Production of methanethiol from methionine by Brevibacterium linens CNRZ 918

The conditions under which Brevibacterium linens CNRZ 918, a strain isolated from the surface smear flora of Gruyère de Comté cheese, produced methanethiol from methionine were studied. Demethiolation was estimated from the methanethiol production capacity of resting cells. Methionine was demethiolated mainly during the exponential growth phase of the organism during which time the cells were rod-shaped and had a generation time of 5 h, and the medium became alkaline. At the end of growth (pH 9) the cells were coccoid, and produced only very little methanethiol. The production of methanethiol required the presence of methionine in the culture medium, this reflecting the probable induction of the enzyme systems involved. Glucose favoured growth and inhibited production of methanethiol. Lactate favoured both growth and methanethiol production. Resting rod cells also produced methanethiol from structural analogues of methionine and from methionine-containing peptides. The apparent kinetic constants of the production of methanethiol for rod and coccoid cells were respectively Km = 14 mM and 46 mM, Vmax = 208 nkat g-1 and 25 nkat g-1. The optimum temperature and pH for production were 30 degrees C and pH 8. Azide or malonate favoured the production of methanethiol by resting cells, whereas chloramphenicol had no effect.     

Cloning and characterization of two Lactobacillus casei genes encoding a cystathionine lyase

Volatile sulfur compounds are key flavor compounds in several cheese types. To better understand the metabolism of sulfur-containing amino acids, which certainly plays a key role in the release of volatile sulfur compounds, we searched the genome database of Lactobacillus casei ATCC 334 for genes encoding putative homologs of enzymes known to degrade cysteine, cystathionine, and methionine. The search revealed that L. casei possesses two genes that putatively encode a cystathionine beta-lyase (CBL; EC 4.4.1.8). The enzyme has been implicated in the degradation of not only cystathionine but also cysteine and methionine. Recombinant CBL proteins catalyzed the degradation of L-cystathionine, O-succinyl-L-homoserine, L-cysteine, L-serine, and L-methionine to form alpha-keto acid, hydrogen sulfide, or methanethiol. The two enzymes showed notable differences in substrate specificity and pH optimum.     

Characterization of a sulfur-regulated oxygenative alkylsulfatase from Pseudomonas putida S-313

The atsK gene of Pseudomonas putida S-313 was required for growth with alkyl sulfate esters as sulfur source. The AtsK protein was overexpressed in Escherichia coli and purified to homogeneity. Sequence analysis revealed that AtsK was closely related to E. coli taurine dioxygenase (38% amino acid identity). The AtsK protein catalyzed the alpha-ketoglutarate-dependent cleavage of a range of alkyl sulfate esters, with chain lengths ranging from C(4) to C(12), required oxygen and Fe(2+) for activity and released succinate, sulfate, and the corresponding aldehyde as products. Enzyme activity was optimal at pH 7 and was strongly stimulated by ascorbate. Unlike most other characterized alpha-ketoglutarate-dependent dioxygenases, AtsK accepted a range of alpha-keto acids as co-substrates, including alpha-ketoglutarate (K(m) 140 microm), alpha-ketoadipate, alpha-ketovalerate, and alpha-ketooctanoate. The measured K(m) values for hexyl sulfate and SDS were 40 and 34 microm, respectively. The apparent M(r) of the purified enzyme of 121,000 was consistent with a homotetrameric structure, which is unusual for this enzyme superfamily, members of which are usually monomeric or dimeric. The properties and amino acid sequence of the AtsK enzyme thus define it as an unusual oxygenolytic alkylsulfatase and a novel member of the alpha-ketoglutarate-dependent dioxygenase family.     

Use of 13C Nuclear Magnetic Resonance and Gas Chromatography To Examine Methionine Catabolism by Lactococci

Formation of methanethiol from methionine is widely believed to play a significant role in development of cheddar cheese flavor. However, the catabolism of methionine by cheese-related microorganisms has not been well characterized. Two independent methionine catabolic pathways are believed to be present in lactococci, one initiated by a lyase and the other initiated by an aminotransferase. To differentiate between these two pathways and to determine the possible distribution between the pathways, ¹³C nuclear magnetic resonance (NMR) performed with uniformly enriched [¹³C]methionine was utilized. The catabolism of methionine by whole cells and cell extracts of five strains of Lactococcus lactis was examined. Only the aminotransferase-initiated pathway was observed. The intermediate and major end products were determined to be 4-methylthio-2-oxobutyric acid and 2-hydroxyl-4-methylthiobutyric acid, respectively. Production of methanethiol was not observed in any of the ¹³C NMR studies. Gas chromatography was utilized to determine if the products of methionine catabolism in the aminotransferase pathway were precursors of methanethiol. The results suggest that the direct precursor of methanethiol is 4-methylthiol-2-oxobutyric acid. These results support the conclusion that an aminotransferase initiates the catabolism of methionine to methanethiol in lactococci.     

Lytic enzyme from lysates of Streptomyces venezuelae infected with actinophage MSP2

A lytic enzyme was purified 600-fold with 12% recovery from lysates of Streptomyces venezuelae S13 infected with actinophage MSP2. The purified enzyme preparation was homogeneous as shown by polyacrylamide electrophoresis. The enzyme was active over a pH range 6.0 to 9.0 with a maximum at pH 7.5. The pH profile for stability was sharp, with an optimum at pH 7.5. Maximal activity occurred between 30 and 35 C. The enzyme was stable at 20 C or less. A 30-min exposure to 25, 30, 35, 40, 45, and 50 C produced an inactivation of 3, 40, 77, 82, 93, and 100%, respectively. Lytic activity was stimulated fivefold by either 5 x 10(-3)m Mg(2+) or Mn(2+) and three- and twofold by Ca(2+) and Ba(2+), respectively. Addition of Na(+), K(+), NH(4) (+), or Li(+) to the tris(hydroxymethyl)aminomethane-hydrochloride buffer did not alter the rate of lysis. Enzyme activity was inhibited 74 and 27% by 10(-4) and 10(-5)m ethylenediaminetetraacetic acid (EDTA), respectively. The inhibition by EDTA was reversed partially by addition of Mg(2+). Lytic activity was abolished by either 5 x 10(-4)m HgCl(2) or p-hydroxymercuribenzoate, whereas 5 x 10(-4)m CuSO(4) inhibited 72%. Cell wall solubilization paralleled the release of N-terminal amino groups and reached a level of 0.23 mumole per mg of cell walls. No release of reducing power was detected in treated or untreated cell wall suspensions. Tests for proteolytic activity were negative.     

Induction and characterization of -galactosidase in an extreme thermophile

A thermostable beta-galactosidase (EC 3.2.1.23; beta-dgalactoside galactohydrolase) was found to be inducible in an extreme thermophile resembling Thermus aquaticus. Enzyme induction was achieved by the addition of lactose, galactose, or the alpha-galactoside, melibiose, to growing cultures. The addition of glucose to induced cultures had a repressive effect on further enzyme synthesis. The enzyme was purified 78-fold, and the optimum temperature and pH for activity were determined to be 80 C and pH 5.0, respectively. The enzyme was activated by both manganese and ferrous iron. Sulfhydryl activation and thermal stabilization indicate that the thermophilic beta-galactosidase is a sulfhydryl enzyme. Kinetic determinations at 80 C established a K(m) of 2.0 x 10(-3)m for the chromogenic substrate o-nitrophenyl beta-d-galactopyranoside (ONPG) and a K(1) of 7.5 x 10(-3)m for lactose. The Arrhenius energy of activation (for the hydrolysis of ONPG) was calculated to be 13.7 kcal/mole. A molecular weight of 5.7 x 10(5) daltons was estimated by elution of the enzyme from Sephadex 4B.     

A novel amidase (half-amidase) for half-amide hydrolysis involved in the bacterial metabolism of cyclic imides

A novel amidase involved in bacterial cyclic imide metabolism was purified from Blastobacter sp. strain A17p-4. The enzyme physiologically functions in the second step of cyclic imide degradation, i.e., the hydrolysis of monoamidated dicarboxylates (half-amides) to dicarboxylates and ammonia. Enzyme production was enhanced by cyclic imides such as succinimide and glutarimide but not by amide compounds which are conventional substrates and inducers of known amidases. The purified amidase showed high catalytic efficiency toward half-amides such as succinamic acid (K(m) = 6.2 mM; k(cat) = 5.76 s(-1)) and glutaramic acid (K(m) = 2.8 mM; k(cat) = 2.23 s(-1)). However, the substrates of known amidases such as short-chain (C(2) to C(4)) aliphatic amides, long-chain (above C(16)) aliphatic amides, amino acid amides, aliphatic diamides, alpha-keto acid amides, N-carbamoyl amino acids, and aliphatic ureides were not substrates for the enzyme. Based on its high specificity toward half-amides, the enzyme was named half-amidase. This half-amidase exists as a monomer with an M(r) of 48,000 and was strongly inhibited by heavy metal ions and sulfhydryl reagents.     

Purification and characterization of two human erythrocyte arylamidases preferentially hydrolysing N-terminal arginine or lysine residues.

Two arylamidases (I and II) were purified from human erythrocytes by a procedure that comprised removal of haemoglobin from disrupted cells with CM-Sephadex D-50, followed by treatment of the haemoglobin-free preparation subsequently with DEAE-cellulose, gel-permeation chromatography on Sephadex G-200, gradient solubilization on Celite, isoelectric focusing in a pH gradient from 4 to 6, gel-permeation chromatography on Sephadex G-100 (superfine), and finally affinity chromatography on Sepharose 4B covalently coupled to L-arginine. In preparative-scale purifications, enzymes I and II were separated at the second gel-permeation chromatography. Enzyme II was obtained as a homogeneous protein, as shown by several criteria. Enzyme I hydrolysed, with decreasing rates, the L-amino acid 2-naphtylamides of lysine, arginine, alanine, methionine, phenylalanine and leucine, and the reactions were slightly inhibited by 0.2 M-NaCl. Enzyme II hydrolysed most rapidly the corresponding derivatives of arginine, leucine, valine, methionine, proline and alanine, in that order, and the hydrolyses were strongly dependent on Cl-. The hydrolysis of these substrates proceeded rapidly at physiological Cl- concentration (0.15 M). The molecular weights (by gel filtration) of enzymes I and II were 85 000 and 52 500 respectively. The pH optimum was approx. 7.2 for both enzymes. The isoelectric point of enzyme II was approx. 4.8. Enzyme I was activated by Co2+, which did not affect enzyme II to any noticeable extent. The kinetics of reactions catalysed by enzyme I were characterized by strong substrate inhibition, but enzyme II was not inhibited by high substrate concentrations. The Cl- activated enzyme II also showed endopeptidase activity in hydrolysing bradykinin.     

Staphylococcal acid phosphatase: preliminary physical and chemical characterization of the loosely bound enzyme

At temperatures between 45 and 50 C, staphylococcal acid phosphatase purified 44-fold had maximal activity at pH 5.2 to 5.3. However, the enzyme was most stable in the alkaline range (pH 8.5 to 9.5) at temperatures below 50 C. Iodoacetate and ethylenediamine-tetraacetic acid were effective inhibitors, whereas mercaptoethanol and Cu(2+) acted as stimulators. The energy of activation for hydrolytic cleavage of the synthetic substrate, p-nitrophenyl phosphate, was 19.5 Kcal/mole. K(m) for the same substrate was 4.5 x 10(-4)m. The purified enzyme was most active against the substrates p-nitrophenyl phosphate and glyceraldehyde 3-phosphate.     

Extracellular Enzyme From Myxobacter AL-1 That Exhibits Both β-1,4-Glucanase and Chitosanase Activities

An enzyme that has both beta-1,4-glucanase and chitosanase activities is characterized. Evidence for homogeneity was obtained from electrophoresis and sedimentation velocity studies; only one N-terminal amino acid, valine, was found. Results of denaturation studies showed that beta-1,4-glucanase and chitosanase activities decreased at equal rates. With carboxymethylcellulose as the substrate, a K(m) of 1.68 g of carboxymethylcellulose per liter of solution and a V(max) of 2.20 x 10(-9) mol/min were found. With chitosan (the beta-1,4-polymer of glucosamine) as the substrate, a K(m) of 0.30 g of chitosan per liter of solution and a V(max) of 0.75 x 10(-9) mol/min were found. A pH optimum of 5.0 was found for beta-1,4-glucanase activity, and pH optima of 5.0 and 6.8 were found for chitosanase activity. beta-1,4-Glucanase activity had a temperature optimum of 38 C, and chitosanase activity had a temperature optimum of 70 C. Chitosan stabilized both enzyme activities at 70 C. Cellotriose was the smallest polymer capable of hydrolysis. Glucosamine was released by action of the enzyme upon cell wall preparations of several fungi.     

Partial purification and characterization of dihydrodipicolinic acid synthetase from sporulating Bacillus megaterium

Sporulation of Bacillus megaterium Km (ATCC 13632) was synchronized by a technique employing three 10% transfers. The culture was harvested when 60% of the cells contained spore forms. Dihydrodipicolinic acid synthetase was purified 150-fold by ammonium sulfate fractionation at pH 6.5, heating for 15 min at 45 C at pH 6.0, ammonium sulfate fractionation at pH 6.0, and subsequent chromatography on diethylaminoethyl cellulose. During the final stage of the purification procedure, the enzyme exhibited sensitivity to refrigeration temperatures. The enzyme had a pH optimum of 7.65 in imidazole buffer. The apparent K(m) values were 4.6 x 10(-4) and 5.0 x 10(-4)m for beta-aspartyl semialdehyde and pyruvate, respectively. All attempts to demonstrate cofactor requirements were unsuccessful. Sulfhydryl inhibiting reagents and lysine did not inhibit the enzymatic reaction. The enzyme exhibited maximal thermal resistance at pH 10.5. The thermal stability of the enzyme at 75 C was increased more than 1,800-fold by the addition of 0.3 m pyruvate. The E(a) was 67,300 cal/mole for the thermal denaturation of the enzyme. At 60 C, the DeltaF, DeltaH, and DeltaS values for the thermal denaturation of the enzyme were 22,250, 66,700, and 133 cal per mole per degree, respectively.     

The extracellular acid phosphatase of the mosquito-parasitizing fungus Lagenidium giganteum

The mosquito-parasitizing fungus Lagenidium giganteum secreted a soluble acid phosphatase and beta-D-glucosidase into the growth medium. The acid phosphatase was isolated and purified to single component, and some of its physicochemical properties were determined. The enzyme exhibited a pH optimum of 5.6 in phthalate buffer with p-nitrophenyl phosphate and was temperature-inactivated at 55 degrees C. Enzyme activity seems to be limited to phenyl-phosphate substrates. A molecular weight of 42,800 was found and the amino acid content was also determined. A Km for p-nitrophenyl phosphate of 1.6 x 10(-7) M was found. The possible involvement of the enzyme in the infective process was discussed.     

Studies on regulatory functions of malic enzymes. VI. Purification and molecular properties of NADP-linked malic enzyme from Escherichia coli W.

NADP-linked malic enzyme [EC 1.1.1.40] was highly purified from Escherichia coli W cells. The purified enzyme was homogeneous as judged by ultracentrifugation and gel electrophoresis. The apparent molecular weights obtained by sedimentation equilibrium analysis, from diffusion and sedimentation constants, and by disc electrophoresis at various gel concentrations were 471,000, 438,000, and 495,000, respectively. The subunit molecular weights obtained by sedimentation equilibrium analysis in the presence of 6 M guanidine hydrochloride and gel electrophoresis in the presence of sodium dodecyl sulfate were 76,000 and 82,000, respectively. The sedimentation coefficient (S(0)20, W) was 13.8S, and the molecular activity was 44,700 min-1 at 30 degrees C. The amino acid composition of the enzyme was determined, and the results were compared with those of NAD-linked malic enzyme from the same organism and those of pigeon liver NADP-linked malic enzyme. The partial specific volume was calculated to be 0.738 ml/g. The Km value for L-malate was 2.3 mM at pH 7.4. Malonate, tartronate, glutarate, and DL-tartrate competitively inhibited the activity. The saturation profile for L-malate exhibited a marked cooperativity in the presence of both chloride ions and acetyl-CoA. However, acetyl-CoA alone did not show cooperativity or produce inhibition in the absence of chloride ions. Vmax and Km were determined as a function of pH. The optimum pH for the reaction was 7.8. Inspection of the Dixon plots suggested that three ionizable groups of the enzyme are essential for the enzyme activity. In addition to the oxidative decarboxylase activity, the enzyme preparation exhibited divalent metal ion-dependent oxaloacetate decarboxylase and alpha-keto acid reductase activities. Based on the above results, the molecular properties of the enzymatic reaction are discussed.     

CDP-diacylglycerol synthase activity in Clostridium perfringens

CTP:phosphatidate cytidylyltransferase (CDP-diacylglycerol synthase; EC 2.7.7.41) was identified in the cell envelope fraction of the gram-positive anaerobe Clostridium perfringens. The association of this enzyme with the cell envelope fraction of cell extracts was demonstrated by glycerol density gradient centrifugation and by activity sedimenting with the 100,000 x g pellet. The enzyme exhibited a broad pH optimum between pH 6.5 and pH 7.5. Enzyme activity was dependent on magnesium (5 mM) or manganese (1 mM) ions. Activity was also dependent on the addition of the nonionic detergent Triton X-100 (5 mM). The apparent Km values for CTP and phosphatidic acid were 0.18 mM and 0.22 mM, respectively. Thioreactive agents inhibited activity, indicating that a sulfhydryl group is essential for activity. Maximal enzyme activity was observed at 50 degrees C.     

CDP-diacylglycerol synthase activity in Clostridium perfringens.

CTP:phosphatidate cytidylyltransferase (CDP-diacylglycerol synthase; EC 2.7.7.41) was identified in the cell envelope fraction of the gram-positive anaerobe Clostridium perfringens. The association of this enzyme with the cell envelope fraction of cell extracts was demonstrated by glycerol density gradient centrifugation and by activity sedimenting with the 100,000 x g pellet. The enzyme exhibited a broad pH optimum between pH 6.5 and pH 7.5. Enzyme activity was dependent on magnesium (5 mM) or manganese (1 mM) ions. Activity was also dependent on the addition of the nonionic detergent Triton X-100 (5 mM). The apparent Km values for CTP and phosphatidic acid were 0.18 mM and 0.22 mM, respectively. Thioreactive agents inhibited activity, indicating that a sulfhydryl group is essential for activity. Maximal enzyme activity was observed at 50 degrees C.     

Identification, purification, and characterization of a thermally stable lipase from rice bran. A new member of the (phospho) lipase family.

A thermally stable lipase (EC 3.1.1.3.) was first identified in rice (Oryza sativa) bran, and the enzyme was purified to homogeneity using octyl-Sepharose chromatography. The enzyme was purified to 7.6-fold with the final specific activity of 0.38 micromol min(-1) mg(-1) at 80 degrees C using [9,10-(3)H]triolein as a substrate. The purified enzyme was found to be a glycoprotein of 9.4 kD. Enzyme showed a maximum activity at 80 degrees C and at pH 11.0. The protein was biologically active and retained most of its secondary structure even at 90 degrees C as judged by the enzymatic assays and far-ultraviolet circular dichroism spectroscopy, respectively. Differential scanning calorimetric studies indicated that the transition temperature was 76 degrees C and enthalpy 1.3 x 10(5) Calorie mol(-1) at this temperature. The purified lipase also exhibited phospholipase A(2) activity. Colocalization of both the hydrolytic activities in reverse-phase high-performance liquid chromatography and isoelectric focusing showed that the dual activity was associated with a single protein. Further, a direct interaction between both the substrates and the purified protein was demonstrated by photoaffinity labeling, using chemically synthesized analogs of triolein and phosphatidylcholine (PC). Apparent K(m) for triolein (6.71 mM) was higher than that for PC (1.02 mM). The enzyme preferentially hydrolyzed the sn-2 position of PC, whereas it apparently exhibited no positional specificity toward triacylglycerol. Diisopropyl fluorophosphate inhibited both lipase and phospholipase activities of the purified enzyme. This enzyme is a new member from plants in the family of lipases capable of hydrolyzing phospholipids.     

Sulfur metabolism in bacteria associated with cheese

Metabolism of sulfur in bacteria associated with cheese has long been a topic of interest. Volatile sulfur compounds, specifically methanethiol, are correlated to desirable flavor in Cheddar cheese, but their definitive role remains elusive. Only recently have enzymes been found that produce this compound in bacteria associated with cheese making. Cystathionine - and -lyase are found in lactic acid bacteria and are capable of producing methanethiol from methionine. Their primary function is in the metabolism of cysteine. Methionine -lyase produces methanethiol from methionine at a higher efficiency than the cystathionine enzymes. This enzyme is found in brevibacteria, bacilli, and pseudomonads. Addition of brevibacteria containing this enzyme improves Cheddar cheese flavor. Despite recent progress in sulfur metabolism more information is needed before cheese flavor associated with sulfur can be predicted or controlled.     

Separation and Some Properties of Two Intracellular beta-Glucosidases of Sporotrichum (Chrysosporium) thermophile

Intracellular, inducible beta-glucosidase from the cellulolytic fungus Sporotrichum (Chrysosporium) thermophile (ATCC 42464) was fractionated by gel chromatography or isoelectric focusing into components A and B. Enzyme A (molecular weight 440,000) had only aryl-beta-glucosidase activity, whereas enzyme B (molecular weight 40,000) hydrolyzed several beta-glucosides but had only low activity against o-nitrophenyl-beta-d-glucopyranoside (ONPG). Both enzymes had temperature optima of about 50 degrees C. The pH optimum was 5.6 for enzyme A and 6.3 for enzyme B, respectively. The K(m) (ONPG) value for enzyme A was 0.5 mM, and the corresponding values for enzyme B were 0.18 mM (ONPG) and 0.28 mM (cellobiose). Enzyme B, when tested with ONPG, showed substrate inhibition at a substrate concentration above 0.4 mM which could be released by cellobiitol and other alditols. Enzyme A was isoelectric at pH 4.48, and enzyme B was isoelectric at pH 4.64. Several inhibitors were tested for their action on the activity of enzymes A and B. Both enzymes were found to be concomitantly induced in cultures with either cellobiose or cellulose as carbon source.     

Ability of thermophilic lactic acid bacteria to produce aroma compounds from amino acids

Although a large number of key odorants of Swiss-type cheese result from amino acid catabolism, the amino acid catabolic pathways in the bacteria present in these cheeses are not well known. In this study, we compared the in vitro abilities of Lactobacillus delbrueckii subsp. lactis, Lactobacillus helveticus, and Streptococcus thermophilus to produce aroma compounds from three amino acids, leucine, phenylalanine, and methionine, under mid-pH conditions of cheese ripening (pH 5.5), and we investigated the catabolic pathways used by these bacteria. In the three lactic acid bacterial species, amino acid catabolism was initiated by a transamination step, which requires the presence of an alpha-keto acid such as alpha-ketoglutarate (alpha-KG) as the amino group acceptor, and produced alpha-keto acids. Only S. thermophilus exhibited glutamate dehydrogenase activity, which produces alpha-KG from glutamate, and consequently only S. thermophilus was capable of catabolizing amino acids in the reaction medium without alpha-KG addition. In the presence of alpha-KG, lactobacilli produced much more varied aroma compounds such as acids, aldehydes, and alcohols than S. thermophilus, which mainly produced alpha-keto acids and a small amount of hydroxy acids and acids. L. helveticus mainly produced acids from phenylalanine and leucine, while L. delbrueckii subsp. lactis produced larger amounts of alcohols and/or aldehydes. Formation of aldehydes, alcohols, and acids from alpha-keto acids by L. delbrueckii subsp. lactis mainly results from the action of an alpha-keto acid decarboxylase, which produces aldehydes that are then oxidized or reduced to acids or alcohols. In contrast, the enzyme involved in the alpha-keto acid conversion to acids in L. helveticus and S. thermophilus is an alpha-keto acid dehydrogenase that produces acyl coenzymes A.     

Leucyl-transfer ribonucleic acid synthetase from a wild-type and temperature-sensitive mutant of Salmonella typhimurium

Leucyl-transfer ribonucleic acid (tRNA) synthetase was purified 100-fold from extracts of Salmonella typhimurium. The partially purified enzyme had the following K(m) values: leucine, 1.1 x 10(-5)m; adenosine triphosphate, 6.5 x 10(-4)m; tRNA(I) (Leu), 4.1 x 10(-8)m; tRNA(II) (Leu), 4.3 x 10(-8)m; tRNA(III) (Leu), 5.3 x 10(-8)m; and tRNA(IV) (Leu), 2.9 x 10(-8)m. The tRNA(Leu) fractions were isolated from Salmonella bulk tRNA by chromatography on reversed-phase columns and benzoylated diethylaminoethyl cellulose. The enzyme had a pH optimum of 8.5 and an activation energy of 10,400 cal per mole, and was inactivated exponentially at 49.5 C with a first-order rate constant of 0.064 min(-1). Strain CV356 (leuS3 leuABCD702 ara-9 gal-205) was isolated as a mutant resistant to dl-4-azaleucine and able to grow at 27 C but not at 37 C. Extracts of strain CV356 had no leucyl-tRNA synthetase activity (charging assay) when assayed at 27 or 37 C. Temperature sensitivity and enzyme deficiency were caused by mutation in the structural gene locus specifying leucyl-tRNA synthetase. A prototrophic derivative of strain CV356 (CV357) excreted branched-chain amino acids and had high pathway-specific enzyme levels when grown at temperatures where its doubling time was near normal. At growth-restricting temperatures, both amino acid excretion and enzyme levels were further elevated. The properties of strain CV357 indicate that there is only a single leucyl-tRNA synthetase in S. typhimurium.