The Cad system consists of the cytoplasmic protein CadA and the transmembrane proteins CadB and CadC [1]. CadA is a lysine decarboxylase that catalyzes decarboxylation

of lysine to cadaverine whereby one proton is consumed resulting in a relief of the intracellular acid stress. The alkaline product cadaverine is concomitantly excreted by the lysine/cadaverine antiporter CadB [2, 3]. The genes cadA and cadB are organized in an operon [3, 4], which is under the control of the P Cad promoter. Expression of the cadBA operon is induced after external acidification, and simultaneous presence of extracellular lysine. CadC is the positive regulator of cadBA expression [5], and binds to two sites within the cadBA promoter [6]. cadC is located upstream of the cadBA operon and encodes a 58 kDa inner membrane protein. CadC, a member of the STI571 purchase ToxR-like transcriptional selleck inhibitor activators [7], consists of a cytoplasmic N-terminal

domain (amino acids 1-158), a single transmembrane domain (amino acids 159-187), and a periplasmic C-terminal domain (amino acids 188-512) [5, 8]. The cytoplasmic domain shows sequence similarity to the ROII-subgroup of DNA-binding domains of response regulators [5]. However, contrary to prototypical response regulators [9] signal transduction in CadC functions without phosphorylation. Thus, CadC and all other ToxR-like proteins represent a one-component stimulus-response system. Based on CadC derivatives with altered sensing properties due to single amino acid replacements within the periplasmic domain, it was suggested that this domain is the signal input domain [8]. Recently, it became clear that CadC senses alterations of the external pH directly [10], but lysine is sensed only indirectly. The lysine-dependent Osimertinib datasheet regulation of CadC is exerted by an interplay with the lysine permease LysP,

and it is proposed that in the absence of lysine, CadC is inactivated by an interaction with LysP [11]. Here, we investigated the role of the three cysteine residues in CadC. The best investigated member of the ToxR-like protein family, ToxR of Vibrio cholerae, contains two cysteines within the periplasmic domain. These cysteines were found to be involved in the formation of an intramolecular disulfide bond but also in the formation of intermolecular disulfide bonds between two ToxR molecules and between ToxR and a second transmembrane protein, ToxS [12, 13]. Although it was shown that ToxR binds to the DNA only in a dimeric form [7], ToxR oligomerization in vivo was independent of environmental changes [14], and thus evidence for the functional importance of the cysteines in ToxR is still lacking. Our studies indicated that a disulfide bond within the periplasmic domain of CadC is formed at pH 7.6, but these cysteines are in the reduced state at pH 5.8. These results give new insights into the switch between inactive and active states of a pH-responsive protein.