Structure of Cry3A delta-endotoxin within phospholipid membranes.

Interaction of delta-endotoxin and its proteolytic fragments with phospholipid vesicles was studied using electron microscopy, scanning microcalorimetry, and limited proteolysis. It was shown that native protein destroys liposomes. The removal of 4 N-terminal alpha-helices and the extreme 56 C-terminal amino acid residues did not affect this ability. The results obtained by limited proteolysis of delta-endotoxin bound to lipid vesicles show essential conformational changes in three or four N-terminal helices and in the C-terminal region. The calorimetric method used in this study provides a unique possibility for the validation of existing models of protein binding and for a more accurate determination of the regions where conformational changes take place. It was found that the binding of the protein to model liposomes does not alter its structure in the regions starting with the fourth alpha-helix of domain I. This can be concluded from the fact that the activation energy of denaturation of the protein remains unchanged upon its binding to the phospholipid membranes. A new structural model has been proposed which agrees with the data obtained.

[Characteristic of mutants of Pseudomonas putida IPM-36 recombinant strains, selected by anticipating growth on a media containing an inducer of cry3A gene expression]. / Kharakteristika mutantov rekombinantnogo shtamma Pseudomonas putida IPM-36, otobrannykh po priznaku operezhaiushchego rosta na srede s induktorom ékspressii cry3A-gena.

Induction of the expression of the delta-endotoxin gene from Bacillus thuringiensis var. tenebrionis in the recombinant strain Pseudomonas putida IPM-36 negatively affected the viability and the growth rate of the culture. In order to optimize the insecticide production by the recombinant strain, mutant clones exhibiting anticipating growth on an inducer-containing medium were selected and studied. These clones differed in such aspects as the localization of mutations (either in plasmid pBTN11, carrying the cry3A gene, or in the chromosome), growth rate, or the level of delta-endotoxin synthesis after induction. Several mutants obtained proved much superior to P. putida IPM-36 in their structural and segregation stability, although they were as efficient as the original strain with respect to the production of the insecticide (protei Cry3A).

Transition state of the rate-limiting step of heat denaturation of Cry3A delta-endotoxin.

Heat denaturation of Cry3A delta-endotoxin from Bacillus thuringiensis var. tenebrionis and its 55 kDa fragment was studied by differential scanning microcalorimetry at low pH. Analysis of the calorimetric data has shown that denaturation of Cry3A delta-endotoxin is a nonequilibrium process at heating rates from 0. 125 to 2 K/min. This means that the stability of delta-endotoxin (the apparent temperature of denaturation Tm) under these conditions is under kinetic control rather than under thermodynamic control. It has been shown that heat denaturation of this protein is a one-step kinetic process. The enthalpy of the process and its activation energy were measured as functions of temperature. The data obtained allow confirmation of the fact that the conformation of delta-endotoxin at the transition state only slightly differs from its native conformation with respect to compactness and extent of hydration. The comparison of the activation energy for intact delta-endotoxin and the 55 kDa fragment showed that the transition of the molecule to a transition state does not cause any changes in the conformation of three N-terminal alpha-helices. Complete removal of the N-terminal domain of delta-endotoxin and 40 amino acids from the C-terminus beta-sheet domain III causes an irreversible loss of the tertiary structure. Thus, during protein folding the nucleation core determining protein stability does not involve its three initial alpha-helices but may include the remaining alpha-helices of the N-terminal domain. The functional significance of peculiarities of structure arrangement of the delta-endotoxin molecule is discussed.

[Features of the production of CryIIIA delta-endotoxin from Bacillus thuringiensis in a recombinant strain of Pseudomonas putida under control of Pm and xylS regulatory elements]. / Osobennosti produktsii CryIIIA delta-éndotoksina Bacillus thuringiensis v rekombinantnom shtamme Pseudomonas putida pod kontrolem reguliatornykh élementov Pm i xylS.

The addition of 3-methyl benzoate to the culture of the recombinant strain Pseudomonas putida IPM-36 (bearing the cryIIIA gene of B. thuringiensis subsp, tenebrionis under the control of the Pm promoter and the regulator gene xylS) slowed down the growth rate of the recombinant strain and increased, under non-selective conditions, the number of plasmid-free cells. Intense synthesis of the Coleoptera-specific delta-endotoxin encoded by the cryIIIA gene began 6-8 h after the addition of the inducer 3-methyl benzoate, no matter whether it was added in the early or late logarithmic phase. Maximal production of endotoxin (0.5-0.6 g/l) was observed when the inducer was added in the early logarithmic phase (3 h of growth). Overproduction of delta-endotoxin impaired cell division, so that filamentous cells became predominant in the culture. delta-Endotoxin accumulated in overproducing cells as irregular crystalloid inclusions.

[Thermodynamic analysis of domain organization of Bacillus thuringiensis toxins]. / Termodinamicheskii analiz domennoi organizatsii toksinov Bacillus thuringiensis.

The structure of delta-endotoxins CryIA(c) and CryIIIA from Bacillus thuringiensis was studied by differential scanning microcalorimerty. The analysis of molecular melting showed that the N- and C-terminal halves of the CryIA(c) protoxin from B. thuringiensis subspecies kurstaki HD-73 are thermodynamically independent subunits, with the C-terminal fragment being denatured at a much lower temperature than the N-terminal fragment. The tertiary structure of the N-terminal fragment undergoes no changes during the protoxin-toxin transition. The melting of the native structure of CryIA(c) at pH 9.7-11.0 suggests that it consists of two domains. In CryIIIA from B. thuringiensis subspecies tenebrionis, the transition from the native to denatured state under alkaline conditions (pH 9.7-11.0) proceeds by the "two-state" principle; i.e., the protein melts as one cooperative domain. The melting of the CryIIIA toxin at pH 2.2-3.5 is described by two transitions overlapping by temperatures, indicating the presence of two domains.

[Cloning and expression of determinants of Bacillus anthracis protective antigen in Escherichia coli, Bacillus subtilis, and Bacillus anthracis cells]. / Klonirovanie i ékspressiia determinanty protektivnogo antigena Bacillus anthracis v kletkakh Escherichia coli, Bacillus subtilis i Bacillus anthracis.

A determinant for a protective antigen (pag) of Bacillus anthracis STI has been cloned. Its expression in Escherichia coli, Bacillus subtilis and Bacillus anthracis cells has been studied. The hybrid plasmids were obtained carrying the different fragments of the gene. The plasmids pPA2 and pPA3 having the 3'-end fragment of pag deleted (the size of 1 kb) still code for a part of protective antigen preserving the immunological and protective properties.

[Study of the nature of determinants for synthesis of delta-endotoxin and beta-exotoxin in Bacillus thuringiensis subsp. thuringiensis strains--bitoxybacillin producers]. / Izuchenie prirody determinant sinteza delta-éndotoksina i beta- ékzotoksina v shtammakh Bacillus thuringiensis subsp. thuringiensis--produtsentakh bitoksibatsillina.

The growth of Bacillus thuringiensis subsp. thuringiensis strains (BT) 202, 1140, 98 producing bitoxybacillin in the presence of novobiocin, mitomycin C and at high temperature 43 degrees C resulted in obtaining of the mutants for the synthesis of crystalline protein (Cry) and exotoxin (Exo). Analysis of the plasmid content of the mutants has shown the Cry Exo phenotyre to correlate with the loss of 55 MD plasmid in the strain BT202 and the loss of 60MD plasmid in BT1140. The transfer of these plasmids into Bacillus cereus leads to the transfer of endo- and exotoxin production properties. The synthesis of Cry in BT98 is controlled by the 56MD plasmid, while the synthesis of Exo is encoded by the chromosome or plasmid that cannot be eliminated or transferred into other strains. Localization of Cry on the 55 and 56 MD plasmids in BT202 and BT98 is confirmed by the hybridization of the plasmid DNA with the DNA-probe.

[Expression of delta-endotoxin gene from Bacillus thuringiensis var. berliner 1715 in Escherichia coli and Bacillus megaterium cells]. / Proiavlenie gena delta-éndotoksina Bacillus thuringiensis var. berliner 1715 v kletkakh Escherichia coli i Bacillus megaterium.

Effects of the structure of plasmids carrying the cloned delta-endotoxin gene (tox) ot Bacillus thuringiensis and of the culture media on the expression of the gene have been studied. The DNA region located upstream from the crystal protein gene promoter inhibited the expression of the tox gene in Escherichia coli cells, but enhanced the expression in Bacillus megaterium cells grown in LB medium. The upstream DNA region did not affect the tox gene expression when Bacillus megaterium cells were grown in SSM medium.

[Structure and biological features of the temperate phage of Bacillus thuringiensis var. galleriae 69/9, sensitive to chloroform]. / Strukturai biologicheskie osobennosti chuvstvitel'nogo k khloroformu umerennogo faga Bacillus thuringiensis var. galleriae 69/6.

A new temperate bacteriophage designated Px1 has been isolated from the culture of Bacillus thuringiensis var. galleriae 69/6 producing enthobacterin. The bacteriophage belongs to morphological group B1 in accordance with the classification by D. Reanney and H. Ackerman. The bacteriophage head has an isometric multifaceted form with 40 nm diameter. The length of its noncontractile transversely lined tail is 130 nm. High sensitivity to chloroform is peculiar of the phage. The lytical specter of the phage Px1 has been studied. The phage is shown to be capable of efficient transduction of plasmids between the bacteria of Bacillus cereus group.

[Comparative analysis of the structure of incompatibility plasmids N, P and W]. / Sravnitel'nyi analiz plazmid N, P i W grupp nesovmestimosti.

Evolutionary relationships of the IncN plasmid R15 and other broad host range plasmids (IncN plasmids N3 and R46, IncP plasmids RP4 and R906, IncW plasmids Sa and R388) were studied by Southern blot hybridization technique. The IncN plasmids were shown to harbour homologous determinants for replication and conjugation. No homology was found between the rep and tra genes in R15 and in the IncW and IncP plasmids, respectively. The second rep region of the N3 plasmid is distinctive from the corresponding determinants in the IncN plasmids. Homology was demonstrated for the plasmid genes that mediate restriction and modification in R15 and N3, mercury resistance in R15 and R906, sulfanilamide resistance in R15, N3, R46, Sa, R388, and R906, streptomycin resistance in R15, R46 and Sa. The latter genes are different from the R906 SmR gene. In addition to the three known mobile elements in the plasmid R15, the fourth one (IS46) that is a part of the transposon Tn2353 was identified in this study. Besides, the third copy of this insertion sequence was found in the N3 plasmid.

Physical and genetic structure of the IncN plasmid R15.

Restriction sites for seven hexanucleotide-specific endonucleases were located on the map of the conjugative IncN plasmid R15 (SmrSurHgr, 62.3 kb). The distribution of the cleavage sites is strongly asymmetric. Twenty-eight of thirty-four sites for BamHI, EcoRI, HindIII, SalI, SmaI, and PstI were located close to or within the sequences of an IS5-like element and the transposons Tn2353 and Tn2354. By analysis of R15::Tn1756 deletion derivatives and recombinant plasmids harboring R15 fragments, the genetic determinants for the streptomycin, sulfonamide, and mercury resistances were mapped, as well as the regions necessary for EcoRII restriction-modification and for plasmid replication and conjugation. The features of physical and genetic structures of the plasmid R15 and other IncN plasmids are discussed.

[Physical and genetic organization of the plasmid R15]. / Fizicheskaia i geneticheskaia organizatsiia plazmidy R15.

The conjugative IncN plasmid R15 (SmrSurHgr, 62.3 kb) is cleaved by the hexanucleotide-specific endonucleases BglII, HindIII, EcoRI, BamHI, SmaI, SalI, PstI and XhoI into 9, 9, 6, 5, 4, 4, 4 and 2 fragments, respectively. The restriction sites were located on the physical map of the R15 genome. Distribution of the cleavage sites is strongly asymmetric. 28 of 32 sites for BamHI, EcoRI, HindIII, SalI, SmaI and PstI were located close to or within the sequences of transposable elements Tn2353 and Tn2354. According to the results of analysis of R15::Tn1756 deletion derivatives and recombinant plasmids harboring fragments of R15, the genetic determinants for resistance to Sm, Su and Hg were mapped, as well as the regions necessary for EcoRII restriction--modification and for plasmid replication and conjugation. The features of physical and genetic structures of R15 and other IncN plasmids are discussed.

IS8- and Tn2353-mediated cointegration of the plasmids R15 and RP4::Tn1.

The plasmids R15 and RP4::Tn1 form fused structures (85 Md and 92 Md cointegrates). The cointegrates do not resolve practically in recA- Escherichia coli cells and have a mean life-time of more than 50 generations in a recA+ background. The 85 Md cointegrates were generated at a frequency of 4 x 10(-4) per R15 transconjugant during a mating between E. coli [R15; RP4::Tn1] and E. coli [F' ColVBtrp :: Tn1755 ]. These plasmids carry two directly repeated copies of the mobile element IS8 at the junctions between R15 and RP4::Tn1. The transposition of IS8 from RP4::Tn1 to the R15 plasmid and the formation of hybrid molecules promoted by this process appear to be induced by the IS8 element of the Tn1755 structure during or after conjugal transfer of F' ColVBtrp :: Tn1755 into E. coli [R15; RP4::Tn1] cells. The formation of the 92 Md cointegrates occurs at a frequency of 2 x 10(-5). The fused molecules of R15 and RP4::Tn1 carry two direct copies of an 8.65 Md R15 fragment at the junctions between these replicons. The fragment has specific features of a new transposon. This element designated Tn2353 determines resistance to Hg, Sm and Su and contains two sites for each BamHI, Bg/II and Sa/I and three sites for both EcoRI and PstI. The physical map and some other characteristics of Tn2353 are presented.

Transposition of DNA fragments flanked by two inverted Tn1 sequences: translocation of the plasmid RP4::Tn1 region harboring the Tcr marker.

We have demonstrated the possibility of transposition of the plasmid RP4::Tn1 fragment (21.2 kb) carrying the tetracycline resistance (Tcr) gene and flanked by two Tn1 copies. The new transposon, designated Tn1756, bears lethal genes that kill host cells. Therefore, its transposition can only be revealed in the presence of lethality-compensating helper regions of the plasmid RP4. Thus, RP4::Tn1 consists of two transposons, Tn1755 (Tn1-Kmr-Tn1) and Tn1756 (Tn1-Tcr-Tn1), sharing the Tn1 sequences. Both of these transposons are capable of recA-independent translocation to other plasmids. Therefore, transposition of DNA fragments flanked by two inverted Tn1 sequences does not depend on Tn1 orientation.

[Site-specific endonuclease BmeI from Bacillus megaterium 216]. / Sait-spetsificheskaia éndonukleaza BmeI iz Bacillus megaterium 216.

A site-specific endonuclease BmeI has been isolated from Bacillus megaterium 216 by gel filtration on ultragel AcA-44 with a subsequent chromatography on heparin-sepharose 6B. On the double-stranded DNA the endonuclease recognizes the pentanucleotide sequence (Formula: see text); and hydrolyzes it in the points shown by arrows. At gel filtration the endonuclease is eluted in the volume corresponding to a molecular mass of 60 000.