Relationships of Arc and Psp systems 23 Fig. 5. Roles of PspB and PspC in transducing the psp-inducing signal(s). psp expression was induced by either pIV, ArcB* or ArcAWT. Cells were grown under microaerobic conditions and expression of psp was measured using b-Gal assays. The DpspB mutation was complemented with either PspB (pAJM1) or PspBLeuZm (pGJ48), and DpspC mutation was complemented by PspC (pAJM2). WT – wild-type strain [MC1061 f(pspA–lacZ); MVA4]; vector (WT panel) – pCA24N; vector (DpspB/C panels) – pBAD18-cam. consistent with the inability of PspBLeuZm to interact with ArcB (Table 2). Importantly, both PspBWT and PspBLeuZm are stably expressed and localized within the IM (Fig. S5). Further, in the absence of PspC (DpspC), psp induction by pIV and by ArcB* was also abolished (Fig. 5). Considering that pIV-dependent induction of psp expression involves ArcB and ArcA activities, we suggest that the Arc and Psp systems are integrated via the PspB leucine zipper motif and as such, Arc conditionally forms part of the PspBC- speciﬁc signal transduction pathway. Discussion Conditional requirement for Arc activation in pIV-dependent induction of psp expression In this work, several lines of evidence demonstrate that the ArcAB system in microaerobiosis is required to facili- tate pIV-dependent induction of psp expression and may potentially contribute to maintaining pmf (via the Psp response). ArcB activation is clearly essential for pIV- dependent induction of psp expression as is the integrity of the phosphorelay signalling cascade (which results in phosphorylation of ArcA) and ArcA itself. However, we doubt that the Arc system directly controls expression of the psp genes, since neither of the two E. coli psp pro- moters contains recognizable ArcA binding sites and the absence of Arc proteins does not affect transcription of psp genes in DpspA cells (i.e. in the absence of negative control). In microaerobiosis, Arc-dependent induction of psp expression in the presence of pIV correlates with a decrease in Dy (pmf), which is dependent both on the phosphorelay activity of ArcB and on ArcA itself. Under pIV stress conditions, the Psp response is not able to conserve pmf when the Arc proteins are absent, therefore we suggest that activation of the ArcAB system helps with generating and/or propagating a pIV-dependent psp- inducing signal threshold needed to mount the Psp response. Since the Psp response helps to maintain the cells pmf (and energy status) and is implicated in the growth and virulence of enterobacteria (reviewed by Darwin, 2005; Rowley et al., 2006; Darwin, 2007), the cross-talk between the Arc and Psp systems is of physi- ological relevance given that changes in oxygen availabil- ity often prevail during pathogenesis. The requirement for ArcAB activation in pIV-dependent induction of psp expression signiﬁcantly decreases in aerobiosis and anaerobiosis, consistent with the pro- posed major importance of the ArcAB regulators in microaerobic growth and in managing transitions between aerobic and anaerobic conditions (Alexeeva et al., 2003; Partridge et al., 2007). Conditional use of ArcAB can explain results in which involvement of Arc in psp induc- tion was not evident under aerobic growth conditions (Seo et al., 2007). We speculate that in anaerobiosis, the more reducing environment in the cytoplasm will decrease the psp- inducing signal threshold resulting in increasingly Arc- independent pIV-dependent psp expression, consistent with the cellular redox state (as deﬁned by the NADH/ NAD ratio) favouring a reducing environment in anaero- biosis versus an oxidizing environment in aerobiosis (de Graef et al., 1999). In addition, under anaerobic condi- tions, the contribution of UQ to the respiratory chain is signiﬁcantly lower (and is replaced by menaquinone) (Gennis and Stewart, 1996) and so may contribute to a decreased requirement for Arc in pIV-dependent induction of psp expression. Hence, Arc-dependent induction of psp expression under pIV stress depends on speciﬁc growth conditions. Other examples of conditional effects of Arc on certain genes are known (Mika and Hengge, 2005). © 2009 The Authors Journal compilation © 2009 Blackwell Publishing Ltd, Molecular Microbiology, 74, 16–28