Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
권호 표지
DOI QR코드

DOI QR Code

H-NS binding on dicA promoter DNA inhibits dicA gene expression

dicA promoter DNA에 붙는 H-NS 단백질에 의한 dicA 유전자의 발현 조절

  • Yun, Sang Hoon (Department of Biological Sciences, College of Biological Sciences and Biotechnology, Chungnam National University) ;
  • Lee, Yonho (Department of Biological Sciences, College of Biological Sciences and Biotechnology, Chungnam National University) ;
  • Lim, Heon M. (Department of Biological Sciences, College of Biological Sciences and Biotechnology, Chungnam National University)
  • 윤상훈 (충남대학교 생명시스템과학대학 생물과학과) ;
  • 이연호 (충남대학교 생명시스템과학대학 생물과학과) ;
  • 임헌만 (충남대학교 생명시스템과학대학 생물과학과)
  • Received : 2019.07.05
  • Accepted : 2019.08.26
  • Published : 2019.09.30
Download PDF
⟨ Previous Next ⟩

Abstract

H-NS binds to promoter DNA and works as a general transcription silencer. DicA protein, by binding to the promoter DNA of dicA, activates dicA expression and at the same time inhibits expression of dicF and dicB, thus, exerting cell division control in Escherichia coli. H-NS complexed with a nucleoid protein Cnu was known to be involved in dicA expression. However, the exact nature of H-NS binding to dicA promoter DNA and the consequences of H-NS binding in expression of dicA is not clear. In this study, we explored the DNA binding activity of H-NS on the promoter DNA of dicA and found that H-NS binding occurs exclusively to the dicA promoter DNA. We never observed, however, H-NS binding at the vicinity of the dicA promoter. Temperature dependent oligomerization of H-NS was observed during DNA binding and the Cnu protein enhances the oligomerization process of H-NS binding. In vivo measurement of dicA expression in an hns deleted strain showed that dicA expression increased. These results demonstrated that H-NS binds specifically to dicA promoter DNA and functions as a transcription silencer.

H-NS는 대장균에서 DNA 결합 단백질로 수많은 유전자의 발현에 영향을 주는 것으로 잘 알려져 있다. DicA 단백질은 dicF, dicB의 발현을 억제하여 대장균의 분열을 조절한다. dicA의 발현에 Cnu, H-NS의 관여 여부는 CnuK9E 돌연변이가 $37^{\circ}C$에서 dicA의 발현을 억제하여 대장균이 길게 자라는 현상을 일으키며 처음 알려졌다. 하지만 Cnu와 H-NS 두 단백질이 어떻게 dicA의 발현을 조절하는지에 대한 분자적인 기작 연구는 잘 되어있지 않다. 본 연구에서 H-NS가 dicA와 dicC 유전자의 프로모터 부근에 염기서열 특이적으로 결합하며, $37^{\circ}C$ 보다 $25^{\circ}C$에서 DNA 더 잘 결합하는 것을 확인하였다. 그리고 EMSA를 통해 Cnu는 H-NS의 DNA 결합의 oligomeric state를 변화시키는 방식으로 작용하는 것을 보여주었다. In vivo transcription assay와 real time PCR을 통해 H-NS가 제거된 대장균에서 dicA 프로모터 활성이 높아지고, 분열 초기 dicA의 발현이 조절 받지 못하고 증가하는 것으로 보아, H-NS는 dicA의 발현에 억제자로서 기능한다.

Keywords

References

  1. Afflerbach H, Schroder O, and Wagner R. 1999. Conformational changes of the upstream DNA mediated by H-NS and FIS regulate E. coli rrnB P1 promoter activity. J. Mol. Biol. 286, 339-353. https://doi.org/10.1006/jmbi.1998.2494
  2. Azam TA and Ishihama A. 1999. Twelve species of the nucleoidassociated protein from Escherichia coli. Sequence recognition specificity and DNA binding affinity. J. Biol. Chem. 274, 33105-33113. https://doi.org/10.1074/jbc.274.46.33105
  3. Bejar S and Bouche JP. 1985. A new dispensable genetic locus of the terminus region involved in control of cell division in Escherichia coli. Mol. Gen. Genet. 201, 146-150. https://doi.org/10.1007/BF00425651
  4. Bouche F and Bouche JP. 1989. Genetic evidence that DicF, a second division inhibitor encoded by the Escherichia coli dicB operon, is probably RNA. Mol. Microbiol. 3, 991-994. https://doi.org/10.1111/j.1365-2958.1989.tb00249.x
  5. Coombes BK, Wickham ME, Lowden MJ, Brown NF, and Finlay BB. 2005. Negative regulation of Salmonella pathogenicity island 2 is required for contextual control of virulence during typhoid. Proc. Natl. Acad. Sci. USA 102, 17460-17465. https://doi.org/10.1073/pnas.0505401102
  6. Cukier-Kahn R, Jacquet M, and Gros F. 1972. Two heat-resistant, low molecular weight proteins from Escherichia coli that stimulate DNA-directed RNA synthesis. Proc. Natl. Acad. Sci. USA 69, 3643-3647. https://doi.org/10.1073/pnas.69.12.3643
  7. Dame RT, Wyman C, Wurm R, Wagner R, and Goosen N. 2002. Structural basis for H-NS-mediated trapping of RNA polymerase in the open initiation complex at the rrnB P1. J. Biol. Chem. 277, 2146-2150. https://doi.org/10.1074/jbc.C100603200
  8. Datsenko KA and Wanner BL. 2000. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA 97, 6640-6645. https://doi.org/10.1073/pnas.120163297
  9. de Boer PA, Crossley RE, and Rothfield LI. 1990. Central role for the Escherichia coli minC gene product in two different cell divisioninhibition systems. Proc. Natl. Acad. Sci. USA 87, 1129-1133. https://doi.org/10.1073/pnas.87.3.1129
  10. Kim MS, Bae SH, Yun SH, Lee HJ, Ji SC, Lee JH, Srivastava P, Lee SH, Chae H, Lee Y, Choi BS, Chattoraj DK, and Lim HM. 2005. Cnu, a novel oric-binding protein of escherichia coli. J Bacteriol. 187, 6998-7008. https://doi.org/10.1128/JB.187.20.6998-7008.2005
  11. La Teana A, Falconi M, Scarlato V, Lammi M, and Pon CL. 1989. Characterization of the structural genes for the DNA-binding protein H-NS in Enterobacteriaceae. FEBS Lett. 244, 34-38. https://doi.org/10.1016/0014-5793(89)81156-1
  12. Lithgow JK, Haider F, Roberts IS, and Green J. 2007. Alternate Slya and H-NS nucleoprotein complexes control hlyE expression in Escherichia coli K-12. Mol. Microbiol. 66, 685-698. https://doi.org/10.1111/j.1365-2958.2007.05950.x
  13. Marsh M and Hillyard DR. 1990. Nucleotide sequence of hns encoding the DNA-binding protein H-NS of Salmonella typhimurium. Nucleic Acids Res. 18, 3397. https://doi.org/10.1093/nar/18.11.3397
  14. Pon CL, Calogero RA, and Gualerzi CO. 1988. Identification, cloning, nucleotide sequence and chromosomal map location of hns, the structural gene for Escherichia coli DNA-binding protein H-NS. Mol. Gen. Genet. 212, 199-202. https://doi.org/10.1007/BF00334684
  15. Prosseda G, Falconi M, Giangrossi M, Gualerzi CO, Micheli G, and Colonna B. 2004. The virF promoter in Shigella: More than just a curved DNA stretch. Mol. Microbiol. 51, 523-537. https://doi.org/10.1046/j.1365-2958.2003.03848.x
  16. Prosseda G, Fradiani PA, Di Lorenzo M, Falconi M, Micheli G, Casalino M, Nicoletti M, and Colonna B. 1998. A role for H-NS in the regulation of the virF gene of Shigella and enteroinvasive Escherichia coli. Res. Microbiol. 149, 15-25. https://doi.org/10.1016/S0923-2508(97)83619-4
  17. Quade N, Mendonca C, Herbst K, Heroven AK, Ritter C, Heinz DW, and Dersch P. 2012. Structural basis for intrinsic thermosensing by the master virulence regulator RovA of Yersinia. J. Biol. Chem. 287, 35796-35803. https://doi.org/10.1074/jbc.M112.379156
  18. Rajkumari K, Kusano S, Ishihama A, Mizuno T, and Gowrishankar J. 1996. Effects of H-NS and potassium glutamate on sigmaS- and sigma70-directed transcription in vitro from osmotically regulated P1 and P2 promoters of proU in Escherichia coli. J. Bacteriol. 178, 4176-4181. https://doi.org/10.1128/jb.178.14.4176-4181.1996
  19. Silphaduang U, Mascarenhas M, Karmali M, and Coombes BK. 2007. Repression of intracellular virulence factors in Salmonella by the Hha and YdgT nucleoid-associated proteins. J. Bacteriol. 189, 3669-3673. https://doi.org/10.1128/JB.00002-07
  20. White-Ziegler CA, Angus Hill ML, Braaten BA, van der Woude MW, and Low DA. 1998. Thermoregulation of Escherichia coli pap transcription: H-NS is a temperature-dependent DNA methylation blocking factor. Mol. Microbiol. 28, 1121-1137. https://doi.org/10.1046/j.1365-2958.1998.00872.x
  21. Yun SH, Ji SC, Jeon HJ, Wang X, Kim SW, Bak G, Lee Y, and Lim HM. 2012. The CnuK9E H-NS complex antagonizes DNA binding of DicA and leads to temperature-dependent filamentous growth in E. coli. PLoS One 7, e45236. https://doi.org/10.1371/journal.pone.0045236