coli EP-CD4 (cadC::Tn10, cadA’::lacZ, ΔlysP). In a lysP – background, wild-type CadC activates cadBA expression in a lysine-independent, but pH-dependent manner [11, 19]. As expected, in the lysP – background, CadC_C208A,C272A induced cadBA expression lysine- and pH-independently revealing that LysP is responsible for the inhibition Romidepsin clinical trial of CadC_C208A,C272A in the absence of lysine at pH 7.6 (data not shown). As discussed below, these experiments revealed that CadC without a disulfide bond is transformed into a semi-active state with respect to both the pH and the lysine

stimuli. Periplasmic disulfide oxidoreductases have no major influence on CadC activation The results described above led to the hypothesis that at physiological pH CadC contains a disulfide bond which is reduced at low pH. Opening and formation of disulfide bonds requires either the corresponding environment (oxidizing or reducing) or enzymes that catalyze these processes. Therefore, we analyzed whether periplasmic proteins known to be involved in formation and opening of disulfide Napabucasin mouse bonds during the protein folding process such as the Dsb proteins [20] have an influence on CadC activation. Six gene deletion mutants were constructed lacking the disulfide bond-modifying proteins DsbA, DsbB, DsbC, DsbD, DsbG and CcmG (also known

as DsbE). CcmG does not belong to the Dsb system, but is a membrane-anchored protein with a periplasmic thiol:disulfide oxidoreductase domain involved in cytochrome c biogenesis [21]. DsbA is a disulfide oxidase responsible for the formation of disulfide bonds and is recycled by the membrane protein DsbB [20]. DsbC is an isomerase that opens wrongly formed disulfide bonds and introduces the correct ones and as such also exhibits a reductase activity. DsbG is a non-essential isomerase that is able to substitute

for DsbC, and seems to protect single cysteines from oxidation that are needed in a reduced state to be catalytically active [22]. Both, DsbC and DsbG, are recycled by DsbD. While DsbB and DsbD are membrane proteins, DsbA, DsbC and DsbG are soluble proteins located in the periplasm. Mutants of E. coli MG1655 each lacking a single dsb Ribonucleotide reductase gene were grown at pH 5.8 and 7.6 in the presence of external lysine, and lysine decarboxylase (CadA) activity was determined as a measurement for the expression level of cadBA and thus of the functionality of CadC (Figure 6). All strains tested exhibited a pH-dependent regulation that was comparable to the wild-type strain, though the fold-induction differed slightly in some mutants. Under inducing conditions (pH 5.8, lysine) CadA activity was more than twice as high in the mutant MG1655ΔdsbA, lacking the disulfide oxidase DsbA, as in the wild-type strain MG1655 [specific CadA activity of 2.96 μmol/(min*mg protein) instead of 1.27].