B1500, a small membrane protein, connects the two-component systems EvgS/EvgA and PhoQ/PhoP in Escherichia coli Yoko Eguchi*, Junji Itou*, Masatake Yamane*, Ryo Demizu*, Fumiyuki Yamato*, Ario Okada*, Hirotada Mori † , Akinori Kato*, and Ryutaro Utsumi* ‡ *Department of Bioscience, Graduate School of Agriculture, Kinki University, 3327-204, Nakamachi, Nara, 631-8505, Japan; and † Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, 630-0101, Japan Edited by Eduardo A. Groisman, Washington University, St. Louis, MO, and accepted by the Editorial Board October 3, 2007 (received for review June 20, 2007) Two-component signal-transduction systems (TCSs) of bacteria are considered to form an intricate signal network to cope with various environmental stresses. One example of such a network in Esch- erichia coli is the signal transduction cascade from the EvgS/EvgA system to the PhoQ/PhoP system, where activation of the EvgS/ EvgA system promotes expression of PhoP-activated genes. As a factor connecting this signal transduction cascade, we have iden- tified a small inner membrane protein (65 aa), B1500. Expression of the b1500 gene is directly regulated by the EvgS/EvgA system, and b1500 expression from a heterologous promoter simultaneously activated the expression of mgtA and other PhoP regulon genes. This activation was PhoQ/PhoP-dependent and EvgS/EvgA-inde- pendent. Furthermore, deletion of b1500 from an EvgS-activated strain suppressed mgtA expression. B1500 is localized in the inner membrane, and bacterial two-hybrid data showed that B1500 formed a complex with the sensor PhoQ. These results indicate that the small membrane protein, B1500, connected the signal trans- duction between EvgS/EvgA and PhoQ/PhoP systems by directly interacting with PhoQ, thus activating the PhoQ/PhoP system. two-component signal transduction T he two-component signal transduction system (TCS) is the major system in bacteria for sensing environmental changes and transducing the information inside the cells to regulate gene expression (1). TCSs control the expression of genes for nutrient acquisition, virulence, antibiotic resistance, and numerous other pathways in diverse bacteria. TCS is composed of a membrane- bound sensor histidine kinase (HK) that perceives environmental stimuli and a cognate cytoplasmic response regulator (RR). The sensor HK monitors the environmental stimuli by autophosphory- lating its conserved histidine residue, which in turn phosphorylates the conserved aspartate residue of its cognate response regulator. In most cases, RR binds to DNA regulatory sequences and affects transcription. In Escherichia coli, 29 HKs, 32 RRs, and one histidine containing phosphotransfer (HPt) domain have been found by analyses of the E. coli K-12 genome (2). Oshima et al. proposed the existence of a network of functional interactions, such as cross-talks (transfer of phosphoryl groups from a sensory HK to a noncognate RR) and cascade signal transduction among multiple TCSs (3). Several examples of in vivo (4, 5, 6) and in vitro (7, 8) cross-talks, as well as signal transduction cascade between TCSs in E. coli (9–11), have been reported. We reported signal transduction cascade between EvgS/EvgA and PhoQ/PhoP TCSs in E. coli. The EvgS/EvgA system confers acid resistance and multidrug resistance to E. coli, and it is very similar to the virulence-related BvgS/BvgA system in Bordetella pertussis (12, 13, 14), whereas the PhoQ/PhoP system responds to external Mg 2+ and Ca 2+ levels, regulating expression of the genes including Mg 2+ transporters and LPS modification genes (15, 16). Cultivation of E. coli in neutral rich media results in activation of the PhoQ/PhoP system but not the EvgS/EvgA system. An addition of high concentration of Mg 2+ turns off the PhoQ/PhoP system, presumably by activating the phosphatase activity of the sensor PhoQ (17). However, in the evgS1 mutant strain, which constitu- tively activates the EvgS/EvgA system, the PhoQ/PhoP system remains active even at high Mg 2+ levels. This signal transduction between the two TCSs can also occur as a result of overproduction of the EvgA regulator, which rules out phosphotransfer between the activated sensor EvgS and the noncognate regulator PhoP. Moreover, enhanced transcription of the phoPQ genes did not further activate expression of the PhoQ/PhoP regulated genes, and no EvgA binding consensus sequence (12, 18) was found in the promoter regions of the PhoP regulon genes. Thus, activation of the EvgS/EvgA system may increase or at least maintain the level of phospho-PhoP by (i) inhibiting the phosphatase activity of PhoQ, (ii) activating the PhoQ kinase, or (iii) protecting the phospho-PhoP against dephosphorylation by PhoQ. It has been reported that some small (200-aa) proteins regulate TCSs to adapt cells for rapid environmental changes. In Salmonella enterica, PmrD, a cytoplasmic protein composed of 85 aa, connects the PhoQ/PhoP and PmrB/PmrA TCSs (19). PmrD binds to the phosphorylated form of the response regulator PmrA at the N- terminal domain, thus preventing both its intrinsic dephosphory- lation and that promoted by its cognate sensor kinase PmrB (20). Another small protein in Salmonella and in E. coli is IraP (86 aa), which enhances RpoS stability by interacting with MviA (Salmo- nella, 21) or RssB (E. coli, 22). Moreover, CpxP, a periplasmic protein (166 aa) of E. coli, interacts with the sensor domain of a histidine kinase CpxA to inhibit the CpxA/CpxR pathway (23). In the present study, we have identified a regulatory protein, B1500, whose expression was enhanced by the EvgS/EvgA system; moreover, this protein activated the PhoQ/PhoP system, thus connecting the two TCSs. B1500, composed of 65 aa, is localized in the inner membrane and forms a complex with the sensor PhoQ. Therefore, B1500 directly interacts with PhoQ to activate the PhoQ/PhoP system. To our knowledge, this is the first report of such a small membrane protein connecting two TCSs. Results Cloning and Sequencing of a Gene Involved in Signal Transduction Between EvgS/EvgA and PhoQ/PhoP Systems. We set up a screening system to identify any additional factor(s) involved in signal trans- Author contributions: Y.E. and J.I. contributed equally to this work; R.U. designed research; Y.E., J.I., M.Y., R.D., F.Y., A.O., H.M., and A.K. performed research; and Y.E. and R.U. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. E.A.G. is a guest editor invited by the Editorial Board. ‡ To whom correspondence should be addressed. E-mail: utsumi@nara.kindai.ac.jp. This article contains supporting information online at www.pnas.org/cgi/content/full/ 0705768104/DC1. © 2007 by The National Academy of Sciences of the USA 18712–18717 | PNAS | November 20, 2007 | vol. 104 | no. 47 www.pnas.orgcgidoi10.1073pnas.0705768104