18 G. Jovanovic, C. Engl and M. Buck Table 1. Biochemical phenotypes of arcB alleles. Protein ArcBWT ArcBH292A ArcBH717A ArcB* ArcB*H292A ArcB*H717A ArcBLeuZm ArcBLHe2u9Z2mA ArcBLHe7u1Z7mA Motif(s) mutation Wild type H292A H717A C180A C241A C180A C241A H292A C180A C241A H717A L87A L90A L94A M97A L87A L90A L94A M97A H292A L87A L90A L94A M97A H717A a. Kinase ‘on’, wild-type C180 and C241 (apo form). b. Kinase constitutively ‘on’. c. Phosphorelay ‘on’. Since Psp effector proteins (PspA, PspD and PspG) alter expression levels of genes associated with aerobic growth (Jovanovic et al., 2006) and ArcAB regulates the switch from aerobic to anaerobic respiration and fermen- tation, we tested whether (i) pIV changes oxygen con- sumption in microaerobiosis (Fig. S1C) and (ii) other global regulators associated with aerobicity (e.g. FNR or Nar; see Fig. S1D) also affect psp induction. The results show that pIV changes oxygen consumption in an ArcB- dependent manner and that among the global regulators, the Arc system has the greatest effect on psp expression, supporting the idea of speciﬁc cross-talk between the Psp and ArcAB systems. Taken together, these results indicate a growth condition-speciﬁc requirement for ArcAB and PspBC proteins in psp induction. In addition, since under anaerobiosis induction of psp is independent of the PspBC-positive regulators we suggest that the PspA–F regulatory complex may itself be capable of perceiving a pIV-dependent inducing signal. Importantly, all subse- quent experiments reported here were conducted under the microaerobic conditions where the roles of PspBC and ArcAB were most prominent. Activation of ArcA facilitates pIV-dependent induction of psp expression To further assess the roles of ArcAB in pIV-dependent induction of psp expression in microaerobiosis, we con- structed mutants of ArcB (see Table 1) and ArcA that: (i) had a constitutively active kinase: ArcBC180A/C241A (termed ArcB*), (ii) disrupted the phosphorelay from ArcB to ArcA: ArcBH292A, ArcBH717A, ArcB*H292A and ArcB*H717A, and (iii) inactivated ArcA (by disrupting phosphorylation and pre- venting binding to target promoters): ArcADΔH54TAH (Fig. 2A). Initially we determined that WT and mutant forms of ArcB proteins were similarly expressed (Fig. S2Ai and data not shown) and appropriately localized within the IM ArcB activationa + + + - - - + + + ArcB constitutively activeb - - - + + + + + + ArcA activationc + - - + - - + - - (Fig. S2Aii and data not shown). As a further measure of functionality, we showed that the WT and certain mutant ArcB and ArcA proteins (only those with intact phospho- relay systems) were able to activate ArcA-P-dependent pﬂ transcription (Fig. S2B). We then assayed the ability of Arc mutants to complement DarcB or DarcA mutations and support pIV-dependent induction of psp expression. As shown in Fig. 2C, ArcBWT and ArcB* or ArcAWT fully complemented DarcB or DarcA mutations, respectively, for pIV-dependent induction of psp expression. None of the phosphorelay mutants in ArcB or the inactive ArcA variant fully supported pIV-dependent induction of psp expression (Fig. 2C), indicating that ArcA-P has a role in promoting pIV-dependent induction. Importantly, control reactions demonstrated that similar levels of pIV secretin were present in all the samples tested (Fig. S2C and data not shown). Overexpression of ArcB proteins in a DarcAB strain showed that in the absence of ArcAWT, the ArcBWT and ArcB* similarly to ArcB* phosphorelay mutants cannot fully support pIV-dependent induction of psp expression (compared with results obtained with a DarcB) and that ArcB* and its phosphorelay mutants promote an ArcA- and pIV-independent low-level induction of psp (Fig. 2C). These data indicate that ArcB has an activity with respect to Psp that is partially ArcA-independent. In contrast, overexpression of ArcAWT in the DarcAB strain led to strong pIV-dependent induction of psp (Fig. 2C), indicat- ing that there are two levels of Arc involvement in psp expression, one mediated by ArcB independent of ArcA, and one directed by ArcA. An active form of ArcB promotes low-level induction of psp expression independent of pIV In the absence of pIV (white bars in Fig. 2C) we observed that overexpression of either ArcBWT or the ArcB phospho- relay mutants (ArcBH292A or ArcBH717A) did not signiﬁcantly © 2009 The Authors Journal compilation © 2009 Blackwell Publishing Ltd, Molecular Microbiology, 74, 16–28