Responses to environmental stress triggers differential expression and cellular damage in Borrelia burgdorferi
Treglown, Jennifer S
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Regulation of coordinated gene expression is required for survival of Borrelia burgdorferi in different hosts. The sigma factor RpoS is involved in the regulation of gene products needed for stationary phase response. Analysis of the upstream promoter region of rpoS revealed a putative ?54-dependent promoter, which was confirmed to be required for expression of rpoS as the cultures entered stationary phase. Interestingly, QRT-PCR and immunoblots indicated the presence of a second promoter that was independent of ?54. 5’RACE and RT-PCR linkage experiments suggested that the second promoter for rpoS was located in flgI, the gene immediately upstream of rpoS. Primer extension mapped this ?54-independent promoter to 13 bp from the 3’-end of flgI. ?54-dependent transcription requires an activator protein to stimulate open complex formation. Analysis of the B. burgdorferi genome revealed a two-component system consisting of a putative ?54-activator (SisR) and the putative cognate histidine kinase (SisK) located directly upstream of SisR. QRT-PCR showed that the histidine kinase is required for the maximum expression of rpoS during stationary phase. In addition, phosphorylation assays demonstrated that SisK and SisR are part of the same two-component system, suggesting that they are necessary for ?54-dependent transcription. Reactive oxygen species (ROS) are encountered in the tick vector and the mammalian host therefore, B. burgdorferi possesses proteins necessary to detoxify these compounds. NapA was first identified as a Dps/Dpr homolog and initial studies showed that expression increased when cells are grown at high levels of oxygen (> 12%) or when anaerobically grown cells are exposed to ROS. NapA complemented an E.coli ahpCF mutant when exposed to t-butyl hydrogen peroxide, suggesting alkyl hydroperoxide reductase activity, however this may not be the primary function of the enzyme. Immunoelectron microscopy revealed that anaerobic B. burgdorferi cultures exposed to ROS develop membrane blebs around the spirochete. Lipid analysis showed the levels of linoleic acid decreased when the cultures were treated with ROS, suggesting that the major target for damage caused by ROS in B. burgdorferi may be lipids, rather than proteins or DNA as occurs in most bacteria.