On the nature of the cytosine-methylated sequence in DNA of Bacillus brevis var. G.-B.

On growing the cells of Bacillus brevis S methionine-auxotroph mutant in the presence of [Me-3H]methionine, practically all the radioactivity incorporated into DNA is found to exist in 5-methylcytosine and N6-methyladenine. The analysis of pyrimidine isopliths isolated from DNA shows that radioactivity only exists in mono- and dinucleotides and the content of 5-methylcytosine in R-m5 C-R and R-m5 C-T-R oligonucleotides is equal. The analysis of dinucleotides isolated from DNA by means of pancreatic DNAase hydrolysis allows the nature of purine residues neighbouring 5-methylcytosine to be identified and shows that 5-methylcytosine localizes in G-m5 C-A and G-m5 C-Tr fragments. B. brevis S DNA methylase modifying cytosine residues recognizes the GCA/TGC degenerate nucleotide sequence which is a part of the following complementary structure with a two-fold rotational axis of symmetry: (5')...N'-G-C-T-G-C-N... (3') (3')...N-C-G-A-C-G-N'... (5') (Methylated cytosine residues are askerisked). Cytosine-modifying DNA methylase activity is isolated from B. brevis cells; it is capable of methylating in vitro homologous and heterologous DNA. Hence DNA in bacterial cells can be undermethylated. This enzyme methylates cytosine residues in native and denatured DNA in the same nucleotide sequences. Specificity of methylation of cytosine residues in vitro and in vivo does not depend on the nature of substrate DNA. DNA methylases of different variants of B. brevis (R, S, P+, P-)) methylate cytosine residues in the same nucleotide sequences. It means that specificity or methylation of DNA cytosine residues in the cells of different variants of B. brevis is the same.

DNA protection with the DNA methylase M . BbvI from Bacillus brevis var. GB against cleavage by the restriction endonucleases PstI and PvuII.

BamHI fragments of the Bacillus brevis var. GB plasmid pAD1 have been cloned in Escherichia coli HB101 using pBR322 plasmid as a vector. The analysis of the recombinant plasmids showed that additional PstI sites had appeared in cloned fragments of pAD1. Methylation of the recombinant plasmids in vitro by enzymes from B. brevis GB cells blocks cleavage at these additional PstI sites of cloned pAD1 fragments and at the PstI site of pBR322. Among DNA methylases of B. brevis GB, the cytosine DNA methylase M . BbvI is the most likely agent modifying the recognition sequences of PstI. The methylase can modify cytosine residues in PstI or PvuII sites if these recognition sequences are linked to G at 5'- or to C at 3'-termini. In particular, in vitro methylation of the SV40 DNA by B. brevis GB methylases protects one of the two PstI sites and two of the three PvuII sites. The described effect of the protection of the specific PstI and PvuII sites may be used for physical mapping of genomes and DNA cloning.

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.

Transposition of a DNA fragment flanked by two inverted Tn1 sequences.

The 32 Md fragment (derived from plasmid RP4::Tn1) carrying the Kmr gene and flanked by two inverted Tn1 elements is capable of recA-independent translocation to other plasmids. We designated this new transposon Tn1755. In various crosses, frequencies of Tn1755 transposition to plasmids Co1B-R3, R15 and F'Co1VBtrp varied from 2.5 to 90% of the frequencies of Tn1 transposition. Tn1755 can integrate into various sites of the recipient plasmids. We failed to observe transposition of another RP4::Tn1 fragment flanked by two opposingly oriented Tn1 transposons and harboring the Tcr gene. Presumably, to form a new transposable structure, other features must also be of importance.

Isolation of transposon TnA from plasmid RP4 carrying two copies of this element.

Employing heteroduplex and restriction analyses, two inverted copies of a 3.2.10(6) dalton transposable sequence, TnA, were found in RP4::TnA, a spontaneously arisen derivative of the plasmid RP4. Integration of the second copy of TnA causes loss of the conjugative properties of RP4. Both TnA sequences in RP4::TnA were localized and found to have opposite orientations. The DNA fragment corresponding to the individual transposon TnA was isolated after the endonuclease S1 digestion of RP4::TnA molecules annealed under conditions favoring intramolecular renaturation. The attempts to transform the cells of Escherichia coli QD5003, HB101[pCRI] and JC7623 with the isolated transposon were unsuccessful.

[Specificity of methylation of cytosine risidues in DNA of Bacillus brevis var. G-B]. / Spetsifichnost' metilirovaniia tsitozinovykh ostatkov v DNK Bacillus brevis var. G-B

On growing the cells of Bacillus brevis S methionine-auxotroph mutant in the presence of (methyl-3H)-methionine practically the total radioactivity included into DNA is found to exist in 5-methylcytosine (MC) and 6N-methyladenine (MA). The analysis of pyrimidine isopliths isolated from DNA shows that radioactivity only exists in mono- and dinucleotides and the content of MC in Pur-MC-Pur and Pur-MC-T-Pur oligonucleotides is equal. The analysis of dinucleotides isolated from DNA by means of pancreatic DNAase hydrolysis allows the nature of purine residues neighbouring with MC to be revealed and shows that MC localizes in G-MC-A and G-MC-T-Pu fragments. Bac. brevis S DNA-methylase modifying cytosine residues recognizes the GCAT GC degenerative nucleotide sequence which is a part of the following complementary structure with rotational symmetry: (5') ... N'--G--MC--T--G--C--N ... (3') (3') ... N--C--G--A--MC--G--N' ... (5') Cytosine modifying DNA-methylase activity is isolated from Bac. brevis cells; it is capable of methylating in vitro homologous and heterologous DNA. Hence, DNA in bacterial cells can be partially undermethylated. This enzyme methylates cytosine residues in native and deneaturated DNA in the same nucleotide sequences. As compared to the native DNA, the denaturated DNA is indicative of a decrease in the level of methylation of adenine, rather than cytosine residues. Specificity of methylation of cytosine residues in vitro and in vivo does not depend on the nature of substrate DNA (calf thymus, Pseudomonas aeruginosa etc.). DNA-methylases of different variants of Bac. brevis (R, S, P+, P-) methylate cytosine residues in the same nucleotide sequences. It means that specificity of methylation of DNA cytosine residues in the cells of different variants of Bac. brevis is the same.

[Some properties of 1 P+f bacteriophage for Bacillus brevis var. G.-B and its nucleic acid]. / Nekotorye svoistva bakteriofaga 1 P+f Bacillus brevis var. G.-B. i ego nukleinovoi kisloty

The 1 P+f phage, a virulent mutant of the moderate P+ phage for Bac. brevis var. G.-B., consists of a hexagonal head (90x90 nm) and a long non-contractile tail (340 nm). This phage is characterized by a relatively long latent period (90-110 min) and a low yield (40-50 particles per cell). The 1P+f phage is quite stable at pH values from 1 to 11, insensitive to osmotic shock, treatment with chloroform and acridine orange. The sensitivity of the phage to thermal treatment and UV-radiation has been studied. The nucleic acid of the P+f phage is double-stranded DNA of AT-type (GC equals 34.5 mole %) which contains 5-methylcytosine (0.18 mole %) and N6-methyladenine (0.32 mole%). The level of methylation of cytosine and adenine residues in DNA of the 1 P+f phage does not depend on the host studied (Bac. brevis, P- and S variants). The specificity of methylation of cytosine residues in the S and P- cells appears to be the same. DNA of the 1 P+f phage strongly differs from DNA of the host in nucleotide composition (GC equals 45.7 mole %). Nevertheless, phage DNA is very similar to DNA from Bac. subtilis in the character of pyrimidine distribution (the amount of different pyrimidine isopliths). This may testify to a somewhat common character of the nucleotide sequence organization in DNA of the phage and its host.

[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.

[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.

[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.

[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.

[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.

[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.

[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.

[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.

Isolation and characterization of plasmid from the Bacillus brevis var. G.-B. cells.

The plasmid designated pAD1 was isolated from the cells of four variants of Bacillus brevis var. G.-B. The plasmid DNA has a molecular weight of about 47.1 x 10(6) daltons and contains 43.4 mole % G+C. The bulk of pAD1 DNA (96--98%) is associated with the fraction of chromosome DNA and membranes. Restriction endonucleases Sma I, Sal I and Bam HI cleaved the plasmid DNA into two, two and six fragments, respectively. The cleavage map of the pAD1 genome has been constructed for these three endonucleases. Restriction enzymes Eco RI, Hind III, Kpn I and Pst I hydrolized the plasmid DNA into 16, 21, 10 and 9 fragments, respectively. The presence of repeated sequences in the plasmid genome was shown based on pAD1 DNA cleavage by these endonucleases.