ABSTRACT
The effect of conjugative plasmids of various incompatibility groups of the enterobacteria family on the activity of the cell restriction-modification system of type I (EcoK) was studied. Twenty-two conjugative plasmids of 15 incompatibility groups were tested. In addition to plasmids of the incI1 and incN groups studied earlier, conjugative plasmids of the incFII, incB/O, and incK groups were also shown to be able to weaken the action of type I restriction enzymes upon nonmodified DNA (Ard phenotype). A hybridization analysis of all the plasmid DNAs studied, using ard gene DNA sequences from the ColIb-P9 (incI1) plasmid as a probe, was performed. The ard locus of the R100 (incFII) plasmid was cloned in the pBR322 and pACYC184 vectors. The ard gene was located 2.5 kb from the oriT site in the leading region on the R100 conjugative plasmid.
Subject(s)
Deoxyribonucleases, Type I Site-Specific/antagonists & inhibitors , Plasmids , Bacteriophage lambda/genetics , Cloning, Molecular , Conjugation, Genetic , DNA, Recombinant , Escherichia coli/genetics , PhenotypeABSTRACT
The bacteriophages lambda:lux and lambda:luxAB have been constructed by ligation of phage arms generated by BamHI or SalGI restriction endonucleases digestion of EMBL4 to BamHI digested plasmid pF1 lux+ or to SalGI digested plasmid pF2 lambda:luxA+B+. Cells of Escherichia coli prototrophic strain Cs were infected with lambda:lux or lambda:luxAB and intensity of bioluminiscence of the samples registered at different time intervals determined. The signal of bioluminiscence was first detected 15 min after infection and its level increased exponentially thereafter demonstrating replication of the lambda:lux bacteriophages. We have used the recombinant lambda:luxAB bacteriophage to detect the enteric indicator bacteria without enrichment in 15 min, provided that they are present at levels higher than 10(4).
Subject(s)
Bacteriophage lambda/genetics , Escherichia coli/genetics , Luciferases/genetics , Cloning, Molecular , Luminescent Measurements , PlasmidsABSTRACT
The pdk gene from Z. mobilis localized on the 4.7-kb SpHI DNA fragment in plasmid pB201 was subcloned using DraI restriction endonuclease into the SmaI site of the phage cloning vector M13mp19. The derivatives of M13mp19 obtained, containing 1.8-kb inserts of the pdk gene in two opposite orientations, were used for DNA sequencing and site-directed mutagenesis. The latter was performed using polymerase chain reaction (PCR) and synthetic deoxyribonucleotides of appropriate structure as primers. In this way a BamHI site near the initial (formylmethionine) codon of the pdk gene was created. After amplification the pdk gene was treated by restriction endonuclease BamHI and cloned into pUC19, and then recloned into shuttle vector pCB20 capable of replicating in both Gram negative and Gram positive bacteria. A recombinant plasmid pCB20pdkI--a derivative of pCB20 carrying the pdk gene under control of the "expression unit" EU19035 containing a bacillar vegetative promoter and an RBS site was obtained. The properties of the pCB20pdkI in E. coli and Bac. subtilis cells were studied. It was shown that pCB20pdkI determines a high level of PDK synthesis in Bac. subtilis. At the same time, it strongly inhibits E. coli cell growth and segregates rapidly from this host.
Subject(s)
Bacillus subtilis/genetics , Plasmids , Pyruvate Decarboxylase/genetics , Zymomonas/enzymology , Base Sequence , Cloning, Molecular , DNA, Bacterial , Molecular Sequence Data , Mutagenesis, Site-DirectedABSTRACT
To elucidate the role of the insA reading frame in transposition of the IS1 element of the Tn9' transposon, the derivatives of plasmids pUC19::Tn9' and pUC19::IS1 have been obtained using oligonucleotide inserts of the length equal or exceeding 9 bp and equal to 10 bp. The ability of mutant variants of the Tn9' transposon and the IS1 element to form simple insertions and plasmid cointegrates was studied. To this end, experiments were performed on mobilization of the derivatives of pUC19 containing mutant variants of the IS1 element and Tn9' as well as of the plasmids pUC19::Tn9' by the conjugative plasmid pRP3.1. According to the data obtained, mutations (inserts) in the insA gene have no influence on the frequency of transposition of the IS1 element and Tn9' from the plasmid pUC19 to pRP3.1. At the same time, the frequency of transposition events of mutant variants of Tn9' from the plasmid pRP3.1 to pBR322 is more than 10 times lower in comparison with the wild type transposon. The data obtained are in accordance with the assumption that the insA gene is not essential for transposition. A hypothesis is put forward explaining the role of the insA gene product in the process of bringing together short inverted repeats of the IS1, which are the sites for the transposase to be recognized at first stages of transposition.
Subject(s)
DNA Transposable Elements/physiology , Genes, Bacterial/genetics , Oligonucleotides/genetics , Plasmids/genetics , MutationABSTRACT
The ability of industrial strains of mesophylic Streptococcus diacetylactis to synthesize the enzyme beta-galactosidase has been studied. Among the 22 studied strains 8 were found to synthesize the enzyme. Plasmid DNA was isolated from the Streptococcus diacetylactis strain 144 possessing the highest level of beta-galactosidase activity. The cells of the strain harbour the 35, 40 and 60 kb plasmids. The alpha-galactosidase genes from this strain was cloned in Escherichia coli cells. The gene is located on the BglIII DNA fragment of the total plasmid DNA from Streptococcus diacetylactis the size of 2.8 kb. Following the Sau3A restriction endonuclease digestion the gene was subcloned on a birepliconed vector plasmid pCB20. The latter is capable of replication in the Gram-negative as well as Gram-positive microorganisms. The pCB20 derivatives carrying the different length fragments with the beta-galactosidase gene were isolated. DNA of an obtained plasmid was used for transformation of Streptococcus diacetylactis cells. The presence of the recombinant plasmid in streptococcus strain 144 results in the 1.8 fold increase in beta-galactosidase production.
Subject(s)
Escherichia coli/genetics , Genes, Bacterial , Streptococcus/genetics , beta-Galactosidase/genetics , Plasmids , Restriction Mapping , Streptococcus/metabolism , beta-Galactosidase/biosynthesisABSTRACT
The ability of the industrial strains of Streptococcus lactis to synthesize the enzyme beta-galactosidase was studied. Five strains among sixteen were found to produce high levels of the enzyme. The beta-galactosidase gene in the most active strain Streptococcus lactis 111 was shown to be located on the 50 kb conjugative plasmid. The plasmid was transferred by conjugation into Streptococcus thermophilus cells and subsequently the gene for beta-galactosidase was studied in transconjugants. The beta-galactosidase gene from Streptococcus lactis 111 was subcloned in Escherichia coli cells on the plasmid pBR322. The gene was localized on the 4.8 kb BgIII fragment of DNA. Following the restriction of DNA by the Sau3A the gene was subcloned on the birepliconed plasmid vector pCB20 capable of replication in the Gram-negative as well as Gram-positive microorganisms. The recombinant derivatives of pCB20 were isolated that carry the beta-galactosidase gene on the DNA fragments of different size.
Subject(s)
Escherichia coli/genetics , Lactococcus lactis/enzymology , Streptococcus/genetics , Transfection , beta-Galactosidase/genetics , Cloning, Molecular , Conjugation, Genetic , DNA, Bacterial/genetics , Electrophoresis, Agar Gel , Gene Expression Regulation, Bacterial , Gene Expression Regulation, Enzymologic , Genes, Bacterial , Lactococcus lactis/genetics , Plasmids , beta-Galactosidase/metabolismABSTRACT
The DNA nucleotide sequence from the central region of the composite transposons Tn9* and Tn9' at the junction with the right copy of IS1 was determined. From the data obtained it follows that both transposons are members of the Tn9 family, although they contain additional DNA segments with regard to Tn9 of the length about 320 and 290 b.p. respectively lying distal to the cat gene. It was proposed that all the transposons of the Tn9 family have been formed as a result of IS1-mediated deletions of the plasmid R100 r-determinant. It was revealed from the data of computer analysis that in the sequenced DNA there are two potential promotors with transcription directed opposite in relation to the transcription of the cat gene.
Subject(s)
Chloramphenicol/pharmacology , DNA Transposable Elements , Drug Resistance/genetics , Base Sequence , DNA/genetics , Molecular Sequence Data , Plasmids , Promoter Regions, Genetic , Restriction Mapping , Transcription, GeneticABSTRACT
In order to elucidate the function of the IS1 insA gene derivatives of plasmid pUC19::Tn9' with insertions of synthetic oligonucleotides were obtained. The latter are equal or multiple of 9 b.p. in length and are located in the Pst1 site within each of the two IS1 copies of the Tn9' transposon. The insertions of the nine base oligonucleotides code for the neutral amino acids and do not shift the reading frame. One of the mutant transposon obtained - Tn9'/X was studied on the ability to form simple insertions and plasmid cointegrates. For this purpose the pUC19 derivatives carrying the wild type and mutant transposon were mobilized by conjugative plasmid pRP3.1. It was found that the damage of the insA gene does not influence the ability of transposon to form simple insertions and plasmid cointegrates in both recA - and rec+ cells of E. coli. However, the frequency of the cointegrate formation in the subsequent transposition of the mutant transposon from pRP3.1::Tn9'/X to pBR322 was by 10-20 times lower in comparison to the wild type transposon. Instable (dissociating) Tn9'/X-mediated plasmid cointegrates formed by interaction pUC19::Tn9'/X and pRP3.1 were obtained. It was shown that in the E. coli recA-cells such cointegrates dissociate, as a rule, "correctly", i.e. they segregate mainly plasmids of types pUC19::Tn9'/X and pUC19::IS1/X. The data obtained correspond with the notion that the gene insA product is not essential for transposition, but is, possibly, involved in the formation of the IS1-generated deletions.
Subject(s)
DNA Transposable Elements/genetics , Escherichia coli Proteins , Open Reading Frames , Base Sequence , DNA, Bacterial/genetics , Electrophoresis, Agar Gel , Escherichia coli/genetics , Gene Expression Regulation, Bacterial , Genes, Bacterial , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutation , Plasmids , Repressor Proteins/genetics , Restriction MappingABSTRACT
The beta-galactosidase gene from the chromosome of Streptococcus thermophilus, strain 6 kb, has been cloned on a vector plasmid pBR322. The corresponding gene has been found to be located on the Pst1 DNA fragment. The restriction map of this 6 kb fragment has been constructed. The shortening of the DNA fragment carrying the beta-galactosidase gene has been achieved by digestion of the recombinant derivative of pBR322 by the restriction endonuclease Sau3A under the conditions of incomplete hydrolysis. The obtained fragments have been cloned into the BamHI site in the berepliconed shuttle vector pCB20 for grampositive and gramnegative bacteria. The obtained recombinant plasmids contained the beta-galactosidase gene in the inserted fragments of different length. Expression of the cloned beta-galactosidase gene in Escherichia coli and Bacillus subtilis cells has been studied.