Abstract
The PhoP-PhoQ two-component system is required for virulence and/or regulatory stress responses in enteric bacteria. The PhoQ protein responds to low concentrations of extracellular divalent cations by activating PhoP-mediated transcription of a set of genes. PhoQ is a member of a family of transmembrane proteins that contain a periplasmic sensor domain coupled to a cytoplasmic transmitter domain. Here, we describe the cloning, purification, and properties of a fragment of Escherichia coli PhoQ corresponding to the sensor domain. This fragment is monomeric in solution and has a circular dichroism spectrum indicative of a mixture of alphahelix and beta-sheet. Divalent cations do not affect the oligomeric state, circular dichroism spectrum, or fluorescence spectrum of the sensor domain but do stabilize this domain to denaturation in a fashion expected for a direct binding model. We have also constructed a mutant in which a cluster of acidic amino acids (EDDDDAE) in the sensor domain is replaced with conservative, uncharged residues (QNNNNAQ). The mutant sensor domain is indistinguishable from wild type in terms of oligomeric form and spectral properties but differs in being substantially more stable to urea denaturation, showing no additional stabilization in the presence of divalent cations, and showing little activation of PhoP-mediated transcription in response to divalent-cation starvation in vivo. These data are consistent with a model in which divalent cations bind to the acidic cluster of the wild-type sensor domain and stabilize a conformation that is inactive in signaling. Substituting uncharged residues for the acidic cluster appears to mimic the effect of divalent-cation binding by stabilizing the inactive conformation.
Highlights
The PhoP-PhoQ two-component system is required for virulence and/or regulatory stress responses in enteric bacteria
We have constructed a mutant in which a cluster of acidic amino acids (EDDDDAE) in the sensor domain is replaced with conservative, uncharged residues (QNNNNAQ)
The six acidic residues in the wild-type cluster have been predicted to form a site for divalent-cation binding [9, 10]. If this prediction is correct, we reasoned that replacing these acidic residues with isosteric but uncharged residues should alter the mutant sensor domain’s ability to bind divalent cations
Summary
Plasmids—Several plasmids were constructed for these studies. pNL3 (Fig. 1A) is a pBR322-derived reporter plasmid used to assay PhoP-mediated transcriptional activation. PAED4Q (Fig. 1A) is a pUC19-derived plasmid used to express the PhoQ sensor domain (residues 43–190) for purification. The resulting plasmid (pAED4Q) has an initiating ATG codon (at the synthetic NdeI site) fused to codon 43 of phoQ and a translational termination codon after codon 190 of phoQ (Fig. 1B) In this plasmid, expression of the sensor domain (residues 43–190) is driven by a plasmid-borne T7 10 promoter and ribosome-binding site. Isolated candidate colonies were screened for CmS, indicating loss of the plasmid, and replacement of wild-type phoQ with the ⌬Q deletion was confirmed by PCR analysis of chromosomal DNA. Protein Purification—The PhoQ sensor domain (residues 43–190) was purified from E. coli strain X90(DE3) transformed with plasmid pAED4Q (or a variant containing the acidic-cluster mutations). The low level of phoPphoQ expression in the absence of IPTG is sufficient for activation of phoN-lacZ
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