1887

Abstract

Compaction of the nucleoid in the cell's centre was associated with the loss of colony-forming ability; these effects were caused by induction of Cyt1Aa, the cytotoxic 27 kDa protein from subsp. . Cyt1Aa-affected compaction of the nucleoids was delayed but eventually more intense than compaction caused by chloramphenicol. The possibility that small, compact nucleoids in Cyt1Aa-expressing cells resulted in DNA replication run-out and segregation following cell division was ruled out by measuring relative nucleoid length. Treatments with membrane-perforating substances other than Cyt1Aa did not cause such compaction of the nucleoids, but rather the nucleoids overexpanded to occupy nearly all of the cell volume. These findings support the suggestion that, in addition to its perforating ability, Cyt1Aa causes specific disruption of nucleoid associations with the cytoplasmic membrane. immunofluorescence labelling with Alexa did not demonstrate a great amount of Cyt1Aa associated with the membrane. Clear separation between Alexa-labelled Cyt1Aa and 4′,6-diamidino-2-phenylindole (DAPI)-stained DNA indicates that the nucleoid does not bind Cyt1Aa. Around 2 h after induction, nucleoids in Cyt1Aa-expressing cells started to decompact and expanded to fill the whole cell volume, most likely due to partial cell lysis without massive peptidoglycan destruction.

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2003-12-01
2020-04-05
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References

  1. Adams L. F., Visick J. E., Whiteley H. R.. 1989; A 20-kilodalton protein is required for efficient production of the Bacillus thuringiensis subsp. israelensis 27-kilodalton crystal protein in Escherichia coli . J Bacteriol171:521–530
    [Google Scholar]
  2. Al-yahyaee S. A. S., Ellar D. J.. 1995; Maximal toxicity of cloned CytA δ -endotoxin from Bacillus thuringiensis subsp. israelensis requires proteolytic processing from both the N- and C-termini. Microbiology141:3141–3148
    [Google Scholar]
  3. Ballesta J. P., Cundliffe E., Daniels M. J., Silverstein J. L., Susskind M. M., Schaechter M.. 1972; Some unique properties of the deoxyribonucleic acid-bearing portion of the bacterial membrane. J Bacteriol112:195–199
    [Google Scholar]
  4. Ben-Dov E., Nissan G., Pelleg N., Manasherob R., Boussiba S., Zaritsky A.. 1999; Refined, circular restriction map of the Bacillus thuringiensis subsp. israelensis plasmid carrying the mosquito larvicidal genes. Plasmid42:186–191
    [Google Scholar]
  5. Binenbaum Z., Parola A. H., Zaritsky A., Fishov I.. 1999; Transcription- and translation-dependent changes in membrane dynamics in bacteria: testing the transertion model for domain formation. Mol Microbiol32:1173–1182
    [Google Scholar]
  6. Bradford M. M.. 1976; A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem72:248–254
    [Google Scholar]
  7. Buddelmeijer N., Aarsman M. E., Kolk A. H., Vicente M., Nanninga N.. 1998; Localization of cell division protein FtsQ by immunofluorescence microscopy in dividing and nondividing cells of Escherichia coli . J Bacteriol180:6107–6116
    [Google Scholar]
  8. Bujard H., Gentz R., Lanzer M., Stueber D., Mueller M., Ibrahimi I., Haeuptle M. T., Dobberstein B.. 1987; A T5 promoter-based transcription–translation system for the analysis of proteins in vitro and in vivo . Methods Enzymol155:416–433
    [Google Scholar]
  9. Butko P., Huang F., Pusztai-Carey M., Surewicz W. K.. 1996; Membrane permeabilization induced by cytolytic δ -endotoxin CytA from Bacillus thuringiensis var. israelensis . Biochemistry35:11355–11360
    [Google Scholar]
  10. Butko P., Huang F., Pusztai-Carey M., Surewicz W. K.. 1997; Interaction of the delta-endotoxin CytA from Bacillus thuringiensis var. israelensis with lipid membranes. Biochemistry36:12862–12868
    [Google Scholar]
  11. Cheong H., Gill S. S.. 1997; Cloning and characterization of a cytolytic and mosquitocidal δ -endotoxin from Bacillus thuringiensis subsp. jegathesan . Appl Environ Microbiol63:3254–3260
    [Google Scholar]
  12. Cormack B. P., Valdivia R. H., Falkow S.. 1996; FACS-optimized mutants of the green fluorescent protein (GFP. Gene173:33–38
    [Google Scholar]
  13. Crickmore N., Bone E. J., Williams J. A., Ellar D. J.. 1995; Contribution of the individual components of the δ -endotoxin crystal to the mosquitocidal activity of Bacillus thuringiensis subsp. israelensis . FEMS Microbiol Lett131:249–254
    [Google Scholar]
  14. Crickmore N., Zeigler D. R., Feitelson J., Schnepf E., Van Rie J., Lereclus D., Baum J., Dean D. H.. 1998; Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins. Microbiol Mol Biol Rev62:807–813
    [Google Scholar]
  15. Deuschle U., Kammerer W., Gentz R., Bujard H.. 1986; Promoters of Escherichia coli : a hierarchy of in vivo strength indicates alternate structures. EMBO J5:2987–2994
    [Google Scholar]
  16. Douek J., Einav M., Zaritsky A.. 1992; Sensitivity to plating of Escherichia coli cells expressing the cryA gene from Bacillus thuringiensis var. israelensis . Mol Gen Genet232:162–165
    [Google Scholar]
  17. Drobniewski F. A., Ellar D. J.. 1988; Investigation of the membrane-lesion induced in vitro by two mosquitocidal δ -endotoxins of Bacillus thuringiensis . Curr Microbiol16:195–199
    [Google Scholar]
  18. Drobniewski F. A., Ellar D. J.. 1989; Purification and properties of a 28-kilodalton hemolytic and mosquitocidal protein toxin of Bacillus thuringiensis subsp. darmstadiensis 73-E10-2. J Bacteriol171:3060–3065
    [Google Scholar]
  19. Dworsky P., Schaechter M.. 1973; Effect of rifampin on the structure and membrane attachment of the nucleoid of Escherichia coli . J Bacteriol116:1364–1374
    [Google Scholar]
  20. Earp D. J., Ellar D. J.. 1987; Bacillus thuringiensis var. morrisoni strain PG14: nucleotide sequence of a gene encoding a 27 kDa crystal protein. Nucleic Acids Res15:3619
    [Google Scholar]
  21. Fishov I., Zaritsky A., Grover N. B.. 1995; On microbial states of growth. Mol Microbiol15:789–794
    [Google Scholar]
  22. Gazit E., Burshtein N., Ellar D. J., Sawyer T., Shai Y.. 1997; Bacillus thuringiensis cytolytic toxin associates specifically with its synthetic helices A and C in the membrane bound state. Implications for the assembly of oligomeric transmembrane pores. Biochemistry36:15546–15554
    [Google Scholar]
  23. Georghiou G. P., Wirth M. C.. 1997; Influence of exposure to single versus multiple toxins of Bacillus thuringiensis subsp. israelensis on development of resistance in the mosquito Culex quinquefasciatus (Diptera: Culicidae. Appl Environ Microbiol63:1095–1101
    [Google Scholar]
  24. Gill S. S., Cowels E. A., Pietrantonio P. V.. 1992; The mode of action of Bacillus thuringiensis endotoxins. Annu Rev Entomol37:615–636
    [Google Scholar]
  25. Goldberg L. J., Margalit J.. 1977; A bacterial spore demonstrating rapid larvicidal activity against Anopheles sergentii , Uranotaenia unguiculata , Culex univitattus , Aedes aegypti and Culex pipiens . Mosq News37:355–358
    [Google Scholar]
  26. Guerchicoff A., Ugalde R. A., Rubinstein C. P.. 1997; Identification and characterization of a previously undescribed cyt gene in Bacillus thuringiensis subsp. israelensis . Appl Environ Microbiol63:2716–2721
    [Google Scholar]
  27. Haugland R. P.. 1996; Handbook of Fluorescent Probes and Research Chemicals , 6th edn. OR, USA: Molecular Probes;
  28. Hofte H., Whiteley H. R.. 1989; Insecticidal crystal proteins of Bacillus thuringiensis . Microbiol Rev53:242–255
    [Google Scholar]
  29. Horton R. M., Hunt H. D., Ho S. N., Pullen J. K., Pease L. R.. 1989; Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene77:61–68
    [Google Scholar]
  30. Johansen C., Verheul A., Gram L., Gill T., Abee T.. 1997; Protamin-induced permeabilization of cell envelopes of gram-positive and gram-negative bacteria. Appl Environ Microbiol63:1155–1159
    [Google Scholar]
  31. Khasdan V., Ben-Dov E., Manasherob R., Boussiba S., Zaritsky A.. 2001; Toxicity and synergism in transgenic Escherichia coli expressing four genes from Bacillus thuringiensis subsp. israelensis . Environ Microbiol3:798–806
    [Google Scholar]
  32. Knowles B. H., Ellar D. J.. 1987; Colloid-osmotic lysis is a general feature of the mechanism of action of Bacillus thuringiensis δ -endotoxins with different insect specificities. Biochim Biophys Acta924:509–518
    [Google Scholar]
  33. Knowles B. H., Blatt M. R., Tester M., Horsnell J. M., Carroll J., Menestrina G., Ellar D. J.. 1989; A cytolytic δ -endotoxin from Bacillus thuringiensis var. israelensis forms cation-selective channels in planar lipid bilayers. FEBS Lett244:259–262
    [Google Scholar]
  34. Knowles B. H., White P. J., Nicholls C. N., Ellar D. J.. 1992; A broad spectrum cytolytic toxin from Bacillus thuringiensis var. kyushuensis . Proc R Soc Lond B Biol Sci248:1–7
    [Google Scholar]
  35. Koni P. A., Ellar D. J.. 1993; Cloning and characterization of a novel Bacillus thuringiensis cytolytic delta-endotoxin. J Mol Biol229:319–327
    [Google Scholar]
  36. Laemmli U. K.. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature227:680–685
    [Google Scholar]
  37. Lee D., Katayama H., Akao T., Maeda M., Tanaka R., Yamashita S., Saitoh H., Mizuki E., Ohba M.. 2001; A 28 kDa protein of the Bacillus thuringiensis serovar shandongiensis isolate 89-T-34-22 induces a human leukemic cell-specific cytotoxicity. Biochim Biophys Acta 1547;57–63
    [Google Scholar]
  38. Li J., Koni P. A., Ellar D. J.. 1996; Structure of the mosquitocidal δ -endotoxin CytB from Bacillus thuringiensis sp. kyushuensis and implications for membrane pore formation. J Mol Biol257:129–152
    [Google Scholar]
  39. Manasherob R., Zaritsky A., Ben-Dov E., Saxena D., Barak Z., Einav M.. 2001; Effect of accessory proteins P19 and P20 on cytolytic activity of Cyt1Aa from Bacillus thuringiensis subsp. israelensis in Escherichia coli . Curr Microbiol43:355–364
    [Google Scholar]
  40. Margalith Y., Ben-Dov E.. 2000; Biological control by Bacillus thuringiensis subsp. israelensis . In Insect Pest Management: Techniques for Environmental Protection pp243–301 Rechcigl J. E., Rechcigl N. A. Boca Raton, FL: CRC Press;
  41. Morgan C., Rosenkranz H. S., Carr H. S., Rose H. M.. 1967; Electron microscopy of chloramphenicol-treated Escherichia coli . J Bacteriol93:1987–2002
    [Google Scholar]
  42. Mulder E., Woldringh C. L.. 1989; Actively replicating nucleoids influence positioning of division sites in Escherichia coli filaments forming cells lacking DNA. J Bacteriol171:4303–4314
    [Google Scholar]
  43. Murphy L. D., Zimmerman S. B.. 2001; A limited loss of DNA compaction accompanying the release of cytoplasm from cells of Escherichia coli . J Struct Biol133:75–86
    [Google Scholar]
  44. Norris V.. 1995; Hypothesis: chromosome separation in Escherichia coli involves autocatalytic gene expression, transertion and membrane-domain formation. Mol Microbiol16:1051–1057
    [Google Scholar]
  45. Parola A. H., Ibdah M., Gill D., Zaritsky A.. 1990; Deviation from homeoviscous adaptation in Escherichia coli membranes. Biophys J57:621–626
    [Google Scholar]
  46. Pato M. L.. 1975; Alterations of the rate of movement of deoxyribonucleic acid replication forks. J Bacteriol123:272–277
    [Google Scholar]
  47. Ryals J., Little R., Bremer H.. 1982; Control of rRNA and tRNA syntheses in Escherichia coli by guanosine tetraphosphate. J Bacteriol151:1261–1268
    [Google Scholar]
  48. Sambrook J., Russell D.. 2001; Molecular Cloning: a Laboratory Manual , 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
  49. Schnepf E., Crickmore N., Van Rie J., Lereclus D., Baum J., Feitelson J., Zeigler D. R., Dean D. H.. 1998; Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev62:775–806
    [Google Scholar]
  50. Service M. W.. editor 1986; Blood-Sucking Insects: Vectors of Disease London, UK: Edward Arnold;
  51. Seth A.. 1984; A new method for linker ligation. Gene Anal Tech1:99–103
    [Google Scholar]
  52. Shellman V. L., Pettijohn D. E.. 1991; Introduction of proteins into living bacterial cells: distribution of labeled HU protein in Escherichia coli . J Bacteriol173:3047–3059
    [Google Scholar]
  53. Sun Q., Margolin W.. 2001; Influence of the nucleoid on placement of FtsZ and MinE rings in Escherichia coli . J Bacteriol183:1413–1422
    [Google Scholar]
  54. Thiery I., Delecluse A., Tamayo M. C., Orduz S.. 1997; Identification of a gene for Cyt1A-like hemolysin from Bacillus thuringiensis subsp. medellin and expression in a crystal-negative B. thuringiensis strain. Appl Environ Microbiol63:468–473
    [Google Scholar]
  55. Thomas W. E., Ellar D. J.. 1983a; Mechanism of action of Bacillus thuringiensis var. israelensis insecticidal δ -endotoxin. FEBS Lett154:362–368
    [Google Scholar]
  56. Thomas W. E., Ellar D. J.. 1983b; Bacillus thuringiensis var. israelensis crystal δ -endotoxin: effects on insect and mammalian cells in vitro and in vivo . J Cell Sci60:181–197
    [Google Scholar]
  57. Van Helvoort J. M. L. M.. 1996; A cytometric study of nucleoid partitioning PhD thesis Institute for Molecular Cell Biology, Section of Molecular Cytology, Biocentrum Amsterdam, University of Amsterdam;
  58. Van Helvoort J. M. L. M., Woldringh C. L.. 1994; Nucleoid partitioning in Escherichia coli during steady-state growth and upon recovery from chloramphenicol treatment. Mol Microbiol13:577–583
    [Google Scholar]
  59. Van Helvoort J. M. L. M., Kool J., Woldringh C. L.. 1996; Chloramphenicol causes fusion of separated nucleoids in Escherichia coli K-12 cells and filaments. J Bacteriol178:4289–4293
    [Google Scholar]
  60. Van Helvoort J. M. L. M., Huls P. G., Vischer N. O. E., Woldringh C. L.. 1998; Fused nucleoids resegregate faster than cell elongation in Escherichia coli pbpB (Ts) filaments after release from chloramphenicol inhibition. Microbiology144:1309–1317
    [Google Scholar]
  61. Vischer N. O. E., Huls P. G., Woldringh C. L.. 1994; object-image: an interactive image analysis program using structured point collection. Binary6:160–166
    [Google Scholar]
  62. Visick J. E., Whiteley H. R.. 1991; Effect of a 20-kilodalton protein from Bacillus thuringiensis subsp. israelensis on production of the CytA protein by Escherichia coli . J Bacteriol173:1748–1756
    [Google Scholar]
  63. Voss J. G.. 1964; Lysozyme lysis of Gram-negative bacteria without production of spheroplasts. J Gen Microbiol35:313–317
    [Google Scholar]
  64. Wirth M. C., Georghiou G. P.. 1997; Cross-resistance among CryIV toxins of Bacillus thuringiensis subsp. israelensis in Culex quinquefasciatus (Diptera: Culicidae. J Econ Entomol90:1471–1477
    [Google Scholar]
  65. Wirth M. C., Georghiou G. P., Federici B. A.. 1997; CytA enables CryIV endotoxins of Bacillus thuringiensis to overcome high levels of CryIV resistance in the mosquito, Culex quinquefasciatus . Proc Natl Acad Sci U S A94:10536–10540
    [Google Scholar]
  66. Woldringh C. L.. 1973; Effects of toluene and phenethyl alcohol on the ultrastructure of Escherichia coli . J Bacteriol114:1359–1361
    [Google Scholar]
  67. Woldringh C. L.. 2002; The role of co-transcriptional translation and protein translocation (transertion) in bacterial chromosome segregation. Mol Microbiol Rev45:17–29
    [Google Scholar]
  68. Woldringh C. L., Jensen P. R., Westerhoff H. V.. 1995; Structure and partitioning of bacterial DNA: determined by a balance of compaction and expansion forces?. FEMS Microbiol Lett131:235–242
    [Google Scholar]
  69. Wu D., Federici B. A.. 1993; A 20-kilodalton protein preserves cell viability and promotes CytA crystal formation during sporulation in Bacillus thuringiensis . J Bacteriol175:5276–5280
    [Google Scholar]
  70. Yokoyama Y., Kohda K., Okamoto M.. 1998; CytA protein, a δ -endotoxin of Bacillus thuringiensis subsp. israelensis is associated with DNA. Biol Pharm Bull21:1263–1266
    [Google Scholar]
  71. Yu Y. M., Ohba M., Gill S. S.. 1991; Characterization of mosquitocidal activity of Bacillus thuringiensis subsp. fukuokaensis crystal proteins. Appl Environ Microbiol57:1075–1081
    [Google Scholar]
  72. Zaritsky A., Parola A. H., Ibdah M., Masalha H.. 1985; Homeoviscous adaptation, growth rate, and morphogenesis in bacteria. Biophys J48:337–339
    [Google Scholar]
  73. Zimmerman S. B.. 1993; Macromolecular crowding effects on macromolecular interactions: some implications for genome structure and function. Biochim Biophys Acta1216:175–185
    [Google Scholar]
  74. Zusman D. R., Carbonell A., Haga J. Y.. 1973; Nucleoid condensation and cell division in Escerichia coli MX74T2 ts52 after inhibition of protein synthesis. J Bacteriol115:1167–1178
    [Google Scholar]
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