, OspC, RpoS|
Copyright in each Dissertation and Thesis is retained by the author. All Rights Reserved
The supplemental file or files you are about to download were provided to ProQuest by the author as part of a
dissertation or thesis. The supplemental files are provided "AS IS" without warranty. ProQuest is not responsible for the
content, format or impact on the supplemental file(s) on our system. in some cases, the file type may be unknown or
may be a .exe file. We recommend caution as you open such files.
Copyright of the original materials contained in the supplemental file is retained by the author and your access to the
supplemental files is subject to the ProQuest Terms and Conditions of use.
Depending on the size of the file(s) you are downloading, the system may take some time to download them. Please be