Relationships of Arc and Psp systems 19 Fig. 2. Activation of ArcAB is required for robustness of pIV-dependent induction of psp expression. A. Schematic representation of ArcB and ArcA (adapted from Malpica et al., 2004) with the locations of the amino acid substitutions used in this study. ArcB: activation mutants – C180A/S and C241A/S substitutions in PAS domain; phosphorelay mutants – H292A or H717A; leucine zipper mutant (LeuZm) – L87A, L90A, L94A and M97A. ArcA: a phosphorylation-defective mutant – D54A, and DNA-binding-deﬁcient mutant (introduction of two stop codons, E129Stop and E130Stop) – DHTH. B. Regulation of the ArcAB system. ArcB is an IM-localized dimer. Its kinase activity is diminished by UQ and elevated in the presence of UQH2 leading to reduction of the ArcB Cys180 and Cys241 thiol groups (in this article functionally equivalent to ArcB* form) in PAS (Per-Arnt-Sim) domain and an increased interaction between the two ArcB TM domains. (PAS domains are signalling domains and they function as input modules in proteins that sense oxygen, redox potential, light and several other stimuli.) Reduction of ArcB is followed by autophosphorylation of H292 (H1) and phosphorelay via D576 (D1) and H717 (H2) to residue D54 (D2) within the response regulator ArcA. C. Bipartite role of ArcB activation in pIV-dependent induction of psp expression. Induction of psp expression in DarcB (MVA63), DarcA (MVA79) and a double mutant DarcAB (MVA94) strains expressing ArcBWT, ArcAWT and their mutants was measured using b-Gal assays under microaerobic conditions in the absence or presence of pIV. WT – wild-type strain (MVA44); vector – pCA24N. induce psp expression. However, overexpression of either to be due to an effect of activated ArcB*, which is com- activated ArcB (ArcB*) or the ArcB* phosphorelay mutants pletely independent of ArcA or ArcA-P action. (e.g. ArcB*H717A) were able to induce a low level of psp expression (compared with ArcBWT). When ArcB* is over- expressed in the DarcAB strain, a similar low level of pIV-independent psp expression was observed (Fig. 2C). We reasoned that the low-level induction of psp expres- sion caused by the ArcB* phosphorelay mutants appears Overexpression of ArcAWT in a DarcA strain in the absence of pIV results in induction of psp expression (Fig. 2C). Notably, this elevation of psp expression by overexpression of ArcAWT is ArcB-dependent (compare DarcA and DarcAB strains) (Fig. 2C). However in the pres- ence of pIV, overexpression of ArcAWT in the DarcAB strain © 2009 The Authors Journal compilation © 2009 Blackwell Publishing Ltd, Molecular Microbiology, 74, 16–28