Abstract
The membrane-integrated transcriptional regulator CadC of Escherichia coli activates expression of the cadBA operon at low external pH with concomitantly available lysine, providing adaptation to mild acidic stress. CadC is a representative of the ToxR-like proteins that combine sensory, signal transduction, and DNA-binding activities within a single polypeptide. Although several ToxR-like regulators such as CadC, as well as the main regulator of Vibrio cholerae virulence, ToxR itself, which activate gene expression at acidic pH, have been intensively investigated, their molecular activation mechanism is still unclear. In this study, a structure-guided mutational analysis was performed to elucidate the mechanism by which CadC detects acidification of the external milieu. Thus, a cluster of negatively charged amino acids (Asp-198, Asp-200, Glu-461, Glu-468, and Asp-471) was found to be crucial for pH detection. These amino acids form a negatively charged patch on the surface of the periplasmic domain of CadC that stretches across its two subdomains. The results of different combinations of amino acid replacements within this patch indicated that the N-terminal subdomain integrates and transduces the signals coming from both subdomains to the transmembrane domain. Alterations in the phospholipid composition did not influence pH-dependent cadBA expression, and therefore, interplay of the acidic surface patch with the negatively charged headgroups is unlikely. Models are discussed according to which protonation of these acidic amino acid side chains reduces repulsive forces between the two subdomains and/or between two monomers within a CadC dimer and thereby enables receptor activation upon lowering of the environmental pH.
Highlights
Bacteria that prefer the human intestine as a natural habitat encounter a broad range of environmental stresses, including acidic pH [1]
One system that is important for the response to mild acidic stress in E. coli is the Cad system [1]
Because all CadC variants were inserted into the membrane (Fig. 1B), the lack of response of CadC-H240Q seemed to be related to defects in sensing and/or signal transduction
Summary
Bacterial Strains and Growth Conditions—E. coli JM109 or DH5␣ [11, 12] was used as a carrier for the plasmids described. Pellets were resuspended and transferred to prewarmed LB medium (pH 5.8 –7.6) by adjusting the A600 to 0.7 After growth for another 1.5 h, cells were harvested by centrifugation. To test CadA activity, a modified spectrophotometric assay for lysine decarboxylase as described by Lemonnier and Lane [20] was used. For this purpose, the cell density was adjusted to A600 ϭ 1 with 20 mM potassium Pi buffer (pH 5.8). The absorption of N,NЈ-bistrinitrophenyl cadaverine (toluene phase) was determined at 340 nm, and the specific activity of CadA (mol/min*mg) was calculated. Calculation of electrostatic surface was carried out with the program GRASP [23]
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