Phosphoryl Group Flow within the Pseudomonas aeruginosa Pil-Chp Chemosensory System: Differential Function of the Eight Phosphotransferase and Three Receiver Domains

Abstract Bacterial chemosensory signal transduction systems that regulate motility by Type IV pili (T4P) can be markedly more complex than related flagellar-based chemotaxis systems. In T4P-based systems, the CheA kinase often contains numerous potential sites of phosphorylation, but the signaling mechanisms of these systems are unknown. In Pseudomonas aeruginosa, the Pil-Chp system regulates T4P-mediated twitching motility and cAMP levels, both of which play roles in pathogenesis. The Pil-Chp histidine kinase (ChpA) has eight ‘Xpt’ domains; six are canonical Hpt (Histidine-containing phosphotransfer) domains and two have a threonine (Tpt) or serine (Spt) in place of the histidine. Additionally, there are a ChpA C-terminal receiver domain (ChpArec) and two stand-alone receiver domains (PilG and PilH). Here, we demonstrate that the ChpA Xpts are functionally divided into three categories: (i) those phosphorylated with ATP (Hpts 4, 5, 6), (ii) those reversibly phosphorylated by ChpArec (Hpts 2-6), and (iii) those with no detectable phosphorylation (Hpt1, Spt, Tpt). There was rapid phosphotransfer from Hpts 2-6 to ChpArec and from Hpt3 to PilH whereas transfer to PilG was slower. ChpArec also had a rapid rate of autodephosphorylation. The biochemical results together with in vivo cAMP and twitching phenotypes of key ChpA phosphorylation site point mutants supported a scheme whereby ChpArec functions both as a phosphate sink and a phosphotransfer element linking Hpts 4,5,6 to Hpts 2,3. Hpt2 and Hpt3 are likely the dominant sources of phosphoryl groups for PilG and PilH, respectively. The data are synthesized in a signaling circuit that contains fundamental features of two-component phosphorelays.