RESUMO
AIMS: To achieve reliable detection of methicillin resistance in clinical isolates of coagulase-negative staphylococci. METHODS AND RESULTS: Strains (105) were evaluated by normatized antimicrobial susceptibility methods, and for the presence of the methicillin resistance-determining mecA gene, using the polymerase chain reaction. Correlation between phenotypic and genotypic methods was obtained in 87.6% of the samples. Six strains, classified as methicillin-susceptible by phenotypic assays, revealed the presence of the mecA gene, indicating that methicillin resistance expression was probably repressed. Another seven isolates failed to show mecA amplification after displaying methicillin resistance in phenotypic evaluations. The susceptibility of the methicillin-resistant isolates to other antimicrobial agents was variable. CONCLUSION: Genotypic determination of the mecA gene proved to be the most reliable method for detection of methicillin resistance. SIGNIFICANCE AND IMPACT OF THE STUDY: Correct assessment of methicillin resistance, such as that attained through genotyping, is essential for defining therapeutic strategies, particularly when treating severely compromised patients.
Assuntos
Proteínas de Bactérias , Coagulase/metabolismo , Hexosiltransferases , Resistência a Meticilina/genética , Meticilina/farmacologia , Peptidil Transferases , Staphylococcus/efeitos dos fármacos , Staphylococcus/genética , Proteínas de Transporte/genética , Genes Bacterianos/genética , Genótipo , Meticilina/uso terapêutico , Testes de Sensibilidade Microbiana , Muramilpentapeptídeo Carboxipeptidase/genética , Proteínas de Ligação às Penicilinas , Fenótipo , Reação em Cadeia da Polimerase , Staphylococcus/classificação , Staphylococcus/enzimologiaRESUMO
The DnaK system is required for the productive folding of pea chloroplast ferredoxin-NADP+ reductase (FNR) expressed in Escherichia coli. The formation of a mature active enzyme was severely impaired in E. coli dnaK, dnaJ or grpE mutants expressing either the cytosolic precursor of the reductase (preFNR) or the mature apoenzyme, and these forms aggregated extensively in these cells. Coexpression of dnaK from a multicopy plasmid in the dnaK-null mutants restored preFNR processing and folding of FNR, rendering a mature-sized active enzyme. Overexpression of GroESL chaperonins failed to prevent preFNR aggregation, but it restored productive folding of FNR in dnaK-null mutants expressing the mature enzyme. Expression of preFNR in OmpT-protease-deficient E. coli cells resulted in the accumulation of the unprocessed precursor in the soluble fraction of the cells. The interaction of this soluble preFNR, but not the mature reductase, with DnaK and GroEL was evidenced by immunoprecipitation studies. We conclude that, in addition to the GroE chaperonins [Carrillo, N., Ceccarelli, E. A., Krapp, A. R., Boggio, S., Ferreyra, R. G. & Viale, A. M. (1992) J. Biol. Chem. 267, 15537-15541], the DnaK chaperone system plays a crucial role in the folding pathway of FNR.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli , Escherichia coli/fisiologia , Ferredoxina-NADP Redutase/biossíntese , Ferredoxina-NADP Redutase/química , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Óperon , Pisum sativum/enzimologia , Dobramento de Proteína , Alelos , Proteínas de Bactérias/genética , Sítios de Ligação , Chaperoninas , Cloroplastos/enzimologia , Clonagem Molecular , Escherichia coli/genética , Proteínas de Choque Térmico HSP40 , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/químicaRESUMO
Ferredoxin-NADP+ reductases (FNR) participate in cellular defense against oxidative damage. Escherichia coli mutants deficient in FNR are abnormally sensitive to methyl viologen and hydrogen peroxide. Tolerance to these oxidants was regained by expression of plant FNR, superoxide dismutase, or catalase genes in the mutant cells. FNR contribution to the concerted defense against viologen toxicity under redox-cycling conditions was similar to that of the two major E. coli superoxide dismutases together, as judged by the phenotypes displayed by relevant mutant strains. However, FNR expression in sodA sodB strains failed to increase their tolerance to viologens, indicating that the FNR target is not the superoxide radical. Sensitivity of FNR-deficient cells to oxidants is related to extensive DNA damage. Incubation of the mutant bacteria with iron chelators or hydroxyl radical scavengers provided significant protection against viologens or peroxide, suggesting that oxidative injury in FNR-deficient cells was mediated by intracellular iron through the formation of hydroxyl radicals in situ. The NADP(H)-dependent activities of the reductase were necessary and sufficient for detoxification, without participation of either ferredoxin or flavodoxin in the process. Possible mechanisms by which FNR may exert its protective role are discussed.
Assuntos
Proteínas de Escherichia coli , Escherichia coli/fisiologia , Ferredoxina-NADP Redutase/metabolismo , Genes de Plantas , Estresse Oxidativo/fisiologia , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Catalase/biossíntese , Catalase/metabolismo , Cloroplastos/enzimologia , Clonagem Molecular , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Ferredoxina-NADP Redutase/biossíntese , Ferredoxina-NADP Redutase/genética , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Proteínas Ferro-Enxofre/biossíntese , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/metabolismo , Solanum lycopersicum/enzimologia , Solanum lycopersicum/genética , Modelos Biológicos , Modelos Estruturais , Oxigênio/toxicidade , Paraquat/farmacologia , Pisum sativum/enzimologia , Pisum sativum/genética , Conformação Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Superóxido Dismutase/biossíntese , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismoRESUMO
The cytosolic precursor of the chloroplast flavoprotein ferredoxin-NADP+ reductase was expressed in Escherichia coli rendering a soluble protein that contained bound FAD and could be imported by isolated chloroplasts. The mechanism of plastid translocation was studied under defined conditions using this recombinant precursor holoprotein and intact pea chloroplasts. The first step in the import pathway, namely, binding of the reductase precursor to isolated chloroplasts, was saturable at about 2000 molecules/plastid, and showed a high-affinity interaction with a dissociation constant Kd of approximately 5 nM. Binding was not affected by the addition of soluble leaf extracts or by prior denaturation of the precursor with urea. Analysis of the initial import rates at different precursor concentrations indicated the existence of a single translocation system for this protein. Inclusion of leaf extracts in the assay resulted in a three-fold increase of the maximal import rates to 14,000 molecules . min-(1).chloroplast-(1), with a concomitant decrease in the apparent Km for the recombinant precursor, from 1 microM to 100-150 nM. Comparison of Km and Kd values under various conditions indicated that the binding step of the translocation process is largely irreversible, favouring import and processing. In the absence of extract, a denatured precursor obtained by incubation with urea was a better substrate for plastid import than the holoprotein. Treatment of the precursor with either extract or urea resulted in similar increases in import efficiency (V/Km), suggesting that stimulation by leaf extracts is probably related to unfolding of the precursor prior to translocation.
Assuntos
Cloroplastos/metabolismo , Ferredoxinas/química , Ferredoxinas/metabolismo , NADP/metabolismo , Transporte Biológico , Cloroplastos/enzimologia , Precursores Enzimáticos/química , Precursores Enzimáticos/metabolismo , NADP/química , Folhas de Planta/química , Conformação Proteica , Desnaturação Proteica , Dobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Fatores de Tempo , Ureia/químicaRESUMO
The precursor of the chloroplast flavoprotein ferredoxin-NADP+ reductase from pea was expressed in Escherichia coli as a carboxyl-terminal fusion to glutathione S-transferase. The fused protein was soluble, and the precursor could be purified in a few steps involving affinity chromatography on glutathione-agarose, cleavage of the transferase portion by protease Xa, and ion exchange chromatography on DEAE-cellulose. The purified prereductase contained bound FAD but displayed marginally low levels of activity. Removal of the transit peptide by limited proteolysis rendered a functional protease-resistant core exhibiting enzymatic activity. The FAD-containing precursor expressed in E. coli was readily transported into isolated pea chloroplasts and was processed to the mature size, both inside the plastid and by incubation with stromal extracts in a plastid-free reaction. Import was dependent on the presence of ATP and was stimulated severalfold by the addition of plant leaf extracts.
Assuntos
Precursores Enzimáticos/metabolismo , Ferredoxina-NADP Redutase/metabolismo , Pisum sativum/enzimologia , Sequência de Bases , Transporte Biológico Ativo , Cloroplastos/enzimologia , DNA Complementar/genética , DNA de Plantas/genética , Precursores Enzimáticos/química , Precursores Enzimáticos/genética , Escherichia coli/genética , Ferredoxina-NADP Redutase/química , Ferredoxina-NADP Redutase/genética , Flavina-Adenina Dinucleotídeo/química , Vetores Genéticos , Dados de Sequência Molecular , Pisum sativum/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Escherichia coli cells carrying the mvrA mutation are unable to grow aerobically in the presence of the radical propagator methyl viologen (MV). Resistance against MV toxicity could be restored by the introduction of cloned DNA sequences encoding pea chloroplast ferredoxin-NADP+ reductase (FNR), a member of a class of flavoenzymes involved in redox pathways in bacteria, plants and animals. Complementation was strictly dependent on the accumulation of a functional transgenic FNR, since mutated reductases showing decreased enzymatic activities only partially rescued the MV-resistant phenotype. These results support recent observations suggesting that the E. coli mvrA gene encodes a ferredoxin (flavodoxin)-NADP+ reductase (V. Bianchi et al. (1993) J. Bacteriol. 175, 1590-1595). The mvrA mutant cells showed a moderate decrease in the flavodoxin-dependent activation of enzymes essential for anaerobic growth of E. coli. This effect is prevented by expression of a functional pea FNR in the mutant cells.
Assuntos
Escherichia coli/enzimologia , Oxirredutases/metabolismo , Paraquat/metabolismo , Pisum sativum/enzimologia , Cloroplastos/enzimologia , Ativação Enzimática , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Flavodoxina/farmacologia , Mutação , Oxirredutases/genética , Paraquat/farmacologiaRESUMO
Complementary DNA sequences encoding the mature form of pea ferredoxin-NADP+ reductase were cloned in-frame at the 3' end of the Schistosoma japonicum glutathione S-transferase gene in the expression vector pGEX-3X (Smith and Johnson, Gene 67, 31-40, 1988). A spacer sequence linking the two genes was modified to provide a proteolytic site just before the first amino acid residue of mature pea reductase. When introduced into competent Escherichia coli cells and induced, the resulting plasmid (pGF205) directed the expression of a 60-kDa immunoreactive peptide that results from the fusion between glutathione S-transferase and ferredoxin-NADP+ reductase sequences. The fused protein could be purified in a single step by selective absorption onto glutathione-agarose beads, followed by elution with free glutathione. It showed both transferase and reductase activities. Removal of the transferase portion by cleavage with the restriction protease Xa rendered ferredoxin-NADP+ reductase electrophoretically homogeneous. The purified transgenic enzyme showed kinetic and spectroscopic properties that were similar to those reported for the plant flavoprotein, indicating that, even when fused to the 27-kDa transferase portion, the reductase was still able to assemble FAD and to acquire an active conformation in the bacterial host. The expression-purification protocol employed here allows the isolation of up to 1 mg of active ferredoxin-NADP+ reductase/g of transformed cells. The system is potentially useful for the purification of activity-impaired forms of the flavoprotein.
Assuntos
Fabaceae/enzimologia , Ferredoxina-NADP Redutase/genética , Ferredoxina-NADP Redutase/isolamento & purificação , Plantas Medicinais , Sequência de Aminoácidos , Sequência de Bases , Endopeptidases/metabolismo , Fabaceae/genética , Ferredoxina-NADP Redutase/biossíntese , Ferredoxina-NADP Redutase/metabolismo , Glutationa Transferase/biossíntese , Glutationa Transferase/genética , Glutationa Transferase/isolamento & purificação , Dados de Sequência Molecular , Plasmídeos/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismoRESUMO
We have recently reported the expression in Escherichia coli of an enzymatically competent ferredoxin-NADP+ oxidoreductase from cloned pea genes encoding either the mature enzyme or its precursor protein (Ceccarelli, E. A., Viale, A. M., Krapp, A. R., and Carrillo, N. (1991) J. Biol. Chem. 266, 14283-14287). Processing to the mature form by bacterial protease(s) and FAD assembly occurred in the bacterial cytosol. Expression of ferredoxin-NADP+ reductase in chaperonin-deficient (groE-) mutants of E. coli resulted in partial reductase assembly at permissive growth temperatures (i.e. 30 degrees C), and in total breakdown of holoenzyme assembly, and accumulation as aggregated inclusion bodies at non-permissive temperatures (i.e. 42 degrees C). Coexpression in these mutants of a cloned groESL operon from the phototrophic bacterium Chromatium vinosum resulted in partial or total recoveries of ferredoxin-NADP+ reductase assembly. The overall results indicate that bacterial chaperonins are required for the productive folding/assembly of eucaryotic ferredoxin-NADP+ reductase expressed in E. coli.
Assuntos
Proteínas de Bactérias/metabolismo , Chaperoninas/metabolismo , Chromatium/genética , Escherichia coli/genética , Ferredoxina-NADP Redutase/genética , Óperon , Proteínas de Bactérias/genética , Chaperonina 10 , Chaperonina 60 , Chromatium/enzimologia , Clonagem Molecular , Escherichia coli/metabolismo , Proteínas de Escherichia coli , Expressão Gênica , Proteínas de Choque Térmico/genética , PlasmídeosRESUMO
The flavoprotein ferredoxin-NADP+ reductase (FNR) catalyzes the final step of the photosynthetic electron transport chain, i.e. the reduction of NADP+ by ferredoxin. A cloned FNR cDNA from a pea library (Newman, B., and Gray, J. (1988) Plant Mol. Biol. 10, 511-520) was used to construct plasmids which express the apoenzyme in Escherichia coli. Two recombinant vectors were prepared, one containing the sequence corresponding to the mature enzyme and another including, in addition, the sequence of the transit peptide that directs FNR to the chloroplast. These proteins were expressed as fusion products to the NH2-terminal portion of beta-galactosidase. In both cases, a 35-kDa immunoreactive polypeptide was the major product, suggesting that the proteins were processed in vivo. NH2-terminal sequence determination of the purified recombinant proteins indicate cleavage at positions -1/-2 with respect to the normal processing site in chloroplasts. The processed enzymes showed enzymatic activities and spectral properties that were similar or identical to those of native plant FNR. When a La protease-deficient E. coli strain was used as a host, the expressed FNR precursor was found to be poorly processed, associated to bacterial pellets, and showed no detectable FNR activity. The overall results indicate that acquisition of the native enzyme conformation and assembly of the prosthetic group takes place in the bacterial host, generating an enzyme that is, as far as studied, indistinguishable from plant FNR.