SummaryIn an acidic (pH 5.8) and lysine-rich environment Escherichia coli induces expression of the cadBA operon which encodes CadA, catalysing the decarboxylation of lysine to cadaverine, and CadB, the lysine/cadaverine antiporter. cadBA expression is dependent on CadC, a membrane-integrated transcriptional activator which belongs to the ToxR-like protein family and directly binds to the DNA in the cadBA promoter region. Here we describe that CadC senses the extracellular lysine not directly but indirectly requiring the interplay with the lysine permease LysP. Biochemical analyses of isolated CadC or the periplasmic domain of CadC (CadC188-512) revealed an unexpectedly low affinity for lysine, making it unlikely that CadC is a direct sensor for this substrate. Moreover, CadC hybrid proteins, in which the transmembrane domain or single amino acids were replaced, supported lysine-independent cadBA expression but retained a pH-dependent regulation. These CadC mutants were resistant to the effect of an overproduction of LysP, which represses cadBA expression in wild-type cells. Our results suggest a model according to which CadC is inactivated by an interaction with LysP at a low external lysine concentration. When lysine is abundantly available, the interaction between LysP and CadC is released, and CadC becomes susceptible to activation by low pH.

Professional phagocytic cells ingest microbial intruders by engulfing them into phagosomes, which subsequently mature into microbicidal phagolysosomes. Phagosome maturation requires sequential fusion of the phagosome with early endosomes, late endosomes, and lysosomes. Although various phosphoinositides (PIPs) have been detected on phagosomes, it remained unclear which PIPs actually govern phagosome maturation. Here, we analyzed the involvement of PIPs in fusion of phagosomes with various endocytic compartments and identified phosphatidylinositol 4-phosphate [PI(4)P], phosphatidylinositol 3-phosphate [PI(3)P], and the lipid kinases that generate these PIPs, as mediators of phagosome–lysosome fusion. Phagosome–early endosome fusion required PI(3)P, yet did not depend on PI(4)P. Thus, PI(3)P regulates phagosome maturation at early and late stages, whereas PI(4)P is selectively required late in the pathway.

The analysis of stress response systems in microorganisms can reveal molecular strategies for regulatory control and adaptation. In this study, we focused on the Cad module, a subsystem of Escherichia coli's response to acidic stress that is conditionally activated at low pH only when lysine is available. When expressed, the Cad system counteracts the elevated H(+) concentration by converting lysine to cadaverine under the consumption of H(+) and exporting cadaverine in exchange for external lysine. Surprisingly, the cad operon displays a transient response, even when the conditions for its induction persist. To quantitatively characterize the regulation of the Cad module, we experimentally recorded and theoretically modeled the dynamics of important system variables. We established a quantitative model that adequately describes and predicts the transient expression behavior for various initial conditions. Our quantitative analysis of the Cad system supports negative feedback by external cadaverine as the origin of the transient response. Furthermore, the analysis puts causal constraints on the precise mechanism of signal transduction via the regulatory protein CadC.

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.