RESUMEN
The beta-tubulin gene of Microbotryum violaceum was sought originally for its potential use in improving the transformation of this organism. The gene was cloned and its nucleotide sequence was determined. The gene was predicted to encode a 444-residue protein with strong sequence similarity to other beta-tubulins. The coding region was 2.85 kb, much larger than the corresponding genes from other organisms. This was due to the large number of introns in this gene, as determined by comparison of the cDNA sequence with that for the genomic clone. This gene contained 14 introns, which is the most introns in a beta-tubulin-encoding gene yet reported for any organism. Intron position comparisons between the M. violaceum gene and those from beta-tubulin genes of other organisms revealed a striking result, since 10 of the 14 introns were unique. An additional feature of the gene's organization was an unusually long 5' untranslated region, predicted to be nearly 1 kb in length. The possible significance of these unusual features of genetic structure is discussed.
Asunto(s)
Basidiomycota/genética , Tubulina (Proteína)/genética , Secuencia de Aminoácidos , Secuencia de Bases , Basidiomycota/metabolismo , Northern Blotting , Southern Blotting , Clonación Molecular , Codón , Cartilla de ADN/química , ADN Complementario , Intrones , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Tubulina (Proteína)/aislamiento & purificaciónRESUMEN
This work describes the characterization of the phosphotransferase enzymatic activity responsible for amikacin resistance in two clinical Pseudomona aeruginosa strains, isolated from a hospital that used amikacin as first-line aminoglycoside. Amikacin-resistant P. aeruginosa PA40 and PA43 (MIC: 128 mg/l) were shown to have APH activity with a substrate profile similar to that of APH(3')-VI. The enzyme from P. aeruginosa PA40 was purified to > 70% homogeneity. The Km of amikacin for this enzyme was 1.4 microM, the Vmax/Km ratio for amikacin was higher than for the other aminoglycosides tested and PCR and DNA sequencing ruled out the presence of aph(3')-IIps. Amikacin resistance in this strain was, therefore, associated with APH(3')-VI and the high affinity of this enzyme for amikacin could explain the high-level resistance that we observed.
Asunto(s)
Amicacina/farmacología , Antibacterianos/farmacología , Farmacorresistencia Microbiana , Fosfotransferasas/metabolismo , Pseudomonas aeruginosa/efectos de los fármacos , Amicacina/metabolismo , Antibacterianos/metabolismo , Secuencia de Bases , Southern Blotting , Cartilla de ADN , Cinética , Pruebas de Sensibilidad Microbiana , Fosfotransferasas/aislamiento & purificación , Reacción en Cadena de la Polimerasa , Pseudomonas aeruginosa/enzimología , Especificidad por SustratoRESUMEN
The aminoglycoside (AG) 3'-phosphotransferases [APH(3')s] are an important class of modifying enzymes which confer high-level resistance to those AGs actively modified by the enzymes. They catalyze the transfer of the terminal phosphate from ATP to the drug, thus preventing the AG s action at the 70S ribosome. These enzymes, which utilize ATP as a co-substrate, appear from amino acid alignments to be part of a much larger superfamily of kinases and ATP-binding proteins. Structure-function analyses have been initiated in our laboratory for APH(3')-II, whose gene was derived from transposon Tn5. Site-directed mutagenesis of the cloned APH(3')-II gene was used to genetically examine the residues in two highly-conserved motifs proposed to participate in ATP binding. Several of these residues, in fact, were shown to affect the enzyme s affinity for ATP. We have also initiated studies using photoaffinity labelling of APH(3')-II with the ATP analogs, 8-azido-ATP and 2-azido-ATP. We have shown that 8-N3ATP and 2-N3ATP can be substituted for ATP in the APH(3')-II catalyzed phosphorylation of kanamycin; such findings indicate that the interaction of these photoaffinity analogs of ATP with APH(3')-II is biologically relevant. One of the best-characterized of the APH(3') enzymes is APH(3')-IIIa, the first of the group whose structure has been analyzed by x- ray crystallography. Several studies have demonstrated that this enzyme functions by a Theorell-Chance mechanism. Moreover, the architecture of the enzyme, crystallized in the presence of ADP has revealed residues in the ATP-binding pocket which are likely to play important roles in catalysis. Once the results from biochemical analyses can be correlated with those from mutagenesis studies and x-ray crystallography, a clearer picture of the active site will be provided for an important class of AG-modifying enzymes and phosphotransferases. This picture will also allow a better understanding of these enzymes within the greater context of kinases and nucleotide-binding proteins.
Asunto(s)
Adenosina Trifosfato/metabolismo , Antibacterianos/metabolismo , Bacterias/enzimología , Farmacorresistencia Microbiana , Kanamicina Quinasa/metabolismo , Secuencia de Aminoácidos , Aminoglicósidos , Kanamicina Quinasa/química , Kanamicina Quinasa/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Etiquetas de Fotoafinidad , Alineación de Secuencia , Análisis de SecuenciaRESUMEN
Previous reports have suggested a common origin for all methicillin resistance (mec) genes from methicillin-resistant Staphylococcus aureus (MRSA) isolates examined so far. The purpose of this study was to explore several molecular methods for screening MRSA isolates from different sources and, in some cases, with varying phenotypes. Eighty MRSA isolates from three teaching hospitals in the University of Louisville Medical Center were compared with MRSAs from a hospital in southern California and with methicillin-sensitive S. aureus isolates. The methods were used to detect the presence of mec gene and to screen for any polymorphisms in these genes for the respective strains. The mec gene for each isolate was amplified via the polymerase chain reaction, and each polymerase chain reaction product was compared to the others by restriction enzyme digestion, denaturing-gradient gel electrophoresis, and mutation detection enhancement. By these criteria, the mec genes from the 80 MRSA strains in this study seemed to be identical. Such a finding was not unexpected and supported the existing hypothesis of a common ancestor for all mec genes isolated in MRSA isolates. However, the combination of methods used in this study may facilitate screening of MRSA strains in population studies as mec gene variants begin to emerge.
Asunto(s)
Genes Bacterianos , Resistencia a la Meticilina/genética , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/genética , Electroforesis en Gel de Agar , Electroforesis en Gel de Poliacrilamida , Expresión Génica , Humanos , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
1. Oligonucleotide-directed mutagenesis of APH(3')-II was used to investigate the functions of key amino acids in the P-loop analogous motif of the enzyme. 2. The mutations of Gly205-->Glu, Gly210-->Ala and Arg211-->Pro considerably reduced the resistance of the resulting strains to KM and to related drugs, e.g. G418. 3. Similarly, enzyme activity in the crude extracts of these mutants was substantially reduced as well as the enzyme's affinity for Mg2+ ATP. 4. Alternatively substitutions at a highly conserved basic residue (Arg211-->Lys and Arg211-->His) were not sufficient for the enzyme to sustain the activity at a level comparable to that of the wildtype. 5. Moreover, an Arg211-->His mutation drastically reduced affinity of the enzyme for Mg2+ ATP. 6. This argues the importance of Arg211 residue in contributing to the formation of the P-loop structure in addition to its involvement in phosphoryl transfer reaction. 7. Computer analysis of the secondary structure predicted that the APH(3')-II loop connects a beta-strand to an alpha-helix and that the above mutations caused varying degrees of structural distortions at the corresponding regions of the protein.
Asunto(s)
Aminoácidos/genética , Mutagénesis Sitio-Dirigida , Nucleótidos/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Secuencia de Aminoácidos , Secuencia de Bases , Western Blotting , Farmacorresistencia Microbiana/genética , Kanamicina Quinasa , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Fenotipo , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Conformación ProteicaRESUMEN
The gene (gamma-tub) encoding gamma-tubulin (gamma-Tub) was isolated from a cosmid library constructed for Ustilago violacea by using a PCR-amplified DNA fragment as a probe. About 2.8 kb of DNA sequence was analyzed and found to encode a protein of 469 amino acids highly homologous to the gamma-Tub from other organisms. There were eight introns interrupting the coding sequence. A 'TATA'-like sequence was found 389 bp upstream from the initial Met codon. No polyadenylation signal was found in the 3' non-coding region. Southern blot analyses indicated that gamma-tub is a single-copy gene. Northern blot analyses indicated that a 1.81-kb RNA species was transcribed. Primer extension experiments determined that the transcription start point (tsp) is at 58 bp downstream from the putative TATA box, with another possible tsp at 95 bp downstream. The long 5' non-coding sequence of the RNA contained several small open reading frames; their possible roles in the regulation of gamma-tub translation are discussed.
Asunto(s)
Intrones , Tubulina (Proteína)/genética , Ustilago/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Southern Blotting , Clonación Molecular , Codón , Cósmidos , ADN de Hongos , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Reacción en Cadena de la Polimerasa , Homología de Secuencia de Aminoácido , Transcripción GenéticaRESUMEN
Mutant aminoglycoside 3'-phosphotransferase II enzymes were produced in which Tyr218 was changed to serine, aspartic acid, or phenylalanine. In each case the mutation resulted in increased bacterial susceptibility to neomycin and kanamycin, while simultaneously increasing the Km values for these substrates. For the Ser and Asp mutants, bacterial resistance to amikacin increased, with a concomitant increase in affinity for this drug. Initial velocity studies indicated that the wild-type and mutant enzymes all followed Michaelis-Menten kinetics. Although these mutagenic substitutions changed the substrate specificity of these enzymes they did not alter the enzyme affinity for Mg(2+)-ATP.
Asunto(s)
Resistencia a la Kanamicina/genética , Neomicina/farmacología , Fosfotransferasas/genética , Fosfotransferasas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Farmacorresistencia Microbiana/genética , Kanamicina Quinasa , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
In order to test the biological importance of amino acids in the C-terminal quarter of aminoglycoside 3'-phosphotransferase II enzyme, seven of the highly conserved residues in this region, His-188, Asp-190, Asp-208, Gly-210, Arg-211, Asp-216 and Asp-220, were changed via site-directed mutagenesis. The phenotype of each mutant was compared to wildtype in terms of antibiotic susceptibilities and enzymatic activities. All of the substitutions either abolished or significantly reduced the resistance of the resulting strains to kanamycin, neomycin, butirosin, ribostamycin, paromomycin, gentamicin A, and G-418. Similarly, enzyme activities in crude extracts were substantially reduced for the mutant strains. Affinity of the enzyme for Mg+2-ATP decreased with His-188, Asp-190, Asp-216 and Asp-220 substitutions as revealed by Km measurements. Secondary structure analysis predicted that substitutions at the conserved residues caused severe conformational distortions at the corresponding regions of the protein.
Asunto(s)
Aminoglicósidos/farmacología , Mutagénesis Sitio-Dirigida , Fosfotransferasas/genética , Fosfotransferasas/metabolismo , Secuencia de Aminoácidos , Bacterias/efectos de los fármacos , Bacterias/enzimología , Bacterias/genética , Western Blotting , Elementos Transponibles de ADN , Kanamicina Quinasa , Cinética , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Fenotipo , Fosfotransferasas/química , Conformación Proteica , Homología de Secuencia de Ácido Nucleico , Especificidad por SustratoRESUMEN
Giardia spp. are waterborne organisms that are the most commonly identified pathogenic intestinal protozoans in the United States. Current detection techniques for Giardia species in water include microscopy and immunofluorescence techniques. Species of the genus Giardia are classified on the basis of taxonomic criteria, such as cell morphology, and on host specificity. We have developed a polymerase chain reaction- and gene probe-based detection system specific for Giardia spp., which can discriminate between the relevant species of the G. duodenalis type pathogenic to humans and other Giardia species that are not human pathogens. This method can detect a single Giardia cyst and is therefore sensitive enough for environmental monitoring.
Asunto(s)
Sondas de ADN , Giardia/clasificación , Reacción en Cadena de la Polimerasa , Animales , Secuencia de Bases , ADN Protozoario/química , Giardia/genética , Datos de Secuencia Molecular , Especificidad de la Especie , Microbiología del AguaRESUMEN
Mutant strains containing APH(3')-II were constructed via site-directed mutagenesis of the cloned gene and by random mutagenesis of a strain containing the APH(3')-II gene on a conjugative plasmid. Substitutions at highly conserved amino acid residues produced APH(3') enzymes which in general showed reduced activity and conferred reduced levels of resistance to their substrates. Substitutions at Tyr 218 altered substrate specificity for the enzymes. Random mutagenesis produced plasmid-borne mutations conferring amikacin resistance. Two of these mutations appeared to be localized to the APH(3')-II structural gene.
Asunto(s)
Fosfotransferasas/química , Secuencia de Aminoácidos , Escherichia coli/genética , Kanamicina Quinasa , Cinética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fosfotransferasas/genética , Fosfotransferasas/metabolismo , Plásmidos , Pseudomonas/genética , Salmonella typhimurium/genética , Homología de Secuencia de Ácido Nucleico , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Plasmid pUCH1 is a 5.2-kb pUC18 construct bearing the hygB gene fused to a promoter from Cochliobolus heterostrophus. Haploid cells of the basidiomycete, Ustilago violacea, were transformed with this plasmid. In addition to multiple integrations of plasmid sequences into U. violacea nuclear DNA, vector sequences independent of the nuclear genome were indicated by Southern-blot analysis using all or part of pUCH1 as a probe. Hybridization also revealed intact pUCH1 and several larger derivatives in satellite bands from CsCl-bis-benzamide gradients of whole cellular DNA and in DNA from purified mitochondria [mitochondrial (mt) DNA preparations] of transformed U. violacea; circular DNAs consistent with the sizes of DNAs in these satellite bands were seen in electron microscope analyses of the same mt DNA preparations as well. The plasmids could be detected in mt DNA preparations even after 30 generations of transformant growth under selective pressure. Transformation of Escherichia coli by these mt DNA preparations produced bacterial transformants bearing intact pUCH1, as well as several pUCH1 derivatives, including pUCH2, an approx. 8.0-kb plasmid. A 2.5-kb EcoRI fragment from pUCH2 showed only weak hybridization with pUCH1. This unique fragment did hybridize strongly with mt DNA from untransformed U. violacea. This derivative thus appears to have acquired mt sequences from U. violacea.
Asunto(s)
Cinamatos , ADN de Hongos/genética , ADN Mitocondrial/genética , Vectores Genéticos , Plásmidos/genética , Transformación Genética , Ustilago/genética , Southern Blotting , Clonación Molecular , Farmacorresistencia Microbiana/genética , Escherichia coli/genética , Genes Fúngicos , Higromicina B/análogos & derivados , Higromicina B/farmacología , Mapeo RestrictivoRESUMEN
A strain of the basidiomycete, Ustilago violacea, was transformed with a prokaryotic plasmid, pMP4-1, which confers resistance to neomycin. U. violacea transformants were selected at a frequency of 5 per microgram pMP4-1 DNA. Such transformants were at least 8-fold more resistant to neomycin than was the untransformed recipient U. violacea. Enzyme activity associated with the neomycin resistance gene was also found in the transformants. Southern DNA-DNA hybridization detected pMP4-1-derived sequences in both nuclear and mitochondrially-associated DNAs from transformants. The patterns of hybridization suggested integration of pMP4-1 sequences into the respective genomes. DNA from the nuclear fraction of U. violacea transformants failed to produce E. coli transformants resistant to neomycin or to carbenicillin. In contrast, DNA from the mitochondrially-associated fraction in U. violacea transformants produced E. coli transformants resistant to neomycin. The E. coli transformants contained a pMP4-1-derivative, pWP8, which was subsequently shown by Southern blot analysis to harbor U. violacea mitochondrial DNA. Thus, a prokaryotic plasmid can be used to transform the eukaryote U. violacea and acquire endogenous sequences from this organism.
Asunto(s)
ADN Bacteriano/genética , Escherichia coli/genética , Factores R , Transformación Genética , Ustilago/genética , Southern Blotting , Sondas de ADN , ADN de Hongos/genética , ADN Mitocondrial/genética , Farmacorresistencia Microbiana/genética , Escherichia coli/efectos de los fármacos , Neomicina/farmacología , Hibridación de Ácido Nucleico , Células Procariotas , Mapeo Restrictivo , Ustilago/efectos de los fármacosRESUMEN
A basidiomycete phytopathogenic fungus, Ustilago violacea, was transformed with pUCH1, a bacterial plasmid containing the hygromycin (Hyg)-resistance hygB gene fused to a promoter from the ascomycete Cochliobolus heterostrophus. After lithium acetate/polyethylene glycol treatment of whole sporidial cells, U. violacea transformants appeared on Hyg-agar at a frequency of 60-80 per microgram pUCH1 DNA. The Hyg phenotype was 100% stable in these transformants for at least 30 generations of mitotic growth under non-selective conditions. Southern DNA-DNA hybridization revealed multiple integrations of the pUCH1 plasmid into the U. violacea nuclear DNA. In addition, Escherichia coli transformants appeared at a frequency of 12 per microgram nuclear fraction DNA from Hyg U. violacea transformants; these E. coli consistently contained a deleted pUCH1 plasmid. This latter result suggested the low-frequency production of circular molecules by recombination within the integrated sequences.
Asunto(s)
Antibacterianos/farmacología , Basidiomycota/genética , Cinamatos , Higromicina B/farmacología , Litio/farmacología , Transformación Genética , Ustilago/genética , ADN de Hongos/genética , Farmacorresistencia Microbiana/genética , Genes Fúngicos , Marcadores Genéticos , Higromicina B/análogos & derivados , PlásmidosRESUMEN
A model suicide vector (pBAP19h), designed for the potential containment of genetically engineered microorganisms, was made by constructing a plasmid with the hok gene, which codes for a lethal polypeptide, under the control of the lac promoter. The vector plasmid also codes for carbenicillin resistance. In the absence of carbenicillin, induction of the hok gene in vitro caused elimination of all detectable cells containing the suicide vector; pBAP19h-free cells of the culture survived and grew exponentially. In the presence of carbenicillin, however, the number of cells containing pBAP19h initially declined after induction of hok but then multiplied exponentially. The surviving cells still had a fully functional hok gene and had apparently developed resistance to the action of the Hok polypeptide. Thus, high selective pressure against the loss of the suicide vector led to a failure of the system. Soil microcosm experiments confirmed the ability of a suicide vector to restrict the growth of a genetically engineered microorganism in the absence of selective pressure against the loss of the plasmid, with 90 to 99% elimination of hok-bearing cells within 24 h of hok induction. However, some pBAP19h-bearing cells survived in the soil microcosms after hok induction. The surviving cells contained an active hok gene but were not capable of normal growth even after elimination of the hok gene; it appears that a mutation that made them Hok resistant also reduced their capacity for membrane functions needed for energy generation and exponential cell growth. Thus, the model suicide vector was shown to be functional in soil as well as in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Proteínas Bacterianas/genética , Escherichia coli/genética , Genes Bacterianos , Vectores Genéticos , Plásmidos , Southern Blotting , Carbenicilina/farmacología , Clonación Molecular , Recuento de Colonia Microbiana , Sondas de ADN , ADN Bacteriano/genética , Escherichia coli/efectos de los fármacos , Regulación de la Expresión Génica , Modelos Genéticos , Mutación , Hibridación de Ácido Nucleico , Resistencia a las Penicilinas , Péptidos/genética , Transformación BacterianaRESUMEN
In order to make accurate kinetic measurements for the substrates of aminoglycoside (AG) phosphotransferases (APHs), we have developed an assay which overcomes many of the limitations of currently used assays. We have adapted the coupled spectrophotometric assay (P. R. Goldman and D. B. Northrop (1976) Biochem. Biophys. Res. Commun. 68, 230-236) for use in a spectrofluorometer. At an excitation wavelength of 340 nm, NADH will emit an intensity peak at 450 nm; NAD does not emit under these conditions. Our assay can accurately measure differences of 0.25 microM. For the APH(3')-II encoded on Tn5, we have redetermined the Km's for the AGs, amikacin (AK), kanamycin (KM), and ribostamycin (Rib), and for ATP. Our values for AK (76 microM) were lower than those derived from the spectrophotometric assay; for KM and Rib we obtained Km values of 5.1 and 3.6 microM, respectively. These values were well below the limit of accuracy (10 microM) for the spectrophotometric assay. In addition, we have begun characterization of an APH from a clinical isolate with a low Km for AK. Thus far, we have determined that this enzyme has Km's of approximately 1 microM for both AK and KM. These results show that the assay is well suited for accurate determinations of kinetic constants for low Km substrates of APH enzymes.
Asunto(s)
Fosfotransferasas/análisis , Farmacorresistencia Microbiana , Escherichia coli/enzimología , Kanamicina Quinasa , Fosfotransferasas/genética , Plásmidos , Pseudomonas aeruginosa/enzimología , Espectrometría de Fluorescencia/métodosRESUMEN
Plasmid pMP1-1 in Escherichia coli L-0 encodes aminoglycoside (AG) 3'-phosphotransferase II [APH(3')-II]. This enzyme modifies and confers high-level resistance to kanamycin. Although amikacin is a substrate for APH(3')-II, strain L-0(pMP1-1) is susceptible to amikacin. Plasmid pMP1-2 is a spontaneous mutant of pMP1-1 which determines increased APH(3')-II activity for amikacin, apparently as a result of an increase in the copy number of the plasmid. From amikacin-susceptible, gentamicin-susceptible transformants and transconjugants that bear the APH(3')-II gene on plasmid pMP1-1 or pMP1-2 or cloned into multicopy plasmid pBR322, we selected spontaneous mutants at concentrations of amikacin or gentamicin that were two to four times higher than the MICs of these antibiotics. In each case, whether they were selected by using amikacin or gentamicin, the mutants exhibited modest (two- to eightfold) increases in the MIC of gentamicin and major (64- to 128-fold) increases in the MIC of amikacin. Using these laboratory strains of E. coli, we examined the effects on AG susceptibility of the interaction of AG-modifying enzyme activity and generalized AG uptake. Increasing the level of activity of an AG phosphotransferase in these strains lowered their susceptibility to AGs which were substrates for which the enzyme had low Kms. However, an increase in AG-modifying activity alone did not result in large increases in the MICs for poor substrates of the enzyme. In strains which lacked AG-modifying enzymes, a decrease in the rate of AG uptake increased the MICs modestly for a broad spectrum of AGs. When a strain bore the phosphotransferase, a decrease in generalized AG uptake could raise the MIC further, not only for low-Km substrates, but even for AG substrates for which the enzyme had high Kms. Thus, increased modifying activity, together with a diminished rate of uptake, could produce even higher MICs for poor AG substrates.
Asunto(s)
Amicacina/farmacología , Aminoglicósidos/metabolismo , Escherichia coli/efectos de los fármacos , Kanamicina/análogos & derivados , Fosfotransferasas/metabolismo , Amicacina/metabolismo , ADN Bacteriano/análisis , Sulfato de Dihidroestreptomicina/metabolismo , Farmacorresistencia Microbiana , Escherichia coli/enzimología , Escherichia coli/genética , Kanamicina/metabolismo , Kanamicina/farmacología , Kanamicina Quinasa , Cinética , Potenciales de la Membrana , Mutación , Fosforilación , Factores RRESUMEN
Accumulation of purified adenylylated dihydrostreptomycin (DHS-AMP) was examined in two strains of Escherichia coli. E. coli JSRO-N was plasmid free and aminoglycoside (AG) susceptible; E. coli JSRO-N(pSAD1) contained a plasmid-encoded AG adenylyltransferase which modifies DHS and streptomycin and confers resistance to both of these drugs. Although both whole cells and spheroplasts of JSRO-N accumulated free DHS, we were not able to demonstrate uptake of DHS-AMP by this strain. Whole cells and spheroplasts of JSRO-N(pSAD1) accumulated DHS at a much slower rate than that observed in JSRO-N. This was presumably due to the activity of the adenylyltransferase in JSRO-N(pSAD1). However, this low rate of accumulation of DHS was still higher than the uptake of DHS-AMP by either JSRO-N or JSRO-N(pSAD1). Thus, the rate of accumulation of DHS-AMP was even lower than that of DHS during the slow, initial, energy-dependent phase of AG uptake seen in JSRO-N(pSAD1). We also found that when either JSRO-N or JSRO-N(pSAD1) was incubated with barely inhibitory or subinhibitory concentrations of DHS, rapid uptake of DHS could be stimulated by the addition of an inhibitory concentration of another AG, such as amikacin. Uptake of DHS-AMP could not be similarly enhanced by the addition of amikacin. Our results indicate that DHS-AMP is not accumulated by whole cells or spheroplasts of E. coli. These results are consistent with the postulated intracellular location of AG-modifying enzymes.
Asunto(s)
Sulfato de Dihidroestreptomicina/análogos & derivados , Escherichia coli/metabolismo , Esferoplastos/metabolismo , Cromatografía en Capa Delgada , Sulfato de Dihidroestreptomicina/aislamiento & purificación , Sulfato de Dihidroestreptomicina/metabolismo , Nucleotidiltransferasas/aislamiento & purificación , Factores de TiempoRESUMEN
Escherichia coli MP6 contains a plasmid that encodes aminoglycoside 3'-phosphotransferase II, which phosphorylates kanamycin and confers high-level kanamycin resistance, Amikacin is a minor substrate of this enzyme, but MP6 is susceptible to amikacin. Strain MP10 has a spontaneous mutation in the plasmid of MP6 that increases the aminoglycoside 3'-phosphotransferase II activity not only against kanamycin but also against amikacin. This mutation is also responsible for the appearance of resistance to amikacin in MP10. Resistance to 4'-deoxy-6'-N-methylamikacin (BB-K311) by enzymatic modification has not been reported previously. As with amikacin, MP6 was susceptible to BB-K311 and its aminoglycoside 3'-phosphotransferase II did not phosphorylate this amikacin derivative appreciably. We found that the plasmid-borne mutation in MP10, however, localized by being cloned with a 3.7-megadalton HindIII fragment containing the aminoglycoside 3'-phosphotransferase II gene, resulted in increased phosphorylation of BB-K311 and resistance to it. Thus, the mutation distinguishing MP6 and MP10 has increased the activity of an existing aminoglycoside-modifying enzyme and produced new bacterial resistance to two previously minor substrates of the enzyme.
Asunto(s)
Amicacina/análogos & derivados , Escherichia coli/genética , Kanamicina/análogos & derivados , Factores R , Amicacina/farmacología , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Genes Bacterianos , Kanamicina Quinasa , Mutación , Fosfotransferasas/genéticaRESUMEN
A plasmid-encoded enzyme reported by us to phosphorylate amikacin in a laboratory strain of Escherichia coli has been localized in the bacterial cell. More than 88% of this amikacin phosphotransferase (APH) activity was retained in spheroplasts formed by ethylenediaminetetraacetate-lysozyme treatment of an APH-containing E. coli transconguant known to form spheroplasts readily. By comparison, the spheroplasts retained 94% of deoxyribonucleic acid polymerase I and 98% of glutamyl-transfer ribonucleic acid synthetase, two internal markers, whereas less than 10% of the activity of a periplasmic marker, acid phosphatase, was present in spheroplasts. Treatment of whole cells of the transconjugant with chemical probes incapable of crossing the plasma membrane obliterated acid phosphatase activity, whereas the internal markers deoxyribonucleic acid polymerase I, glutamyl-transfer ribonucleic acid synthetase, and beta-galactosidase were virtually unaffected after treatment for 5 min; more than 60% of the APH activity remained. As a control, similar chemical treatment of sonic extracts, in which enzymes were not protected by bacterial compartmentalization, produced more extensive reduction in the activities of all test enzymes, including APH. Spheroplasts preincubated with adenosine triphosphatase were shown by thin-layer chromatography to phosphorylate amikacin. Spheroplasts of cells grown in the presence of H(3) (32)PO(4) were shown to utilize internally generated adenosine 5'-triphosphate in the phosphorylation of amikacin. The absence of (32)P-phosphorylated amikacin after incubation of [gamma-(32)P]adenosine 5'-triphosphate with spheroplasts confirmed that exogenous adenosine 5'-triphosphate was not used in the reaction. These results suggest an internal location for APH. This conclusion has implications for the role of such enzymes in aminoglycoside resistance of gram-negative bacteria.