RESUMEN
Two bacterial species, Gram-negative Escherichia coli and Gram-positive Bacillus subtilis, were exposed to different auxins to examine possible effects of these substances on bacterial stress tolerance. Bacterial resistance to UV irradiation, heat shock, and streptomycin was assessed with and without previous exposure to the following auxins: indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), and 1-naphthalene acetic acid (NAA). Escherichia coli and B. subtilis cultures pretreated with any of the 3 auxins survived UV irradiation better than the untreated cultures. Also, B. subtilis cultures pretreated with IBA or NAA survived prolonged heat exposure better than the untreated cultures, while IAA pretreatment had no effect on heat shock survival. In contrast, auxin pretreatment rendered E. coli more sensitive to heat shock. Escherichia coli cultures pretreated with auxins were also more sensitive to streptomycin, while auxin pretreatment had no effect on sensitivity of B. subtilis to streptomycin. These results show that auxins may either enhance or reduce bacterial tolerance to different stressors, depending on the bacterial species and the type and level of the stress. Auxins usually had similar effects on the same bacterial species in cases when the same type and level of stress were applied.
Asunto(s)
Bacillus subtilis/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Ácidos Indolacéticos/farmacología , Estrés Fisiológico/efectos de los fármacos , Antibacterianos/farmacología , Bacillus subtilis/efectos de la radiación , Escherichia coli/efectos de la radiación , Respuesta al Choque Térmico/efectos de los fármacos , Indoles/farmacología , Ácidos Naftalenoacéticos/farmacología , Estreptomicina/farmacología , Rayos UltravioletaRESUMEN
Some phages survive irradiation much better upon multiple than upon single infection, a phenomenon known as multiplicity reactivation (MR). Long ago MR of UV-irradiated lambda red phage in E. coli cells was shown to be a manifestation of recA-dependent recombinational DNA repair. We used this experimental model to assess the influence of helicase II on the type of recombinational repair responsible for MR. Since helicase II is encoded by the SOS-inducible uvrD gene, SOS-inducing treatments such as irradiating recA(+) or heating recA441 cells were used. We found: i) that MR was abolished by the SOS-inducing treatments; ii) that in uvrD background these treatments did not affect MR; and iii) that the presence of a high-copy plasmid vector carrying the uvrD(+) allele together with its natural promoter region was sufficient to block MR. From these results we infer that helicase II is able to antagonize the type of recA-dependent recombinational repair acting on multiple copies of UV-damaged lambda DNA and that its anti-recombinogenic activity is operative at elevated levels only.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas Bacterianas/genética , Bacteriófago lambda/efectos de la radiación , ADN Helicasas/metabolismo , Reparación del ADN/efectos de los fármacos , Proteínas de Escherichia coli , Respuesta SOS en Genética/genética , Factores de Transcripción , Rayos Ultravioleta , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/farmacología , Proteínas Bacterianas/efectos de los fármacos , Bacteriófago lambda/efectos de los fármacos , Bacteriófago lambda/genética , ADN Helicasas/genética , ADN Helicasas/farmacología , Reparación del ADN/genética , Escherichia coli/enzimología , Escherichia coli/genética , Regulación de la Expresión Génica , Rec A Recombinasas/farmacología , Rec A Recombinasas/efectos de la radiación , Recombinación Genética/efectos de los fármacos , Rayos Ultravioleta/efectos adversosRESUMEN
Earlier studies have revealed a radiation-induced process leading to the loss of lambda prophage recombinogenicity. The process takes place in UV-irradiated Escherichia coli cells, and renders the prophage incapable of site-specific recombination with the host chromosome, and of general recombination with an infecting homologous phage. It was found that the inhibition of prophage recombinogenicity depends on functional RecBCD enzyme of E. coli. In this work, the role of ruvABC and recG genes in the inhibitory process was assessed. The products of these genes are known to act at the last step of homologous recombination and recombinational DNA repair by catalyzing the resolution of recombination intermediates (the Holliday junctions). Irradiated prophage retained its ability to recombine in ruvA, ruvB, ruvC, and recG mutants. These results suggest that in addition to RecBCD enzyme, RuvABC and RecG proteins are also involved in the inhibition of prophage recombinogenicity. We infer that RuvABC and RecG act in this process before RecBCD, probably by processing the Holliday junctions formed upon replication arrest, and thereby providing double-stranded DNA breaks as substrate for RecBCD-mediated recombinational repair of UV-damaged bacterial chromosome.
Asunto(s)
Bacteriófago lambda/fisiología , ADN Helicasas , Proteínas de Escherichia coli , Escherichia coli/virología , Proteínas Bacterianas/genética , Bacteriófago lambda/genética , Reparación del ADN , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/efectos de la radiación , Endodesoxirribonucleasas/genética , Escherichia coli/genética , Escherichia coli/efectos de la radiación , Exodesoxirribonucleasa V , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/efectos de la radiación , Mutación , Recombinación Genética , Rayos Ultravioleta , Activación ViralRESUMEN
RecBCD enzyme is involved in the radiation-induced process known as prophage inactivation. The process leads to the inability of lambda prophage to excise itself from the Escherichia coli chromosome via site-specific recombination. In this work we sought to further characterize the role of RecBCD enzyme in this process. In addition, we examined the ability of irradiated prophage to recombine with the infecting homologous phage. We used several E. coli mutants differentially altered in RecBCD's activities. The results showed that in the mutants carrying either recB2109 or recD1903, which do not exhibit significant nuclease activities, the prophage progressively loses its capacity for both site-specific and general recombination. In the recB268 null mutant, however, prophage recombinogenicity remained fully preserved. We also showed that the prophage unable to recombine retained its ability to complement the mutant infecting phage and that the recombination frequencies in phage x phage crosses were not affected by postirradiation incubation. Our results suggest that the helicase activity of RecBCD is responsible for the progressive loss of prophage recombinogenicity. This loss is most probably a consequence of the unsuccessful RecBCD-dependent recombinational repair of double-stranded breaks in the cell chromosome, during which some structures unsuitable for further recombination reactions may be produced.
Asunto(s)
Bacteriófago lambda/genética , Escherichia coli/virología , Exodesoxirribonucleasas/metabolismo , Recombinación Genética , Rayos Ultravioleta , Bacteriófago lambda/patogenicidad , Bacteriófago lambda/fisiología , Reparación del ADN , Escherichia coli/enzimología , Escherichia coli/crecimiento & desarrollo , Escherichia coli/efectos de la radiación , Exodesoxirribonucleasa V , Activación ViralRESUMEN
The efficiency of Xenopus laevis egg extract to repair T:G and A:C mismatched base pairs in unmethylated, hemimethylated and fullymethylated heteroduplexes was investigated. Filamentous phage M13mp18 and its derivative M13mp18/MP-1 (C changed to T inside the sequence dCC*C GGG, at the position 6248) were used for heteroduplexes construction. The three origins of mismatched base-pairs in the eukaryotic DNA are mimicked by in vitro methylation: hemimethylated DNA (me-/me+) for replication errors; unmethylated (me-/me-) and fully methylated DNA (me+/me+) for recombination heteroduplexes, and fullymethylated also for locally, spontaneously deaminated 5-methylcytosine (5meC) to T, generating the exclusively T:G mismatch. The methylations were in CpG dinucleotides, mostly characteristic ofeukaryotic cells [5, 24]. In vitro methylation was done by HpaII methylase which methylate central C of dCCGG sequence in the manner of eukaryotic methylation. The position of mismatched bases was chosen so that correction of mismatched bases in any strand would create the sequence for one of the "diagnostic" restriction endonucleases, either BstNI or MspI. Correction efficiency was about 10(8) repair events per egg equivalent. Correction in favor of C:G base pair restoration occurred regardless of the T:G or C:A mispairs, with almost equal efficiency. Repair of T:G to T:A was up to 10 times less efficient comparing to C:G, and repair of C:A to T:A was in our experimental system undetectable. No significant difference in repair efficiency of mismatched bases situated in unmethylated, hemimethylated or fullymethylated heteroduplexes indicate methylation-independent repair of mismatched bases in X. laevis oocite extracts.
Asunto(s)
Disparidad de Par Base , Metilación de ADN , Reparación del ADN , Óvulo/metabolismo , Animales , Islas de CpG , ADN-Citosina Metilasas/metabolismo , Análisis Heterodúplex , Plásmidos/metabolismo , XenopusRESUMEN
The RuvC protein is important for DNA recombination and repair in Escherichia coli. The present work shows that a ruvC null mutation introduced into a recBC sbcBC background causes severe defects in chromosome segregation and cell division. Both defects were found to result from abortive recombination initiated by the RecA protein.