Mutational Analysis of the Escherichia coli PhoQ Sensor Kinase: Differences with the Salmonella enterica Serovar Typhimurium PhoQ Protein and in the Mechanism of Mg ²⁺ and Ca ²⁺ Sensing

Abstract

The PhoP-PhoQ two-component system plays a role in Mg 2؉ homeostasis and/or the virulence properties of a number of bacterial species. A Salmonella enterica serovar Typhimurium PhoQ sensor kinase mutant, in which the threonine at residue 48 in the periplasmic sensor domain is changed to an isoleucine, was shown previously to result in elevated expression of PhoP-activated genes and to affect mouse virulence, epithelial cell invasion, and sensitivity to macrophage killing. We characterized a complete set of proteins having amino acid substitutions at position 48 in the closely related Escherichia coli PhoQ protein. Numerous mutant proteins having amino acid substitutions with side chains of various sizes and characters displayed signaling phenotypes similar to that of the wild-type protein, indicating that interactions mediated by the wild-type threonine side chain are not required for normal protein function. Changes to amino acids with aromatic side chains had little impact on signaling in response to extracellular Mg 2؉ but resulted in reduced sensitivity to extracellular Ca 2؉ , suggesting that the mechanisms of signal transduction in response to these two divalent cations are different. Surprisingly, the Ile48 protein displayed a defective phenotype rather than the hyperactive phenotype seen with the S. enterica serovar Typhimurium protein. We also describe a mutant PhoQ protein lacking the extracellular sensor domain with a defect in the ability to activate PhoP. The defect does not appear to be due to reduced autokinase activity but rather appears to be due to an effect on the stability of the aspartylphosphate bond of phospho-PhoP.

Isolation and Molecular Characterization of the Locked-on Mutant of Mg2+Sensor PhoQ inEscherichia coli

“…8) The equivalent mutation in the E. coli PhoQ protein resulted in a strain that responded to Mg 2þ , 9) this being consistent with our results (Fig. 1).…”

“…Regelman et al have constructed a set of 19 mutants with mutations covering the full spectrum of possible amino acid substitutions at residue 48 in E. coli PhoQ protein. 9) They consequently found mutants that showed less than 30% of the wild-type level of reporter expression in the absence of Mg 2þ and Ca 2þ , and the defects displayed by the mutants were not the result of any significant reduction in protein level. 9) Similar results were observed in Mg1307/pHO47L, pHO48A and pHO150I, whose mutations might affect the autophosphorylation or dephosphorylation activity of PhoQ more than the others (Fig.…”

Crystal Structure of a Functional Dimer of the PhoQ Sensor Domain

“…␤-Galactosidase Assays-␤-Galactosidase assays were performed as described (14) in N-media with additions of MgCl 2 as indicated using strain CSH26⌬Q/FЈ lacI Q kan/ pNL3 containing derivatives of pLPQ2 with wild-type phoQ or mutant variants.…”

“…Q kan/pNL3 strains containing derivatives of pLPQ2 with wild-type phoQ or mutant variants were grown to mid-log phase in N-media with appropriate antibiotics as described previously (14) and then induced for 2 h at 37°C with the addition of 1 mM isopropyl-␤-D-thiogalactopyranoside. Cells were harvested by centrifugation, resuspended in resuspension buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl), and lysed by sonication on ice.…”

Evidence for Serine/Threonine and Histidine Kinase Activity in the Tobacco Ethylene Receptor Protein NTHK2

“…EnvZ, also possess activity in the presence of Ca 21 (Park et al, 1998), and others may need different divalent cations for their kinase activity (Koo and Stephens, 2003). A bacterial HK PhoQ can even sense Mg 21 and Ca 21 (Regelmann et al, 2002). In plants, the HK-like ethylene receptor ETR1 depends on Mn 21 for its HK activity.…”