Abstract
The PhoP-PhoQ two-component system is present in a number of Gram-negative bacteria where it has roles in Mg(2+) homeostasis and virulence. PhoQ is a transmembrane histidine kinase that activates PhoP-mediated regulation of a set of genes when the extracellular concentration of divalent cations is low. Divalent cations are thought to interact directly with the periplasmic PhoQ sensor domain. The PhoP-PhoQ systems of Escherichia coli and Pseudomonas aeruginosa are similar in their biological response to extracellular divalent cations; however, their sensor domains display little sequence identity. Here we have begun to explore the consequences of this sequence divergence by comparing the biophysical properties of the P. aeruginosa PhoQ sensor domain with the corresponding E. coli sensor domain. Unlike the E. coli protein, the P. aeruginosa PhoQ sensor domain undergoes changes in the circular dichroism and fluorescence spectra as well as destabilization of its dimeric form in response to divalent cations. These results suggest that distinct mechanisms of signal detection are utilized by these proteins. A hybrid protein in which the E. coli sensor domain has been substituted with the corresponding P. aeruginosa sensor domain responds normally to the presence of extracellular divalent cations in vivo in E. coli. Thus, despite apparent differences in the structural response to its stimulus, the P. aeruginosa sensor domain transduces signals to the E. coli PhoQ cytoplasmic kinase domain in a manner that mimics normal E. coli PhoQ function.
Highlights
The PhoP-PhoQ two-component system is present in a number of Gram-negative bacteria where it has roles in Mg2؉ homeostasis and virulence
In this paper we show that the P. aeruginosa PhoQ sensor domain responds to divalent cations in a manner that is distinct from that of the E. coli PhoQ sensor domain and is characterized by changes in its oligomeric state and secondary and tertiary structures
Plasmids and Bacterial Strains—PEMQ2a [7] is a pUCP20-derived plasmid that contains the phoP-phoQ operon from P. aeruginosa. pLPQ2 [12] is a pSC101-derived plasmid in which expression of the E. coli phoP-phoQ operon is driven by the lacUV5 promoter. pNL3 [12] is a pBR322-derived reporter plasmid for assaying PhoP-mediated transcriptional activation that contains the phoN promoter fused to lacZ. pNL2 is a low copy number reporter plasmid in which the phoN-lacZ fusion has been subcloned into pGB2 [15]. pLPQ*2 is a derivative of pLPQ2 in which DNA encoding the sensor domain of PhoQ has been replaced with the corresponding region of the P. aeruginosa phoQ gene
Summary
Unlike the E. coli protein, the P. aeruginosa PhoQ sensor domain undergoes changes in the circular dichroism and fluorescence spectra as well as destabilization of its dimeric form in response to divalent cations These results suggest that distinct mechanisms of signal detection are utilized by these proteins. The transmembrane PhoQ histidine kinase consists of an extracellular sensor domain coupled to an intracellular kinase domain, and the cytoplasmic PhoP response-regulator is composed of a receiver domain and a transcriptional regulator domain Extracellular divalent cations such as Mg2ϩ and Ca2ϩ have been shown to repress PhoPmediated transcriptional regulation in E. coli, S. typhimurium, and P. aeruginosa [7, 9, 10, 12]. The P. aeruginosa PhoQ sensor domain can regulate the enzymatic output of the E. coli PhoQ kinase domain in a manner that mimics the normal function of the E. coli protein
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