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. 2007 Nov 20;104(47):18712-7.
doi: 10.1073/pnas.0705768104. Epub 2007 Nov 12.

B1500, a small membrane protein, connects the two-component systems EvgS/EvgA and PhoQ/PhoP in Escherichia coli

Yoko Eguchi  1 , Junji Itou, Masatake Yamane, Ryo Demizu, Fumiyuki Yamato, Ario Okada, Hirotada Mori, Akinori Kato, Ryutaro Utsumi
Affiliations

B1500, a small membrane protein, connects the two-component systems EvgS/EvgA and PhoQ/PhoP in Escherichia coli

Yoko Eguchi et al. Proc Natl Acad Sci U S A. .

Abstract

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 Escherichia 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 identified 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-independent. 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 transduction between EvgS/EvgA and PhoQ/PhoP systems by directly interacting with PhoQ, thus activating the PhoQ/PhoP system.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Characterization of pYE5 plasmid obtained from shotgun screening. (A) Transcriptional activity of mgtA in MG1601/pUC18, MG1601/pYE5, MG1601evgS1/pUC18, and MG1601evgS1/pYE5 strains in the presence and absence of 30 mM MgCl2. (B) Nucleic acid sequence of the insert portion of pYE5 corresponds to AE000247 (complementary, 1581559–1582293). ORFs (underlined) included in the insert were the C-terminal part of ydeP, the complete b1500, and the N-terminal part of ydeO. Corresponding amino acids are shown under the line, and the stop codons are shown as ***. The EvgA binding consensus sequence (18) is boxed, and the inverted repeat is indicated by the bold arrows.
Fig. 2.
Fig. 2.
Functional analysis of b1500. (A) Deletion of b1500 suppresses mgtA transcription. S1 nuclease assay of mgtA was performed against the following strains grown in the presence of 30 mM MgCl2: lanes 1, MG1655/pUC18; lane 2, MG1655 b1500/pUC18; lane 3, MG1655 evgS1/pUC18; lane 4, MG1655 evgS1 b1500/pUC18; lane 5, MG1655/pYE5; lane 6, MG1655 b1500/pYE5; lane 7, MG1655 evgS1/pYE5; and lane 8, MG1655 evgS1 b1500/pYE5. A+G, Maxam–Gilbert sequencing ladder. (B) Radioactivity intensity of each lane in A relative to the radioactivity of lane 1. (C) Overexpression of b1500 promotes mgtA transcription in a PhoQ/PhoP-dependent and EvgS/EvgA-independent manner. Column 1, MG1601/pQE80L; column 2, MG1601/pQE-b1500; column 3, MG1607/pQE80L; column 4, MG1607/pQE-b1500; column 5, MG1622/pQE80L; column 6, MG1622/pQE-b1500; column 7, MG1601 evgS/pQE80L; column 8, MG1601 evgS/pQE-b1500; column 9, MG1601 evgA/pQE80L; and column 10, MG1601 evgA/pQE-b1500. Cells were grown in the presence of 30 mM MgCl2. For induction of b1500, IPTG, at a final concentration of 1 mM, was added to the culture at OD600 = 0.4.
Fig. 3.
Fig. 3.
Transcriptional activation of the PhoP regulon by b1500. S1 nuclease assay of phoPQ, mgtA, mgrB and rstAB was performed against cells grown in the presence of 30 mM MgCl2, with and without the addition of 1 mM IPTG at OD600 = 0.4. Lanes 1 and 2 are MG1601/pQE80L and Lanes 3 and 4 are MG1601/pQE-b1500.
Fig. 4.
Fig. 4.
B1500 is localized in the inner membrane. (A) Western blot analysis against anti-His tag antibody for B1500 detection. MG1601/pQE-b1500 was grown in the presence of 30 mM MgCl2, and IPTG, at a final concentration of 1 mM, was added to the culture at OD600 = 0.4 for induction of B1500. Cells were lysed by sonication, followed by brief centrifugation and ultracentrifugation. (B) Sarcosyl treatment of the membrane fraction. Total membrane fraction after ultracentrifugation was treated with sarcosyl at different concentrations, followed by ultracentrifugation for the separation of inner and outer membrane. Anti-TolC antibody was used for the detection of TolC, an outer membrane protein control. (C) Predicted topology of B1500. Bold letters indicate the transmembrane domain predicted by the programs MEMSTAT and HMMTOP. Lysines and arginines are underlined.
Fig. 5.
Fig. 5.
A bacterial two-hybrid assay reveals interaction between B1500 and PhoQ. β-Galactosidase activity was determined for adenylate cyclase-deficient E. coli strain, DHP1, harboring plasmids expressing B. pertussis adenylate cyclase fragment T18 and T25 either unfused or fused to b1500, phoP, and phoQ. β-Galactosidase activity above background levels indicates an interaction between the coexpressed hybrid constructs. Column 1, pT25/pT18; column 2, pT25/pT18-PhoP; column 3, pT25/pT18-PhoQ; column 4, pT25/pT18c-B1500; column 5, pT25-B1500/pT18; column 6, pT25-B1500/pT18-PhoP; column 7, pT25-B1500/pT18-PhoQ; and column 8, pT25-B1500/pT18c-B1500.
Fig. 6.
Fig. 6.
A model of acid resistance controlled by EvgS/EvgA and PhoQ/PhoP TCSs connected by B1500.
See this image and copyright information in PMC

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