Abstract
Histone-like nucleoid structuring protein (H-NS) is a small but abundant protein present in enteric bacteria and is involved in compaction of the DNA and regulation of the transcription. Recent reports have suggested that H-NS binds to a specific AT rich DNA sequence than to intrinsically curved DNA in sequence independent manner. We detected two high-specificity H-NS binding sites in LEE5 promoter of EPEC centered at -110 and -138, which were close to the proposed consensus H-NS binding motif. To identify H-NS binding sequence in LEE5 promoter, we took a random mutagenesis approach and found the mutations at around -138 were specifically defective in the regulation by H-NS. It was concluded that H-NS exertsmaximumrepression via the specific sequence at around -138 and subsequently contacts a subunit of RNAP through oligomerization.
Similar content being viewed by others
Refrerences
Blot, N., Mavathur, R., Geertz, M., Travers, A., and Muskhelishvili, G. 2006. Homeostatic regulation of supercoiling sensitivity coordinates transcription of the bacterial genome. EMBO Reports 7, 710–715.
Bouffartigues, E., Buckle, M., Badaut, C., Travers, A., and Rimsky, S. 2007. H-NS cooperative binding to high-affinity sites in a regulatory element results in transcriptional silencing. Nat. Struct. Mol. Biol. 14, 441–448.
Choy, H.E. and Adhya, S. 1993. RNA polymerase idling and clearance in gal promoters: use of supercoiled minicircle DNA template made in vivo. Proc. Natl. Acad. Sci. USA 90, 472–476.
Cordeiro, T.N., Schmidt, H., Madrid, C., Juárez, A., Bernadó, P., Griesinger, C., García, J., and Pons, M. 2011. Indirect DNA readout by an H-NS related protein: structure of the DNA complex of the C-terminal domain of Ler. PLoS Pathog. 7, e1002380.
Dorman, C.J. 2004. H-NS: a universal regulator for a dynamic genome. Nat. Rev. Microbiol. 2, 391–400.
Dorman, C. 2007. H-NS, the genome sentinel. Nat. Rev. Microbiol. 5, 157–161.
Falconi, M., Colonna, B., Prosseda, G., Micheli, G., and Gualerzi, C.O. 1998. Thermoregulation of Shigella and Escherichia coli EIEC pathogenicity. A temperature-dependent structural transition of DNA modulates accessibility of virF promoter to transcriptional repressor H-NS. EMBO J. 17, 7033–7043.
Falconi, M., Prosseda, G., Giangrossi, M., Beghetto, E., and Colonna, B. 2001. Involvement of FIS in the H-NS-mediated regulation of virF gene of Shigella and enteroinvasive Escherichia coli. Mol. Microbiol. 42, 439–452.
Fang, F.C. and Rimsky, S. 2008. New insights into transcriptional regulation by H-NS. Curr. Opin. Microbiol. 11, 113–120.
Haack, K.R., Robinson, C.L., Miller, K.J., Fowlkes, J.W., and Mellies, J.L. 2003. Interaction of Ler at the LEE5 (tir) operon of enteropathogenic Escherichia coli. Infect. Immun. 71, 384–392.
Laaberki, M.H., Janabi, N., Oswald, E., and Repoila, F. 2006. Concert of regulators to switch on LEE expression in enterohemorrhagic Escherichia coli O157:H7: interplay between Ler, GrlA, HNS and RpoS. Int. J. Med. Microbiol. 296, 197–210.
Lang, B., Blot, N., Bouffartigues, E., Buckle, M., Geertz, M., Gualerzi, C.O., Mavathur, R., Muskhelishvili, G., Pon, C.L., Rimsky, S., and et al. 2007. High-affinity DNA binding sites for H-NS provide a molecular basis for selective silencing within proteobacterial genomes. Nucleic Acids Res. 35, 6330–6337.
Lingbeck, J., Kubinec, M.G., Miller, J., Reid, B.R., Drobny, G.P., and Kennedy, M.A. 1996. Effect of adenine methylation on the structure and dynamics of TpA steps in DNA: NMR structure determination of [d(CGAGGTTTAAACCTCG)]2 and its A9-methylated derivative at 750 MHz. Biochemistry 35, 719–734.
Martin, C.S.-S., Bustamante, V.H., Calva, E., and Puente, J.L. 2001. Transcriptional regulation of the orf19 gene and the tir-cesT-eae operon of enteropathogenic Escherichia coli. J. Bacteriol. 183, 2823–2833.
Miller, J.H. 1972. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., USA.
Noom, M.C., Navarre, W.W., Oshima, T., Wuite, G.J.L., and Dame, R.T. 2007. H-NS promotes looped domain formation in the bacterial chromosome. Curr. Biol. 17, R913–914.
Oshima, T., Ishikawa, S., Kurokawa, K., Aiba, H., and Ogasawara, N. 2006. Escherichia coli histone-like protein H-NS preferentially binds to horizontally acquired DNA in association with RNA polymerase. DNA Res. 13, 141–153.
Putnam, S.L. and Koch, K.A. 1975. Complications in the simplest cellular enzyme assay: lysis of Escherichia coli for the assay of beta-galactosidase. Anal. Biochem. 63, 350–360.
Rimsky, S. 2004. Structure of the histone-like protein H-NS and its role in regulation and genome superstructure. Curr. Opin. Microbiol. 7, 109–114.
Sette, M., Spurio, R., Trotta, E., Brandizi, C., Brandi, A., Pon, C.L., Barbato, G., Boelens, R., and Gualerzi, C.O. 2009. Sequencespecific recognition of DNA by the C-terminal domain of nucleoid- associated protein H-NS. J. Biol. Chem. 284, 30453–30462.
Shin, M., Lagda, A.C., Lee, J.W., Bhat, A., Rhee, J.H., Kim, J.S., Takeyasu, K., and Choy, H.E. 2012. Gene silencing by H-NS from distal DNA site. Mol. Microbiol. 86, 707–719.
Simons, R.W., Houman, F., and Kleckner, N. 1987. Improved single and multicopy lac-based cloning vectors for protein and operon fusions. Gene 53, 85–96.
Squires, C., Krainer, A., Barry, G., Shen, W.F., and Squires, C.L. 1981. Nucleotide sequence at the end of the gene for the RNA polymerase β’ subunit (rpoC). Nucleic Acids Res. 9, 6827–6840.
Umanski, T., Rosenshine, I., and Friedberg, D. 2002. Thermoregulated expression of virulence genes in enteropathogenic Escherichia coli. Microbiology 148, 2735–2744.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Bhat, A.P., Shin, M. & Choy, H.E. Identification of high-specificity H-NS binding site in LEE5 promoter of enteropathogenic Esherichia coli (EPEC). J Microbiol. 52, 626–629 (2014). https://doi.org/10.1007/s12275-014-3562-x
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12275-014-3562-x