Phosphorylation assays of chemotaxis two-component system proteins in Borrelia burgdorferi

Borrelia burgdorferi has a complex chemotaxis signal transduction system with multiple chemotaxis gene homologs similar to those found in Escherichia coli and Bacillus subtilis. The B. burgdorferi genome sequence encodes two cheA, three cheY, three cheW, two cheB, two cheR, but no cheZ genes. Instead of cheZ, B. burgdorferi contains a different CheY-P phosphatase, referred to as cheX. The multiple B. burgdorferi histidine kinases (CheA1 and CheA2) and response regulators (CheY1, CheY2, and CheY3) possess all the domains and functional residues found in E. coli CheA and CheY, respectively. Understanding protein phosphorylation is critical to unraveling many biological processes, including chemotaxis signal transduction, motility, growth control, metabolism, and disease processes. E. coli, Salmonella enterica serovar Typhimurium, and B. subtilis chemotaxis systems have been studied extensively, providing models to understand chemotaxis signaling in the Lyme disease spirochete B. burgdorferi. Both genetic approaches and biochemical analyses are essential in understanding its complex two-component chemotaxis systems. Specifically, gene inactivation studies assess the importance of specific genes in chemotaxis and motility under certain conditions. Furthermore, biochemical approaches help determine the following in vitro reactions: (1) the extent that the histidine kinases, CheA1 and CheA2, are autophosphorylated using ATP; (2) the transfer of phosphate from CheA1-P and CheA2-P to each CheY species; and (3) the dephosphorylation of each CheY-P species by CheX. We hypothesize that characterizing protein phosphorylation in the B. burgdorferi two-component chemotaxis system will facilitate understanding of how the periplasmic flagellar bundles located near each end of B. burgdorferi cells are coordinately regulated for chemotaxis. During chemotaxis, these bacteria run, pause (stop/flex), and reverse (run again). This chapter describes protocols for assessing B. burgdorferi CheA autophosphorylation, transfer of phosphate from CheA-P to CheY, and CheY-P dephosphorylation.