Regulation of expression of P66, a β3-chain integrin ligand in Borrelia burgdorferi

• Main Author: Medrano, Melisa S
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2008
• Subjects: Microbiology; Molecular biology
• ISBN: 9780549358725; 0549358722

Borrelia burgdorferi, the bacterium that causes Lyme disease, encodes the β3-chain integrin ligand P66. P66 is expressed in the mammal, in medium containing serum and other mammalian factors, and by B. burgdorferi as they are acquired or transmitted by the tick, but is not expressed by the bacterium in the midguts of unfed ticks. We attempted to mimic this expression pattern in cultured bacteria to allow us to differentially purify a regulator of P66 expression. We examined P66 expression at different growth phases and temperatures, and in different types and pH of media, but P66 was expressed in all conditions investigated. Thus we did not identify the in vitro conditions necessary to purify a regulator of P66 expression. As an alternative approach, we identified candidate regulators in a search of the Borrelia genome for homologues to other bacterial transcription factors. We cloned the genes and cotransformed the expression plasmids in E. coli strains carrying a p66 promoter-signal sequence-phoA (alkaline phosphatase or AP) fusion. The p66 promoter fusion alone resulted in basal AP activity. We performed AP assays to identify candidates that acted as potential activators or repressors by increasing or decreasing AP activity, respectively. We identified three candidate transcription factors - two that decreased AP activity (BB0232 and BB0527), and one that increased AP activity (BBA23). We then investigated whether these proteins interact with p66 and other promoters in vitro using gel shift assays. Promoters from differentially regulated genes (ospA, ospC); from a constitutively active gene (flaB), and from the oriC region of DNA, which was previously shown to bind BB0232 (Hbb), were used. We found that while no DNAs were bound at meaningful levels of BBA23 or BB0527, each was bound strongly by Hbb. In agreement with what others have reported, we believe that Hbb plays multiple and varied roles in B. burgdorferi, including binding, bending, and unwinding DNA, in addition to its role at the origin of replication of the chromosome. However, it is also possible that Hbb may play a more direct role in transcriptional regulation by interacting with RNA polymerase.