Relationships of Arc and Psp systems 21 Table 2. Interactions of Psp and ArcB proteins in vivo. T18 fusions T18C–ArcBWT T18C–ArcB* PspB–T18 PspBLeuZm–T18 T18C–PspA T18C–PspC T18C–ArcBLeuZm T18C–PspF +/- ND ++ - +/- +/- ND - T25 fusions T25–ArcBWT T25–ArcB* T25–ArcBLeuZm T25–PspC ND + ++ - +/- +/- ND - ND ND + - +/- +/- +/- - +/- +/- ++ ++ + - +/- - The BACTH system was used to detect the protein–protein interactions between the Psp and ArcB proteins. Negative control: BTH101/pKT25+pUT18C vectors alone (74 4 MU); positive control: BTH101/pKT25-zip+pUT18C-zip (1017 34 MU); interaction estimates including SD were as follows: (+/-) weak interaction (235–350 MU); (+) interaction (350–500 MU); (++) strong interaction (>700 MU); (-) no interaction ( 234 MU); ND, not determined. For construction of fusion proteins, growth conditions and mean values with SD, see Experimental procedures. cross-linking assay, we demonstrate that a higher- molecular-weight cross-linked ‘Psp complex’ (labelled cross-linked species in Fig. S3C) can be detected with PspB antibodies in WT and DarcA strains but not in a DarcB deletion strain (Fig. S3C), providing further evi- dence for an interaction between ArcB and PspB. We also found evidence for a weak, but detectable, interaction between ArcBWT or ArcB* and N-terminal T18 fusions to either PspA or PspC (Table 2), but no interac- tion between ArcB, ArcB* and PspF was detected (Table 2). Since interactions between PspF, PspA, PspB, PspC have previously been established (Adams et al., 2003; Elderkin et al., 2005; Maxson and Darwin, 2006), we conclude that (potentially regardless of its state) ArcB could interact with members of a Psp[FABC] complex via PspB. Since both ArcB and PspB contain putative leucine zipper motifs that are most likely positioned at the IM/cytoplasm interface (see Fig. 2A and Fig. S3A), we reasoned that the interaction observed between ArcB and PspB may be occurring directly through these sequences. To determine whether this was indeed the case, we mutated the leucine zipper motifs (LeuZm) of both ArcB (ArcBLeuZm; L87A, L90A, L94A, M97A) and PspB (Psp- BLeuZm; L10A L15A L18A) and fused these mutants to T25– and –T18 respectively (see Experimental procedures and Table S1). Importantly, the PspBLeuZm–T18 fusion protein remained localized within the IM (Fig. S3Bii); however, PspBLeuZm is no longer able to interact with either ArcBWT or ArcB* (Table 2). Interestingly, PspBLeuZm retained the ability to interact with PspC (Table 2), indicating that the interaction with PspC is not dependent on the leucine zipper sequences. These results support a physical inter- action between ArcB and PspB mediated by the PspB leucine zipper motif. Like PspBLeuZm–T18, the T25–ArcBLeuZm fusion protein remained localized within the IM (Fig. S4Aii) and impor- tantly retained the ability to activate pﬂ transcription (Fig. S4B), similar to T25–ArcB*. However, T25–ArcBLeuZm showed reduced pIV-independent and pIV-dependent induction of psp expression compared with T25–ArcB* (Fig. S4B). Interestingly, the ArcBLeuZm mutant was still able to interact with PspB (albeit not as strongly, Table 2), implying that the site of PspB interaction on ArcB does not solely require the integrity of the putative ArcB leucine zipper motif. Like ArcBWT, ArcBLeuZm self-associates (Table 2), suggesting that the ArcB leucine zipper motif does not play a major role in self-association. Finally, ArcBLeuZm retained a very weak interaction with PspA and PspC (Table 2), at a similar level to those observed with ArcBWT or ArcB*, suggesting no role for the ArcB leucine zipper in these interactions. ArcBLeuZm is in an active kinase form Since the primary role of the putative LeuZm in ArcB is not in binding to PspB, we decided to investigate the role of this sequence. Initially, we tested the activity of ArcBLeuZm in a DarcB strain and found that, in contrast to ArcBWT, ArcBLeuZm is able to signiﬁcantly upregulate the pﬂ pro- moter (Fig. 4A) as does ArcB* (see also Fig. S2B). Impor- tantly, upregulation of the pﬂ promoter by ArcBLeuZm is dependent on the phosphorelay residues H292 and H717; the ArcBLHe2u9Z2mA and ArcBLHe7u1Z7mA (see Experimental proce- dures, Table S1) do not show elevated pﬂ–lacZ activities (Fig. 4A), but are expressed at similar levels (Fig. S4Ai and data not shown). Overexpression of ArcBLeuZm in a DarcB strain resulted in elevated psp expression in the absence of pIV and full induction in the presence of pIV (Fig. 4A), similar to ArcB* (Fig. 2C). Consistent with the pﬂ–lacZ ﬁndings, phosphorelay in the ArcBLeuZm (Fig. 4A, compare ArcBLeuZm with ArcBLHe2u9Z2mA and ArcBLHe7u1Z7mA) appears critically important for pIV-dependent induction of psp expression, but less so for pIV-independent elevation of © 2009 The Authors Journal compilation © 2009 Blackwell Publishing Ltd, Molecular Microbiology, 74, 16–28