either (i) that PspA (and/or PspF) can directly receive the inducing signal and relieve the PspA-imposed negative regulation or (ii) that other factors substitute for PspBC and ArcAB. That a minimal PspA–PspF system is capable of responding to extracytoplasmic stimulus is consistent with a regulated PspA stress response in Gram-positive bacteria and archebacteria where PspBC are absent [(reviewed by Darwin, 2005); Bidle et al., 2008; Vrancken et al., 2008]. PspA homologues are found among Gram- positive bacteria, cyanobacteria, archea and higher plants [(reviewed by Darwin, 2005); Bidle et al., 2008; Vrancken et al., 2008]. In all these organisms, PspA homologues often respond to stress conditions similar to those described for inducing the Psp response (e.g. via extreme heat shock, ethanol treatment, hyperosmotic shock, CCCP) in a partially PspBC-dependent or -independent manner in enterobacteria. Experimental procedures Bacterial strains and growth conditions The bacterial strains used in this study are listed in Table S1. Strains were constructed by transduction using the P1vir bac- teriophage (Miller, 1992) (see Table S1) and were routinely grown in Luria–Bertani (LB) broth or on LB agar plates at 37°C (Miller, 1992). For BACTH assays, strains were grown in LB at 30°C. For in vivo cross-linking experiments, strains were grown in minimal medium [50 mM MOPS (pH 7), 2 mM MgSO4, 0.5% Glucose (w/v), 10 mM NH4Cl, 0.75 mM Na2SO4, 1.2 mM NH4NO3, 0.5 mM KH2PO4] at 30°C. For aerobic growth, overnight cultures of cells were diluted 100- fold into 5 ml of LB in a universal tube with loose ﬁtting caps and shaken at 200 r.p.m. For microaerobic growth, overnight cultures of cells were diluted 100-fold into 5 ml of LB and shaken at 100 r.p.m. For an anaerobic growth, overnight cultures grown at 37°C in a universal tube with tightly closed caps without shaking were transferred (100-fold dilution) into a fully LB-ﬁlled suba-sealed universal tube (to avoid any air space) and incubated overnight at 37°C without shaking. The cells were then taken by syringe for b-galactosidase (b-Gal) assays. The pIV secretin was constitutively expressed from pGJ4. For induction of the pBAD ara promoter, 0.001% ﬁnal arabinose (Ara) was added for 1 h. For induction of the lac promoter, 0.1 mM isopropyl-b-D-galactopyranoside (IPTG) was added for 1 h. For scoring the lacZ + colonies, indicator plates containing 40 ml of 20 mg ml-1 stock solution of 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside (X-gal) and 0.5 mM IPTG were used. Antibiotics were routinely used at the following concentrations: ampicillin (Amp; 100 mg ml-1), kanamycin (Kan; 25 mg ml-1), chloramphenicol (Cam; 30 mg ml-1) and tetracycline (Tet; 10 mg ml-1). Construction of the DarcB59 mutant To eliminate the Kan cassette from a DarcB::Kan mutant (strain MVA59) and construct the marker-less arcB59 variant (strain MVA92), we used plasmid pCP20 and the method Relationships of Arc and Psp systems 25 described by Cherepanov and Wackernagel (1995). The resulting strain MVA92 had lost the FRT-ﬂanked Kan resis- tance gene and the FLP helper plasmid (pCP20) was used to construct the strains MVA93 and MVA94 (see Table S1). The PCR and ParcB/elbB pair of primers were used to verify that arcB59 mutant in MVA92–94 strains had the correct structure (see Fig. S2D). PCR reactions were carried out either with isolated chromosomal DNA (Qiagen, DNeasy) or with a freshly grown colony suspended in 15 ml of water. Measurement of oxygen consumption in bacterial cultures Oxygen content in bacterial cultures was measured using a non-invasive OxySense 4000B system (Air Monitors). Bacte- rial cultures grown either in aerobiosis, microaerobiosis or anaerobiosis (see below) were prepared as described above, except that the tubes for dilution of overnight cultures con- tained an O2xyDot sensor (OxySense, Dallas, TX, USA) to detect O2 concentrations. The output ﬂuorescence was mea- sured by an OxySense Reader Pen (through the growth tube) and analysed using the OxySense 4000B system expressed either as a percentage or p.p.b. (1000 p.p.b. = 1 mg of O2 l-1). For oxygen consumption experiments, autocalibrated O2xyDot sensors were used and control measurements of ambient air (20.446 1.005%) and the LB medium under either aerobic (8.845 0.965%), microaerobic (4.901 0.181%; air above the medium 17.147 0.624%) or anaero- bic conditions (0.075 0.010%) were taken. An optimal signal-to-noise ratio was achieved by measuring in reduced ambient light conditions. Mean values of the oxygen con- sumption were calculated from 10 measurements with 100 signals captured per sample taken from technical duplicates of three independently grown cultures of each strain. DNA manipulations Plasmids used in this study are shown in Table S1. Using PCR-based site-speciﬁc mutagenesis (Quickchange mutagenesis kit, Stratagene) of the plasmid templates pJW5536(-) (ArcB), pJW4364(-) (ArcA) and pAJM1, we con- structed plasmids pGJ21-23/27/29-31/33, pGJ45 and pGJ48 respectively (see Table S1). The positions of substituted resi- dues in ArcB and ArcA and the mutant proteins activities are shown in Fig. 2A and Table 1. Plasmid pGJ46 that carries the f(pﬂ–lacZ) transcription fusion was constructed as shown in Table S1. A DNA fragment carrying the pﬂ promoter region was chosen according to Drapal and Sawers (1995). All con- structs were veriﬁed by DNA sequencing. For the BACTH system experiments, the ArcB, ArcB* or ArcBLeuZm proteins were fused to either T25 (plasmid pKT25), T18C (pUT18C) or T18 (pUT18) Cya domains. The corre- sponding genes were ampliﬁed from plasmids pJW5536(-), pGJ23 and pGJ22 using primers that introduce either XbaI– KpnI, HindIII–KpnI or XbaI–EcoRI restriction sites, cloned in pGEM-T Easy (Promega), and then subcloned in frame into multiple cloning sites (MCS) of pKT25, pUT18 and pUT18C respectively. The genes for PspA, PspB, PspC and PspF were ampliﬁed by PCR using primers that introduce XbaI– KpnI restriction sites, cloned in pGEM-T Easy and then sub- © 2009 The Authors Journal compilation © 2009 Blackwell Publishing Ltd, Molecular Microbiology, 74, 16–28