20 G. Jovanovic, C. Engl and M. Buck Fig. 3. Activation of ArcAB reduces the electron potential (Dy). Cells were grown microaerobically, treated with JC-1 dye, and the change in Dy calculated as the 530/590 nm (green/red) ratio. ArcBWT, ArcAWT and key variants were expressed in the absence or presence of pIV. An increase in the 530/590 ratio indicates a decrease in Dy compared with WT (E. coli MG1655) with vector plasmid pCA24N. results in strong psp expression even in the absence of ArcB (although we note that this activity could be due to ArcA-P formation arising from cross-talk or acetyl phosphate). Together with the results implying that low- level induction of psp expression can occur in an ArcA- independent manner in the presence of the ArcB* phosphorelay mutants, these data again strongly suggest that activation of the Arc system results in two distinct outcomes (one via ArcA-P, the other via an active form of ArcB) that generate the Arc-dependences in the signalling pathway for pIV-dependent induction of psp expression. Activation of ArcAB results in decreased electron potential (Dy) To address the nature of the signal that is responsible for low-level induction of psp expression by ArcB* and to potentially discriminate between roles of ArcB* and ArcA-P, we measured the electron potential (Dy) of strains expressing either WT or mutant ArcB or ArcA proteins in the presence and absence of pIV (Fig. 3). In the absence of a functioning Psp system (i.e. no effectors present, DpspAG; or no Psp proteins present, DpspF), a clear decrease in Dy under pIV stress is observed (Fig. 3) that appears to be ArcB-dependent (Jovanovic et al., 2006). As shown in Fig. 3, overexpres- sion of ArcBWT, ArcB* or ArcAWT in DpspF DarcB or DpspF DarcA strains resulted in a clear drop in Dy under pIV stress conditions. Interestingly, when we analysed the phospho- relay ArcB and ArcA mutants we no longer observed a drop in Dy, suggesting that phosphorylation of ArcA could there- fore be a prerequisite for the pIV-dependent drop in pmf. Notably, in the absence of pIV, overexpression of ArcB* or ArcAWT (which also results in psp expression; Fig. 2C) was also sufficient to signiﬁcantly reduce Dy (Fig. 3). In contrast, overexpression of ArcBWT, ArcB* phosphorelay mutants (e.g. ArcB*H717A) or inactive ArcA (ArcADΔH54TAH) did not reduce Dy in the absence of stress (Fig. 3). ArcB and PspB interact in vivo To understand how ArcB*-dependent signalling might be integrated into the PspBC-speciﬁc signal transduction pathway, we used an adenylate cyclase (Cya)-based bac- terial two-hybrid system (BACTH) to study potential Arc– Psp protein–protein interactions in vivo (Karimova et al., 1998; 2005; and see Experimental procedures). We con- structed ArcBWT, ArcB*, PspA, PspB, PspC and PspF fusion proteins with Cya T18, T18C and T25 fragments (see Experimental procedures). As a control we ﬁrst analysed the ability of ArcB and ArcB* to self-associate (since ArcB forms dimers; reviewed by Malpica et al., 2006) and show that the ArcBWT and ArcB* fusion proteins are capable of self- associating (Table 2). Next, we tested whether ArcBWT or ArcB* could interact with several key members of the Psp regulon (PspA, B, C, F). Additionally we conﬁrmed that known interactions between PspA–PspF, PspA–PspC and PspB–PspC (Table 2 and data not shown) were detected with our fusion proteins (reviewed by Darwin, 2005). We demonstrated that a clear interaction between either ArcBWT or ArcB* (T25-fused protein) occurs with the C-terminal PspB fusion protein (PspB–T18) (Table 2). A Triton X-100 fractionation and Western blotting method was used to demonstrate that the interaction between ArcB, ArcB* and PspB occurs in the IM (using antibodies against Cya T25, Fig. S3Bi or PspB, Fig. S3Bii). As a control, an N-terminal T18C (T18C–PspB) PspB fusion (where T18C is now embedded in the IM) did not detect- ably interact with ArcB (data not shown). Using an in vivo © 2009 The Authors Journal compilation © 2009 Blackwell Publishing Ltd, Molecular Microbiology, 74, 16–28