Two-component systems, composed of a sensor kinase and a response regulator, are a major tool used by microorganisms to adapt to environmental conditions. Typically, phosphorylation of the response regulator by the sensor kinase results in gene activation. In the case of B. pertussis, the response regulator BvgA is phosphorylated by the sensor kinase BvgS to yield a form (BvgA-P) that binds to the promoter regions of the virulence genes, activating their transcription during infection. These genes include adhesins, needed to adhere to the ciliated epithelial cells within the upper respiratory tract, and toxins, which cause major symptoms of the whooping cough disease. Despite the importance of two-component systems in cell growth and virulence, quantifying the level of a phosphorylated response regulator relative to its unphosphorylated form has been challenging, in part because the phospho-Asp acylphosphate bond within the response regulator is easily hydrolysed. Furthermore, current methods to assess in vivo phosphorylation have been cumbersome or have involved radioactive labeling of the response regulator. In collaboration with the lab of S. Stibitz (FDA), we have used protein electrophoresis through polyacrylamide gels derivatized with the proprietary ligand Phos-tag to separate the response regulator BvgA from its phosphorylated counterpart BvgA-P. This approach has allowed us to readily ascertain the degree of phosphorylation of BvgA in in vitro reactions or in crude lysates of Bordetella pertussis grown under varying laboratory conditions. We have used this technique to examine the kinetics of BvgA phosphorylation after shift of B. pertussis cultures from non-permissive to permissive conditions, or of its dephosphorylation following a shift from permissive to non-permissive conditions. Our results provide the first direct evidence that levels of BvgA-P in vivo correspond temporally to the expression of early and late BvgA-regulated virulence genes. We have also examined a number of other aspects of BvgA function predicted from previous studies and by analogy with other two-component response regulators. These include the site of BvgA phosphorylation, the exclusive role of the cognate BvgS sensor kinase in its phosphorylation in Bordetella pertussis, and the effect of the T194M mutation on phosphorylation. We also detected the phosphorylation of a small but consistent fraction of BvgA purified after expression in Escherichia coli.
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