RESUMO
Bacterial plasmids propagate through microbial populations via the directed process of conjugative plasmid transfer (CPT). Because conjugative plasmids often encode antibiotic resistance genes and virulence factors, several approaches to inhibit CPT have been described. Bisphosphonates and structurally related compounds (BSRCs) were previously reported to disrupt conjugative transfer of the F (fertility) plasmid in Escherichia coli. We have further investigated the effect of these compounds on the transfer of two additional conjugative plasmids, pCU1 and R100, between E. coli cells. The impact of BSRCs on E. coli survival and plasmid transfer was found to be dependent on the plasmid type, the length of time the E. coli were exposed to the compounds, and the ratio of plasmid donor to plasmid recipient cells. Therefore, these data indicate that BSRCs produce a range of effects on the conjugative transfer of bacterial plasmids in E. coli. Since their impact appears to be plasmid type-dependent, BSRCs are unlikely to be applicable as broad inhibitors of antibiotic resistance propagation.
Assuntos
Quelantes/farmacologia , Conjugação Genética/efeitos dos fármacos , Difosfonatos/farmacologia , Escherichia coli/efeitos dos fármacos , Fator F/efeitos dos fármacos , Fatores R/efeitos dos fármacos , Quelantes/química , Difosfonatos/química , Escherichia coli/genética , Fator F/genética , Estrutura Molecular , Plasmídeos/efeitos dos fármacos , Plasmídeos/genética , Fatores R/genéticaRESUMO
The RepE protein (251 residues, 29 kDa) of mini-F plasmid, mostly found as dimers, plays a key role in mini-F replication. Whereas monomers bind to the origin to initiate replication, dimers bind to the repE operator to repress its own transcription. Among the host factors required for mini-F replication, a set of molecular chaperones (DnaK, DnaJ and GrpE) is thought to facilitate monomerization of RepE dimers. To further understand the structural basis of functional differentiation between the two forms of RepE, we examined the region(s) critical for dimerization by isolation and characterization of RepE mutants that were defective in autogenous repressor function. Such mutations were isolated from two separate regions of RepE, the central region (residues 111 to 161) and the C-terminal region (residues 195 to 208). The central region overlapped the region where the chaperone-independent copy-up mutations were previously isolated (residues 93 to 135). Likewise the mini-F mutant plasmids, carrying the mutations in the central region, could replicate in a dnaK null mutant host. One of them, S111P (111th serine changed to proline), showed a very high origin-binding activity vis-à-vis a severely reduced operator-binding activity, much like the RepE54 (R118P) mutant previously shown to form only monomers. Gel filtration and chemical crosslinking studies with purified RepE revealed that S111P primarily formed monomers, whereas other mutant proteins formed mostly dimers. On the other hand, analysis of deletion mutants revealed that the N-terminal 42 and the C-terminal 57 residues were dispensable for dimerization. Thus, the region spanning residues 93 to 161 of RepE (including Ser111 and Arg118) appeared to be primarily involved in dimerization, contributing to the negative regulation of plasmid replication.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Replicação do DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Proteínas de Escherichia coli , Fator F/fisiologia , Proteínas Repressoras/genética , Proteínas Repressoras/fisiologia , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Cromatografia em Gel , Reagentes de Ligações Cruzadas , Replicação do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Dimerização , Fator F/efeitos dos fármacos , Dosagem de Genes , Chaperonas Moleculares/fisiologia , Mutagênese Insercional , Ligação Proteica/genética , Proteínas Repressoras/isolamento & purificação , Proteínas Repressoras/metabolismo , Análise de Sequência de DNARESUMO
Several antimicrobial agents including mitomycin C and molecules belonging to the 4-quinolone, aminoglycoside and beta-lactam groups inhibited plasmid transfer to a varying extent, in actively growing Escherichia coli. In contrast, the same antibiotics did not prevent effective conjugation in nongrowing bacteria with the exception of mitomycin C. These results indicate that the drugs inhibit plasmid transfer by interfering with bacterial host functions rather than by recognizing a specific plasmid-mediated target. Several drugs are therefore capable, in principle, of reducing the spread of plasmid-mediated antibiotic resistance.
Assuntos
Antibacterianos/farmacologia , Conjugação Genética/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Fator F/efeitos dos fármacos , Resistência Microbiana a Medicamentos , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Fator F/fisiologiaRESUMO
Estrogens are believed to be major contributors to many cancers of the human female genital tract, but the mechanism of their carcinogenic action is not well-understood. While a tumor-promoting role for estrogens is well-supported, whether they also act as tumor initiators has remained controversial. Here, we have sought to examine the mutagenic potential of diethylstilbestrol, a synthetic estrogen that is a powerful carcinogen in hamsters, and is suspected to be a human carcinogen. Phage M13 single-stranded DNA was treated in vitro with diethylstilbestrol quinone (DES Q: 1.25 mM) and transfected into Escherichia coli cells. DES Q treatment resulted in an apparent enhancement of mutagenesis in the LacZ(alpha) gene segment. DNA sequence analysis of LacZ(alpha) mutants obtained by transfection of DES Q-treated DNA revealed that the major effect of DES Q treatment has been a 6-fold elevation of recombination between the phage-borne LacZ(alpha) sequence and the LacZ delta M15 sequence on the E. coli fertility plasmid F. To confirm whether DES Q treatment is recombinagenic, we used an experimental system that allows the detection of recombination between a defective E. coli chromosomal LacY gene and a normal counterpart borne on a plasmid. Transfection of DES Q (0.06-12 mM) treated plasmid DNA showed significant enhancement (2-100-fold) in recombination, but not in mutagenesis. These results raise the possibility that estrogen quinones may induce recombinagenic DNA damage.
Assuntos
Dano ao DNA , Dietilestilbestrol/análogos & derivados , Mutagênese , Mutagênicos/toxicidade , Recombinação Genética/efeitos dos fármacos , Bacteriófago M13/efeitos dos fármacos , Bacteriófago M13/genética , Sequência de Bases , Análise Mutacional de DNA , DNA Bacteriano/efeitos dos fármacos , DNA de Cadeia Simples/efeitos dos fármacos , DNA Viral/efeitos dos fármacos , Dietilestilbestrol/toxicidade , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Fator F/efeitos dos fármacos , Óperon Lac/efeitos dos fármacos , Dados de Sequência Molecular , Plasmídeos , Mutação Puntual , Deleção de Sequência , TransfecçãoRESUMO
With help of nitrosoguanidine 60 mutants of F-like plasmids pAP18-1 drd::Tn 5 and pAP18-1::Tn 9 were induced which determined resistance of E. coli cells of specific phage MS2. Mutational changes in fin-locus of those plasmids were accompanied by phenotypic reversion Fin(-)-Fin+.
Assuntos
Escherichia coli/genética , Fator F/genética , Genes Bacterianos/genética , Mutação/genética , Conjugação Genética/efeitos dos fármacos , Conjugação Genética/genética , Elementos de DNA Transponíveis/efeitos dos fármacos , Elementos de DNA Transponíveis/genética , Escherichia coli/efeitos dos fármacos , Fator F/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/genética , Genes Bacterianos/efeitos dos fármacos , Mutação/efeitos dos fármacos , Nitrosoguanidinas/farmacologiaRESUMO
Five-hundred and twenty seven strains of Salmonella isolated from different patients admitted to hospitals in Rome from 1982 to 1985 were screened for their resistance to antimicrobial drugs. Sixty-one strains (11.6%) were found to be resistant to two or more antibiotics; the most frequent resistances were to sulfathiazole, streptomycin, tetracycline, chloramphenicol and ampicillin. Of the thirty-eight strains showing resistance to three or more antibiotics, 17 were able to transfer their resistance to E. coli K 12. The isolates were heterogeneous in plasmid population: only few strains harbored a sole plasmid, most harbored many plasmids ranging between 20 and 120 megadaltons in weight. Most strains were found to carry a conjugative plasmid of incompatibility group Inc H of 100-120 megadaltons and Inc I alpha of 60-70 megadaltons.
Assuntos
Fatores R/genética , Salmonella/genética , Antibacterianos/farmacologia , Colicinas/biossíntese , Resistência Microbiana a Medicamentos , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Fator F/efeitos dos fármacos , Gastroenterite/microbiologia , Humanos , Técnicas In Vitro , Salmonella/efeitos dos fármacosRESUMO
Three representative groups of tricyclic psychopharmacons were shown to have antiplasmid activity. The first group, e.g., comprises promazine, chlorpromazine, promethazine, levo- and dextromepromazine, thioridazine, diethazine, thiethylperazine. There are two heteroatoms (S and N) in the ring system of these compounds. In the second group, which includes desipramine, imipramine, however, there is only one heteroatom N, or S in chlorprothixene, chlorpenthixols and in the third group amitriptyline, maprotiline contain no heteroatoms. The medical significance of plasmid elimination by tricyclic psychopharmacons and related compounds in vitro is that this opens up a new perspective in the struggle against bacterial resistance to antibiotics. This approach utilizes the beneficial side-effect of the plasmid curing ability of these psychopharmacons, and may serve as a guideline for drug design in the future, to develop related substances which are potent in eliminating the drug resistance-carrying plasmids of bacteria, but which have no other pharmacological effects. Here we propose a new mechanism for plasmid curing by tricyclic drugs.
Assuntos
Antidepressivos Tricíclicos/farmacologia , Plasmídeos/efeitos dos fármacos , Conjugação Genética/efeitos dos fármacos , DNA Bacteriano/efeitos dos fármacos , Escherichia coli/genética , Fator F/efeitos dos fármacos , Fatores R/efeitos dos fármacos , EstereoisomerismoRESUMO
Relation between induced mutations of plasmid pAP18-1 (Tc, Col) and alterations in it's restriction map was studied. Nitrosoguanidine induced mutations of transfer regulation system and incompatibility of this plasmid related with alteration in the situation of recognition sites for restrictases EcoR1 and Sal1 in map positions 42.2-4.3 and 12.9-17.9 MD. Insertions of transposons Tn5 and Tn9 into the plasmid DNA resulted in a decrease of incompatibility level.
Assuntos
Elementos de DNA Transponíveis , Fator F/efeitos dos fármacos , Mutação , Nitrosoguanidinas/farmacologia , Sequência de Bases/efeitos dos fármacos , Mapeamento Cromossômico , Conjugação Genética/efeitos dos fármacos , Enzimas de Restrição do DNA , DNA Bacteriano/efeitos dos fármacos , DNA Bacteriano/genética , Resistência Microbiana a Medicamentos/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Marcadores Genéticos/efeitos dos fármacosRESUMO
Citrinin, a mycotoxin of Penicillium citrinum and other species of the genera Penicillium and Aspergillus, caused the following effects at different concentrations in Escherichia coli. In vivo at 100 micrograms/ml single-strand breaks were caused in the chromosomal DNA. In the presence of 100 micrograms/ml, UV (254 nm)-induced DNA damage was repaired in the bacterial cells without need for a complete growth medium. At 300 micrograms/ml lambda ts prophage was induced in a lysogenic E. coli strain. In an E. coli strain carrying a F' lac plasmid, 4.7% of the cells displayed the Lac- phenotype after treatment with 200 micrograms of citrinin per ml, suggesting elimination of the F' factor. In vitro, DNA repair synthesis was observed at 5 micrograms of citrinin per ml in permeabilized cells, and replicative DNA synthesis was inhibited at 200 micrograms/ml. In these systems synthesis of stable RNAs was slightly diminished at 300 micrograms/ml, and protein synthesis was not affected at concentrations up to 450 micrograms/ml. Lambda and ColE1 plasmid DNA were cleaved in vitro when small amounts of copper ions were present. This DNA-attacking activity was prevented by NADPH, catalase, and superoxide dismutase and by higher concentrations of hydroxyl radical scavengers, suggesting the involvement of free radicals in the mechanism of action of citrinin on DNA.
Assuntos
Benzopiranos/farmacologia , Citrinina/farmacologia , Dano ao DNA , DNA Bacteriano/efeitos dos fármacos , Plasmídeos/efeitos dos fármacos , Plasmídeos de Bacteriocinas/efeitos dos fármacos , Bacteriófago lambda/crescimento & desenvolvimento , Catalase/farmacologia , Reparo do DNA/efeitos dos fármacos , DNA Bacteriano/biossíntese , DNA Viral/efeitos dos fármacos , Dimetil Sulfóxido/farmacologia , Escherichia coli/efeitos dos fármacos , Fator F/efeitos dos fármacos , Superóxido Dismutase/farmacologia , Ativação Viral/efeitos dos fármacosRESUMO
A transfer function derepressed mutant of the F-like plasmid pAP18-1 (Tc, ColV) was induced with the help of N-methyl-N'-nitro-N-nitrozoguanidine. The mutant plasmid pAP18-1drd belongs to the FVII incompatibility group of the F-like plasmids. The plasmid pAP18-1drd is characterized by the loss of the capacity for inhibiting the tra-genes functions of the Flac plasmid and is sensitive to the Tra-function inhibitors of the reference plasmids of the FinV and FinW groups.
Assuntos
Escherichia coli/genética , Fator F , Regulação da Expressão Gênica , Código Genético , Mutação , Tetraciclina/antagonistas & inibidores , Conjugação Genética/efeitos dos fármacos , Elementos de DNA Transponíveis/efeitos dos fármacos , Resistência Microbiana a Medicamentos , Escherichia coli/efeitos dos fármacos , Fator F/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Bacterianos/efeitos dos fármacos , Código Genético/efeitos dos fármacos , Metilnitronitrosoguanidina/farmacologiaAssuntos
Fator F , Laranja de Acridina/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Divisão Celular , Replicação do DNA , DNA Bacteriano/biossíntese , DNA Bacteriano/genética , Escherichia coli/genética , Escherichia coli/fisiologia , Escherichia coli/ultraestrutura , Fator F/efeitos dos fármacos , Regulação da Expressão Gênica , Genes Bacterianos , RepliconRESUMO
Based on the calculated molecular weights of EcoR1, HindIII, and SalI fragments of the genetic transfer factor pAP42 the restriction map of this plasmid was designed. Sites recognizing restrictases are mostly located in the plasmid fragment with a molecular weight of 5.7 MD.
Assuntos
Mapeamento Cromossômico , Enzimas de Restrição do DNA/farmacologia , Desoxirribonucleases de Sítio Específico do Tipo II , Escherichia coli/genética , Fator F/efeitos dos fármacos , Sítios de Ligação Microbiológicos/efeitos dos fármacos , DNA Bacteriano/genética , Desoxirribonuclease EcoRI , Desoxirribonuclease HindIII , Eletroforese em Gel de Ágar , Escherichia coli/efeitos dos fármacos , Peso MolecularAssuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Animais , Antibacterianos/classificação , Bactérias/genética , Infecções Bacterianas/tratamento farmacológico , Resistência Microbiana a Medicamentos , Tolerância a Medicamentos , Fator F/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana/métodos , Mutação , Fatores R/efeitos dos fármacosRESUMO
Replication of F (including mini-F) and some related plasmids is known to be specifically inhibited in mafA mutants of Escherichia coli K-12. We have now isolated and characterized mini-F mutants that can overcome the replication inhibition. Such plasmids, designated pom (permissive on maf), were obtained spontaneously or after mutagenesis with hydroxylamine or by transposon (Tn3) insertion. In addition to their ability to replicate in mafA mutant bacteria, the pom mutant plasmids exhibit an increased copy number and resistance to "curing" by acridine dye in the mafA+ host. In agreement with these results, Tn3-induced pom mutants were found to carry Tn3 inserted at the incC region of mini-F DNA, known to be involved in incompatibility, control of copy number, and sensitivity to acridine dye. Furthermore, three of the seven mini-F plasmids tested that carry Tn3 within the tandem repeat sequences of the incC region (previously isolated by other workers) exhibit all the phenotypes of pom plasmids, the ability to replicate in the mafA strain, and high copy number and acridine resistance in the mafA+ strain. The rest of the plasmids that contain Tn3 just outside the tandem repeats remain wild type in all these properties. These results strongly suggest that the putative mafA gene product of host bacteria controls mini-F replication through interaction with the incC region.
Assuntos
Replicação do DNA , Escherichia coli/genética , Fator F , Genes Bacterianos , Genes , Laranja de Acridina/farmacologia , Sequência de Bases , Enzimas de Restrição do DNA , Elementos de DNA Transponíveis , Fator F/efeitos dos fármacos , Hidroxilamina , Hidroxilaminas/farmacologia , Mutação , FenótipoRESUMO
Novobiocin eliminated (cured) F'lac and three low-copy-number mini-F plasmids (pML31, pMF21, and pMF45) from Escherichia coli to different extents. F'lac was cured 0 to 3%. pML31, whose replication region is contained on the 9-kilobase f5 EcoRI restriction enzyme fragment of F, was eliminated 10 to 92%. pMF21, deleted of the origin of mini-F replication at 42.6 kilobases on the F map and known to initiate from an origin at 45.1 kilobases, and its closely related derivative pMF45 were cured to the greatest extent (greater than 97%). pMF45 was eliminated from a wild-type bacterial strain but not from an isogenic novobiocin-resistant gyrB mutant strain, indicating involvement of the B subunit of DNA gyrase in the curing phenomenon. The number of bacteria containing pMF45 halved with each generation of growth in the presence of novobiocin, as is predicted for complete inhibition of plasmid DNA replication.
Assuntos
Replicação do DNA/efeitos dos fármacos , Escherichia coli/genética , Fator F/efeitos dos fármacos , Novobiocina/farmacologia , DNA Topoisomerases Tipo II/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismoAssuntos
Ampicilina/antagonistas & inibidores , Elementos de DNA Transponíveis/efeitos dos fármacos , Escherichia coli/genética , Fator F/efeitos dos fármacos , Translocação Genética/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Marcadores Genéticos/efeitos dos fármacos , Plasmídeos/efeitos dos fármacosRESUMO
Methylene blue enhanced the plasmid curing efficiency of chlorpromazine, imipramine and amitriptyline with strains of Escherichia coli K12 carrying F-prime lac or the resistance factor R-144. In contrast, methylene blue inhibited the elimination of plasmids by acridine orange and ethydium bromide at all concentrations tested. Two metabolic derivatives of chlorpromazine, chlorpromazine sulphoxide and 7.8-dioxochlorpromazine had no plasmid curing effect even in the presence of methylene blue. Amitriptyline, 7,8-dioxochlorpromazine and acridine orange were effective inhibitors of the conjugal transfer of the resistance plasmid, R-144, whilst methylene blue, chlorpromazine sulphoxide, and imipramine had only slight effects. We were therefore unable to demonstrate a simple correlation between curing ability and inhibition of plasmid transfer amongst the psychoactive drugs tested. A mechanism of plasmid curing by surface action of the drugs is suggested as an alternative to direct intercalation of the drugs into plasmid DNA.
Assuntos
Amitriptilina/farmacologia , Clorpromazina/farmacologia , Fator F/efeitos dos fármacos , Imipramina/farmacologia , Fatores R/efeitos dos fármacos , Laranja de Acridina/farmacologia , Conjugação Genética/efeitos dos fármacos , Escherichia coli/genética , Etídio/farmacologia , Azul de Metileno/farmacologiaRESUMO
Antibiotic resistance plasmids from organisms that had caused serious epidemics, including those responsible for epidemics of chloramphenicol-resistant typhoid fever and dysentery in Central America, were transferred to a strain of Salmonella typhimurium and of Salmonella gallinarum. The virulence and infectivity of these R(+) forms were then compared with the R(-) parent forms in orally inoculated chickens.None of the R(+) forms were more virulent than their R(-) parent forms. The mortality rates they produced were either the same as or less than that of their R(-) parent forms. The mortality rates were not increased by feeding the chickens on diets containing antibiotics against which the plasmids provided resistance.The removal of the plasmids from some R(+) forms of decreased virulence was not accompanied by any alteration in virulence, indicating that they were less virulent mutants of the parent strain that had conjugated preferentially. In other cases their virulence was increased, indicating that the very possession of the plasmid was involved in their decreased virulence. Of four forms of the S. gallinarum strain harbouring the plasmid that had been incriminated in the Central American dysentery outbreak, one was as virulent as the parent R(-) form and the other three were less virulent. Preferential conjugation by an avirulent mutant was responsible for the lack of virulence of one of them but the very possession of the plasmid appeared responsible for the decreased virulence of the other two. The decreased virulence of de-repressed F(+) and I(+) forms of the S. typhimurium strain was increased to that of repressed F(+) form and of the parent form by plasmid removal.Organisms of the R(+) forms of the S. typhimurium strain were not excreted in larger amounts or for longer periods of time by infected chickens than organisms of the R(-) parent form were. Neither did organisms of the R(+) forms of this strain or the S. gallinarum strain spread more rapidly or more extensively from infected chickens to in-contact chickens than organisms of the R(-) parent forms did. When antibiotics against which the infecting R(+) organisms provided resistance were included in the diet of these chickens the R(+) organisms were usually excreted in greater amounts, for longer periods of time and spread more rapidly and more extensively from the infected chickens to the in-contact chickens.