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A S Solonin

Publications and source records attributed to A S Solonin.

At least 19 recordsLinked to original sources
PubMed2003

Structural plasmid evolution as a result of coupled recombinations at bom and cer sites.

We have studied the recombination of plasmids bearing bom and cer sites. The bom ( basis of mobilization) site is required for conjugative transfer, while the cer ( Col E1 resolution) site is involved in the resolution of plasmid multimers, which increases plasmid stability. We constructed a pair of parent plasmids in such a way as to allow us select clones containing recombinant plasmids directly. Clone selection was based on the McrA sensitivity of recipient host DNA modified by M. Ecl18kI, which is encoded by one of the parent plasmids. The recombinant plasmid contains segments originating from both parental DNAs, which are bounded by bom and cer sites. Its structure is in accordance with our previously proposed model for recombination mediated by bom and cer sequences. The frequency of recombinant plasmid formation coincided with the frequency of recombination at the bom site. We also show that bom-mediated recombination in trans, unlike in cis, is independent of other genetic determinants on the conjugative plasmids.

Base Sequence↗
PubMed2002

Characterization of pECL18 and pKPN2: a proposed pathway for the evolution of two plasmids that carry identical genes for a Type II restriction-modification system.

The primary structures of the plasmids pECL18 (5571 bp) and pKPN2 (4196 bp) from Escherichia coli and Klebsiella pneumoniae, respectively, which carry genes for a Type II restriction-modification system (RMS2) with the specificity 5'-CCNGG-3', were determined in order to elucidate the structural relationship between them. The data suggest a possible role for recombination events at bom (basis of mobility) regions and the sites of resolution of multimer plasmid forms (so-called cer sequences) in the structural evolution of multicopy plasmids. Analysis of the sequences of pECL18 and pKPN2 showed that the genes for RM* Ecl18kI and RM* Kpn2kI, and the sequences of the rep (replication) regions in the two plasmids, are almost identical. In both plasmids, these regions are localized between the bom regions and the cer sites. The rest of the pECL18 sequence is almost identical to that of the mob (mobilization) region of ColE1, and the corresponding segment of pKPN2 is almost identical to part of pHS-2 from Shigella flexneri. The difference in primary structures results in different mobilization properties of pECL18 and pKPN2. The complete sequences of pECL18, pKPN2 and the pairwise comparison of the sequences of pECL18, pKPN2, ColE1 and pHS-2 suggest that plasmids may exchange DNA units via site-specific recombination events at bom and cer sites. In the course of BLASTN database searches using the cer sites of pECL18 and pKPN2 as queries, we found twenty cer sites of natural plasmids. Alignment of these sequences reveals that they fall into two classes. The plasmids in each group possess related segments between their cer and bom sites.

Base Sequence↗
PubMed2000

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.

Base Sequence↗
PubMed1999

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.

Amino Acid Sequence↗
PubMed1998

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.

Base Sequence↗
PubMed1998

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.

Base Sequence↗
PubMed1998

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.

Amino Acid Sequence↗
PubMed1998

[Novel isoschizomers of restriction endonucleases from Pseudomonas aeruginosa strains].

Testing of Pseudomonas aeruginosa strains from the collection of the L. V. Gromashevsky Institute of Epidemiology and Infectious Diseases (Kiev) for specific endonucleases resulted in isolation of five class II restriction endonucleases, which were partially purified and their recognition targets were determined. Two of these endonucleases, Pae2kI and Pae18kI, are isoschizomers of Bg1II (5'-AGACTC-3'). Pae5kI and Pae14kI recognize the 5'-CCGC/GG-3' sequence and are therefore true isoschizomers of SacII. Hence, Pae17kI is an isoschizomer of PvuII and cleaves the DNA within the recognition sequence 5'-CAG/CTG-3'. Bg1II and PvuII are for the first time detected in Pseudomonas aeruginosa.

Deoxyribonucleases, Type II Site-Specific↗
PubMed1998

[High homology of plasmids carrying class II restriction modification systems, EcoRV isoschizomers, and their prevalence in natural Escherichia coli strains].

Screening of 660 clinical Enterbacteriaceae strains from the collection of L. V. Gromashevsky Kiev Institute of Epidemiology and Infectious Diseases for specific endonuclear activity revealed site-specific endonucleases, EcoRV isoschizomers in 6 E. coli strains. Genes coding for endonucleases and methyltransferases were localized on small (6.2 kb) multicopy Hsd+ plasmids. All plasmids were successfully transferred in laboratory strain E. coli K802. Restriction analysis and subcloning showed no differences in the structural and functional organization of studied plasmids and a previously revealed pLB1 plasmid [3,10], this reflecting their high homology, if not identity. These data allow us to propose effective horizontal transfer of EcoRV plasmids among natural E. coli isolates in the region studied.

Chromosome Mapping↗
PubMed1997

Isolation of a strain overproducing endonuclease Eco29kI: purification and characterization of the homogeneous enzyme.

The physical map of the plasmid pSACII1 carrying the genes of restriction-modification system Eco29kI (isoschizomer of SacII) was determined. The cloning of the Eco29kI endonuclease and methylase genes into the plasmid vector pUC129 produced recombinant strain Escherichia coli K802[pECO29A15] with Eco29kI synthesis level about 100 times higher than in the parent strain. The restriction endonuclease was purified from Escherichia coli K802 [pECO29A15] cells to near homogeneity using column chromatography sequentially on phosphocellulose, hydroxyapatite, and heparin-Sepharose and rechromatography on phosphocellulose. Biochemical characterization of the homogeneous R Eco29kI is given. The enzyme has molecular mass 24.5 kD and is present in the solution as a monomer.

Cloning, Molecular↗
PubMed1993

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.

Amino Acid Sequence↗
PubMed1993

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, Molecular↗
PubMed1990

[Cloning of Shigella flexneri 2a genes coding for restriction- modification system Sfl2aI].

The Sfl2aI system of restriction-modification (RM) was revealed in the cells of Shigella flexneri encoded by pKMR114 plasmid belonging to the IncN incompatibility group. The genes for Sfl2aI RM system were cloned. The system was ascribed to the enzymes of the EcoRII specificity, as shown by in vivo and in vitro experiments. Restriction analysis of these genes' region and antigenic properties of the Sfl2aI endonuclease pointed to significant differences between this and EcoRII RM systems.

Cloning, Molecular↗
PubMed1987

[Plasmid vectors of "insertion inactivation" of the trimethoprim resistance marker].

We demonstrate the possibility of using the T4 phage frd gene as an insertion inactivation marker within pBR322, in plasmids with changing copy number and expression of foreign genes under control. The structural part of the frd genes contains unique recognition sites for EcoRI and SalI endonucleases. Transformants with recombinant plasmids carrying the frd gene grow on media with up to 500 mkg/ml trimethoprim, whatever the gene dosage.

DNA Restriction Enzymes↗