RESUMEN
Predominant enterobacteria from infants with necrotizing enterocolitis (NEC) were examined for an unusual ability to ferment lactose. One such isolate, a Klebsiella pneumoniae strain, was partially induced for lactose operon expression in tryptone containing media, and was also pathogenic in a rabbit ileal loop model for NEC. A spontaneous segregant of this strain was no longer partially induced for lactose operon expression, and was no longer pathogenic in the rabbit model. The gene responsible for this phenotype was cloned. The resulting plasmid was shown to cause both partially induced lactose operon expression and pathogenicity when introduced into a laboratory K. pneumoniae strain. A K. pneumoniae mutant deficient in lactose repressor synthesis was also pathogenic in the rabbit model. These results and previous studies on the intraluminal biochemistry of infants with NEC support the hypothesis that an increased ability for lactose fermentation may be a bacterial pathogenic trait with respect to NEC.
Asunto(s)
Enterocolitis Seudomembranosa/microbiología , Klebsiella pneumoniae/aislamiento & purificación , Animales , Clonación Molecular , Enterobacter/enzimología , Inducción Enzimática , Escherichia coli/enzimología , Humanos , Íleon/microbiología , Íleon/patología , Lactante , Klebsiella/enzimología , Klebsiella pneumoniae/enzimología , Klebsiella pneumoniae/patogenicidad , Masculino , Plásmidos , Conejos , beta-Galactosidasa/biosíntesis , beta-Galactosidasa/metabolismoRESUMEN
We examined the effect of miaA, a mutation conferring a deficiency in 2-methylthio-N6-isopentenyladenosine in tRNA, on patterns of peptidyl-tRNA accumulation in Escherichia coli strains deficient in peptidyl-tRNA hydrolase activity. A specific reduction in peptidyl-tRNA accumulation was seen for tRNAs which normally contain the 2-methylthio-N6-isopentenyladenosine modification. These results provide new evidence in support of the ribosome editor model, which links peptidyl-tRNA release to mistranslation events.
Asunto(s)
Adenosina/análogos & derivados , Escherichia coli/metabolismo , Isopenteniladenosina/análogos & derivados , Biosíntesis de Proteínas , Aminoacil-ARN de Transferencia/metabolismo , ARN de Transferencia/metabolismo , Sistema Libre de Células , Escherichia coli/genética , Isopenteniladenosina/deficiencia , Mutación , ARN de Transferencia/genética , Aminoacil-ARN de Transferencia/genética , Ribosomas/metabolismoRESUMEN
We report the genetic mapping of pheU , an Escherichia coli gene for phenylalanine tRNA. This gene was located near 94.5 min on the E. coli map. There are no other known tRNA or ribosomal genes in its immediate vicinity.
Asunto(s)
Cromosomas Bacterianos , Escherichia coli/genética , Genes Bacterianos , Aminoacil-ARN de Transferencia/genética , Mapeo Cromosómico , Escherichia coli/ultraestructura , Transducción Genética , Transformación BacterianaRESUMEN
Novel E. coli mutants deficient in biosynthesis of 5- methylaminomethyl -2-thiouridine were isolated based on a phenotype of reduced readthrough at UAG codons. They define 2 new loci trmE and trmF , near 83' on the E. coli map. These mutants are different from strains carrying trmC mutations, which are known to confer a methylation deficiency in biosynthesis of 5- methylaminomethyl -2-thiouridine. tRNA from mutants carrying trmE or trmF mutations was shown to carry 2-thiouridine instead of 5- methylaminomethyl -2-thiouridine. This deficiency affects the triplet binding properties of the mutant tRNA. Our results suggest that the 5- methylaminomethyl group stabilizes the basepairing of this modified nucleotide with G, most likely through direct interaction with the ribosomal binding site(s).
Asunto(s)
Escherichia coli/genética , Mutación , ARN de Transferencia/genética , Tiouridina/análogos & derivados , Secuencia de Bases , Codón , Genotipo , Fenotipo , Especificidad de la Especie , Tiouridina/metabolismo , beta-Galactosidasa/genéticaRESUMEN
A recombinant plasmid (designated pID2) carrying the E. coli gene for tRNAPhe has been isolated from a plasmid bank constructed by the ligation of a total EcoRI digest of E. coli K12 DNA into the EcoRI site of pACYC184 DNA. The plasmid was selected by virtue of its ability to complement a temperature-sensitive lesion in the gene (PheS) for the alpha-subunit of phenylalanyl-tRNA synthetase. Crude tRNA isolated from such transformants exhibited elevated levels of phenylalanine acceptor activity. The tRNAPhe gene has been localized within the first 300 base pairs of a 3.6 kb SalI fragment of pID2. The sequence of the gene and its flanking regions is presented.
Asunto(s)
Clonación Molecular , Escherichia coli/genética , Genes Bacterianos , Genes , Aminoacil-ARN de Transferencia/genética , Secuencia de Bases , Enzimas de Restricción del ADN , Hibridación de Ácido Nucleico , PlásmidosRESUMEN
Readthrough and suppression of nonsense codons was compared in Escherichia coli strains with and without a miaA mutation, which confers a loss of the isopentenyladenosine modification in transfer RNA. Generally speaking, our results conform to predictions based on previous literature. In addition, we showed that the miaA mutation in strain TRPX is itself a UAA mutation. An antagonism between miaA and rpsL mutations, which confer streptomycin resistance, was also discovered. Our data further suggest that slight alterations of the translation apparatus are easily detectable by monitoring readthrough and suppression of nonsense codons. Our findings are discussed in the context of old and recent reports.
Asunto(s)
Adenosina/análogos & derivados , Escherichia coli/genética , Regulación de la Expresión Génica , Isopenteniladenosina/análogos & derivados , Biosíntesis de Proteínas , Supresión Genética , Codón , Proteínas de Escherichia coli , Genes Bacterianos , Isopenteniladenosina/genética , Isopenteniladenosina/fisiología , Mutación , Proteína Ribosómica S9 , Proteínas Ribosómicas/genética , beta-Galactosidasa/biosíntesisRESUMEN
A 22-kilobase fragment of the Escherichia coli chromosome which contains the genes for translation initiation factor 3, phenylalanyl-tRNA synthetase, and threonyl-tRNA synthetase was cloned into plasmid pACYC184. The hybrid plasmid (designated pID1) complements a temperature-sensitive pheS lesion in E. coli NP37. pID1-transformed NP37 overproduce initiation factor 3 and phenylalanyl-tRNA synthetase. Gene expression from pID1 was studied in vitro in a coupled transcription-translation system and in minicells. The results suggest that the genes for initiation factor 3 and phenylalanyl- and threonyl-tRNA synthetase are regulated by different mechanisms.
Asunto(s)
Aminoacil-ARNt Sintetasas/genética , Clonación Molecular , Regulación de la Expresión Génica , Genes Bacterianos , Factores de Iniciación de Péptidos/genética , Escherichia coli/genética , Factores de Iniciación de Péptidos/biosíntesis , Fenilalanina-ARNt Ligasa/biosíntesis , Fenilalanina-ARNt Ligasa/genética , Plásmidos , Factor 3 Procariótico de Iniciación , Treonina-ARNt Ligasa/biosíntesis , Treonina-ARNt Ligasa/genéticaRESUMEN
galU and rpsL mutations restore plating efficiency of bacteriophage T7 in male Escherichia coli without suppressing leakage of adenosine 5'-triphosphate pools.
Asunto(s)
Adenosina Trifosfato/metabolismo , Escherichia coli/fisiología , Fagos T/genética , Permeabilidad de la Membrana Celular , Proteínas de Escherichia coli , Espacio Extracelular/metabolismo , Factor F , Genes Reguladores , Mutación , Proteína Ribosómica S9RESUMEN
We have isolated two regulatory mutants altered in the leader region of the Escherichia coli tryptophan (trp) operon. In one mutant, trpL29, the AUG translation start codon for the trip leader peptide is replaced by AUA. The other mutant, trpL75, has a G leads to A change at residue 75, immediately after the UGA translation stop codon for the trp leader peptide. In vivo, trpL29 and trpL75 increase the efficiency of transcription termination at the trp attenuator 3- to 5-fold. trpL29 and trpL75 also fail to respond fully to tryptophan starvation and other conditions that normally relieve transcription termination at the trp attenuator. The trpL29 mutation, which presumably reduces synthesis of the trp leader peptide, is cis dominant. The effect of starvation for a number of the amino acids in the trp leader peptide was determined. Only starvation for tryptophan and arginine, amino acids that occur at residues 10, 11, and 12 of the 14-residue trp leader peptide, elicits relief of transcription termination. Our findings suggest that translation of trp leader RNA is involved in regulation of transcription termination at the attenuator. A model is discussed in which the location of the ribosome synthesizing the leader peptide is communicated to the RNA polymerase transcribing the leader region.
Asunto(s)
Escherichia coli/genética , Triptófano/genética , Secuencia de Bases , ARN Polimerasas Dirigidas por ADN/metabolismo , Genes Dominantes , Mutación , Conformación de Ácido Nucleico , Operón , Biosíntesis de Proteínas , Aminoacil-ARN de Transferencia/metabolismo , Transcripción GenéticaRESUMEN
Two genes for ornithinetranscarbamylase exist in strain Escherichia coli K-12, argI, at 85 min, and argF, at 7 min. In an attempt to compare the deoxyribonucleic acid material of these two genes, the lambda transducing phages carrying a portion of the argI region, lambda dvalS argI, lambda pvalS, and lambda dvalS pyrB, and of the argF region, lambda dargF, have been isolated. Their structure, including that of phi 80dargF previously isolated, was studied by the method of heteroduplex mapping. In this paper, the results of this mapping are reported.
Asunto(s)
Colifagos , Escherichia coli/enzimología , Genes , Ornitina Carbamoiltransferasa/biosíntesis , Transducción Genética , Centrifugación por Gradiente de Densidad , Mapeo Cromosómico , Colifagos/análisis , Colifagos/aislamiento & purificación , Virus ADN , ADN Viral/análisis , Prueba de Complementación Genética , Lisogenia , Microscopía Electrónica , Modelos Biológicos , Mutación , Conformación de Ácido NucleicoRESUMEN
We describe a method for the direct selection of E. coli mutants restricting efficiency of suppression and misreading levels using a T4-coded nonsense suppressor. One mutant isolated has the phenotype expected for a restrictive mutant and may be ribosomal. Other possibilities are discussed.
Asunto(s)
Escherichia coli , Mutación , Supresión Genética , Transcripción Genética , Bacteriófagos , Mapeo Cromosómico , Cromosomas Bacterianos , Escherichia coli/aislamiento & purificación , Genotipo , FenotipoRESUMEN
16 lac frameshift mutants induced by an acridine derivative, ICR-191D, in E. coli are leaky for beta-galactosidase activity. Activities of all mutants differ from each other and from the wild type in their stability to thermal denaturation. The leakiness is under ribosomal control, since it is strongly reduced by strA restrictive mutations and is restored by ram mutations that reverse restriction. Addition of streptomycin during growth has an effect similar to the presence of the ram mutation. These ribosomal alterations do not modify the thermal stability of the enzyme.It is suggested that the leakiness is due to an infrequent 2- or 4-base reading close to the frameshift mutation site. The possibility that not only the ribosome, but also the reading context in the messenger, plays a role in securing code fidelity is discussed.