RESUMEN
AIM: Determine the possibility of Iysogenization of Escherichia coli single strain DNA (ssDNA) by 1ø7 bacteriophage from the Microviridae family and determine the role of phage lø7 lysogeny in genetic variability of these bacteria. MATERIALS AND METHODS: A method of E. coli K12 lysogenization by phage lø7 was developed. A spot-test for the control of resistance of the obtained lysogens against phage lø7 and determination of lysogen lø7 spontaneous production was worked out. Criteria for phage lø7 identification, that is spontaneously produced by E. coli K12 lysogens, were proposed. A kit of isogenic E. coli strains, that vary by mutations in ptsI, ptsH and fruA genes, that code phosphoenolpyruvate (PEP): carbohydrate phosphotransferase system (PTS) proteins, was constructed. RESULTS: The ability of highly virulent bacteriophage lø7 to lysogenize E. coli was shown. A reduction of lø7 titers in ptsI, ptsH and fruA E. coli K12 mutants was demonstrated compared with titers in wild-type bacteria. Lytic bacteriophage lø7 was also able to lysogenize ptsI, ptsH and fruA mutants at a high frequency. Lysogens are resistant to phages lø7, phiX174 of Microvirus genus and spontaneously produce lø7. CONCLUSION: Bacteriophage lø7 of the Microviridae family is able to lysogenize E. coli K12 and vertically transfer genome of this lytic phage. As a result, lytic phage lø7 takes part in bacterial variability as a factor of lysogen selection in bacteria population corresponding to PTS mutants by phenotype.
Asunto(s)
Cromosomas Bacterianos , Colifagos/genética , Escherichia coli K12/virología , Regulación Bacteriana de la Expresión Génica , Variación Genética , Lisogenia/genética , Proteínas Bacterianas/genética , Mapeo Cromosómico , Colifagos/patogenicidad , ADN Bacteriano/genética , ADN de Cadena Simple/genética , Escherichia coli K12/genética , Proteínas de Escherichia coli/genética , Transferencia de Gen Horizontal , Genotipo , Interacciones Huésped-Patógeno , Proteínas de Transporte de Monosacáridos/genética , Mutación , Fenotipo , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/genéticaRESUMEN
Induction of transcription by the plasmid pKM101 (mutability mediating derivate of the plasmid R46) of the sfiA gene controlling cell division and of the fruA gene encoding the fructose specific enzyme II of the phosphoenolpyruvate-phosphotransferase system in intact cultures of Escherichia coli was studied. The genes under study were fused to the bacteriophage Mu dl (Ap lac). Activation of the sfiA gene, a typical member of the SOS-regulon, was demonstrated to depend on the key genes of the SOS-system-recA and lexA. In contrast, the fruA gene that is non-inducible by the UV-light, a classical SOS-inducing agent, is not activated by the presence of the plasmid pKMIO1 in the bacterial cells. The data obtained suggest that the presence of pKMIO1 plasmid in the Escherichia coli cells induces a SOS-signal as a consequence of the plasmid DNA replication or its conjugative transfer.
Asunto(s)
Escherichia coli K12/genética , Plásmidos/fisiología , Respuesta SOS en Genética , Proteínas Bacterianas/genética , División Celular , Proteínas de Escherichia coli/genética , Mutación , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/genética , Rec A Recombinasas/genética , Proteínas Represoras/genética , Serina Endopeptidasas/genética , beta-Galactosidasa/genéticaRESUMEN
The genome of lambda phage with thermosensitive repressor was integrated into the pts region of the E. coli chromosome. Such a lysogenic culture behaves as a pts mutant at 30 degrees. Heating of cells of this strain leads to the induction of lambda prophage and formation of deletions in the pts region. A mutant with a deletion covering ptsH gene was isolated after prophage induction. The deletion nature of pts mutation was confirmed in genetic and biochemical experiments. It was shown that the deletion is small and does not involve ptsI and lig genes. The isolated deltaptsH mutant possesses all characteristics of pts mutants: pleiotropic impairment of transport and utilization of a number of carbohydrates, repression of the enzyme inducible synthesis and resistance to catabolite repression with glucose. These data (together with earlier ones) allow us to conclude that the phosphorylated form of HPr is involved (in direct of indirect manner/ in activation of DNA transcription.
Asunto(s)
Cromosomas Bacterianos , Inducción Enzimática , Represión Enzimática , Escherichia coli/metabolismo , Colifagos/genética , Escherichia coli/genética , Lisogenia , Mutación , Transcripción GenéticaRESUMEN
Induction of the tetracycline-resistance genes function by the inducer of the DNA-repair and mutability SOS-system, UV-light, has been tested. Activity of the tet-genes residing on the plasmid RP4 in Escherichia coli cells has been shown to be inducible by the low doses of tetracycline as well as by the mutagenic doses of UV-light. The induction was quantitatively registered by measuring the activity of beta-galactosidase of bacteriophage Mud1 (Ap, Lac) inserted into the tet-genes of the plasmid RP4. The bacteriophage integration inactivates the tet-genes function of the constructed plasmid fusing the lac-operon to a promoter of inactivated genes. Precise excision of bacteriophage restores the activity of the tet-genes proving together with the plasmid DNA-restriction analysis the fusion of tet-promoter with Iac-operon. The tet-genes of RP4 are concluded to be a part of the SOS-regulon, a set of genes inducible by the conditions harming the bacterial cell. Preliminary data on the mutagenic activity of tetracycline obtained in the bacterial test-system of mutagens are discussed.
Asunto(s)
Reparación del ADN , Genes Bacterianos , Plásmidos , Respuesta SOS en Genética , Resistencia a la Tetraciclina/genética , Elementos Transponibles de ADN , Escherichia coli/genética , Mutación , Salmonella typhimurium/genética , beta-Galactosidasa/genéticaRESUMEN
A pair of isogenic strains-E. coli K-12 and E. coli B/r differing by the status of umuDC genes and presence of pKM101 plasmid-were constructed and the relationship between UV induction of transposons Tn5 and Tn 10 and the gene umuDC function shown. This relationship is not absolute, in contrast to that of point mutations. Induction of precise excision of these transposons can be inhibited by pKM101 plasmid. Induction of precise excision of Tn5 and Tn 10 from the sites under study is absolutely lexA- and recA- dependent.
Asunto(s)
Elementos Transponibles de ADN , Escherichia coli/genética , Genes Bacterianos/efectos de la radiación , Plásmidos/efectos de la radiación , Rayos Ultravioleta , Escherichia coli/efectos de la radiación , Mutagénesis Insercional , Mutación PuntualRESUMEN
Comparative analysis of UV induction of tandem chromosome (Mtc+) duplications and reversions of point mutations (base pair substitutions) and of insertion mutations (precise excision of Tn10) was carried out. Salmonella (umuST) genes deficiency preventing dose-dependent UV induction of point mutation reversion were shown to have a less pronounced effect on the induction of tandem chromosome duplications. UV induction of tandem chromosome duplications is similar to UV induction of insertion mutation reversion: dose-dependent UV induction of both occurs in umuST Salmonella strains. E. coli umuDC+ genes included in Salmonella genome appreciably enhance the UV induction of both tandem duplications and insertion mutation reversion. The presence of umuDC+ genes ensures an expressed dose-dependent UV induction of point mutation reversion.
Asunto(s)
Aberraciones Cromosómicas , Genes Bacterianos , Salmonella/genética , Mutagénesis Insercional , Mutación Puntual , Rayos UltravioletaRESUMEN
The study focused on plasmid pKM101, which is a necessary component of the short-term test of Eim's system (Salmonella-microsome test), to detect the potential carcinogens through their mutagen activity. We found a previously unknown feature of the plasmid to enhance the expression of certain plasmid and chromosome genes. The purpose of the present study was to examine and specify the role of operon mucAB responsible for the mutation properties of the plasmid in activating the expression of bacterial genes. An ultraviolet-induction examination of bacterial genes, with the mutants of plasmid pKM101 affecting operon mucAB being used, showed that the function of genes mucAB did activate, but, on the contrary, suppressed the induction of genes elt (i.e. of genes controlling the formation of LT-toxin of Escherichia coli) and of sfiA (SOS-regulated gen E. col controlling the cell division.
Asunto(s)
Escherichia coli/genética , Escherichia coli/efectos de la radiación , Genes Bacterianos/efectos de la radiación , Plásmidos/genética , Salmonella typhimurium/genética , Toxinas Bacterianas/genética , División Celular , Enterotoxinas/genética , Proteínas de Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica/efectos de la radiación , Mutación , Operón , Salmonella typhimurium/efectos de la radiación , Supresión Genética , Rayos UltravioletaRESUMEN
The plasmid elt-operon pVZ14 was constructed by fusing of the eltoperon of the plasmid pVZ357 with the lac-gene of the bacteriophage Mud1 (Amp, Lac). lacZ gene has been proven to be fused with an elt-promoter by the loss of toxin production coded by pVZ357 and acquiring of Lac+ phenotype by pVZ14 containing cells, as well as by HindIII fragments hybridization of pVZ357 and pVZ14 with the labelled elt-probe. The kinetics of beta-galactosidase synthesis in E. coli cells harboring pVZ14 shows an elt-operon promoter to have expressed constitutive activity and to be activated by a SOS-inducing agent, UV-light.
Asunto(s)
Toxinas Bacterianas/genética , Enterotoxinas/genética , Proteínas de Escherichia coli , Operón/efectos de la radiación , Autorradiografía , Southern Blotting , Células Cultivadas , Escherichia coli/genética , Plásmidos , Mapeo Restrictivo , Rayos Ultravioleta , beta-Galactosidasa/biosíntesis , beta-Galactosidasa/metabolismoRESUMEN
Fine genetic mapping of the pts region coding for general components of PTS was performed. Over 30 spontaneous pts mutations were investigated. By means of the complementation test using the F'trp+ purC+ ptsI episome, both ptsI and ptsH mutations were revealed among them. With the help of reciprocal three point transduction crosses, 8 of them were situated in the pts region. Two of them were in ptsH gene, the rest being in ptsI gene. The lysogenic strain was obtained with lambda inserted in the pts region. Heat curing of the lysogene led to a number of deletions and insertions. Six of them were mapped with the help of the point mutations studied.
Asunto(s)
Proteínas Bacterianas , Proteínas Portadoras/genética , Mapeo Cromosómico , Escherichia coli/genética , Genes Bacterianos , Mutación , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/genética , Fosfotransferasas (Aceptor del Grupo Nitrogenado) , Bacteriófago lambda/genética , Proteínas Portadoras/biosíntesis , Inducción Enzimática , Escherichia coli/enzimología , Lisogenia , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/biosíntesis , Transducción GenéticaRESUMEN
UV induction of the elt operon (the LT-toxin operon in Escherichia coli) was demonstrated in experiments using fusion of elt::lac operons with the help of Mud1(Ap lac) phage. UV induction of the elt operon is lexA-dependent; thus, the possibility of SOS regulation of this process may be assumed. However, UV induction of the elt operon turned out to be recA-independent, which makes it impossible to consider this induction as a typical SOS response. UV induction of the elt operon is also observed in Salmonella typhimurium, which differs from E. coli in the product of umuD, which suggests that the UV induction of the elt operon is umuD independent.
Asunto(s)
Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Enterotoxinas/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Operón , Rec A Recombinasas/genética , Serina Endopeptidasas , ADN Polimerasa Dirigida por ADN , Genes Bacterianos , Respuesta SOS en Genética/efectos de la radiación , Salmonella typhimurium/genética , Rayos UltravioletaRESUMEN
The Salmonella typhimurium strain AG262 has been constructed for screening of SOS-mutagens, such as UV-light and 4NQO, the Ames plasmidless tester strains of S. typhimurium being mutation-proof under the action of this class of mutagens. The strain AG262 has also been successfully used for screening of induced base pair change mutations occurring independently of cellular SOS-system of mutagenesis (MNNG, EMS, nitrofuran derivatives), and of the mutations induced by frame-shift mutagens (benzo/a/pyrene 7,12-dimethylbenzo/a/antracene ICR-191, 9AA, DDDTDP).
Asunto(s)
Mutágenos , Mutación , Salmonella typhimurium/genética , Genes Bacterianos , Genes Letales , Pruebas de Mutagenicidad , Respuesta SOS en Genética , Salmonella typhimurium/efectos de los fármacos , Rayos UltravioletaRESUMEN
An experimental system ensuring fusion of bacterial genes to the lac operon of the Mu dl(Aplac) phage was used. Fusion operons in which the lac operon was under the control of promoters of the elt gene, responsible for synthesis of the LT toxin, of the tetracyclin-resistance tet gene, and sfiA gene encoding filament production, was studied. Using this experimental system, plasmid pKM101 was shown to be capable of activating the expression of the above Escherichia coli and Salmonella typhimurium genes, which is manifested as the activation of beta-galactosidase synthesis. The activation of the elt gene expression by the pKM101 plasmid was also confirmed in experiments on detecting the LT toxin synthesized by bacteria carrying this plasmid. Effect of the plasmid on the activation of elt operon expression, unlike the effect of this plasmid on mutability, does not depend on the functioning of the lexA and recA genes, i.e., this is not a SOS-regulated process. The mutant plasmid pGW12, a derivative of pKM101, deficient in the mucAB genes responsible for mutagenesis, causes a more pronounced activation of the elt gene than plasmid pKM101.
Asunto(s)
Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Plásmidos/genética , Salmonella typhimurium/genética , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Genes Reporteros , Operón Lac/genética , Transfección , beta-Galactosidasa/genéticaAsunto(s)
Proteínas Bacterianas/genética , Proteínas Portadoras/genética , Mapeo Cromosómico , Escherichia coli/genética , Genes Bacterianos , Mutación , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/genética , Fosfotransferasas (Aceptor del Grupo Nitrogenado) , Deleción Cromosómica , Escherichia coli/enzimología , Código Genético , Recombinación Genética , Transducción GenéticaAsunto(s)
Compuestos Bicíclicos con Puentes/farmacología , Hidrocarburos Aromáticos con Puentes/farmacología , Insecticidas/farmacología , Mutación , Salmonella/efectos de los fármacos , Medios de Cultivo , Fungicidas Industriales/farmacología , Técnicas In Vitro , Resistencia a los Insecticidas , Pruebas de Sensibilidad Microbiana/métodos , Pruebas de Mutagenicidad/métodos , Mutágenos , Salmonella/genéticaRESUMEN
V. cholerae strain VT5104 capable of donor activity in conjugation has been constructed by the genetic technique based on plasmid RP4::Mucts62 integration into V. cholerae chromosome due to plasmid homology with Mucts62 inserted into the chromosome. The gene for histidine synthesis has been mobilized and transferred into the recipient cells from VT5104 donor. The conjugants obtained are able to efficiently transfer his+ gene included into the plasmid structure in conjugation with eltor recipient. Thus, the constructed strain VT5104 generates R' plasmids carrying V. cholerae chromosomal genes.
Asunto(s)
Histidina/genética , Factores R , Vibrio cholerae/genética , Conjugación Genética , Farmacorresistencia Microbiana/genética , Genes Bacterianos , Marcadores Genéticos , Recombinación Genética , TemperaturaRESUMEN
In experiments on random bred mice and mice of various strains it was shown that when administered parenterally typhoid bacteria O-somatic antigen polysaccharide possesses the immunomodulatory properties. It stimulates the non-specific resistance of the organism to bacterial infection, produces the polyclonal activation of beta-lymphocytes, possesses the adjuvant properties, activates cells of the mononuclear phagocytic system. At administration in therapeutic doses the drug is not toxic, possesses no carcinogenic, mutagenic and allergenic properties.