Molecular Microbiology (2009) 74(1), 16–28 doi:10.1111/j.1365-2958.2009.06809.x First published online 4 August 2009 Physical, functional and conditional interactions between ArcAB and phage shock proteins upon secretin-induced stress in Escherichia coli mi_680916.28 Goran Jovanovic,† Christoph Engl† and Martin Buck* Division of Biology, Sir Alexander Fleming Building, Imperial College London, South Kensington Campus, London SW7 2AZ, UK. Summary The phage shock protein (Psp) system found in enterobacteria is induced in response to impaired inner membrane integrity (where the Psp response is thought to help maintain the proton motive force of the cell) and is implicated in the virulence of patho- gens such as Yersinia and Salmonella. We provided evidence that the two-component ArcAB system was involved in induction of the Psp response in Escheri- chia coli and now report that role of ArcAB is conditional. ArcAB, predominantly through the action of ArcA regulated genes, but also via a direct ArcB– Psp interaction, is required to propagate the protein IV (pIV)-dependent psp-inducing signal(s) during microaerobiosis, but not during aerobiosis or anaerobiosis. We show that ArcB directly interacts with the PspB, possibly by means of the PspB leucine zipper motif, thereby allowing cross-communication between the two systems. In addition we demonstrate that the pIV-dependent induction of psp expression in anaerobiosis is independent of PspBC, establishing that PspA and PspF can function as a minimal Psp system responsive to inner membrane stress. Introduction Bacteria in their native habitats encounter a plethora of stresses, which challenge their cellular integrity. Robust- ness of the cytoplasmic membrane is required for it to function as a barrier between the inside of the cell and the environment. Bacteria have therefore evolved a number Accepted 14 July, 2009. *For correspondence. E-mail m.buck@ imperial.ac.uk; Tel. (+44) 207 594 5442; Fax (+44) 207 594 5419. †These authors contributed equally to this work. Re-use of this article is permitted in accordance with the Terms and Conditions set out at http://www3.interscience.wiley.com/ authorresources/onlineopen.html © 2009 The Authors Journal compilation © 2009 Blackwell Publishing Ltd of systems to maintain membrane integrity, one of which is the phage shock protein (Psp) response, highly con- served in Gram-negative bacteria. In Escherichia coli, Psp genes are organized in a regulon (pspF pspABCDE and pspG) that is under the control of a sigma54-dependent promoter activated by PspF, positively controlled by PspBC and negatively regulated by PspA (reviewed by Model et al., 1997; Darwin, 2005; Wigneshweraraj et al., 2008). Psp expression is induced upon inner membrane (IM) stress which dissipates the proton motive force (pmf) (potentially resulting in a reduced energy status of the cell). Psp effector proteins (e.g. PspA – effector upon stress, PspD and PspG) subsequently help to conserve pmf (Kleerebezem et al., 1996; Jovanovic et al., 2006; Kobayashi et al., 2007). Understanding the mechanism of Psp induction and its biological function is important since the Psp system is signiﬁcant in protein translocation and in the growth and virulence of pathogenic enterobacteria (reviewed by Darwin, 2005; 2007; Rowley et al., 2006). The PspA homologue Vipp1 has been shown to be essen- tial in plants and Synechocystis for thylakoid biogenesis and hence photosynthesis (Westphal et al., 2001). Further, PspA homologues have been reported in Gram- positive bacteria and archea (Bidle et al., 2008; Vrancken et al., 2008). PspA therefore appears to have a funda- mental role in the three domains of life. The nature of the stress signal and how it is detected by the Psp system remains unknown (reviewed by Darwin, 2005). Previously we observed that ArcB (the sensor kinase of the two-component ArcAB system) is required for full protein IV (pIV)-dependent psp expression in E. coli and proposed that psp-inducing stresses that dissi- pate pmf may activate ArcB (Jovanovic et al., 2006). The ArcAB system regulates the transition from aerobic to anaerobic respiration and fermentation (reviewed by Malpica et al., 2006) where ArcB has been shown to sense the redox state of the cell through changes in the ubiquinone : ubiquinol (UQ : UQH2) ratio (see Fig. 2A and B) (Georgellis et al., 2001; Malpica et al., 2004). Increased levels of UQH2 attenuate inhibition on ArcB kinase activity imposed by UQ, thus enabling ArcB to phosphorylate ArcA (the response regulator). Phosphory- lated ArcA (ArcA-P) then represses genes involved in