26 G. Jovanovic, C. Engl and M. Buck cloned in frame into MCS of pKT25, pUT18 and pUT18C. The PspBLeuZm mutant was ampliﬁed from plasmid pGJ48 using primers that introduce HindIII–KpnI restriction sites, cloned in pGEM-T Easy and then subcloned in frame into MCS of pUT18. All constructs were veriﬁed by DNA sequencing. The production of fusion proteins, following induction by 0.5 mM IPTG at 30°C, was veriﬁed using Western blots (see below) with antibodies corresponding to either the fused proteins or Cya T25 (for pKT25 constructs). Transformation of bacteria was performed as described by Miller (1992). In vivo BACTH The Cya-based BACTH allows detection of protein–protein interactions in vivo and is particularly appropriate for studying interactions among membrane proteins (Karimova et al., 1998; 2005). Proteins of interest are fused to the T18 and T25 fragments (which do not function when physically separated) of the catalytic domain of Cya, and co-transformed into the host strain BTH101 (cya -). Upon interactions of fused pro- teins, cAMP synthesis is restored leading to transcriptional activation of CAP/cAMP-dependent genes, such as the lac operon on the chromosome of the host reporter strain. The topologies of ArcB (Fig. 2A) (reviewed by Malpica et al., 2006), PspA, PspB and PspC (Fig. S3A) (reviewed by Darwin, 2005) were taken into account for construction of the fusion proteins and analysis of results (e.g. a Cya domain fused to the PspB region embedded in IM could interfere with localization of PspB). As a negative control we used pKT25 and pUT18C vectors in the absence of fusion proteins, as a positive control we used pKT25-zip and pUT18C-zip plas- mids carrying fused GCN4 leucine-zipper sequence (Kari- mova et al., 1998). After co-transformation of the BTH101 strain with the two plasmids expressing the fusion proteins, selection plates (Kan, Amp, X-gal and IPTG) were incubated at room temperature for 72 h. The levels of the interactions were quantiﬁed by b-Gal activity in liquid cultures (see below). LacZ expression threefold above the negative control value was considered a positive interaction signal. For this measurement bacteria were grown in LB medium containing 100 mg ml-1 Amp and 50 mg ml-1 Kan at 30°C for 16 h, then cultures were diluted 1:25 and grown until the OD600 reached ~0.3, then 0.5 mM IPTG was added and the cells incubated for a further 1 h at 30°C. b-Gal assays Activity from a single-copy chromosomal f(pspA–lacZ) tran- scriptional fusion was assayed to gauge the level of psp expression. The f(pspA–lacZ) transcriptional reporter fusion was introduced as a single copy into the chromosomal att site to retain the native pspA locus and permit the WT psp response. Activity from the plasmid-derived f(pﬂ–lacZ) tran- scriptional fusion (pGJ46) was assayed to gauge activity of ArcA-P. Cells were grown overnight at 37°C in LB broth containing the appropriate antibiotic and then diluted 100-fold (initial OD600 ~ 0.025) into the same medium (5 ml). Following incubation to OD600 ~ 0.2–0.3, cultures were induced with Ara or IPTG for 1 h (where appropriate), and then assayed for b-Gal activity as described by Miller (1992). For anaerobically grown cells, b-Gal activity was measured following overnight growth at 37°C with no shaking. For all b-Gal assays, mean values of six independent assays taken from technical dupli- cates of three independently grown cultures of each strain were used to calculate activity. The data are shown as a mean values with SD error bars. Bacterial cell fractionation The bacterial cultures (5 ml) were separated into soluble and membrane fractions by a lysozyme-EDTA-osmotic shock pro- tocol and IM and OM were selectively extracted with Triton X-100 (Russel and Kaz´mierczak, 1993). All the samples were air-dried before adding 30 ml of 4% SDS and 30 ml of Laemlli buffer (Sigma). Samples were analysed by Western blotting (see below). Western blot analysis For the Western blot analysis, bacterial cells were collected (1 ml) at OD600 ~ 0.5. Proteins were separated on either 10%, 12.5% or 15% (SDS)-PAGE and transferred onto PVDF membranes using a semi-dry transblot system (Bio-Rad). Western blotting was performed as described (Elderkin et al., 2002) using antibodies to PspF (1:4000 with anti-mouse; Ammersham), PspA (1:10 000 with anti-mouse) (Jones et al., 2003), PspB (a gift from P. Model) (1:5000 with anti-rabbit; Ammersham), PspC (a gift from P. Model) (1:5000 with anti- rabbit), pIV (a gift from M. Russel) (1:10 000 with anti-rabbit), ArcB (a gift from D. Georgellis) (1:10 000 with anti-rabbit) and Cya T25 (a gift from D. Ladant) (1:5000 with anti-rabbit). The proteins were detected using ECL plus Western Blotting Detection Kit according to manufacturer’s guidelines (GE Healthcare). Images were captured in a FujiFilm – intelligent Dark Box by an image analyser with a charge-coupled device camera (LAS-3000). Densitometry analysis was performed with MultiGauge 3.0 software (FujiFilm USA, Valhalla, NY, USA) and quantiﬁcation (results expressed in arbitrary units) performed using the AIDA software. In vivo cross-linking In vivo cross-linking was performed as described (Adams et al., 2003) using the thiol-reactive cross-linking reagent dithiobis (succinimydylpropionate) DSP (PIERCE). Cells were grown in minimal medium at 30°C to an OD600 ~ 1.0, harvested and washed in 0.9% NaCl. Proteins were exposed to the cross-linker [100 mM DSP in 125 mM HEPES (pH 7.3)] for 30 min at 25°C. To quench the reaction, 50 ml of 1 M Tris-HCl (pH 8.0) was added to the reaction mix and the samples then transferred to ice for 5 min. Proteins were separated on a 7.5% SDS-PAGE (run at 200 V for 50 min) and the cross-linked species detected using antibodies spe- ciﬁc to PspB (a-PspB) as described above. Confocal ﬂuorescence microscopy to assess membrane electron potential (Dy) The Dy component of pmf was measured as described by Becker et al. (2005), with the following modiﬁcations. Cells © 2009 The Authors Journal compilation © 2009 Blackwell Publishing Ltd, Molecular Microbiology, 74, 16–28