RESUMO
Three methods to enumerate Escherichia coli in food were compared. They were based on AOAC methods using lauryl tryptose broth (LST) medium, LST-4-methylumbelliferyl-beta-D-glucuronide (MUG) medium, and a proposed method using regular LST in combination with E. coli (EC)-MUG medium. An efficacious and cost-effective method is needed that can detect E. coli and does not produce false presumptive positives. We tested 170 cheeses, 40 frozen processed seafood samples, 210 tree nuts, and 40 other samples. The method of choice for enumerating E. coli depends on the commodity itself. For a product, such as hard cheese or processed seafood, with a history of being negative for E. coli and other lactose-fermenting organisms, the proposed method using regular LST/EC-MUG is a good choice. These samples were seldom presumptive positive in the primary LST medium. If gas was produced, EC-MUG was an effective secondary medium. No false positives (fluorescence) or negatives were detected in EC-MUG medium. For a product with a history of being positive for E. coli and/or other lactose fermenting organisms, such as tree nutmeats or cheeses that are ripened by bacteria or mold, the method using LST-MUG is the method of choice. A presumptive positive in the LST-MUG medium was highly correlative with the biochemical tests that confirmed a sample contain E. coli. For samples spiked with E. coli, the results from each of these 3 methods were identical, and were consistent in enumerating E. coli.
Assuntos
Contagem de Colônia Microbiana/métodos , Escherichia coli , Microbiologia de Alimentos , Himecromona , Bebidas/microbiologia , Meios de Cultura , Corantes Fluorescentes , Contaminação de Alimentos , Conservação de Alimentos , Congelamento , Himecromona/análogos & derivados , Nozes/microbiologia , Alimentos Marinhos/microbiologia , Raios UltravioletaRESUMO
The polymerase chain reaction (PCR), a rapid, sensitive technique for amplifying target DNA sequences of pathogenic microorganisms, was used to amplify Clostridium botulinum type E neurotoxin gene fragments in smoked fish. Other botulinal neurotoxin-producing strains, nontoxigenic strains, and food-related microorganisms did not yield nonspecific amplification products with this PCR assay. PCR products were analyzed by capillary electrophoresis (CE) using a low-viscosity entangled polymer system. Resolution, sensitivity, and DNA sizing accuracy were improved, and analytical times were markedly shortened. The PCR/CE assay detected the C. botulinum type E neurotoxin gene in as few as 10 cells. The technique to other foods may also be a valuable tool for detecting foodborne pathogens.
Assuntos
Toxinas Botulínicas/genética , Clostridium botulinum/genética , DNA Bacteriano/análise , Produtos Pesqueiros/microbiologia , Regulação Bacteriana da Expressão Gênica/genética , Animais , Sequência de Bases , Toxinas Botulínicas/análise , Toxinas Botulínicas/metabolismo , Clostridium botulinum/metabolismo , Cyprinidae , DNA Bacteriano/química , DNA Bacteriano/genética , Eletroforese Capilar , Contaminação de Alimentos , Conservação de Alimentos , Dados de Sequência Molecular , Peso Molecular , Neurotoxinas/química , Neurotoxinas/genética , Neurotoxinas/metabolismo , Reação em Cadeia da Polimerase , Salmão , Sensibilidade e Especificidade , ViscosidadeRESUMO
The short-chain organic acids (SCOAs), acetic and propionic acids, are used widely as food preservatives. The production of these two acids plus butyric acid in the colon by anaerobes serves as a mechanism for controlling the numbers of enterobacteria (which can be pathogens) in this organ. It has been found in this study that the acid tolerance of cells initially grown at near neutral pH (6.5) to a lethal pH of 3.5 is enhanced by their exposure to 0.1% propionate or butyrate. The data also indicate that the inducible arginine and lysine decarboxylases are important for the survival of Escherichia coli exposed to a combination of mildly acidic pH (5.5) and 0.5% butyrate. This study suggests that the presence of SCOAs could trigger an adaptive survival response which may be important in the survival of food-borne pathogens.
Assuntos
Acetatos/farmacologia , Carboxiliases/genética , Escherichia coli/fisiologia , Genes Bacterianos , Propionatos/farmacologia , Ácido Acético , Escherichia coli/genética , Concentração de Íons de HidrogênioRESUMO
The lysyl-tRNA synthetase (LysRS) system of Escherichia coli K-12 consists of two genes, lysS, which is constitutive, and lysU, which is inducible. It is of importance to know how extensively the two-gene LysRS system is distributed in procaryotes, in particular, among members of the family Enterobacteriaceae. To this end, the enterics E. coli K-12 and B; E. coli reference collection (ECOR) isolates EC2, EC49, EC65, and EC68; Shigella flexneri; Salmonella typhimurium; Klebsiella pneumoniae; Enterobacter aerogenes; Serratia marcescens; and Proteus vulgaris and the nonenterics Pseudomonas aeruginosa and Bacillus megaterium were grown in AC broth to a pH of 5.5 or less or cultured in SABO medium at pH 5.0. These growth conditions are known to induce LysRS activity (LysU synthesis) in E. coli K-12. Significant induction of LysRS activity (twofold or better) was observed in the E. coli strains, the ECOR isolates, S. flexneri, K. pneumoniae, and E. aerogenes. To demonstrate an association between LysRS induction and two distinct LysRS genes, Southern blotting was performed with a probe representing an 871-bp fragment amplified from an internal portion of the coding region of the lysU gene. In initial experiments, chromosomal DNA from E. coli K-12 strain MC4100 (lysS+ lysU+) was double digested with either BamHI and HindIII or BamHI and SalI, producing hybridizable fragments of 12.4 and 4.2 kb and 6.6 and 5.2 kb, respectively. Subjecting the chromosomal DNA of E. coli K-12 strain GNB10181 (lysS+ delta lysU) to the same regimen established that the larger fragment from each digestion contained the lysU gene. The results of Southern blot analysis of the other bacterial strains revealed that two hybridizable fragments were obtained from all of the E. coli and ECOR collection strains examined and S. flexneri, K. pneumoniae, and E. aerogenes. Only one lysU homolog was found with S. typhimurium and S. marcescens, and none was obtained with P. vulgaris. A single hybridizable band was found with both P. aeruginose and B, megaterium. These results show that the dual-gene LysRS system is not confined to E. coli K-12 and indicate that it may have first appeared in the genus Enterobacter.
Assuntos
Escherichia coli/genética , Genes Bacterianos , Lisina-tRNA Ligase/genética , Sequência de Bases , Southern Blotting , Enterobacteriaceae/enzimologia , Escherichia coli/enzimologia , Lisina-tRNA Ligase/metabolismo , Dados de Sequência Molecular , Família MultigênicaRESUMO
A general role for chaperonin ring structures in mediating folding of newly translated proteins has been suggested. Here we have directly examined the role of the E. coli chaperonin GroEL in the bacterial cytoplasm by production of temperature-sensitive lethal mutations in this essential gene. After shift to nonpermissive temperature, the rate of general translation in the mutant cells was reduced, but, more specifically, a defined group of cytoplasmic proteins--including citrate synthase, ketoglutarate dehydrogenase, and polynucleotide phosphorylase--were translated but failed to reach native form. Similarly, a monomeric test protein, maltose-binding protein, devoid of its signal domain, was translated but failed to fold to its native conformation. We conclude that GroEL indeed is a machine at the distal end of the pathway of transfer of genetic information, assisting a large and specific set of newly translated cytoplasmic proteins to reach their native tertiary structures.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/biossíntese , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas de Transporte de Monossacarídeos , Ornitina Carbamoiltransferase/biossíntese , Dobramento de Proteína , Proteínas de Bactérias/genética , Bacteriófago lambda/genética , Bacteriófago lambda/metabolismo , Proteínas de Transporte/genética , Chaperonina 60 , Citrato (si)-Sintase/biossíntese , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Choque Térmico/genética , Complexo Cetoglutarato Desidrogenase/biossíntese , Maltose/metabolismo , Proteínas Ligantes de Maltose , Metionina , Óperon , Ornitina Carbamoiltransferase/genética , Plasmídeos , Polirribonucleotídeo Nucleotidiltransferase/biossíntese , Regiões Promotoras Genéticas , Biossíntese de Proteínas , Sinais Direcionadores de Proteínas/metabolismo , Deleção de Sequência , Temperatura , Transdução GenéticaRESUMO
The induction of the inducible lysyl-tRNA synthetase, LysU, and the inducible lysine and arginine decarboxylases of Escherichia coli K-12 grown in AC broth to a pH of 5.5 or less is temperature dependent, being distinctly lower at 24 than at 37 degrees C. This induction does not appear to be under HtpR control.
Assuntos
Escherichia coli/metabolismo , Proteínas Virais/biossíntese , Escherichia coli/genética , Genes Bacterianos , Concentração de Íons de Hidrogênio , Lisina-tRNA Ligase/biossíntese , Lisina-tRNA Ligase/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Temperatura , Proteínas Virais/genética , Proteínas Virais/isolamento & purificação , beta-Galactosidase/biossíntese , beta-Galactosidase/genéticaRESUMO
In Escherichia coli K-12, expression of the lysU gene is regulated by the lrp gene product, as indicated by an increase in the level of lysyl-tRNA synthetase activity and LysU protein in an lrp mutant. Comparison of the patterns of protein expression visualized by two-dimensional gel electrophoresis indicated that LysU is present at higher levels in an lrp strain than in its isogenic lrp+ parent. The purified lrp gene product was shown to bind to sites upstream of the lysU gene and to protect several sites against DNase I digestion. A region extending over 100 nucleotides, between 60 and 160 nucleotides upstream from the start of the lysU coding sequence, showed altered sensitivity to DNase I digestion in the presence of the Lrp protein. The extent of protected DNA suggests a complex interaction of Lrp protein and upstream lysU DNA.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/genética , Lisina-tRNA Ligase/genética , Sequências Reguladoras de Ácido Nucleico/genética , Fatores de Transcrição , Sequência de Bases , Clonagem Molecular , DNA Bacteriano/genética , Proteínas de Escherichia coli , Proteína Reguladora de Resposta a Leucina , Dados de Sequência Molecular , Mutagênese , Biossíntese de ProteínasRESUMO
The Escherichia coli K-12 strain GNB10181 shows no inducible lysyl-tRNA synthetase (LysRS) activity. Two-dimensional gel electrophoretic analysis of the polypeptides synthesized by this strain indicates that the normal lysU gene product, LysU, is absent. When both GNB10181 and its parent, MC4100, were grown at elevated temperatures (42 to 45 degrees C) no significant difference between their growth rates was observed. The lysU mutation was transferred to other E. coli K-12 backgrounds by using P1 transduction. The lysU transductants behaved comparably to their lysU+ parents at different growth temperatures. Therefore, the LysU proteins does not appear to be essential for growth at high temperatures, at least under the conditions examined here. In addition, lysU transductants were found to be defective for inducible lysine decarboxylase, (LDC), inducible arginine decarboxylase (ADI), and melibiose utilization (Mel), which are all missing in GNB10181. Complementation of the above missing functions was achieved by using the Clarke-Carbon plasmids pLC4-5 (LysU LDC) and pLC17-38 (LysU Mel ADI). From these experiments, it appears that GNB10181 has suffered a chromosomal deletion between 93.4 and 93.7 min, which includes the lysU gene. By using plasmid pLC17-38, the position of ADI on two-dimensional gels was identified. Finally, lysS delta lysU double mutants were constructed which can potentially be used as positive selection agents for the isolation of LysRS genes from other sources.
Assuntos
Escherichia coli/genética , Proteínas de Choque Térmico/genética , Lisina-tRNA Ligase/genética , Clonagem Molecular , Eletroforese em Gel Bidimensional , Indução Enzimática , Regulação Bacteriana da Expressão Gênica , Teste de Complementação Genética , Temperatura Alta , MutaçãoRESUMO
Escherichia coli and Salmonella typhimurium were grown in a supplemented minimal medium (SMM) at a pH of 7.0 or 5.0 or were shifted from pH 7.0 to 5.0. Two-dimensional gel electrophoretic analysis of proteins labeled with H2(35)SO4 for 20 min during the shift showed that in E. coli, 13 polypeptides were elevated 1.5- to 4-fold, whereas in S. typhimurium, 19 polypeptides were increased 2- to 14-fold over the pH 7.0 control. Upon long-term growth at pH 5.0, almost double the number of polypeptides were elevated twofold or more in S. typhimurium compared with E. coli. In E. coli, there was no apparent induction of heat shock proteins upon growth at pH 5.0 in SMM. However, growth of E. coli in a complex broth to pH 5.0, or subsequent growth of fresh E. coli cells in the filtrate from this culture, showed that a subset of five polypeptides is uniquely induced by low pH. Two of these polypeptides, D60.5, the inducible lysyl-tRNA synthetase, and C62.5, are known heat shock proteins. Measurements of the internal pH (pHi) and growth rates of both organisms were made during growth in SMM at pH 7.0, pH 5.0, and upon the pH shift. The data show that the pHi of E. coli decreases more severely than that of S. typhimurium at an external pH of 5.0; the growth rate of E. coli is about one-half that of S. typhimurium at this pH, whereas the two organisms have the same growth rate at pH 7.0. The two-dimensional gel, growth, and pHi experiments collectively suggest that, at least in SMM, S. typhimurium is more adaptive to low-pH stress than is E. coli.
Assuntos
Proteínas de Bactérias/biossíntese , Escherichia coli/metabolismo , Salmonella typhimurium/metabolismo , Adaptação Fisiológica , Proteínas de Bactérias/isolamento & purificação , Meios de Cultura , Eletroforese em Gel Bidimensional , Concentração de Íons de Hidrogênio , Especificidade da EspécieRESUMO
The proteins of highly purified rat liver mitochondria were resolved by two-dimensional polyacrylamide gel electrophoresis, and detected by staining with either Coomassie blue or silver. Approximately 250 polypeptides were detected with silver staining which is 2- to 3-times that observed with Coomassie blue. Silver staining was especially more effective than Coomassie blue for detecting polypeptides of less than 50 000 daltons. A two-dimensional gel pattern of rat liver microsomes was distinct from that of the mitochondria. The mitochondrial outer membrane was prepared from purified mitochondria either with digitonin or by swelling in a hypotonic medium. As assessed by marker enzymes, the latter method yielded a considerably purer outer membrane preparation (20-fold purification) than the former (2.6-fold purification). Approximately 50 polypeptides were observed in a two-dimensional gel (pH 3-10) of the highly purified outer membrane fraction. Three isoelectric forms of the pore (VDAC) protein were observed with pI values of 8.2, 7.8 and 7.1. Monoamine oxidase was identified as a polypeptide of Mr 60 000. About 50 polypeptides were also resolved in a reverse polarity non-equilibrium pH gradient electrophoresis gel of the outer membrane, pH 3-10, with at least six isoelectric forms of the VDAC protein observed under these conditions. The six isoforms of the VDAC protein were also observed in a non-equilibrium gel with 2 micrograms of the purified protein.
Assuntos
Proteínas de Membrana/análise , Mitocôndrias Hepáticas/análise , Peptídeos/análise , Animais , Membrana Celular/análise , Eletroforese em Gel Bidimensional , Concentração de Íons de Hidrogênio , Focalização Isoelétrica , Masculino , Dilatação Mitocondrial , Ratos , Ratos EndogâmicosRESUMO
Aseptic isolation of the facultative gut microflora of the tobacco hornworm, Manduca sexta, yielded four microorganisms. Two were gram-positive Bacillus spp., one was Serratia plymuthica, and another was the yeast Candida guilliermondii. The three bacterial species were screened for extrachromosomal DNA, and S. plymuthica was found to have a 6.4-kilobase plasmid, which was designated pCP-1.
RESUMO
The constitutive lysyl-tRNA synthetase gene (lysS) was mapped at 62.1 min on the Escherichia coli chromosome by a combination of conjugation and transduction, with physical confirmation by two-dimensional gel electrophoresis. Revertant analysis suggests that the altered isoelectric point and the low amount of the mutant LysS protein may be due to a single mutational event.
Assuntos
Aminoacil-tRNA Sintetases/genética , Escherichia coli/genética , Genes Bacterianos , Genes , Lisina-tRNA Ligase/genética , Mapeamento Cromossômico , Cromossomos Bacterianos/fisiologia , Escherichia coli/enzimologia , Genótipo , FenótipoRESUMO
Fast-growing revertants have been selected from a slow-growing lysyl-tRNA synthetase mutant. All of the revertants had increased lysyl-tRNA synthetase activity compared with the mutant (5- to 85-fold), and in some revertants this amounted to two to three times the wild-type synthetase activity. Two-dimensional gel electrophoresis of a whole-cell extract of revertant IH2018 (1.5- to 2-fold wild-type synthetase activity) showed that the increase in synthetase activity is due to the induction of cryptic lysyl-tRNA synthetase forms and not to a change in the constitutive lysyl-tRNA synthetase. Genetic studies have shown that a locus termed rlu (for regulation of lysU ) which is cotransducible with purF at 49.5 min influences the amount of the cryptic lysyl-tRNA synthetase.
Assuntos
Aminoacil-tRNA Sintetases/genética , Escherichia coli/enzimologia , Genes Bacterianos , Genes Reguladores , Lisina-tRNA Ligase/genética , Conjugação Genética , Escherichia coli/genética , Lisina-tRNA Ligase/biossíntese , Mutação , Temperatura , Transdução GenéticaRESUMO
Lysyl-transfer ribonucleic acid synthetase (EC 6.1.1.6) was identified as four polypeptide spots after two-dimensional polyacrylamide gel electrophoresis of whole-cell lysates of Escherichia coli. Identification was made by migration with partially purified enzyme preparations, by peptide map patterns, by mutant analysis, and by correlation of spot intensities with changes in enzyme levels under different growth conditions. Wild-type cells growing at 37 degrees C in glucose minimal medium displayed the enzyme predominantly as two spots (spots I and III). Growth at 46 degrees C, growth in the presence of alanine or glycyl-L-leucine, or growth of a strain with a mutational deficiency in S-adenosylmethionine synthetase (metK) greatly increased the synthesis of two other spots (spots II and IV). Polypeptides I and III, but not polypeptides II and IV, had altered isoelectric points in a lysyl-transfer ribonucleic acid synthetase mutant. These data suggest that multiple forms of lysyl-transfer ribonucleic acid synthetase exist in vivo and that they may be encoded by more than one gene.
Assuntos
Aminoacil-tRNA Sintetases/genética , Escherichia coli/enzimologia , Lisina-tRNA Ligase/genética , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Lisina-tRNA Ligase/metabolismoRESUMO
Lysyl-transfer ribonucleic acid (tRNA) synthetase activity was compared in three independently isolated Escherichia coli K-12 mutants of the enzyme S-adenosyl-L-methionine synthetase (metK mutants) and their isogenic parents. In all three cases the activity of the lysyl-tRNA synthetase was elevated two- to fourfold in the mutant strains. Glycyl-L-leucine (3 mM) usually enhanced lysyl-tRNA synthetase activity two- to threefold in wild-type cells but did not further stimulate the synthetase activity in metK mutants. By two other criteria, the lysyl-tRNA synthetase from wild-type cells grown with the peptide and from the metK mutant RG62, grown in minimal medium, were similar. These criteria are enhanced resistance to thermal inactivation and altered susceptibility to endogenous proteases when compared with the synthetase from wild-type cells grown in minimal medium. In a separate set of experiments, the activities of the lysyl-, arginyl-, seryl-, and valyl-tRNA synthetases were measured in an isogenic pair of relt and rel strains of E. coli grown in a relatively poor growth medium (acetate) and in enriched medium. In the rel+ strain the level of all four synthetases was higher (two- to fourfold) in the enriched medium as expected. In the rel strain the difference in the activities of the synthetases between the two media were diminished. In all four cases the activities of the synthetases were higher in acetate medium in the rel strain. Evidence is presented that these two modes of metabolic regulation act independently.
Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Escherichia coli/enzimologia , Lisina-tRNA Ligase/metabolismo , Cromatografia em Gel , Meios de Cultura , Dipeptídeos/metabolismo , Dipeptídeos/farmacologia , Escherichia coli/crescimento & desenvolvimento , Genes , Temperatura Alta , Metionina Adenosiltransferase/metabolismo , Metilação , MutaçãoRESUMO
Lysyl-tRNA synthetase was purified to 70-90% of homogeneity from Escherichia coli K-12. The enzyme was purified from wild-type cells grown in minimal medium, or minimal medium containing either 20 mM L-alanine or 3 mM glycly-L-leucine. The synthetase was similarly purified from a mutant strain grown in minimal medium plus 20 mM L-alanine. Results based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration, and trypsin inactivation studies indicate (A) that the presence of L-alanine of glycyl-L-leucine in the culture medium alters the properties of the wild-type enzyme; (B) that the alteration of the synthetase by l-alanine and glycyl-L-leucine is different; and (c) that the molecular weight of lysyl-tRNA synthetase is at least 135000--140000. The results suggest that most likely the metabolites modify the structure of lysyl-tRNA synthetase, but the possibility that the metabolites induce the synthesis of a new lysyl-tRNA synthetase cannot be completely eliminated.
Assuntos
Alanina/farmacologia , Aminoacil-tRNA Sintetases , Dipeptídeos/farmacologia , Escherichia coli/enzimologia , Lisina-tRNA Ligase , Aminoacil-tRNA Sintetases/metabolismo , Glicina , Leucina , Lisina-tRNA Ligase/metabolismo , Substâncias Macromoleculares , Peso Molecular , Mutação , Conformação Proteica , Especificidade da Espécie , TripsinaRESUMO
Wild-type Escherichia coli K-12 was grown in minimal medium alone or with the addition of 20 mM L-alanine or 3 mM glycyl-L-leucine. A lysyl-tRNA synthetase mutant strain was grown in minimal medium containing 20mM L-alanine. The lysyl-tRNA synthetase from these strains was purified to 70-90% of homogeneity. Kinetic studies comparing the effect of thermal and urea inactivation on these different lysyl-tRNA synthetase preparations and measurement of the Michaelis constant for lysine and transfer RNA indicated that growth of Escherichia coli in the presence of alanine and glycyl-L-leucine induces an alteration in the properties of the synthetase. Measurement of the apparent Km for ATP at pH 7.25 indicates lysyl-tRNA synthetase has two two binding sites for this substrate, and further studies indicated a dependence of the apparent Km for lysine on the ATP concentration.
Assuntos
Alanina/farmacologia , Aminoacil-tRNA Sintetases/farmacologia , Dipeptídeos/farmacologia , Escherichia coli/enzimologia , Lisina-tRNA Ligase/farmacologia , Sítios de Ligação , Glicina , Temperatura Alta , Cinética , Leucina , Mutação , Concentração Osmolar , Desnaturação Proteica , Cloreto de Sódio/farmacologia , Especificidade da Espécie , UreiaRESUMO
Studies on the utilization of leucine peptide amides as a source of leucine for a leucine auxotroph showed that in general compounds with the structure leu-chi amide (where chi is any amide) are utilized as well as the free peptide, but that compounds with the structure chi-leu amide (where chi is not leucine) are used less effectively than the free peptide. Growth and enzymological experiments indicated that the lower capacity of Escherichia coli to utilize amides of the structure chi-leu amide is not a result of poor transport of these compounds, but rather the inability to rapidly liberate leucine from the amide when it is supplied to the cell in the form of a peptide. Competition studies indicated that the peptide amides enter the cell via the oligopeptide permease system.
Assuntos
Amidas/metabolismo , Escherichia coli/metabolismo , Peptídeos/metabolismo , Transporte Biológico Ativo , Sistema Livre de Células , Eletroforese em Papel , Escherichia coli/crescimento & desenvolvimento , Isoleucina/metabolismo , Leucina/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Mutação , Oligopeptídeos , Fenilalanina/metabolismoRESUMO
A mutant of E. coli K-12 has been isolated which has only 1-3% of the wild-type lysyl-tRNA synthetase activity [L-lysine:tRNA ligase (AMP forming), EC 6.1.1.6]. Additions of 20 mM L-alanine or 6 mM leucine dipeptides to the culture medium can restore the activity of lysyl-tRNA synthetase in the mutant strain to the wild-type level. Experiments on the in vivo charging of lysine tRNA in the mutant show that in the absence of the metabolites lysine tRNA is charged 15-23%. Upon the addition of 3 mM L-leucyl-L-alanine to the medium the lysyl tRNA synthetase activity increases 25-fold and the in vivo charging of lysine tRNA returns to the wild-type level. Experiments with antibody against lysyl-tRNA synthetase show that the stimulation of lysyl-tRNA synthetase activity by the metabolites is the result of new protein synthesis.