24 G. Jovanovic, C. Engl and M. Buck ArcB communicates with PspB There appear to be two separate actions of the Arc system in pIV-dependent induction of psp expression. One is related to activation of ArcA (and subsequent control of ArcA-P target genes) required for full psp induction under pIV stress. The other action relates to a more direct effect of activated ArcB that promotes low- level induction of psp expression, where ArcB*, in the absence of pIV, can induce low-level psp expression in a DarcA strain as can ArcB* phosphorelay mutants in DarcB cells. We demonstrated that ArcB interacts with PspB and that this interaction is dependent on the integ- rity of the PspB leucine zipper motif. Further, mutational analysis revealed that the PspB leucine zipper is impor- tant for pIV-dependent induction of psp expression, but not required for direct binding interactions between PspB and PspC, suggesting that PspB may be directly involved in receiving (or transducing) an inducing signal from activated ArcB. The linker region of ArcB connects the trans-membrane (TM) domain with the catalytic domain of ArcB (see Fig. 2A and B) and contains a putative leucine zipper motif (conserved leucine residues at positions 73, 80, 87 and 94) and a PAS domain, the former of unknown function for ArcB activity. It has been suggested that Leu87 functions in intramolecular signal propagation in ArcB kinase acti- vation (reviewed by Malpica et al., 2006). Here we show that the ArcB leucine zipper motif is functionally important in intramolecular activation of ArcB. Our results indicate that the ArcB leucine zipper mutant (ArcBLeuZm) is in a constitutive ‘kinase on’ state, capable of inducing the pﬂ promoter, but not if the phosphorelay residues H292 or H717 are compromised (e.g. ArcBLHe7u1Z7mA). ArcBLeuZm also facilitates pIV-dependent and increases pIV-independent induction of psp expression. However, induction of psp expression by ArcBLHe7u1Z7mA (for example) is signiﬁcantly lower when compared with ArcB*H717A. In addition, the ArcBLeuZm mutant exhibits a decreased affinity for PspB, further suggesting that under speciﬁc psp-inducing condi- tions, the leucine zipper motifs of both ArcB and PspB are involved in ArcB–PspB communication. The PAS domain of ArcB may allow ArcB to respond to a number of factors related to changes in, e.g. oxygen supply and associated metabolic processes. To date only the interaction between ArcB and UQ has been reported (reviewed by Malpica et al., 2006). Since ArcB speciﬁcally interacts with PspB, in principle this would allow PspB to respond to signals received by ArcB, and vice versa. A similar relationship between signalling and control com- ponents has been described for other systems (e.g. for PhoP and PmrD) (Kato et al., 2007) where communica- tion often occurs through membrane interactions (Szur- mant et al., 2008). Integration of the Arc system in the PspB/C-speciﬁc signalling pathway In vivo it has been shown that PspB, PspC and PspA and PspA and PspF interactions occur under non-stress con- ditions (reviewed by Darwin, 2005). Here, we show that ArcB and PspB interact, potentially within one or more ArcB–Psp[B/C/A/F] regulatory complexes or sub- complexes. Stress-induced activation of ArcB may result in conformational changes in either PspB or PspC (or both) and presumably through altered protein–protein interactions releases the PspA-imposed negative regula- tion of PspF. The precise nature of the signal(s) that induces psp expression remains to be determined. It is possible that under stress conditions, besides changes in the integrity of the IM and/or Dy (pmf), the redox state of respiratory chain components speciﬁcally quinones, may also con- tribute to Arc-dependent induction of psp expression. One possibility is that alongside Arc-independent signalling, psp-inducing stimuli reduce UQ and, following an increased diffusion of H+ into the cytosol, the consequent dissipation of the pmf, may then activate ArcB. This view is supported by the observation that both UQ and ArcAB are required to detect a drop in Dy in the presence of pIV, although UQ may be required for the structural integrity ArcB (Jovanovic et al., 2006; this study). Notably induc- tion of psp expression and a signiﬁcant downregulation of aerobic respiration and pmf occur simultaneously in cells undergoing contact-dependent growth inhibition (Aoki et al., 2009). In microaerobiosis, activation of the Arc system greatly facilitates pIV-dependent induction of psp expression in a PspBC-dependent manner. However, since part of the induction process is Arc-independent but still PspBC- dependent (Fig. 1) it seems likely that one (or both) of the psp encoded sensors (PspB and/or PspC) can receive a signal. Therefore, a ‘double check point’ potentially exists to regulate pIV-dependent induction of psp expression. This arrangement of sensors would help the Psp system maintain speciﬁcity to stimuli, as described for unorthodox two-component systems which maintain their robustness to noises (Kim and Cho, 2006). PspA–PspF is a minimal Psp system This study clearly shows that in anaerobiosis, pIV- dependent induction of psp expression is PspBC- independent, consistent with the observation that some psp-inducing stimuli are PspBC-independent (e.g. extreme heat shock, CCCP) or only partially PspBC- dependent (ethanol treatment and hyperosmotic shock) [(reviewed by Model et al., 1997; Darwin, 2005); Jovanovic et al., 2006]. These results strongly suggest © 2009 The Authors Journal compilation © 2009 Blackwell Publishing Ltd, Molecular Microbiology, 74, 16–28