Phosphotransfer Profiling Method

<div><p>(A) Phosphotransfer profile experiments involve three separate reactions: (1) autophosphorylation of the histidine kinase (HK) by radiolabeled ATP, (2) phosphotransfer to a response regulator (RR), and (3) dephosphorylation of the response regulator.</p> <p>(B) Schematic of the phosphotransfer profiling technique. A single preparation of purified, autophosphorylated kinase (HK∼³²P) is mixed with each response regulator from a given organism and analyzed for phosphotransfer by SDS-PAGE and autoradiography. The first lane shows a single band corresponding to the autophosphorylated histidine kinase and is used as a comparison for every other lane. Lanes 2–4 illustrate the three possible outcomes of a phosphotransfer reaction. In lane 2, phosphotransfer from HK to RR1 leads to the appearance of a band corresponding to RR1. In lane 3, phosphotransfer from HK to RR2 also occurs, but owing to high phosphatase activity (either autophosphatase or catalyzed by a bifunctional HK), the net result is production of inorganic phosphate (Pi) and the depletion of radiolabel from both the HK and RR2. In lane 4, no phosphotransfer occurs, and the lane is indistinguishable from lane 1.</p> <p>(C–H) Phosphotransfer profiling was performed for three <i>E. coli</i> kinases (EnvZ, CheA, and CpxA) against all 32 purified <i>E. coli</i> response regulators, with phosphotransfer incubation times of either 1 h (C, E, and G) or 10 s (D, F, and H). For these three histidine kinases, a comparison of the short and long time point profiles indicates a kinetic preference for only their in vivo cognate regulators: OmpR (C and D), CheY and CheB (E and F), and CpxR (G and H). After being examined for phosphotransfer, all gels are stained with Coomassie to verify equal loading of histidine kinase and response regulator in each lane (data not shown). For each kinase profiled, we purified only its soluble, cytoplasmic domain, either as a thioredoxin-His₆ or a His₆-MBP fusion, using standard metal affinity chromatography (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030334#s4" target="_blank">Materials and Methods</a>). When necessary, we made successive N-terminal truncations until we identified a construct that produced active kinase in vitro, always preserving the H-box and ATP binding domain (details on constructs used are in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030334#st003" target="_blank">Table S3</a>). All response regulators were purified as full-length fusions to a thioredoxin-His₆ tag. Purity was assessed by Coomassie staining, with each purified kinase domain and response regulator, except for <i>E. coli</i> FimZ, yielding an intense band of the correct approximate molecular weight (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030334#sg005" target="_blank">Figure S5</a>; <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030334#st003" target="_blank">Table S3</a>).</p>