Two-component signal transduction involves phosphoryl transfer between a histidine kinase sensor and a response regulator effector. The nitrate-responsive two-component signal transduction systems in represent a paradigm for a cross-regulation network, in which the paralogous sensor–response regulator pairs, NarX–NarL and NarQ–NarP, exhibit both cognate (e.g. NarX–NarL) and non-cognate (e.g. NarQ–NarL) interactions to control output. Here, we describe results from bacterial adenylate cyclase two-hybrid (BACTH) analysis to examine sensor dimerization as well as interaction between sensor–response regulator cognate and non-cognate pairs. Although results from BACTH analysis indicated that the NarX and NarQ sensors interact with each other, results from intragenic complementation tests demonstrate that they do not form functional heterodimers. Additionally, intragenic complementation shows that both NarX and NarQ undergo intermolecular autophosphorylation, deviating from the previously reported correlation between DHp (imerization and istidyl hosphotransfer) domain loop handedness and autophosphorylation mode. Results from BACTH analysis revealed robust interactions for the NarX–NarL, NarQ–NarL and NarQ–NarP pairs but a much weaker interaction for the NarX–NarP pair. This demonstrates that asymmetrical cross-regulation results from differential binding affinities between different sensor–regulator pairs. Finally, results indicate that the NarL effector (DNA-binding) domain inhibits NarX–NarL interaction. Missense substitutions at receiver domain residue Ser-80 enhanced NarX–NarL interaction, apparently by destabilizing the NarL receiver–effector domain interface.


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  1. Albanesi D., Martín M., Trajtenberg F., Mansilla M.C., Haouz A., Alzari P.M., de Mendoza D., Buschiazzo A. (2009). Structural plasticity and catalysis regulation of a thermosensor histidine kinaseProc Natl Acad Sci U S A 1061618516190 [View Article][PubMed]. [Google Scholar]
  2. Ashenberg O., Keating A.E., Laub M.T. (2013). Helix bundle loops determine whether histidine kinases autophosphorylate in cis or in trans J Mol Biol 42511981209 [View Article][PubMed]. [Google Scholar]
  3. Baikalov I., Schröder I., Kaczor-Grzeskowiak M., Grzeskowiak K., Gunsalus R.P., Dickerson R.E. (1996). Structure of the Escherichia coli response regulator NarLBiochemistry 351105311061 [View Article][PubMed]. [Google Scholar]
  4. Barbieri C.M., Mack T.R., Robinson V.L., Miller M.T., Stock A.M. (2010). Regulation of response regulator autophosphorylation through interdomain contactsJ Biol Chem 2853232532335 [View Article][PubMed]. [Google Scholar]
  5. Bartolomé B., Jubete Y., Martínez E., de la Cruz F. (1991). Construction and properties of a family of pACYC184-derived cloning vectors compatible with pBR322 and its derivativesGene 1027578 [View Article][PubMed]. [Google Scholar]
  6. Bordoli L., Kiefer F., Arnold K., Benkert P., Battey J., Schwede T. (2009). Protein structure homology modeling using swiss-model workspaceNat Protoc 4113 [View Article][PubMed]. [Google Scholar]
  7. Brickman E., Soll L., Beckwith J. (1973). Genetic characterization of mutations which affect catabolite-sensitive operons in Escherichia coli, including deletions of the gene for adenyl cyclaseJ Bacteriol 116582587[PubMed]. [Google Scholar]
  8. Cai S.J., Inouye M. (2003). Spontaneous subunit exchange and biochemical evidence for trans-autophosphorylation in a dimer of Escherichia coli histidine kinase (EnvZ)J Mol Biol 329495503 [View Article][PubMed]. [Google Scholar]
  9. Casino P., Rubio V., Marina A. (2009). Structural insight into partner specificity and phosphoryl transfer in two-component signal transductionCell 139325336 [View Article][PubMed]. [Google Scholar]
  10. Chang A.C.Y., Cohen S.N. (1978). Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmidJ Bacteriol 13411411156[PubMed]. [Google Scholar]
  11. Cheung J., Hendrickson W.A. (2009). Structural analysis of ligand stimulation of the histidine kinase NarXStructure 17190201 [View Article][PubMed]. [Google Scholar]
  12. Da Re S., Schumacher J., Rousseau P., Fourment J., Ebel C., Kahn D. (1999). Phosphorylation-induced dimerization of the FixJ receiver domainMol Microbiol 34504511 [View Article][PubMed]. [Google Scholar]
  13. Darwin A.J., Tyson K.L., Busby S.J., Stewart V. (1997). Differential regulation by the homologous response regulators NarL and NarP of Escherichia coli K-12 depends on DNA binding site arrangementMol Microbiol 25583595 [View Article][PubMed]. [Google Scholar]
  14. Egan S.M., Stewart V. (1991). Mutational analysis of nitrate regulatory gene narL in Escherichia coli K-12J Bacteriol 17344244432[PubMed]. [Google Scholar]
  15. Eldridge A.M., Kang H.S., Johnson E., Gunsalus R., Dahlquist F.W. (2002). Effect of phosphorylation on the interdomain interaction of the response regulator, NarLBiochemistry 411517315180 [View Article][PubMed]. [Google Scholar]
  16. Falord M., Karimova G., Hiron A., Msadek T. (2012). GraXSR proteins interact with the VraFG ABC transporter to form a five-component system required for cationic antimicrobial peptide sensing and resistance in Staphylococcus aureus Antimicrob Agents Chemother 5610471058 [View Article][PubMed]. [Google Scholar]
  17. Gao R., Stock A.M. (2009). Biological insights from structures of two-component proteinsAnnu Rev Microbiol 63133154 [View Article][PubMed]. [Google Scholar]
  18. Goodman A.L., Merighi M., Hyodo M., Ventre I., Filloux A., Lory S. (2009). Direct interaction between sensor kinase proteins mediates acute and chronic disease phenotypes in a bacterial pathogenGenes Dev 23249259 [View Article][PubMed]. [Google Scholar]
  19. Grebe T.W., Stock J.B. (1999). The histidine protein kinase superfamilyAdv Microb Physiol 41139227 [View Article][PubMed]. [Google Scholar]
  20. Gueguen E., Flores-Kim J., Darwin A.J. (2011). The Yersinia enterocolitica phage shock proteins B and C can form homodimers and heterodimers in vivo with the possibility of close association between multiple domainsJ Bacteriol 19357475758 [View Article][PubMed]. [Google Scholar]
  21. Guo Q., Shen Y., Lee Y.-S., Gibbs C.S., Mrksich M., Tang W.-J. (2005). Structural basis for the interaction of Bordetella pertussis adenylyl cyclase toxin with calmodulinEMBO J 2431903201 [View Article][PubMed]. [Google Scholar]
  22. Hazelbauer G.L., Falke J.J., Parkinson J.S. (2008). Bacterial chemoreceptors: high-performance signaling in networked arraysTrends Biochem Sci 33919 [View Article][PubMed]. [Google Scholar]
  23. Heikaus C.C., Pandit J., Klevit R.E. (2009). Cyclic nucleotide binding GAF domains from phosphodiesterases: structural and mechanistic insightsStructure 1715511557 [View Article][PubMed]. [Google Scholar]
  24. Huynh T.N., Stewart V. (2011). Negative control in two-component signal transduction by transmitter phosphatase activityMol Microbiol 82275286 [View Article][PubMed]. [Google Scholar]
  25. Huynh T.N., Noriega C.E., Stewart V. (2010). Conserved mechanism for sensor phosphatase control of two-component signaling revealed in the nitrate sensor NarXProc Natl Acad Sci U S A 1072114021145 [View Article][PubMed]. [Google Scholar]
  26. Huynh T.N., Noriega C.E., Stewart V. (2013). Missense substitutions reflecting regulatory control of transmitter phosphatase activity in two-component signallingMol Microbiol 88459472 [View Article][PubMed]. [Google Scholar]
  27. Ishihama A., Kori A., Koshio E., Yamada K., Maeda H., Shimada T., Makinoshima H., Iwata A., Fujita N. (2014). Intracellular concentrations of 65 species of transcription factors with known regulatory functions in Escherichia coli J Bacteriol 19627182727 [View Article][PubMed]. [Google Scholar]
  28. Karimova G., Pidoux J., Ullmann A., Ladant D. (1998). A bacterial two-hybrid system based on a reconstituted signal transduction pathwayProc Natl Acad Sci U S A 9557525756 [View Article][PubMed]. [Google Scholar]
  29. Karimova G., Dautin N., Ladant D. (2005). Interaction network among Escherichia coli membrane proteins involved in cell division as revealed by bacterial two-hybrid analysisJ Bacteriol 18722332243 [View Article][PubMed]. [Google Scholar]
  30. Laub M.T., Goulian M. (2007). Specificity in two-component signal transduction pathwaysAnnu Rev Genet 41121145 [View Article][PubMed]. [Google Scholar]
  31. Leonard P.G., Golemi-Kotra D., Stock A.M. (2013). Phosphorylation-dependent conformational changes and domain rearrangements in Staphylococcus aureus VraR activationProc Natl Acad Sci U S A 11085258530 [View Article][PubMed]. [Google Scholar]
  32. Lin A.V., Stewart V. (2010). Functional roles for the GerE-family carboxyl-terminal domains of nitrate response regulators NarL and NarP of Escherichia coli K-12Microbiology 15629332943 [View Article][PubMed]. [Google Scholar]
  33. Lin-Chao S., Chen W.-T., Wong T.-T. (1992). High copy number of the pUC plasmid results from a Rom/Rop-suppressible point mutation in RNA IIMol Microbiol 633853393 [View Article][PubMed]. [Google Scholar]
  34. Maris A.E., Sawaya M.R., Kaczor-Grzeskowiak M., Jarvis M.R., Bearson S.M., Kopka M.L., Schröder I., Gunsalus R.P., Dickerson R.E. (2002). Dimerization allows DNA target site recognition by the NarL response regulatorNat Struct Biol 9771778 [View Article][PubMed]. [Google Scholar]
  35. McCallum N., Meier P.S., Heusser R., Berger-Bächi B. (2011). Mutational analyses of open reading frames within the vraSR operon and their roles in the cell wall stress response of Staphylococcus aureus Antimicrob Agents Chemother 5513911402 [View Article][PubMed]. [Google Scholar]
  36. Miller J.H. (1972). Experiments in Molecular Genetics., Cold Spring Harbor, NYCold Spring Harbor Laboratory. [Google Scholar]
  37. Ninfa E.G., Atkinson M.R., Kamberov E.S., Ninfa A.J. (1993). Mechanism of autophosphorylation of Escherichia coli nitrogen regulator II (NRII or NtrB): trans-phosphorylation between subunitsJ Bacteriol 17570247032[PubMed]. [Google Scholar]
  38. Noriega C.E., Lin H.-Y., Chen L.-L., Williams S.B., Stewart V. (2010). Asymmetric cross-regulation between the nitrate-responsive NarX-NarL and NarQ-NarP two-component regulatory systems from Escherichia coli K-12Mol Microbiol 75394412 [View Article][PubMed]. [Google Scholar]
  39. Parkinson J.S. (2010). Signaling mechanisms of HAMP domains in chemoreceptors and sensor kinasesAnnu Rev Microbiol 64101122 [View Article][PubMed]. [Google Scholar]
  40. Peña-Sandoval G.R., Georgellis D. (2010). The ArcB sensor kinase of Escherichia coli autophosphorylates by an intramolecular reactionJ Bacteriol 19217351739 [View Article][PubMed]. [Google Scholar]
  41. Podgornaia A.I., Laub M.T. (2013). Determinants of specificity in two-component signal transductionCurr Opin Microbiol 16156162 [View Article][PubMed]. [Google Scholar]
  42. Punta M., Coggill P.C., Eberhardt R.Y., Mistry J., Tate J., Boursnell C., Pang N., Forslund K., Ceric G., other authors. (2012). The Pfam protein families databaseNucleic Acids Res 40(D1), D290D301 [View Article][PubMed]. [Google Scholar]
  43. Rabin R.S., Stewart V. (1993). Dual response regulators (NarL and NarP) interact with dual sensors (NarX and NarQ) to control nitrate- and nitrite-regulated gene expression in Escherichia coli K-12J Bacteriol 17532593268[PubMed]. [Google Scholar]
  44. Scheu P., Sdorra S., Liao Y.F., Wegner M., Basché T., Unden G., Erker W. (2008). Polar accumulation of the metabolic sensory histidine kinases DcuS and CitA in Escherichia coli Microbiology 15424632472 [View Article][PubMed]. [Google Scholar]
  45. Scheu P.D., Witan J., Rauschmeier M., Graf S., Liao Y.F., Ebert-Jung A., Basché T., Erker W., Unden G. (2012). CitA/CitB two-component system regulating citrate fermentation in Escherichia coli and its relation to the DcuS/DcuR system in vivo J Bacteriol 194636645 [View Article][PubMed]. [Google Scholar]
  46. Sivanesan D., Hancock M.A., Villamil Giraldo A.M., Baron C. (2010). Quantitative analysis of VirB8-VirB9-VirB10 interactions provides a dynamic model of type IV secretion system core complex assemblyBiochemistry 4944834493 [View Article][PubMed]. [Google Scholar]
  47. Stewart V. (2003). Biochemical Society Special Lecture. Nitrate- and nitrite-responsive sensors NarX and NarQ of proteobacteriaBiochem Soc Trans 31110 [View Article][PubMed]. [Google Scholar]
  48. Stewart V., Chen L.L. (2010). The shelix mediates signal transmission as a HAMP domain coiled-coil extension in the NarX nitrate sensor from Escherichia coli K-12J Bacteriol 192734745 [View Article][PubMed]. [Google Scholar]
  49. Stewart V., Parales J. Jr (1988). Identification and expression of genes narL narX of the nar (nitrate reductase) locus in Escherichia coli K-12J Bacteriol 17015891597[PubMed]. [Google Scholar]
  50. Stewart V., Rabin R.S. (1995). Dual sensors and dual response regulators interact to control nitrate- and nitrite-responsive gene expression in Escherichia coli . In Two-component Signal Transduction, pp. 233252. Edited by Hoch J. A., Silhavy T. J. Washington, DCAmerican Society for Microbiology.[CrossRef] [Google Scholar]
  51. Stewart G.S., Lubinsky-Mink S., Jackson C.G., Cassel A., Kuhn J. (1986). pHG165: a pBR322 copy number derivative of pUC8 for cloning and expressionPlasmid 15172181 [View Article][PubMed]. [Google Scholar]
  52. Stewart V., Chen L.-L., Wu H.C. (2003). Response to culture aeration mediated by the nitrate and nitrite sensor NarQ of Escherichia coli K-12Mol Microbiol 5013911399 [View Article][PubMed]. [Google Scholar]
  53. Stewart V., Bledsoe P.J., Chen L.L., Cai A. (2009). Catabolite repression control of napF (periplasmic nitrate reductase) operon expression in Escherichia coli K-12J Bacteriol 1919961005 [View Article][PubMed]. [Google Scholar]
  54. Stynen B., Tournu H., Tavernier J., Van Dijck P. (2012). Diversity in genetic in vivo methods for protein-protein interaction studies: from the yeast two-hybrid system to the mammalian split-luciferase systemMicrobiol Mol Biol Rev 76331382 [View Article][PubMed]. [Google Scholar]
  55. Swanson R.V., Bourret R.B., Simon M.I. (1993). Intermolecular complementation of the kinase activity of CheAMol Microbiol 8435441 [View Article][PubMed]. [Google Scholar]
  56. Trajtenberg F., Graña M., Ruétalo N., Botti H., Buschiazzo A. (2010). Structural and enzymatic insights into the ATP binding and autophosphorylation mechanism of a sensor histidine kinaseJ Biol Chem 2852489224903 [View Article][PubMed]. [Google Scholar]
  57. Wanner B.L., Kodaira R., Neidhardt F.C. (1978). Regulation of lac operon expression: reappraisal of the theory of catabolite repressionJ Bacteriol 136947954[PubMed]. [Google Scholar]
  58. Wayne K.J., Sham L.T., Tsui H.C., Gutu A.D., Barendt S.M., Keen S.K., Winkler M.E. (2010). Localization and cellular amounts of the WalRKJ (VicRKX) two-component regulatory system proteins in serotype 2 Streptococcus pneumoniae J Bacteriol 19243884394 [View Article][PubMed]. [Google Scholar]
  59. Williams S.B., Stewart V. (1997a). Nitrate- and nitrite-sensing protein NarX of Escherichia coli K-12: mutational analysis of the amino-terminal tail and first transmembrane segmentJ Bacteriol 179721729[PubMed]. [Google Scholar]
  60. Williams S.B., Stewart V. (1997b). Discrimination between structurally related ligands nitrate and nitrite controls autokinase activity of the NarX transmembrane signal transducer of Escherichia coli K-12Mol Microbiol 26911925 [View Article][PubMed]. [Google Scholar]
  61. Yang Y., Inouye M. (1991). Intermolecular complementation between two defective mutant signal-transducing receptors of Escherichia coli Proc Natl Acad Sci U S A 881105711061 [View Article][PubMed]. [Google Scholar]
  62. Yanofsky C., Horn V., Bonner M., Stasiowski S. (1971). Polarity and enzyme functions in mutants of the first three genes of the tryptophan operon of Escherichia coli Genetics 69409433[PubMed]. [Google Scholar]
  63. You C., Okano H., Hui S., Zhang Z., Kim M., Gunderson C.W., Wang Y.P., Lenz P., Yan D., Hwa T. (2013). Coordination of bacterial proteome with metabolism by cyclic AMP signallingNature 500301306 [View Article][PubMed]. [Google Scholar]
  64. Zhang J.H., Xiao G., Gunsalus R.P., Hubbell W.L. (2003). Phosphorylation triggers domain separation in the DNA binding response regulator NarLBiochemistry 4225522559 [View Article][PubMed]. [Google Scholar]

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