Monoclonal Antibody HlyIIC­15 to C-End Domain HlyII B. cereus Interacts with the Trombin Recognition Site.

Among the panel of monoclonal antibodies to the recombinant protein HlyIICTD Bacillus cereus an antibody was found capable of forming an immune complex with a thrombin recognition region, the amino acid sequence of which is located inside the recombinant HlyIICTD. Localization of the epitope was carried out using peptide phage display methods, as well as enzyme immunoassay and immunoblotting for interaction with recombinant proteins, either containing or not containing individual components HlyIICTD. The identified epitope is located in the region of the thrombin site and retains the ability to interact with the antibody after the proteolyotic attack of the protein by thrombin.

[Purification of recombinant Bacillus cereus ResD-ResE proteins expressed in Escherichia coli strains].

Recombinant E. coli strains expressing the Bacillus cereus ATCC 14579T resD and resEgenes fused with the ubiquitin gene were constructed, and purification of the ResD and ResE proteins was performed. The approach used in the study allowed us to increase the protein yield of the electrophoretic homogeneous ResD andResE proteins without denaturation steps up to 150 mg per gram of wet cell weight.

Binding of DNA methyltransferase M.Ecl18kI [corrected] to operator-promoter region decreases its methylating activity.

The type II bifunctional DNA methyltransferase (MTase) Ecl18 that is able to control transcription of its own gene was studied kinetically. Based on initial velocity dependences from S-adenosyl-L-methionine (AdoMet) and target DNA and substrate preincubation assays, it is proposed that the enzyme apparently works by a rapid equilibrium ordered bi-bi mechanism with DNA binding first. By measuring the enzyme activity depending on DNA and AdoMet at different fixed concentrations of the operator sequence oligonucleotide, it was found that its binding has noncompetitive inhibitory effect on Ecl18 MTase activity.

SsoII-like DNA-methyltransferase Ecl18kI: interaction between regulatory and methylating functions.

The interaction of DNA-methyltransferase Ecl18kI (M.Ecl18kI) with a fragment of promoter region of restriction-modification system SsoII was studied. It is shown that dissociation constants of M.Ecl18kI and M.SsoII complexes with DNA ligand carrying a regulatory site previously characterized for M.SsoII have comparable values. A deletion derivative of M.Ecl18kI, Delta(72-379)Ecl18kI, representing the N-terminal protein region responsible for regulation, was obtained. It is shown that such polypeptide fragment has virtually no interaction with the regulatory site. Therefore, the existence of a region responsible for methylation is necessary for maintaining M.Ecl18kI regulatory function. The properties of methyltransferase NlaX, which is actually a natural deletion derivative of M.Ecl18kI and M.SsoII lacking the first 70 amino acid residues and not being able to regulate gene expression of the SsoII restriction-modification system, were studied. The ability of mutant forms of M.Ecl18kI incorporating single substitutions in regions responsible for regulation and methylation to interact with both sites of DNA recognition was characterized. The data show a correlation between DNA-binding activity of two M.Ecl18kI regions-regulatory and methylating.

[Contraction of the disordered loop located within C-terminal domain of the transcriptional regulator HlyIIR causes its structural rearrangement].

HlyIIR is the negative transcriptional regulator of the hemolysin II gene from Bacillus cereus. Previous X-ray studies showed that HlyIIR contains a disordered loop (a.a. 170-185) located within the C-terminal domain near dimerization interface. To understand the influence of this region on HlyIIR properties and for a potential improvement in the crystallogenesis of the HlyIIR, we constructed a mutant of HlyIIR in which this disordered region is substituted by a single alanine residue. Biochemical analysis of the mutant indicated that it still forms a dimer but the DNA-binding activity is lost. HlyIIR mutant displayed improved crystallization properties and its structure was determined by X-ray crystallography at 2.1 A resolution. Unexpectedly, the structure shows that the HlyIIR mutant forms an alternative dimer with subunits rotated by 160 degrees. Moreover, there are also changes in the conformation of individual subunits. These dramatic structural rearrangements are caused by changes in the conformation of the segment Pro161-Ser169. We conclude that correct conformation of this segment is principal for maintaining the structure and activity of HlyIIR.

Pore-forming proteins and adaptation of living organisms to environmental conditions.

Pore-forming proteins are powerful "tools" for adaptation of living organisms to environmental conditions. A wide range of these proteins isolated from various sources, from viruses to mammals, has been used for the analysis of their role in the processes of intra- and inter-species competition, defense, attack, and signaling. Here we review a large number of pore-forming proteins from the perspective of their functions, structures, and mechanisms of membrane penetration. Various mechanisms of cell damage, executed by these proteins in the course of formation of a pore and after its passing to conducting state, have been considered: endo- and exocytosis, lysis, necrosis, apoptosis, etc. The role of pore-forming proteins in evolution is discussed. The relevance of practical application of pore formers has been shown, including application in nanotechnological constructions.

[Regulation of gene expression in type II restriction-modification system].

Type II restriction-modification systems are comprised of a restriction endonuclease and methyltransferase. The enzymes are coded by individual genes and recognize the same DNA sequence. Endonuclease makes a double-stranded break in the recognition site, and methyltransferase covalently modifies the DNA bases within the recognition site, thereby down-regulating endonuclease activity. Coordinated action of these enzymes plays a role of primitive immune system and protects bacterial host cell from the invasion of foreign (for example, viral) DNA. However, uncontrolled expression of the restriction-modification system genes can result in the death of bacterial host cell because of the endonuclease cleavage of host DNA. In the present review, the data on the expression regulation of the type II restriction-modification enzymes are discussed.

DNA methylation at the CfrBI site is involved in expression control in the CfrBI restriction-modification system.

We have previously found that genes of the CFR:BI restriction-modification (R-M) system from Citrobacter freundii are oriented divergently and that their promoter regions overlap. The overlapping promoters suggest regulation of gene expression at the transcriptional level. In this study the transcription regulation of CFR:BI R-M genes was analyzed in vivo and in vitro in Escherichia coli. It was shown that in the presence of CFR:BI methyltransferase (M.CFR:BI), cell galactokinase activity decreases 10-fold when the galactokinase gene (galK) is under the control of the cfrBIM promoter and increases 20-fold when galK is under the control of the cfrBIR promoter. The CFR:BI site, proven to be unique for the entire CFR:BI R-M gene sequence, is located in the -35 cfrBIM promoter region and is in close vicinity of the -10 cfrBIR promoter region. A comparison of the cfrBIM and the cfrBIR promoter activities in the in vitro transcription system using methylated and unmethylated DNA fragments as templates demonstrated that the efficiency of CFR:BI R-M gene transcription is regulated by enzymatic modification at the N-4-position of cytosine bases of the CFR:BI site by M.CFR:BI. From the results of the in vivo and in vitro experiments we suggest a new model of gene expression regulation in type II R-M systems.

[Laccase of the lignolytic fungus Trametes hirsuta: purification and characterization of the enzyme, and cloning and primary structure of the gene].

The main physicochemical characteristics of the major isoform of the laccase secreted by the fungu, Trametes hirsuta 072 were studied. The enzyme belongs to the group of high redox potential laccases (E(T1) = 790 +/- 5), and it oxidizes with high efficiency various substrates of phenolic nature. The gene of this isoform was cloned, and its nucleotide sequence was determined. The length of the complete gene is 2134 bp. It comprises 11 exons and 10 introns. Analysis of the amino acid sequence of T. hirsuta 072 laccase demonstrated a high homology (to 96.9%) to the other laccases secreted by fungi of the genus Trametes.

Overproduction of the EcoRII endonuclease and methylase by Escherichia coli strains carrying recombinant plasmids constructed in vitro.

Recombinant DNA molecules were constructed from the plasmid pIL203 and the EcoRI-fragment of N3 plasmid containing EcoRII endonuclease and methylase genes and also a gene for resistance to sulfanilamide. The pIL203 plasmid, used as a vector, consisted of the Bam HI-EcoRI-fragment of the plasmid pBR322 conferring resistance to ampicillin and the Bam HI-EcoRI-fragment of lambda phage containing promoters, a thermosensitive mutation in the cI gene and a suppressible amber mutation in the cro gene. Ampicillin-sulfanilamide-resistant clones were selected and tested for their restriction and modification phenotype. The recombinant plasmid DNA, isolated from ApRSuR-resistant clones, which restricted and modified phage lambda imm21 with EcoRII specificity, had the EcoRI-fragment with EcoRII genes in a single orientation. The recombinant plasmid pSK323 was transferred into E. coli strains with su-, su1, su2 or su3 phenotypes. The synthesis of products of EcoRII genes by these strains grown at 37 degrees C is increased by 10--50-fold.

Complete nucleotide sequence of the Hsd plasmid pECO29 and identification of its functional regions.

The complete nucleotide sequence of the Hsd plasmid pECO29 has been determined. The plasmid DNA consists of 3895 base pairs. These include 4 genes and 5 sites. Two genes encoding the proteins (restriction endonuclease and DNA methyltransferase) have been fully characterized. The pECO29 comprises a Co1El-type replication system coding for untranslated genes RNAI and RNAII, the emr recombination site containing palindromic sequences and involved in stable maintenance of the plasmid, two pseudo oriT sites homologous to the oriT site of R64 and F plasmids, as well as the bom locus of a Co1El-like plasmid. There are no genes involved in the mobilization of pECO29 plasmid.

Cloning of a recA-like gene of Proteus mirabilis.

A gene of Proteus mirabilis that can substitute for functions of the recA gene of Escherichia coli has been cloned into the plasmid pBR322, using shotgun experiments. The recA-like gene (recAp.m.) has been localized by restriction mapping within a 1.5-Md PstI fragment that is a part of two cloned HindIII fragments of the chromosome of P. mirabilis. The restriction map of the recAp.m. gene differs from that of the recA gene of E. coli. Functionally, the recombinant plasmids containing the recAp.m. gene restore a nearly wild-type level of UV-resistance to several point and deletion mutants in the recA gene of E. coli.

Cloning and sequences of the genes encoding the CfrBI restriction-modification system from Citrobacter freundii.

The genes encoding the CfrBI restriction and modification (R-M) systems from Citrobacter freundii and recognizing the sequence 5'-CCWWGG-3' (W = A or T) were cloned in Escherichia coli McrBC- cells. The nucleotide (nt) sequences of the genes were determined. Two large open reading frames were found. Deletion analysis showed that one of them [1128 nt coding for 376 amino acids (aa)] corresponds to a methyltransferase (MTase)-encoding gene and the other (1065 nt coding for 355 aa) to a restriction endonuclease-encoding gene. The genes are oriented divergently and separated by 76 bp. A CfrBI site (5'-m4CCATGG) was found in the intergenic region of the cfrBIRM genes. Analysis of the deduced aa sequence of M.CfrBI made it possible to determine the typical features of a m4C-specific MTase. Limited homology between the M.CfrBI and R.CfrBI proteins was also found.

The cleavage sites and localization of genes encoding the restriction endonucleases Eco1831I and EcoHI.

The restriction endonucleases Eco1831I and EcoHI cleave before the first 5'-cytosine in the recognition sequence 5'-decreases CCSGG--3'/3'--GGSCC increases-5' (where S = G or C), generate 5-base 5' cohesive ends, and are encoded by homologous plasmids that are restricted in McrA+ hosts. Thus, they differ in their cleavage specificity from that of the BcnI isoschizomer, which cleaves after the second 5' cytosine.

Cloning and sequence analysis of the plasmid-borne genes encoding the Eco29kI restriction and modification enzymes.

The Eco29kI restriction-modification system (RMS2) has been found to be localized on the plasmid pECO29 occurring naturally in the Escherichia coli strain 29k (Pertzev, A.V., Ruban, N.M., Zakharova, M.V., Beletskaya, I.V., Petrov, S.I., Kravetz, A.N., Solonin, A.S., 1992. Eco29kI, a novel plasmid encoded restriction endonuclease from Escherichia coli. Nucleic Acids Res. 20, 1991). The genes coding for this RMS2, a SacII isoschizomer recognizing the sequence CCGCGG have been cloned in Escherichia coli K802 and sequenced. The DNA sequence predicts the restriction endonuclease (ENase) of 214 amino acids (aa) (24,556 Da) and the DNA-methyltransferase (MTase) of 382 aa (43,007 Da) where the genes are separated by 2 bp and arranged in tandem with eco29kIR preceding eco29kIM. The recombinant plasmid with eco29kIR produces a protein of expected size. MEco29kI contains all the conserved aa sequence motifs characteristic of m5C-MTases. Remarkably, its variable region exhibits a significant similarity to the part of the specific target-recognition domain (TRD) from MBssHII--multispecific m5C-MTase (Schumann, J.J., Walter, J., Willert, J., Wild, C., Koch D., Trautner, T.A., 1996. MBssHII: a multispecific cytosine-C5-DNA-methyltransferase with unusual target recognizing properties. J. Mol. Biol. 257, 949-959), which recognizes five different sites on DNA (HaeII, MluI, Cfr10I, SacII and BssHII), and the comparison of the nt sequences of its variable regions allowed us to determine the putative TRD of MEco29kI.

The Ecl18kI restriction-modification system: cloning, expression, properties of the purified enzymes.

Ecl18kI is a type II restriction-modification system isolated from Enterobacter cloaceae 18kI strain. Genes encoding Ecl18kI methyltransferase (M.Ecl18kI) and Ecl18kI restriction endonuclease (R.Ecl18kI) have been cloned and expressed in Escherichia coli. These enzymes recognize the 5'.../CCNGG...3' sequence in DNA; M.Ecl18kI methylates the C5 carbon atom of the inner dC residue and R.Ecl18kI cuts DNA as shown by the arrow. The restriction endonuclease and the methyltransferase were purified from E. coli B834 [p18Ap1] cells to near homogeneity. The restriction endonuclease is present in the solution as a tetramer, while the methyltransferase is a monomer. The interactions of M.Ecl18kI and R.Ecl18kI with 1,2-dideoxy-D-ribofuranose containing DNA duplexes were investigated. The target base flipping-out mechanism is applicable in the case of M.Ecl18kI. Correct cleavage of the abasic substrates by R.Ecl18kI is accompanied by non-canonical hydrolysis of the modified strand.

Complete nucleotide sequence and molecular characterization of hemolysin II gene from Bacillus cereus.

Hemolysin II gene from Bacillus cereus VKM-B771 has been sequenced. The deduced primary translation product consists of 412 amino acid residues which corresponds to the protein with an M(r) of 45.6 kDa. The predicted mature Hly-II protein (residues 32 to 412) is of 42.3 kDa, which is in close agreement with the mini-cell electrophoresis analysis. Hly-II deletion variant lacking 96 C-terminal residues still has hemolytic activity. The protein primary structure analysis revealed no homology with any known Bacillus cytolysins. Significant general homology (31-28% identity) was found between the hemolysin II and Staphylococcus aureus alpha-toxin, gamma-hemolysin (HlgB), and leukocidins (LukF, LukF-R, LukF-PV). The data suggest that hemolysin II belongs to the group of beta-channel forming cytolysins.

[Properties of the replicator region of the natural plasmid pLG13 containing genes of the EcoRV restriction-modification system]. / Svoistva raiona replikatora prirodnoi plazmidy pLG13, soderzhashchei geny sistemy restriktsii-modifikatsii EcoRV.

The previously constructed plasmid pILRV8 that induces endonuclease EcoRV gene overexpression kills cells of some E. coli strains under the induction of this enzyme synthesis. Cell transformation by natural plasmid pLG13 carrying genes of the EcoRV restriction--modification system was found to appreciably enhance cell viability ("survival") under endonuclease overproduction. A plasmid pLG13 region located in immediate proximity to the methylase gene was shown to be responsible for the above effect. This region was also capable for autonomous replication. The analysis of the DNA primary structure in the found replicator region allowed to refer the pLG13 to ColE1 family plasmids. Perturbations in the region lead to loss of the "survival" effect and change of the plasmid replicative properties. A relationship between the replicon elements, the EcoRV genes region and "survival" effect is discussed. Based on the replicon found multicopy vector molecules have been constructed.

[C1 and cro repressors of lambda phages. Isolation of strains of bacteriophage lambda imm434 superproducing repressor cro]. / Repressory c1 i cro liambdoidnykh fagov. II. Poluchenie shtammov-sverkhprodutsentov repressora cro bakteriofaga lambda imm434.

A collection of recombinant plasmids directing the increased synthesis of the cro-repressor of lambda imm434 bacteriophage under control of the lacZ promoter of Escherichia coli has been constructed. About 87 000 molecules of cro-repressor monomer per cell, e. g. 0,7% of total cell protein are synthesized by the overproducers. Strain bearing one of such plasmids--pBS71-1. Several recombinant plasmids in which the cro-gene is placed under the control of early genes promoter of lambda phage (pl, pr, prm) have been also prepared. Plasmid pIL206, constructed on the basis of pIL203 vector, was used as a source of lambda-promoters.

[EcoRV restrictase: physical and catalytic properties of homogenous enzyme]. / Restriktaza EcoRV: fizicheskie i kataliticheskie svoistva gomogennogo fermenta.

With the use of the strain-overproducer restriction endonuclease R.EcoRV was isolated and purified to homogeneity. The molecular mass of the enzyme was determined by gel filtration and polyacrylamide gel electrophoresis to be 25 000 daltons. According to the data of immunological tests R.EcoRV differs in its antigenic characteristics from restriction endonucleases R.EcoRI and R.EcoRII. Dependence of enzyme activity on pH, ionic strength, temperature, presence of divalent cations (Mn2+, Mg2+, Co2+, Zn2+, Ni2+ and Cd2+) and organic solvents (glycerol, dimethylsulfoxide, ethanol) has been studied. It was shown that under conditions of replacement of Mg2+ for Mn2+ or after addition of organic solvents relaxation of R.EcoRV specificity takes place. It was shown also that R.EcoRV is able to digest T-even bacteriophage DNAs with different types and extents of modification. DNA modified by the action of MR.EcoRV system in vivo is susceptible to R.EcoRV in vitro. Under conditions of relaxed specificity noncanonical sites are susceptible to R.EcoRV attack. The fragments resulted may be cloned in canonical pBR322 EcoRV site.