Compaction of the Escherichia coli 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 Bacillus thuringiensis subsp. israelensis. 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. In situ 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.
AdamsL. F.,
VisickJ. E.,
WhiteleyH. 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 Bacteriol 171:521–530
Al-yahyaeeS. A. S.,
EllarD. J.1995; Maximal toxicity of cloned CytA δ -endotoxin from Bacillus thuringiensis subsp. israelensis requires proteolytic processing from both the N- and C-termini. Microbiology 141:3141–3148
BallestaJ. P.,
CundliffeE.,
DanielsM. J.,
SilversteinJ. L.,
SusskindM. M.,
SchaechterM.1972; Some unique properties of the deoxyribonucleic acid-bearing portion of the bacterial membrane. J Bacteriol 112:195–199
BinenbaumZ.,
ParolaA. H.,
ZaritskyA.,
FishovI.1999; Transcription- and translation-dependent changes in membrane dynamics in bacteria: testing the transertion model for domain formation. Mol Microbiol 32:1173–1182
BradfordM. M.1976; A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72:248–254
BuddelmeijerN.,
AarsmanM. E.,
KolkA. H.,
VicenteM.,
NanningaN.1998; Localization of cell division protein FtsQ by immunofluorescence microscopy in dividing and nondividing cells of Escherichia coli . J Bacteriol 180:6107–6116
BujardH.,
GentzR.,
LanzerM.,
StueberD.,
MuellerM.,
IbrahimiI.,
HaeuptleM. T.,
DobbersteinB.1987; A T5 promoter-based transcription–translation system for the analysis of proteins in vitro and in vivo . Methods Enzymol 155:416–433
ButkoP.,
HuangF.,
Pusztai-CareyM.,
SurewiczW. K.1997; Interaction of the delta-endotoxin CytA from Bacillus thuringiensis var. israelensis with lipid membranes. Biochemistry 36:12862–12868
CheongH.,
GillS. S.1997; Cloning and characterization of a cytolytic and mosquitocidal δ -endotoxin from Bacillus thuringiensis subsp. jegathesan . Appl Environ Microbiol 63:3254–3260
CrickmoreN.,
BoneE. J.,
WilliamsJ. A.,
EllarD. J.1995; Contribution of the individual components of the δ -endotoxin crystal to the mosquitocidal activity of Bacillus thuringiensis subsp. israelensis . FEMS Microbiol Lett 131:249–254
CrickmoreN.,
ZeiglerD. R.,
FeitelsonJ.,
SchnepfE.,
Van RieJ.,
LereclusD.,
BaumJ.,
DeanD. H.1998; Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins. Microbiol Mol Biol Rev 62:807–813
DeuschleU.,
KammererW.,
GentzR.,
BujardH.1986; Promoters of Escherichia coli : a hierarchy of in vivo strength indicates alternate structures. EMBO J 5:2987–2994
DouekJ.,
EinavM.,
ZaritskyA.1992; Sensitivity to plating of Escherichia coli cells expressing the cryA gene from Bacillus thuringiensis var. israelensis . Mol Gen Genet 232:162–165
DrobniewskiF. A.,
EllarD. J.1988; Investigation of the membrane-lesion induced in vitro by two mosquitocidal δ -endotoxins of Bacillus thuringiensis . Curr Microbiol 16:195–199
DrobniewskiF. A.,
EllarD. J.1989; Purification and properties of a 28-kilodalton hemolytic and mosquitocidal protein toxin of Bacillus thuringiensis subsp. darmstadiensis 73-E10-2. J Bacteriol 171:3060–3065
DworskyP.,
SchaechterM.1973; Effect of rifampin on the structure and membrane attachment of the nucleoid of Escherichia coli . J Bacteriol 116:1364–1374
EarpD. J.,
EllarD. J.1987; Bacillus thuringiensis var. morrisoni strain PG14: nucleotide sequence of a gene encoding a 27 kDa crystal protein. Nucleic Acids Res 15:3619
GazitE.,
BurshteinN.,
EllarD. J.,
SawyerT.,
ShaiY.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. Biochemistry 36:15546–15554
GeorghiouG. P.,
WirthM. 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 Microbiol 63:1095–1101
GuerchicoffA.,
UgaldeR. A.,
RubinsteinC. P.1997; Identification and characterization of a previously undescribed cyt gene in Bacillus thuringiensis subsp. israelensis . Appl Environ Microbiol 63:2716–2721
HortonR. M.,
HuntH. D.,
HoS. N.,
PullenJ. K.,
PeaseL. R.1989; Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77:61–68
JohansenC.,
VerheulA.,
GramL.,
GillT.,
AbeeT.1997; Protamin-induced permeabilization of cell envelopes of gram-positive and gram-negative bacteria. Appl Environ Microbiol 63:1155–1159
KhasdanV.,
Ben-DovE.,
ManasherobR.,
BoussibaS.,
ZaritskyA.2001; Toxicity and synergism in transgenic Escherichia coli expressing four genes from Bacillus thuringiensis subsp. israelensis . Environ Microbiol 3:798–806
KnowlesB. H.,
EllarD. 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 Acta 924:509–518
KnowlesB. H.,
BlattM. R.,
TesterM.,
HorsnellJ. M.,
CarrollJ.,
MenestrinaG.,
EllarD. J.1989; A cytolytic δ -endotoxin from Bacillus thuringiensis var. israelensis forms cation-selective channels in planar lipid bilayers. FEBS Lett 244:259–262
LeeD.,
KatayamaH.,
AkaoT.,
MaedaM.,
TanakaR.,
YamashitaS.,
SaitohH.,
MizukiE.,
OhbaM.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 Acta154757–63
LiJ.,
KoniP. A.,
EllarD. J.1996; Structure of the mosquitocidal δ -endotoxin CytB from Bacillus thuringiensis sp. kyushuensis and implications for membrane pore formation. J Mol Biol 257:129–152
ManasherobR.,
ZaritskyA.,
Ben-DovE.,
SaxenaD.,
BarakZ.,
EinavM.2001; Effect of accessory proteins P19 and P20 on cytolytic activity of Cyt1Aa from Bacillus thuringiensis subsp. israelensis in Escherichia coli . Curr Microbiol 43:355–364
MargalithY.,
Ben-DovE.2000; Biological control by Bacillus thuringiensis subsp. israelensis . In Insect Pest Management: Techniques for Environmental Protection pp 243–301RechciglJ. E.,
RechciglN. A.
Boca Raton, FL: CRC Press;
MurphyL. D.,
ZimmermanS. B.2001; A limited loss of DNA compaction accompanying the release of cytoplasm from cells of Escherichia coli . J Struct Biol 133:75–86
ShellmanV. L.,
PettijohnD. E.1991; Introduction of proteins into living bacterial cells: distribution of labeled HU protein in Escherichia coli . J Bacteriol 173:3047–3059
ThieryI.,
DelecluseA.,
TamayoM. C.,
OrduzS.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 Microbiol 63:468–473
ThomasW. E.,
EllarD. J.1983b; Bacillus thuringiensis var. israelensis crystal δ -endotoxin: effects on insect and mammalian cells in vitro and in vivo . J Cell Sci 60:181–197
Van HelvoortJ. M. L. M.1996A cytometric study of nucleoid partitioning PhD thesis Institute for Molecular Cell Biology, Section of Molecular Cytology, Biocentrum Amsterdam, University of Amsterdam;
Van HelvoortJ. M. L. M.,
WoldringhC. L.1994; Nucleoid partitioning in Escherichia coli during steady-state growth and upon recovery from chloramphenicol treatment. Mol Microbiol 13:577–583
Van HelvoortJ. M. L. M.,
KoolJ.,
WoldringhC. L.1996; Chloramphenicol causes fusion of separated nucleoids in Escherichia coli K-12 cells and filaments. J Bacteriol 178:4289–4293
Van HelvoortJ. M. L. M.,
HulsP. G.,
VischerN. O. E.,
WoldringhC. L.1998; Fused nucleoids resegregate faster than cell elongation in Escherichia coli pbpB (Ts) filaments after release from chloramphenicol inhibition. Microbiology 144:1309–1317
VischerN. O. E.,
HulsP. G.,
WoldringhC. L.1994; object-image: an interactive image analysis program using structured point collection. Binary 6:160–166
VisickJ. E.,
WhiteleyH. R.1991; Effect of a 20-kilodalton protein from Bacillus thuringiensis subsp. israelensis on production of the CytA protein by Escherichia coli . J Bacteriol 173:1748–1756
WirthM. C.,
GeorghiouG. P.,
FedericiB. 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 A 94:10536–10540
WoldringhC. L.2002; The role of co-transcriptional translation and protein translocation (transertion) in bacterial chromosome segregation. Mol Microbiol Rev 45:17–29
WoldringhC. L.,
JensenP. R.,
WesterhoffH. V.1995; Structure and partitioning of bacterial DNA: determined by a balance of compaction and expansion forces?. FEMS Microbiol Lett 131:235–242
WuD.,
FedericiB. A.1993; A 20-kilodalton protein preserves cell viability and promotes CytA crystal formation during sporulation in Bacillus thuringiensis . J Bacteriol 175:5276–5280
YuY. M.,
OhbaM.,
GillS. S.1991; Characterization of mosquitocidal activity of Bacillus thuringiensis subsp. fukuokaensis crystal proteins. Appl Environ Microbiol 57:1075–1081
ZimmermanS. B.1993; Macromolecular crowding effects on macromolecular interactions: some implications for genome structure and function. Biochim Biophys Acta 1216:175–185
ZusmanD. R.,
CarbonellA.,
HagaJ. Y.1973; Nucleoid condensation and cell division in Escerichia coli MX74T2 ts52 after inhibition of protein synthesis. J Bacteriol 115:1167–1178