Advertisement

Molecular and General Genetics MGG

, Volume 231, Issue 2, pp 201–211 | Cite as

Lethal overproduction of the Escherichia coli nucleoid protein H-NS: ultramicroscopic and molecular autopsy

  • Roberto Spurio
  • Markus Dürrenberger
  • Maurizio Falconi
  • Anna La Teana
  • Cynthia L. Pon
  • Claudio O. Gualerzi
Article

Summary

The Escherichia coli hns gene, which encodes the nucleoid protein H-NS, was deprived of its natural promoter and placed under the control of the inducible lambda PL promoter. An hns mutant yielding a protein (H-NSΔ12) with a deletion of four amino acids (Gly112-Arg-Thr-Pro115) was also obtained. Overproduction of wild-type (wt) H-NS, but not of H-NSΔ12, resulted in a drastic loss of cell viability. The molecular events and the morphological alterations eventually leading to cell death were investigated. A strong and nearly immediate inhibition of both RNA and protein synthesis were among the main effects of overproduction of wt H-NS, while synthesis of DNA and cell wall material was inhibited to a lesser extent and at a later time. Upon cryofixation of the cells, part of the overproduced protein was found in inclusion bodies, while the rest was localized by immunoelectron microscopy to the nucleoids. The nucleoids appeared condensed in cells expressing both forms of H-NS, but the morphological alterations were particularly dramatic in those overproducing wt H-NS; their nucleoids appeared very dense, compact and almost perfectly spherical. These results provide direct evidence for involvement of H-NS in control of the organization and compaction of the bacterial nucleoid in vivo and suggest that it may function, either directly or indirectly, as transcriptional repressor and translational inhibitor.

Key words

Nucleoid structure DNA compaction Cryofixation Transcriptional repression Translational inhibition 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Acetarin JD, Carlemalm E, Villiger W (1986) Development of new Lowicryl resins for embedding biological specimens at even lower temperatures. J Microsc 143:81–88Google Scholar
  2. Bernard H-U, Remaut E, Hershfield MV, Das HK, Helinski DR, Yanofsky C, Franklin N (1979) Construction of plasmid cloning vehicles that promote gene expression from the bacteriophage lambda PL promoter. Gene 5:59–76Google Scholar
  3. Bertin P, Lejeune P, Laurent-Winter C, Danchin A (1990) Mutations in bglY, the structural gene for the DNA-binding protein H1 affect expression of several E. coli genes. Biochimie 72:889–891Google Scholar
  4. Carlemalm E, Garavito RM, Villinger W (1982) Resin development for electron microscopy and an analysis of embedding at low temperatures. J Microsc 126:123–143Google Scholar
  5. Defez R, De Felice M (1981) Cryptic operon for β-glucoside metabolism in Escherichia coli K12: genetic evidence for a regulatory protein. Genetics 97:11–25Google Scholar
  6. Diderichsen B (1980) cur-1, a mutation affecting the phenotype of sup + strains of E. coli. Mol Gen Genet 180:425–428Google Scholar
  7. Dorman CJ, Bhriain NN, Higgins CF (1990) DNA supercoiling and environmental regulation of virulence gene expression in Shigella flexneri. Nature 344:789–792Google Scholar
  8. Drlica K, Rouviere-Yaniv J (1987) Histonelike proteins of bacteria. Microbiol Rev 51:301–319Google Scholar
  9. Dürrenberger M (1989) Removal of background label in immunocytochemistry with the apolar Lowicryls by using washed protein A-gold-precoupled antibodies in a one-step procedure. J Electron Microsc Techn 11:109–116Google Scholar
  10. Dürrenberger M, La Teana A, Citro G, Venanzi F, Gualerzi CO, Pon CL (1991) Escherichia coli DNA-binding protein H-NS is localized in the nucleoid. Res Microbiol 142:373–380Google Scholar
  11. Escaig J (1982) New instrument which facilitates rapid freezing at 83°K and 6°K. J Microsc 126:221–229Google Scholar
  12. Falconi M, Gualtieri MT, La Teana A, Losso MA, Pon CL (1988) Proteins from the prokaryotic nucleoid: primary and quaternary structure of the 15-kD Escherichia coli DNA binding protein H-NS. Mol Microbiol 2:323–329Google Scholar
  13. Falconi M, McGovern V, Gualerzi C, Hillyard D, Higgins NP (1991) Mutations altering chromosome protein H-NS induce mini-Mu transposition. New Biol 3:615–625Google Scholar
  14. Friedrich K, Gualerzi CO, Lammi M, Losso MA, Pon CL (1988) Proteins from the prokaryotic nucleoid. Interaction of nucleic acids with the 15 kDa Escherichia coli histone-like protein HNS. FEBS Lett 229:197–202Google Scholar
  15. Göransson M, Sondén B, Nilsson P, Dagberg B, Forsman K, Emanuelsson K, Uhlin BE (1990) Transcriptional silencing and thermoregulation of gene expression in E. coli. Nature 344:682–685Google Scholar
  16. Gualerzi C, Losso M, Lammi M, Friedrich K, Pawlik R, Canonaco MA, Gianfranceschi GL, Pingoud A, Pon CL (1986) Proteins from the prokaryotic nucleoid. Structural and functional characterization of the Escherichia coli DNA-binding proteins NS(HU) and H-NS. In: Gualerzi CO, Pon CL (eds) Bacterial chromatin. Springer, Heidelberg, pp 101–134Google Scholar
  17. Gualerzi C, Spurio R, La Teana A, Calogero R, Celano B, Pon CL (1989) Site-directed mutagenesis of E. coli translation initiation factor IF1. Identification of the amino acids involved in its ribosomal binding and recycling. Protein Eng 3:133–138Google Scholar
  18. Heuser J, Reese T, Dennis M, Jan Y, Jan L, Evans L (1979) Synaptic vesicle exocytosis captured by quick freezing and correlated with quantal transmitter release. J Cell Biol 81:275–300Google Scholar
  19. Higgins CF, Dorman CJ, Stirling DA, Waddell L, Booth IR, May G, Bremer E (1988) A physiological role for DNA supercoiling in the osmotic regulation of gene expression in S. typhimurium and E. coli. Cell 52:569–684Google Scholar
  20. Higgins CF, Hinton JCD, Hulton, CSJ, Owen-Hughes T, Pavitt GD, Seirafi A (1990) Protein-H1 — a role for chromatin structure in the regulation of bacterial gene expression and virulence. Mol Microbiol 4:2007–2012Google Scholar
  21. Hromocky AE, Maurelli AT (1989) Identification of an Escherichia coli gene homologous to virR, a regulator of Shigella virulence. J Bacteriol 171:2879–2881Google Scholar
  22. Hulton CSJ, Seirafi A, Hinton JCD, Sidebotham JM, Waddell L, Pavitt GD, Owen-Hughes T, Spassky A, Buc H, Higgins CF (1990) Histone-like protein H1 (H-NS), DNA supercoiling and gene expression in bacteria. Cell 63:631–642Google Scholar
  23. Kellenberger E, Ryter A, Séchaud J (1958) Electron microscopy study of DNA-containing plasmas. II. Vegetative and mature phage DNA as compared with normal bacterial nucleoids in different physiological states. J Biophys Biochem Cytol 4:671–678Google Scholar
  24. Kellenberger E, Carlemalm E, Sechaud J, Ryter A, De Haller G (1986) Considerations on the condensation and the degree of compactness of non-eukaryotic DNA-containing plasmas. In: Gualerzi CO, Pon CL (eds) Bacterial chromatin. Springer, Heidelberg, pp 11–25Google Scholar
  25. Kellenberger E, Dürrenberger M, Villiger W, Carlemalm E, Wurtz M (1987) Efficiency of immunolabel on Lowicryl sections compared to theoretical predictions. J Histochem Cytochem 35:959–969Google Scholar
  26. Lammi M, Paci M, Pon CL, Losso MA, Miano A, Pawlik RT, Gianfranceschi GL, Gualerzi C (1984) Proteins from the prokaryotic nucleoid. Biochemical and 1H-NMR studies on three bacterial histone-like proteins. In: Hubscher H, Spadari S (eds) Proteins involved in DNA replication. Plenum, New York, pp 467–478Google Scholar
  27. La Teana A, Falconi M, Scarlato V, Lammi M, Pon CL (1989) Characterization of the structural genes for the DNA-binding protein H-NS in Enterobacteriaceae. FEBS Lett 244:34–38Google Scholar
  28. La Teana A, Brandi A, Falconi M, Spurio R, Pon CL, Gualerzi CO (1990) Identification of a cold shock transcriptional enhancer of the Escherichia coli gene encoding nucleoid protein H-NS. Proc Natl Acad Sci USA (in press)Google Scholar
  29. Lejeune P, Danchin A (1990) Mutations in the bglY gene increase the frequency of spontaneous deletions in Escherichia coli K-12. Proc Natl Acad Sci USA 87:360–363Google Scholar
  30. May G, Dersch P, Haardt M, Middendorf A, Bremer E (1990) The osmZ (bglY) gene encodes the DNA-binding protein H-NS (H1a), a component of the Escherichia coli K12 nucleoid. Mol Gen Genet 224:81–90Google Scholar
  31. Pettijohn DE (1988) Histone-like proteins and bacterial chromosome structure. J Biol Chem 263:12793–12796Google Scholar
  32. Pon CL, Calogero RA, 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 212:199–202Google Scholar
  33. Remaut E, Tsao H, Fiers W (1983) Improved plasmid vectors with a thermoinducible expression and temperature-regulated runaway replication. Gene 22:103–113Google Scholar
  34. Rimsky S, Spassky A (1990) Sequence determinants for H1 binding on E. coli lac and gal promoters. Biochemistry 29:3765–3771Google Scholar
  35. Roth J (1982) The preparation of protein A-gold complexes with 3 nm and 5 nm gold particles and their use in labeling multiple agents on ultrathin sections. Histochem J 14:791–801Google Scholar
  36. Spassky A, Rimsky S, Garreau H, Buc H (1984) H1a, an Escherichia coli DNA-binding protein which accumulates in stationary phase, strongly compacts DNA in vitro. Nucleic Acids Res 12:5321–5339Google Scholar
  37. Spears PA, Schauer D, Orndorff PE (1986) Metastable regulation of type 1 piliation in Escherichia coli and isolation and characterization of a phenotypically stable mutant. J Bacteriol 168:179–185Google Scholar
  38. Tanaka K, Muramatsu S, Yamada H, Mizuno T (1991) Systematic characterization of curved DNA segments randomly cloned from Escherichia coli and their functional significance. Mol Gen Genet 226:367–376Google Scholar
  39. Yamada H, Muramatsu S, Mizuno T (1990) An Escherichia coli protein that preferentially binds to sharply curved DNA. J Biochem 108:420–425Google Scholar

Copyright information

© Springer-Verlag 1992

Authors and Affiliations

  • Roberto Spurio
    • 1
  • Markus Dürrenberger
    • 2
  • Maurizio Falconi
    • 1
  • Anna La Teana
    • 3
  • Cynthia L. Pon
    • 1
  • Claudio O. Gualerzi
    • 1
  1. 1.Laboratory of Genetics, Dept. of BiologyUniversity of CamerinoCamerino (MC)Italy
  2. 2.Elektronenmikroskopisches Zentrallaboratorium der Universität ZürichSwitzerland
  3. 3.Max-Planck-Institut für Molekulare GenetikBerlin-DahlemGermany

Personalised recommendations