RESUMO
In this study, the consumption of 4-bromobenzoic acid and 4-chlorobenzoic acid by the fungus Penicillium brasilianum, an endophyte from Melia azedarach is evaluated. This fungus metabolizes these halobenzoic acids to produce three new brominated compounds, which have been isolated and characterized, and three new chlorinated derivatives identified by HRMS. Among these products, (4-bromobenzoyl)proline has been also chemically synthesized and employed in biological assays, thus providing insights for the elucidation of the defense mechanism of P. brasilianum towards these halobenzoic acids.
Assuntos
Antifúngicos/metabolismo , Bromobenzoatos/metabolismo , Clorobenzoatos/metabolismo , Endófitos/metabolismo , Melia azedarach/microbiologia , Penicillium/metabolismo , Antifúngicos/química , Biotransformação , Bromobenzoatos/química , Clorobenzoatos/química , Endófitos/química , Halogenação , Melia azedarach/metabolismo , Simulação de Acoplamento Molecular , Penicillium/química , Penicillium/enzimologiaRESUMO
The destruction of the herbicide chloramben in 0.050â¯M Na2SO4 solutions at natural pH has been studied by photoelectro-Fenton with UVA light (PEF). The trials were carried out in a cell equipped with an air-diffusion cathode for H2O2 generation and different electrocatalytic anodes, namely active IrO2-based and RuO2-based electrodes and non-active boron-doped diamond (BDD) and PbO2 ones. Similar removal rates were found regardless of the anode nature because the herbicide was mainly oxidized by OH formed from Fenton's reaction, which was enhanced by UVA-induced photo-Fenton reaction. The use of an IrO2-based anode led to almost total mineralization at high current density, as also occurred with the powerful BDD anode, since photoactive intermediates originated from OH-mediated oxidation were degraded under irradiation with UVA light. The good performance of the IrO2-based anode in PEF was confirmed at different current densities and herbicide concentrations. The presence of Cl- in the medium caused a slight deceleration of herbicide removal as well as mineralization inhibition, owing to the production of active chlorine with consequent formation of persistent chloroderivatives. Seven aromatic products along with oxalic and oxamic acids were identified in sulfate medium. Five aromatic derivatives were detected in Cl--containing matrix, corroborating the generation of organochlorine compounds. In secondary effluent, larger mineralization was achieved by PEF with a BDD anode due to its high oxidation ability to destroy the chloroderivatives, although an acceptable performance was also obtained using an IrO2-based anode.
Assuntos
Clorobenzoatos/isolamento & purificação , Poluentes Químicos da Água/isolamento & purificação , Purificação da Água , Boro , Clorobenzoatos/química , Diamante , Eletroquímica , Eletrodos , Herbicidas , Peróxido de Hidrogênio , Oxirredução , Água , Poluentes Químicos da Água/químicaRESUMO
Dihydroergotamine is a semisynthetic natural product derived from ergotamine, an ergot alkaloid. It is used to treat migraines, a neurological disease characterized by recurrent moderate to severe headaches. In this work, the in vitro metabolism of dihydroergotamine was evaluated in a biomimetic phase I reaction, aiming to verify all possible formed metabolites. Dihydroergotamine was submitted to an in vitro metabolism assay using rat liver microsomes, and the metabolites were analyzed by HPLC-MS/MS. The biomimetic reactions were performed with Jacobsen catalyst for scaling up production of oxidized metabolites. Two hydroxylated metabolites were isolated and characterized by MS/MS and 1H NMR analysis.
Assuntos
Di-Hidroergotamina/metabolismo , Di-Hidroergotamina/farmacocinética , Microssomos Hepáticos/metabolismo , Animais , Clorobenzoatos/metabolismo , Cromatografia Líquida de Alta Pressão , Humanos , Hidroxilação , Inativação Metabólica , Espectroscopia de Ressonância Magnética , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Oxirredução , Ratos Wistar , Espectrometria de Massas em TandemRESUMO
Understanding of functional diversity of microbial populations has lagged description of their molecular diversity. Differences in substrate specificity, kinetics, products, and regulation can dramatically influence phenotypic variation among closely related strains, features that are missed when the strains studied are the fastest-growing and most easily isolated from serial enrichments. To investigate the broader bacterial diversity underlying degradation of anthropogenic chemicals in nature, we studied the 3-chlorobenzoate (3-CBA) degradation rate in a collection of aerobic 3-CBA degraders previously isolated from undisturbed soils in two representative ecosystems: (i) Mediterranean sclerophyllous woodlands in California, Chile, South Africa, and Australia and (ii) boreal forests in Canada and Russia. The majority of isolates degraded 3-CBA slowly and did not completely mineralize 1.0 mM 3-CBA within 1 week. Those with intermediate degradation rates had incomplete degradation pathways and produced colored intermediates indicative of chlorocatechol, a product likely metabolized by other members of the community. About 10% of the isolates grew rapidly and mineralized greater than 90% of the 3-CBA, but because of population heterogeneity in soil, they are likely not large contributors to a soil's total transformation capacity. This suggests that xenobiotic degradation in nature is carried out by a community of cometabolic generalists and not by the efficient specialists that have been traditionally studied in the laboratory. A subset of 58 genotypically distinct strains able to degrade >80% of the 3-CBA was examined for their catabolic versatility using 45 different compounds: mono- and dichlorinated benzoates, phenols, anilines, toluenes, nitrobenzenes, chlorobenzenes, and 2,4-dichlorophenoxyacetic acid. The isolates degraded from 2 to more than 30 compounds with a median of 7, but there was no correlation to habitat of isolation or 3-CBA activity. However, these findings were indicative of finer-scale functional diversity.
Assuntos
Clorobenzoatos/análise , Ecossistema , Microbiologia do Solo , Austrália , Biodegradação Ambiental , California , Canadá , Chile , Clorobenzoatos/metabolismo , Genótipo , Federação Russa , África do Sul , Sulfatos/metabolismo , Árvores/microbiologiaRESUMO
An indigenous strain of Pseudomonas putida capable of degrading 3-chlorobenzoic acid as the sole carbon source was isolated from the Riachuelo, a polluted river in Buenos Aires. Aerobic biodegradation assays were performed using a 2-l microfermentor. Biodegradation was evaluated by spectrophotometry, chloride release, gas chromatography and microbial growth. Detoxification was evaluated by using Vibrio fischeri, Pseudokirchneriella subcapitata and Lactuca sativa as test organisms. The indigenous bacterial strain degrades 100 mg l(-1) 3-chlorobenzoic acid in 14 h with a removal efficiency of 92.0 and 86.1% expressed as compound and chemical oxygen demand removal, respectively. The strain was capable of degrading up to 1,000 mg of the compound l(-1). Toxicity was not detected at the end of the biodegradation process. Besides initial concentration, the effect of different factors, such as initial pH, initial inoculum, adaptation to the compound and presence of other substrates and toxic related compounds, was studied.
Assuntos
Clorobenzoatos/metabolismo , Pseudomonas putida/isolamento & purificação , Pseudomonas putida/metabolismo , Rios/microbiologia , Poluentes Químicos da Água/metabolismo , Aerobiose , Aliivibrio fischeri/efeitos dos fármacos , Biotransformação , Carbono/metabolismo , Clorobenzoatos/toxicidade , Clorófitas/efeitos dos fármacos , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Lactuca/efeitos dos fármacos , Pseudomonas putida/classificação , Pseudomonas putida/genética , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Espectrofotometria , Poluentes Químicos da Água/toxicidadeRESUMO
Cupriavidus necator JMP134 has been extensively studied because of its ability to degrade chloroaromatic compounds, including the herbicides 2,4-dichlorophenoxyacetic acid (2,4-D) and 3-chlorobenzoic acid (3-CB), which is achieved through the pJP4-encoded chlorocatechol degradation gene clusters: tfdCIDIEIFI and tfdDIICIIEIIFII. The present work describes a different tfd-genes expression profile depending on whether C. necator cells were induced with 2,4-D or 3-CB. By contrast, in vitro binding assays of the purified transcriptional activator TfdR showed similar binding to both tfd intergenic regions; these results were confirmed by in vivo studies of the expression of transcriptional lacZ fusions for these intergenic regions. Experiments aimed at investigating whether other pJP4 plasmid or chromosomal regulatory proteins could contribute to the differences in the response of both tfd promoters to induction by 2,4-D and 3-CB showed that the transcriptional regulators from the benzoate degradation pathway, CatR1 and CatR2, affected 3-CB- and 2,4-D-related growth capabilities. It was also determined that the ISJP4-interrupted protein TfdT decreased growth on 3-CB. In addition, an ORF with 34% amino acid identity to IclR-type transcriptional regulator members and located near the tfdII gene cluster module was shown to modulate the 2,4-D growth capability. Taken together, these results suggest that tfd transcriptional regulation in C. necator JMP134 is far more complex than previously thought and that it involves proteins from different transcriptional regulator families.
Assuntos
Proteínas de Bactérias/metabolismo , Cupriavidus necator/fisiologia , Regulação Bacteriana da Expressão Gênica , Fatores de Transcrição/metabolismo , Ácido 2,4-Diclorofenoxiacético/metabolismo , Fusão Gênica Artificial , Clorobenzoatos/metabolismo , DNA Intergênico , Ensaio de Desvio de Mobilidade Eletroforética , Perfilação da Expressão Gênica , Ordem dos Genes , Genes Bacterianos , Genes Reporter , Ligação Proteica , Regulon , Ativação Transcricional , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
Maleylacetate reductases (MAR) are required for biodegradation of several substituted aromatic compounds. To date, the functionality of two MAR-encoding genes (tfdF(I) and tfdF(II)) has been reported in Cupriavidus necator JMP134(pJP4), a known degrader of aromatic compounds. These two genes are located in tfd gene clusters involved in the turnover of 2,4-dichlorophenoxyacetate (2,4-D) and 3-chlorobenzoate (3-CB). The C. necator JMP134 genome comprises at least three other genes that putatively encode MAR (tcpD, hqoD and hxqD), but confirmation of their functionality and their role in the catabolism of haloaromatic compounds has not been assessed. RT-PCR expression analyses of C. necator JMP134 cells exposed to 2,4-D, 3-CB, 2,4,6-trichlorophenol (2,4,6-TCP) or 4-fluorobenzoate (4-FB) showed that tfdF(I) and tfdF(II) are induced by haloaromatics channelled to halocatechols as intermediates. In contrast, 2,4,6-TCP only induces tcpD, and any haloaromatic compounds tested did not induce hxqD and hqoD. However, the tcpD, hxqD and hqoD gene products showed MAR activity in cell extracts and provided the MAR function for 2,4-D catabolism when heterologously expressed in MAR-lacking strains. Growth tests for mutants of the five MAR-encoding genes in strain JMP134 showed that none of these genes is essential for degradation of the tested compounds. However, the role of tfdF(I)/tfdF(II) and tcpD genes in the expression of MAR activity during catabolism of 2,4-D and 2,4,6-TCP, respectively, was confirmed by enzyme activity tests in mutants. These results reveal a striking example of genetic redundancy in the degradation of aromatic compounds.
Assuntos
Proteínas de Bactérias/metabolismo , Cupriavidus necator/enzimologia , Cupriavidus necator/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Ácido 2,4-Diclorofenoxiacético/metabolismo , Proteínas de Bactérias/genética , Benzoatos/metabolismo , Biodegradação Ambiental , Clorobenzoatos/metabolismo , Clorofenóis/metabolismo , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , RNA Bacteriano/genéticaRESUMO
Cupriavidus necator JMP134(pJP4) is able to grow on 3-chlorobenzoate (3-CB), a model chloroaromatic pollutant. Catabolism of 3-CB is achieved via the expression of the chromosomally encoded benABCD genes and the tfd genes from plasmid pJP4. Since passive diffusion of benzoic acid derivatives at physiological pH is negligible, the uptake of this compound should be facilitated by a transport system. However, no transporter has so far been described to perform this function, and identification of chloroaromatic compound transporters has been limited. In this work, uptake experiments using 3-[ring-UL-(14)C]CB showed an inducible transport system in strain JMP134, whose expression is activated by 3-CB and benzoate. A similar level of 3-CB uptake was found for a mutant strain of JMP134, defective in chlorobenzoate degradation, indicating that metabolic drag is not an important component of the measured uptake rate. Competitive inhibitor assays showed that uptake of 3-CB was inhibited by benzoate and, to a lesser degree, by 3-CB and 3,5-dichlorobenzoate, but not by any of 12 other substituted benzoates tested. The expression of several gene candidates for this transport function was analysed by RT-PCR, including both permease-type and ABC-type ATP-dependent transporters. Induction of a chromosomally encoded putative permease transporter (benP gene) was found specifically in the presence of 3-CB or benzoate. A benP knockout mutant of strain JMP134 displayed an almost complete loss of 3-CB transport activity. This is to our knowledge the first report of a 3-CB transporter.
Assuntos
Clorobenzoatos/metabolismo , Cromossomos/metabolismo , Cupriavidus necator/genética , Cupriavidus necator/metabolismo , Anti-Infecciosos/farmacologia , Benzoatos/farmacologia , Transporte Biológico/efeitos dos fármacos , Cupriavidus necator/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Família Multigênica , Plasmídeos , RNA Bacteriano/análise , RNA Bacteriano/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ativação TranscricionalRESUMO
Goniothalamin oxide (1) is a styryl lactone which was isolated from bark and leaves of several Goniothalamus species. This natural product has some interesting biological properties such as larvicidal and tripanocidal activities. However, no studies on the antiproliferative profile of goniothalamin oxide (1) and its stereoisomers have been reported yet. Here, goniothalamin epoxide (1), isogoniothalamin epoxide (2) and their enantiomers were prepared via epoxidation of (R)-and (S)-goniothalamin (4). A 3:2 molar ratio in favor of goniothalamin oxide (1) and ent-1 was observed from (R)- and (S)-4, respectively, when 3-chloroperbenzoic acid (mCPBA) was employed while an increase to 6:1 molar ratio was achieved with (S,S)-Jacobsen's catalyst. Antiproliferative activity of these epoxides revealed that ent-isogoniothalamin oxide (ent-2) was the most active against the eight cancer cell lines studied. These results indicate that 6S, 7R and 8R absolute configurations are beneficial for the activity of these epoxides.
Assuntos
Antineoplásicos/síntese química , Compostos de Epóxi/síntese química , Pironas/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Catálise , Linhagem Celular Tumoral , Clorobenzoatos/química , Ensaios de Seleção de Medicamentos Antitumorais , Compostos de Epóxi/química , Compostos de Epóxi/farmacologia , Humanos , Pironas/química , Pironas/farmacologia , EstereoisomerismoRESUMO
Aerobic bacteria, such as Burkholderia xenovorans LB400, are able to degrade a wide range of polychlorobiphenyls (PCBs). Generally, these bacteria are not able to transform chlorobenzoates (CBAs), which accumulate during PCB degradation. In this study, the effects of CBAs on the growth, the morphology and the proteome of Burkholderia xenovorans LB400 were analysed. 4-CBA and 2-CBA were observed to inhibit the growth of strain LB400 on glucose. Strain LB400 exposed to 4-CBA exhibited increased number and size of electron-dense granules in the cytoplasm, which could be polyphosphates. Two-dimensional (2-D) polyacrylamide gel electrophoresis was used to characterise the molecular response of strain LB400 to 4-CBA. This compound induced the enzymes BenD and CatA of benzoate and catechol catabolic pathways. The induction of molecular chaperones DnaK and HtpG by 4-CBA indicated that the exposure to this compound constitutes a stressful condition for this bacterium. Additionally, the induction of some Krebs cycle enzymes was observed, probably as response to cellular energy requirements. This study contributes to the knowledge on the effects of CBA on the PCB-degrader Burkholderia xenovorans LB400.
Assuntos
Burkholderia/efeitos dos fármacos , Clorobenzoatos/farmacologia , Proteínas de Choque Térmico/biossíntese , Bifenilos Policlorados/farmacocinética , Proteoma/metabolismo , Biodegradação Ambiental , Burkholderia/enzimologia , Burkholderia/crescimento & desenvolvimento , Burkholderia/metabolismo , Eletroforese em Gel Bidimensional/métodos , Proteínas de Choque Térmico/antagonistas & inibidores , Proteínas de Choque Térmico/metabolismo , Microscopia Eletrônica/métodos , Fator G para Elongação de Peptídeos/genética , Fator G para Elongação de Peptídeos/metabolismo , Bifenilos Policlorados/metabolismo , Análise de Sequência de ProteínaRESUMO
The polychlorinated biphenyl (PCB)-degrading Pseudomonas sp. B4 was tested for its motility and ability to sense and respond to biphenyl, its chloroderivatives and chlorobenzoates in chemotaxis assays. Pseudomonas sp. B4 was attracted to biphenyl, PCBs and benzoate in swarm plate and capillary assays. Chemotaxis towards these compounds correlated with their use as carbon and energy sources. No chemotactic effect was observed in the presence of 2- and 3-chlorobenzoates. Furthermore, a toxic effect was observed when the microorganism was exposed to 3-chlorobenzoate. A nonmotile Pseudomonas sp. B4 transformant and Burkholderia xenovorans LB400, the laboratory model strain for PCB degradation, were both capable of growing in biphenyl as the sole carbon source, but showed a clear disadvantage to access the pollutants to be degraded, compared with the highly motile Pseudomonas sp. B4, stressing the importance of motility and chemotaxis in this environmental biodegradation.
Assuntos
Clorobenzoatos/farmacologia , Bifenilos Policlorados/farmacologia , Pseudomonas/efeitos dos fármacos , Fenômenos Fisiológicos Bacterianos/efeitos dos fármacos , Quimiotaxia/efeitos dos fármacos , Pseudomonas/fisiologiaRESUMO
Cupriavidus necator JMP134(pJP4) harbors a catabolic plasmid, pJP4, which confers the ability to grow on chloroaromatic compounds. Repeated growth on 3-chlorobenzoate (3-CB) results in selection of a recombinant strain, which degrades 3-CB better but no longer grows on 2,4-dichlorophenoxyacetate (2,4-D). We have previously proposed that this phenotype is due to a double homologous recombination event between inverted repeats of the multicopies of this plasmid within the cell. One recombinant form of this plasmid (pJP4-F3) explains this phenotype, since it harbors two copies of the chlorocatechol degradation tfd gene clusters, which are essential to grow on 3-CB, but has lost the tfdA gene, encoding the first step in degradation of 2,4-D. The other recombinant plasmid (pJP4-FM) should harbor two copies of the tfdA gene but no copies of the tfd gene clusters. A molecular analysis using a multiplex PCR approach to distinguish the wild-type plasmid pJP4 from its two recombinant forms, was carried out. Expected PCR products confirming this recombination model were found and sequenced. Few recombinant plasmid forms in cultures grown in several carbon sources were detected. Kinetic studies indicated that cells containing the recombinant plasmid pJP4-FM were not selectable by sole carbon source growth pressure, whereas those cells harboring recombinant plasmid pJP4-F3 were selected upon growth on 3-CB. After 12 days of repeated growth on 3-CB, the complete plasmid population in C. necator JMP134 apparently corresponds to this form. However, wild-type plasmid forms could be recovered after growing this culture on 2,4-D, indicating that different plasmid forms can be found in C. necator JMP134 at the population level.
Assuntos
Burkholderiaceae/genética , Burkholderiaceae/metabolismo , Plasmídeos/genética , Recombinação Genética , Ácido 2,4-Diclorofenoxiacético/metabolismo , Sequência de Bases , Clorobenzoatos/metabolismo , DNA Bacteriano/análise , DNA Bacteriano/química , DNA Bacteriano/genética , Genes Bacterianos , Dados de Sequência Molecular , Família Multigênica , Reação em Cadeia da Polimerase , Seleção Genética , Análise de Sequência de DNARESUMO
The acrosome reaction (AR) is a special exocytotic process promoted by signal transduction pathways studied in many laboratories. Progesterone (P4) is one of the trigger molecules proposed. Upon the binding of P4 to its receptor, several molecules could be activated, including G-proteins, phospholipase A(2) (PLA(2)), and phospholipase C (PLC). The role of these molecules was analyzed in this study using the Chlortetracycline (CTC) protocol to detect and quantify the AR. Incubation of capacitated sperm cells with GTPgammas (GTPgammas, a mimetic of G-protein activation), arachidonic acid (AA, product of PLA(2) action), or phorbol ester (PMA, an activator of PLC) for 15 min increased the AR to a similar percentage as P4. Conversely, a decrease in the AR was detected when sperm cells were incubated with P4 after preincubation with: GDPbetaS (GDP, an inhibitor of G-protein activation), ONO RS-82 (ONO, an inhibitor of PLA(2)), or neomycin (Neo, an inhibitor of PLC) for 15 min. To analyze the activation sequence of G proteins, PLA(2), and PLC combinations of these mimetic/inhibitors were used during successive incubation periods. Inhibition promoted by GDP, ONO, and Neo were overcome by 15-min incubation with GTPgammas, AA, or PMA, respectively. But GTPgammas or P4 did not reverse the inhibition due to incubation with Neo and ONO. Interestingly, this dual inhibition was reverted by another 15-min incubation with AA or PMA. Results presented here could indicate that the AR triggered by P4 is driven by activation of G-proteins, that in turn activate PLA(2) and PLC simultaneously, that finally promote acrosomal exocytosis.
Assuntos
Reação Acrossômica/fisiologia , Proteínas Heterotriméricas de Ligação ao GTP/fisiologia , Fosfolipases A/metabolismo , Progesterona/fisiologia , Acetato de Tetradecanoilforbol/análogos & derivados , Fosfolipases Tipo C/metabolismo , Aminobenzoatos/farmacologia , Animais , Clorobenzoatos , Cinamatos/farmacologia , Proteínas Heterotriméricas de Ligação ao GTP/antagonistas & inibidores , Masculino , Camundongos , Fosfolipases A/antagonistas & inibidores , Fosfolipases A2 , Espermatozoides/metabolismo , Espermatozoides/fisiologia , Acetato de Tetradecanoilforbol/farmacologia , Fosfolipases Tipo C/antagonistas & inibidores , ortoaminobenzoatosRESUMO
Physicochemical characterization of hazardous compounds often is required for the development of structure-reactivity correlations. Physical, chemical, and toxicological properties of target pollutants require determination for an efficient application of wastewater treatments. In the present work, we chose a chloro-nitro-aromatic derivative (4-chloro-3,5-dinitrobenzoic acid [CDNBA]), as a model compound on which to perform physicochemical and toxicological studies. Several properties of CDNBA are not available in the literature, although many aromatic nitro-compounds are considered hazardous materials. Measurements of solubility in water, acid dissociation constant, and kinetic parameters for the nucleophilic substitution of chlorine atom in alkaline media are reported. We also performed cytotoxicity studies of CDNBA and ultraviolet-irradiated CDNBA solutions. From the analysis of CDNBA solubility in water at different temperatures, an enthalpy of solution of 23.2 +/- 2.5 kJ/mol was found. The study of the acid dissociation constant Kc by using conductivity measurements and the modified Gran's method yielded values for the equilibrium constant Ka of 2.36 x 10(-3) and 2.26 x 10(-3), respectively. The bimolecular rate constant for the reaction of CDNB- and hydroxyl ion (HO) measured at room temperature and 0.1 M of ionic strength was 5.92/M x s, and the activation energy for this process was 70.7 +/- 3.4 kJ/mol. Cytotoxicity assays with aqueous suspensions of Tetrahymena pyriformis showed lethal effects due to the pH change induced by CDNBA. On the other hand, in buffered solutions, a value of 104.47 microM was observed for the median effective concentration, that is, the concentration of CDNBA at which the proliferation was restricted to one half of the blank. Irradiation of CDNBA solutions increased the toxicity, suggesting the formation of intermediate products with higher cytotoxicity effects.
Assuntos
Clorobenzoatos/química , Clorobenzoatos/toxicidade , Água/química , Equilíbrio Ácido-Base , Ácidos/química , Ácidos/toxicidade , Soluções Tampão , Fenômenos Químicos , Físico-Química , Cloro/química , Concentração de Íons de Hidrogênio , Hidróxidos/química , Cinética , Fotólise , Solubilidade , Soluções , Relação Estrutura-Atividade , Temperatura , Eliminação de Resíduos Líquidos/métodosRESUMO
Ralstonia eutropha JMP134(pJP4) degrades 3-chlorobenzoate (3-CB) by using two not completely isofunctional, pJP4-encoded chlorocatechol degradation gene clusters, tfdC(I)D(I)E(I)F(I) and tfdD(II)C(II)E(II)F(II). Introduction of several copies of each gene cluster into R. eutropha JMP222, which lacks pJP4 and thus accumulates chlorocatechols from 3-CB, allows the derivatives to grow in this substrate. However, JMP222 derivatives containing one chromosomal copy of each cluster did not grow in 3-CB. The failure to grow in 3-CB was the result of accumulation of chlorocatechols due to the limiting activity of chlorocatechol 1,2-dioxygenase (TfdC), the first enzyme in the chlorocatechol degradation pathway. Micromolar concentrations of 3- and 4-chlorocatechol inhibited the growth of strains JMP134 and JMP222 in benzoate, and cells of strain JMP222 exposed to 3 mM 3-CB exhibited a 2-order-of-magnitude decrease in viability. This toxicity effect was not observed with strain JMP222 harboring multiple copies of the tfdC(I) gene, and the derivative of strain JMP222 containing tfdC(I)D(I)E(I)F(I) plus multiple copies of the tfdC(I) gene could efficiently grow in 3-CB. In addition, tfdC(I) and tfdC(II) gene mutants of strain JMP134 exhibited no growth and impaired growth in 3-CB, respectively. The introduction into strain JMP134 of the xylS-xylXYZL genes, encoding a broad-substrate-range benzoate 1,2-dioxygenase system and thus increasing the transformation of 3-CB into chlorocatechols, resulted in derivatives that exhibited a sharp decrease in the ability to grow in 3-CB. These observations indicate that the dosage of chlorocatechol-transforming genes is critical for growth in 3-CB. This effect depends on a delicate balance between chlorocatechol-producing and chlorocatechol-consuming reactions.
Assuntos
Catecóis/metabolismo , Clorobenzoatos/metabolismo , Cupriavidus necator/genética , Cupriavidus necator/metabolismo , Dioxigenases , Endo-1,4-beta-Xilanases , Oxirredutases atuantes sobre Doadores de Grupo CH-CH , Proteínas de Bactérias , Sequência de Bases , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Divisão Celular/genética , Cupriavidus necator/crescimento & desenvolvimento , Proteínas de Ligação a DNA , Dosagem de Genes , Dados de Sequência Molecular , Família Multigênica , Oxirredutases/genética , Oxirredutases/metabolismo , Oxigenases/genética , Oxigenases/metabolismo , Transativadores/genética , Transativadores/metabolismo , Xilosidases/genética , Xilosidases/metabolismoRESUMO
Ralstonia eutropha JMP134 (pJP4) grows on 3-chlorobenzoate (3-CB) or 2,4-dichlorophenoxyacetate (2,4-D). The copy number of chlorocatechol genes has been observed to be important for allowing growth of bacterial strains on chloroaromatic compounds. Despite the fact that two functional chlorocatechol degradation tfd gene clusters are harbored on plasmid pJP4, a single copy of the region comprising all tfd genes in strain JMP134-F was insufficient to allow growth on 3-CB, whereas growth on 2,4-D was only slightly retarded compared to the wild-type strain. Using competitive PCR, approximately five copies of pJP4 per genome were observed to be present in the wild-type strain, whereas only one copy of pJP4 was present per chromosome in strain JMP134-F. Therefore, several copies of pJP4 per chromosome are required for full expression of the tfd-encoded growth abilities in the wild-type R. eutropha strain.
Assuntos
Clorobenzoatos/metabolismo , Cupriavidus necator/crescimento & desenvolvimento , Dosagem de Genes , Plasmídeos/genética , Ácido 2,4-Diclorofenoxiacético/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biodegradação Ambiental , Cupriavidus necator/genética , Família Multigênica , Reação em Cadeia da PolimeraseRESUMO
The enzymes chlorocatechol-1,2-dioxygenase, chloromuconate cycloisomerase, dienelactone hydrolase, and maleylacetate reductase allow Ralstonia eutropha JMP134(pJP4) to degrade chlorocatechols formed during growth in 2,4-dichlorophenoxyacetate or 3-chlorobenzoate (3-CB). There are two gene modules located in plasmid pJP4, tfdC(I)D(I)E(I)F(I) (module I) and tfdD(II)C(II)E(II)F(II) (module II), putatively encoding these enzymes. To assess the role of both tfd modules in the degradation of chloroaromatics, each module was cloned into the medium-copy-number plasmid vector pBBR1MCS-2 under the control of the tfdR regulatory gene. These constructs were introduced into R. eutropha JMP222 (a JMP134 derivative lacking pJP4) and Pseudomonas putida KT2442, two strains able to transform 3-CB into chlorocatechols. Specific activities in cell extracts of chlorocatechol-1,2-dioxygenase (tfdC), chloromuconate cycloisomerase (tfdD), and dienelactone hydrolase (tfdE) were 2 to 50 times higher for microorganisms containing module I compared to those containing module II. In contrast, a significantly (50-fold) higher activity of maleylacetate reductase (tfdF) was observed in cell extracts of microorganisms containing module II compared to module I. The R. eutropha JMP222 derivative containing tfdR-tfdC(I)D(I)E(I)F(I) grew four times faster in liquid cultures with 3-CB as a sole carbon and energy source than in cultures containing tfdR-tfdD(II)C(II)E(II)F(II). In the case of P. putida KT2442, only the derivative containing module I was able to grow in liquid cultures of 3-CB. These results indicate that efficient degradation of 3-CB by R. eutropha JMP134(pJP4) requires the two tfd modules such that TfdCDE is likely supplied primarily by module I, while TfdF is likely supplied by module II.
Assuntos
Clorobenzoatos/metabolismo , Cupriavidus necator/enzimologia , Cupriavidus necator/genética , Genes Bacterianos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biodegradação Ambiental , Meios de Cultura , Cupriavidus necator/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Plasmídeos/genéticaRESUMO
Phospholipase A2 (PLA2, EC 3.1.1.4) is involved in the cascade of signalling events leading to the acrosome reaction in human spermatozoa. In order to study the role of PLA2 in the acrosome reaction triggered by GTP gamma S, a non-hydrolizable analogue of GTP, two well-known PLA2 inhibitory reagents were used: dexamethasone (1 mM, a synthetic glucocorticoid), and 2-(p-amylcinnamoyl)amino-4-chlorobenzoic acid (ONO-RS-082, 320 micrograms/ml). Normal human spermatozoa were incubated for 3 h under capacitating conditions and treated with several reagents [GTP gamma S, dexamethasone, ONO-RS-082, arachidonic acid (AA) and lysophosphatidylcholine (LPC)], alone or in different combinations. In confirmation of earlier reports, GTP gamma S induced the acrosome reaction. On the other hand, dexamethasone and ONO-RS-082 were both able to inhibit the acrosome reaction induced by GTP gamma S. However, when AA or LPC was added after dexamethasone or ONO-RS-082, the acrosome reaction reached values close to those obtained using GTP gamma S alone. It is concluded that PLA2 probably plays an active role in the acrosome reaction triggered by GTP-binding proteins.
Assuntos
Acrossomo/efeitos dos fármacos , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Guanosina Trifosfato/análogos & derivados , Fosfolipases A/antagonistas & inibidores , Espermatozoides/efeitos dos fármacos , Aminobenzoatos/farmacologia , Ácido Araquidônico/farmacologia , Clorobenzoatos , Cinamatos/farmacologia , Dexametasona/farmacologia , Humanos , Lisofosfatidilcolinas/farmacologia , Masculino , Fosfolipases A2 , Espermatozoides/fisiologia , ortoaminobenzoatosRESUMO
Pseudomonas putida P111 was isolated by enrichment culture on 2,5-dichlorobenzoate and was also able to grow on 2-chloro-, 3-chloro-, 4-chloro-, 2,3-dichloro-, 2,4-dichloro-, and 2,3,5-trichlorobenzoates. However, 3,5-dichlorobenzoate completely inhibited growth of P111 on all ortho-substituted benzoates that were tested. When 3,5-dichlorobenzoate was added as a cosubstrate with either 3- or 4-chlorobenzoate, cell yields and chloride release were greater than those observed from growth on either monochlorobenzoate alone. Moreover, resting cells of P111 grown on 4-chlorobenzoate released chloride from 3,5-dichlorobenzoate and produced no identifiable intermediate. In contrast, resting cells grown on 2,5-dichlorobenzoate metabolized 3,5-dichlorobenzoate without release of chloride and accumulated a degradation product, which was identified as 1-carboxy-1,2-dihydroxy-3,5-dichlorocyclohexadiene on the basis of gas chromatography-mass spectrometry confirmation of its two acid-hydrolyzed products, 3,5- and 2,4-dichlorophenol. Since 3,5-dichlorocatechol was rapidly metabolized by cells grown on 2,5-dichlorobenzoate, it is apparent that 1-carboxy-1,2-dihydroxy-3,5-dichlorocyclohexadiene is not further metabolized by these cells. Moreover, induction of a functional dihyrodiol dehydrogenase would not be required for growth of P111 on other ortho-chlorobenzoates since the corresponding chlorodihydrodiols produced from a 1,2-dioxygenase attack would spontaneously decompose to the corresponding catechols. In contrast, growth on 3-chloro-, 4-chloro-, or 3,5-dichlorobenzoate requires a functional dihydrodiol dehydrogenase, yet only the two monochlorobenzoates appear to induce for it.
Assuntos
Clorobenzoatos/metabolismo , Pseudomonas putida/crescimento & desenvolvimento , Biodegradação Ambiental , Clorobenzoatos/química , Clorobenzoatos/farmacologia , Meios de Cultura , Poluentes Ambientais/metabolismo , Poluentes Ambientais/farmacologia , Pseudomonas putida/efeitos dos fármacos , Pseudomonas putida/metabolismoRESUMO
The aim of this work was to learn the toxicity of acaricides: monocrotophos, chlorobenzilate and chlorphenamidine when used as an immersion and as a spray on the phytophagous mites, Tetranychus (T) urticae, Tetranychus (T) cinnabarinus and Tetranychus (T) ludeni under laboratory conditions. It was concluded that the mite T. urticae was sensitive to chlorphenamidine at least when used as a spray without killing them in a significant level. However the mites T. cinnabarinus and T. ludeni were sensitive to chlorphenamidine when using immersion method. The monocrotophos and the chlorobenzilate were toxic of the three species of mites using though the employed methods.