RESUMO
Conjugative-like transfer of hybrid plasmid RP4::Mucts62 from Escherichia coli to plasmid-free Bacillus polymyxa was carried out. Bacillus transcipients are detected by the markers of kanamycin and tetracyclin resistance RP4 and thermal sensitivity to growth at 40-42 degrees C, determined by prophage Mucts62 in the plasmid. The technique of transception using millipore filters on solid media has been improved. For comparison with experimental samples, restriction mapping of the native plasmid in donor E. coli GA570 strain was carried out with identification of the prophage location in point 30.5 n. p. of RP4 map with counter-clockwise orientation of the right terminal towards IS21 element. Two phenotypes of bacillus transcipients selected for restriction mapping were singled out. Phenotype KmRTcRTS retains all markers of donor strain plasmid, and by the sum of restricts electrophoretically either corresponds to intact hybrid plasmid or contains deletions in RP4 sites (1-8 n. p.) adjacent to the left arm of prophage or rarely on the right arm (up to 3 n. p.). KmRTcRTS transcipients lose kanamycin resistance and on restriction map show greater prolongation of deletions from the prophage right terminal to kan RP4 gene, shortening it to 13 n. p. without involving the prophage proper. Its left terminal is retained, but 2-7 n. p. sites are deleted in the RP4 area adjacent to it. The possibility of transception in nature and horizontal routes of drug resistance dissemination among genetically remote bacteria are discussed.
Assuntos
Bacillus/isolamento & purificação , Bacillus/genética , Fagos Bacilares/genética , Conjugação Genética , Escherichia coli/genética , Resistência a Canamicina/genética , Fenótipo , Plasmídeos , Mapeamento por Restrição , Deleção de Sequência , Resistência a Tetraciclina/genéticaRESUMO
The plasmid RP4::Mu cts62 in stably inherited by Erwinia carotovora 268 strain. Under the conditions of thermoinduction bacteriophage Mu is segregated and completely eliminated more intensively than in Escherichia coli cells. At thermoinduction the transposition of bacteriophage Mu cts62 into different chromosomal sites takes place, causing the induction of chlorate resistant and auxotrophic mutants with the frequency of 10(-4). Two clones deficient in production of 2 of the 4 resident prophages of Erwinia carotovora 268 strain were found among Mu-induced mutants. The deleted prophages are E105 and 59. DNA-DNA hybridization has revealed the complete and partial deletions of bacteriophage E105 with the level of L-asparaginase production in the cells remaining intact. The damage of the prophage 59 is probably caused by point mutations or short deletions.
Assuntos
Bacteriófagos , Cromossomos Bacterianos , Deleção de Genes , Mutação , Pectobacterium carotovorum/genética , Plasmídeos , Genes BacterianosRESUMO
The plasmid RP4::Mu cts62 is transferred from Escherichia coli cells into a recipient strain Erwinia carotovora 268 by conjugation with the frequency 1.5-5 x 10(-7) per donor cell. The maximal frequencies of transfer are obtained by cultivation of donor and recipient cells for 3-5 h on the filters. Structural and functional validity of the plasmid in transconjugants is expressed in preservation of all antibiotic-resistant markers of RP4, thermosensitivity to growth at 42 degrees C as well as spontaneous and thermally-induced production and zygotic induction of bacteriophage determined by the genome of Mu cts62, total length of the plasmid restricts. Location and orientation of Mu cts62 genome in the plasmid restricts. Location and orientation of Mu cts62 genome in the plasmid RP4::Mu cts62 in Erwinia carotovora transconjugant cells has been determined. A single bacteriophage genome has been shown to transpose into the chromosome of the cell with the elimination of RP4 fragment under the conditions of thermal induction.
Assuntos
Bacteriófago mu/genética , Erwinia/genética , Plasmídeos , Asparaginase/biossíntese , Conjugação Genética , Elementos de DNA Transponíveis , Erwinia/metabolismo , Engenharia GenéticaRESUMO
Transfer of conjugative hybrid plasmid RP4::Mu cts 62 from Escherichia coli into Bac. cereus, Bac. thuringiensis, Bac. mesentericus and Bac. polymyxa cells led to the multiple effects on the structure and physiology of bacillus cells. It has resulted in a decrease of the bacillus vitality, in the accelerated autolytic decay of cells, in the delay of cell growth and reproduction rate in liquid and solid media, in the disruption of ultrastructure of the cell membrane and its surface layer.
Assuntos
Bacillus/genética , Plasmídeos/genética , Transformação Bacteriana/genética , Bacillus/ultraestrutura , Conjugação Genética , Resistência Microbiana a Medicamentos/genética , Escherichia coli/genética , Microscopia EletrônicaRESUMO
The transcipients were obtained in intrageneric matings of E.coli donor harbouring the plasmid PR4::Mu cts 62 with Bac. cereus GP7 recipient cells with the frequency 10(-9). The transcipient clone Bac. cereus 682 was selected having stably inherited and expressed the hybrid plasmid PR4::Mu cts 62 genes for antibiotic resistance and temperature sensitivity. Production of the bacteriophage Mu cts 62 particles was not registered in the bacillary transcipient cells. The plasmid RP4::Mu cts 62 genes were localized in the chromosome of Bac. cereus 682 transcipient by the blot-hybridization technique with 32P-labelled DNA of the bacteriophage Mu cts 62 and the plasmid PR4. The transcipient of Bac. cereus 682 has the donor properties and transfers the RP4::Mu cts 62 genes to recipient cells of Bac. cereus DSM 318 with the frequency of 10(-6)-10(-7). The expression and transfer of the gram-negative plasmid genes in gram-positive bacterial cells are discussed.
Assuntos
Bacillus cereus/genética , Regulação Bacteriana da Expressão Gênica/genética , Genes Bacterianos/genética , Plasmídeos/genética , Transformação Bacteriana , Escherichia coli/genética , Marcadores Genéticos , Fenótipo , Especificidade da EspécieRESUMO
Possibility of cryotransformation of Bacillus anthracis cells by the DNA of pUB110 plasmid has been established. The parameters of cryotransformation process have been optimized permitting one to increase the efficiency of transformation up to 3.1 . 10(2) transformants per 1 mkg of transforming DNA. The factors affecting the efficiency of cryotransformation and its reproducibility have been studied including the treatment of recipient cells by glycine, the procedure of freeze-thawing, the composition of freezing medium. The recipient activity of Bacillus anthracis cells has been shown to depend on the set of their own plasmids.
Assuntos
Bacillus anthracis/genética , DNA Bacteriano/genética , Plasmídeos , Transformação Bacteriana , Bacillus anthracis/crescimento & desenvolvimento , Bacillus subtilis/genética , Meios de Cultura , CongelamentoRESUMO
Incubation of bacterial cells in 0.1 M CaCl2 at 0 degrees C considerably increases the amount of phospholipids susceptible to action of a specific enzyme of phospholipid metabolism phospholipase C (hydrolysis to diacylglycerides). In process of incubation in CaCl2 solutions at 0 degrees C the expressed activity of an endogenous enzyme phospholipase A has been registered in cellular samples. Binding of the enzyme by the cells under conditions unfavourable for phospholipids hydrolysis (0 degrees C) suppresses strongly and reversibly cellular ability to DNA transformation without affecting cellular survival. As calculated, the enzyme molecules cover about 10% of cellular surface while inhibiting 90% of transmembrane transfer. The obtained data are considered to be a solid argument supporting the important role of the membrane phospholipids in the mechanism of cation-induced DNA transfer into the cell.
Assuntos
DNA Bacteriano/metabolismo , Escherichia coli/metabolismo , Lipídeos de Membrana/metabolismo , Fosfolipídeos/metabolismo , Fosfolipases Tipo C/fisiologia , Transporte Biológico , Cátions Bivalentes/farmacologia , Escherichia coli/enzimologia , Escherichia coli/genética , Hidrólise , Transformação BacterianaRESUMO
The level of plasmid transformation and transfection by the high molecular mass DNA was studied for Escherichia coli mutants having increased efficiency of plasmid transformation by low molecular mass DNA. Decreased level of plasmid transformation and transfection registered in some mutants as compared to the one in wild type strain suggests the specificity of Escherichia coli cells penetration for DNA of different molecular mass.
Assuntos
DNA Bacteriano/genética , Escherichia coli/genética , Plasmídeos , Transformação Bacteriana , Peso Molecular , MutaçãoRESUMO
The process of polyethyleneglycol-induced plasmid transformation of Bacillus cereus protoplasts was studied. Plasmid transfer into Bacillus cereus strains was demonstrated with the frequencies 1.3.10(1)-1.6.10(2) transformants per 1 mkg of plasmid DNA. The plasmids transferred are stably inherited by Bacillus cereus cells causing tetracycline resistance (pBC16) or kanamycin resistance (pUB110 and pBD64). The proposed method can be used for construction of Bacillus cereus strains having the plasmid determined characteristics.
Assuntos
Bacillus cereus/genética , Plasmídeos , Transformação Bacteriana , Polietilenoglicóis , ProtoplastosRESUMO
Polyethyleneglycol induced fusion of Bacillus cereus protoplasts and its genetic consequences have been investigated. The technique used allows the transfer of a small plasmid pBC16 between Bacillus cereus cells. Fusion has resulted in isolation of hybrid cells having acquired TcR phenotype (harbouring pBC16) with high frequencies (10(-2)-10(-3)). However, the genetic instability of hybrid cells and segregation effects have influenced dramatically the final results of plasmid transfer. Nevertheless, the fusion of protoplasts proves to be useful in construction of BAcillus cereus strains inheriting plasmid determinants.
Assuntos
Bacillus cereus/genética , Protoplastos , Resistência Microbiana a Medicamentos , Mutação , Plasmídeos , Recombinação GenéticaRESUMO
The Escherichia coli K12 mutant having the increased efficiency for plasmid DNA transformation has been shown to possess the different protein composition of the outer membrane of the cellular wall, as compared with that of the wild type strain. Correlation between the level of calcium-dependent plasmid transformation and the portion of infections DNA bound with cytoplasmic membranes is demonstrated for the Escherichia coli cells mutant for outer membrane structure and ability to be transformed by plasmid DNA.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Permeabilidade da Membrana Celular , DNA Bacteriano/genética , Escherichia coli/genética , Plasmídeos , DNA Bacteriano/metabolismo , Escherichia coli/metabolismo , Mutação , Transformação BacterianaRESUMO
The action of tetracaine hydrochloride, a local anesthetic, on the effectiveness of plasmid transformation and transfection in variants of E. coli K-12 has been studied. The concentrations of tetracaine hydrochloride used in the experiment (0.002 M) affect the level of the synthesis of most proteins in the outer membrane of these bacteria. This seems to be one of the causes of changes in the effectiveness of plasmid transformation and transfection in E. coli K-12.
Assuntos
Anestésicos Locais/farmacologia , Escherichia coli/efeitos dos fármacos , Plasmídeos/efeitos dos fármacos , Tetracaína/farmacologia , Transfecção/efeitos dos fármacos , Transformação Bacteriana/efeitos dos fármacos , Proteínas da Membrana Bacteriana Externa/biossíntese , Bacteriófago lambda/efeitos dos fármacos , Bacteriófago lambda/genética , Membrana Celular/efeitos dos fármacos , DNA Bacteriano/genética , Escherichia coli/genética , MutaçãoRESUMO
The study of the plasmid transformation of E. coli K-12, carried out in mutants with the known damages in the main proteins of the outer membrane, made it possible to reveal the essential role of OmpA protein in this process. The damage of lipoprotein led to a considerable increase in the level of plasmid transformation. The mutants with the enhanced efficiency of plasmid transformation, obtained by the authors, were not connected with lesions in the main proteins of the outer membrane: OmpF, OmpC and OmpA.
Assuntos
Proteínas de Bactérias/genética , DNA Bacteriano/genética , Proteínas de Membrana/genética , Mutação , Plasmídeos , Transformação Bacteriana , Proteínas da Membrana Bacteriana ExternaRESUMO
Escherichia coli K-12 mutants with an enhanced efficiency of plasmid transformation were obtained. In all the mutants, the efficiency of transfection with lambda vir phage DNA was changed, in comparison to the parent strain. However, these changes did not always correlate strictly with plasmid transformation alterations. For instance, two mutants with an increased plasmid transformation efficiency demonstrated 50-fold decrease in the level of transfection with lambda phage DNA. Polyacrylamide gel electrophoresis points to both quantitative and qualitative differences in protein composition of the mutant cell envelopes, as compared with the parent strain.
Assuntos
Escherichia coli/genética , Mutação , Plasmídeos , Transformação Bacteriana , Proteínas de Bactérias/análise , Proteínas de Bactérias/genética , Bacteriófago lambda/genética , DNA Viral/genética , Eletroforese em Gel de Poliacrilamida , Proteínas de Membrana/análise , Proteínas de Membrana/genética , TransfecçãoRESUMO
A new rapid method for plasmid transformation of Escherichia coli K-12 cells has been devised. It consists in application of plasmid pMB9 DNA to the surface of an agar medium with 0.05 M CaCl2 and tetracycline (50 micrograms/ml). The recipient cells treated with nitrosoguanidine were drifted on by sectors on the plates with pMB9 DNA. The method enabled the obtaining of 12 mutants with high efficiency of plasmid transformation.
