Isoleucine and Valine Metabolism in Escherichia coli XVIII. Induction of Acetohydroxy Acid Isomeroreductase

The regulation by substrate induction of the acetohydroxy acid isomeroreductase was studied in Escherichia coli. Induction was inhibited by chloramphenicol and rifampin. The addition of rifampin resulted in a decay of the capacity to form isomeroreductase. This was attributed to the breakdown of the isomeroreductase messenger, which had a half-life of about 45 sec at 37 C. Induction of isomeroreductase was enhanced by including glucose in the medium. This effect was shown to be due in part to the lowering of the pH of the medium, which presumably made inducer entry more rapid.     

Branched-Chain Amino Acid Biosynthesis in Salmonella typhimurium: a Quantitative Analysis

We report here the first quantitative study of the branched-chain amino acid biosynthetic pathway in Salmonella typhimurium LT2. The intracellular levels of the enzymes of the pathway and of the 2-keto acid intermediates were determined under various physiological conditions and used for estimation of several of the fluxes in the cells. The results led to a revision of previous ideas concerning the way in which multiple acetohydroxy acid synthase (AHAS) isozymes contribute to the fitness of enterobacteria. In wild-type LT2, AHAS isozyme I provides most of the flux to valine, leucine, and pantothenate, while isozyme II provides most of the flux to isoleucine. With acetate as a carbon source, a strain expressing AHAS II only is limited in growth because of the low enzyme activity in the presence of elevated levels of the inhibitor glyoxylate. A strain with AHAS I only is limited during growth on glucose by the low tendency of this enzyme to utilize 2-ketobutyrate as a substrate; isoleucine limitation then leads to elevated threonine deaminase activity and an increased 2-ketobutyrate/2-ketoisovalerate ratio, which in turn interferes with the synthesis of coenzyme A and methionine. The regulation of threonine deaminase is also crucial in this regard. It is conceivable that, because of fundamental limitations on the specificity of enzymes, no single AHAS could possibly be adequate for the varied conditions that enterobacteria successfully encounter.     

Regulation of Homocysteine Biosynthesis in Salmonella typhimurium

The regulation of the homocysteine branch of the methionine biosynthetic pathway in Salmonella typhimurium has been reexamined with the aid of a new assay for the first enzyme. The activity of this enzyme is subject to synergistic feedback inhibition by methionine plus S-adenosylmethionine. The synthesis of all three enzymes of the pathway is regulated by noncoordinate repression. The enzymes are derepressed in metJ and metK regulatory mutants, suggesting the existence of regulatory elements common to all three. Experiments with a methionine/vitamin B(12) auxotroph (metE) grown in a chemostat on methionine or vitamin B(12) suggested that the first enzyme is more sensitive to repression by methionine derived from exogenous than from endogenous sources. metB and metC mutants grown on methionine in the chemostat did not show hypersensitivity to repression by exogenous methionine. Therefore, it appears that the metE chemostat findings are peculiar to the phenotype of this mutant; such evidence suggests a possible role for a functional methyltetrahydrofolate-homocysteine transmethylase in regulating the synthesis of the first enzyme. Thus there appear to be regulatory elements which are common to the repression of all three enzymes, as well as some that are unique to the first enzyme. The nature of the corepressor is not known, but it may be a derivative of S-adenosylmethionine. metJ and metK mutants of Salmonella have a normal capacity for S-adenosylmethionine synthesis but may be blocked in synthesis or utilization of a corepressor derived from it.     

flrB, a Regulatory Locus Controlling Branched-Chain Amino Acid Biosynthesis in Salmonella typhimurium

Salmonella typhimurium strain CV123 (ara-9 gal-205 flrB1), isolated as a mutant resistant to trifluoroleucine, has derepressed and constitutive levels of enzymes forming branched-chain amino acids. This strain grows more slowly than the parent at several temperatures, both in minimal medium and nutrient broth. It overproduces and excretes sizeable amounts of leucine, valine, and isoleucine in comparison with the parental strain. Both leuS (coding for leucyl-transfer ribonucleic acid [tRNA]synthetase) and flrB are linked to lip (min 20 to 25) by P1 transduction, whereas only leuS is linked to lip by P22 transduction. Strain CV123 containing an F' lip(+) episome from Escherichia coli has repressed levels of leucine-forming enzymes, indicating that flrB(+) is dominant to flrB. Leucyl-tRNA synthetase from strain CV123 appears to be identical to the leucyl-tRNA synthetase in the parent. No differences were detected between strain CV123 and the parent with respect to tRNA acceptor activity for a number of amino acids. Furthermore, there was no large difference between the two strains in the patterns of leucine tRNA isoaccepting species after fractionation on several different columns. Several other flrB strains exhibited temperature-sensitive excretion of leucine, i.e., they excreted leucine at 37 C but not 25 C. In one such strain, excretion at 37 C was correlated with derepression of some enzymes specified by ilv and leu. These latter results suggest that flrB codes for a protein.     

Biosynthesis During Recovery of Heat-Injured Salmonella typhimurium

Salmonella typhimurium 7136 incorporated label from glucose-U-(14)C into nucleic acids, lipids, and pool material during recovery from heat injury. There was very little incorporation of label into protein during recovery.     

First Enzyme of Histidine Biosynthesis and Repression Control of Histidyl-Transfer Ribonucleic Acid Synthetase of Salmonella typhimurium

The regulation of formation of histidyl-transfer ribonucleic acid (tRNA) synthetase was examined in strains of Salmonella typhimurium. When the first of the histidine-forming enzymes was wild type, the presence of 2-thiazolealanine in the growth medium prevented repression of histidyl-tRNA synthetase formation elicited by the addition of 1, 2, 4-triazole-3-alanine to these cultures. Conversely, thiazolealanine had no effect on repression of histidyl-tRNA synthetase formation by triazolealanine in hisG mutant strains. These data suggest a relationship between the control of histidyl-tRNA synthetase formation and the functional state of the histidine operon.     

Biosynthesis of Branched-Chain Amino Acids in Schizosaccharomyces pombe: Properties of Acetohydroxy Acid Synthetase1

The regulatory properties of acetohydroxy acid synthetase (AHAS), the first enzyme in the biosynthetic pathway to valine and the second in the isoleucine pathway, were investigated in the fission yeast Schizosaccharomyces pombe. The enzyme was partially purified from crude extracts by protamine sulfate treatment, ammonium sulfate fractionation, and gel filtration through Sephadex G-25. AHAS from S. pombe is unique in that its activity shows a single peak around pH 6.5; high sensitivity to feedback inhibition by valine at this pH (K(i) = 0.1 mM) indicates that the enzyme is involved in valine biosynthesis. Pyruvate saturation kinetics of AHAS extracted from cells grown on glycerol as sole carbon and energy source were normal and hyperbolic. In contrast, the enzyme from glucose-grown cells exhibited sigmoidal saturation kinetics, an effect which disappeared when the synthetase from such cells was partially purified. This phenomenon was shown to be due to competition for pyruvate between AHAS and pyruvate decarboxylase; the latter enzyme is present in large amounts in cells fermenting glucose. Valine inhibition is noncompetitive in nature, and this effector exhibits homotropic cooperative effects; isoleucine is a less-potent inhibitor of AHAS activity. Mercurial treatment reversibly desensitized the enzyme to valine inhibition. On the basis of these data, the S. pombe AHAS appears to be an allosteric regulatory enzyme with the properties of a negative V system.     

Interaction Between Histidyl Transfer Ribonucleic Acid and the First Enzyme for Histidine Biosynthesis of Salmonella typhimurium

Previous studies showed that the enzyme (phosphoribosyltransferase) which catalyzes the first step of the histidine pathway in Salmonella typhimurium plays a role in regulation of the histidine operon. Since histidyl transfer ribonucleic acid (His-tRNA) is required for repression of the histidine operon, we considered the possibility that the role of phosphoribosyltransferase might be realized through an interaction with His-tRNA. One prediction inherent in this idea is that the enzyme should interact with His-tRNA in vitro. Evidence is presented for such an interaction. Binding of (3)H-His-tRNA to purified phosphoribosyltransferase was tested on Sephadex columns and on nitrocellulose filters. The enzyme was found to have a high affinity for tRNA. Comparing the binding of (3)H-His-tRNA with that of tRNA aminoacylated with other (3)H-amino acids disclosed that the binding of the histidyl species of tRNA is favored over that of other species and is dependent upon magnesium-ion concentration.     

Biosynthesis of tRNA in histidine regulatory mutants of Salmonella typhimurium.

HisU mutants of Salmonella typhimurium are depressed in the histidine operon since they have lower intracellular concentration of histidyl-tRNAHis. In this paper we present evidences showing that a strain carrying a hisU mutation (hisUl206) is altered in a nucleolytic enzyme involved in tRNA maturation process. The analysis of several hisU mutants indicates that hisU region of bacterial genome may account for more than one function involved in tRNA biosynthesis.     

Role of Histidine Transfer Ribonucleic Acid in Regulation of Synthesis of Histidyl-Transfer Ribonucleic Acid Synthetase of Salmonella typhimurium

The role of histidine transfer ribonucleic acid (tRNA) in repression of synthesis of histidyl-tRNA synthetase was examined in two strains of Salmonella typhimurium, one of which was a histidine tRNA (hisR) mutant possessing 52% of the wild-type (hisR(+)) histidine tRNA and a derepressed level of the histidine biosynthetic enzymes during histidine-unrestricted growth. Histidine-restricted growth caused a derepression of the rate of formation of histidyl-tRNA synthetase in both strains. In the case of the wild-type strain, addition of histidine to the derepressed culture caused a repression of synthesis of histidyl-tRNA synthetase for at least one generation of growth. In contrast, when histidine was restored to the derepressed hisR mutant culture, synthesis of histidyl-tRNA synthetase was continued at the initial derepressed rate. These results suggest that histidine must be attached to histidine tRNA for repression of synthesis of histidyl-tRNA synthetase.     

Transformation of Salmonella typhimurium by Plasmid Deoxyribonucleic Acid

A modified transformation procedure that is effective for the introduction of plasmid deoxyribonucleic acid at high frequency into Salmonella typhimurium, as well as into Escherichia coli, is described. Transformed bacteria acquire a circular deoxyribonucleic acid species having the genetic and molecular characteristics of the transforming plasmid.     

Pyrimidine Nucleotide Metabolism and Pathways of Thymidine Triphosphate Biosynthesis in Salmonella typhimurium

The nucleoside triphosphate pools of two cytidine auxotrophic mutants of Salmonella typhimurium LT-2 were studied under different conditions of pyrimidine starvation. Both mutants, DP-45 and DP-55, are defective in cytidine deaminase and cytidine triphosphate (CTP) synthase. In addition, DP-55 has a requirement for uracil (uridine). Cytidine starvation of the mutants results in accumulation of high concentrations of uridine triphosphate (UTP) in the cells, while the pools of CTP and deoxy-CTP drop to undetectable levels within a few minutes. Addition of deoxycytidine to such cells does not restore the dCTP pool, indicating that S. typhimurium has no deoxycytidine kinase. From the kinetics of UTP accumulation during cytidine starvation, it is concluded that only cytidine nucleotides participate in the feedback regulation of de novo synthesis of UTP; both uridine and cytidine nucleotides participate in the regulation of UTP synthesis from exogenously supplied uracil or uridine. Uracil starvation of DP-55 in presence of cytidine results in extensive accumulation of CTP, suggesting that CTP does not regulate its own synthesis from exogenous cytidine. Analysis of the thymidine triphosphate (dTTP) pool of DP-55 labeled for several generations with (32)P-orthophosphate and (3)H-uracil in presence of (12)C-cytidine shows that only 20% of the dTTP pool is derived from uracil (via the methylation of deoxyuridine monophosphate); 80% is apparently synthesized from a cytidine nucleotide.     

Deoxyribonucleic Acid Adenine and Cytosine Methylation in Salmonella typhimurium and Salmonella typhi

The methylations of adenine in the sequence -GATC- and of the second cytosine in the sequence - [Formula: see text] - were studied in Salmonella typhimurium and in Salmonella typhi. The study was carried out by using endonucleases which restrict the plasmid pBR322 by cleavage at the sequences -GATC- (DpnI and MboI) and - [Formula: see text] - (EcoRII). The restriction patterns obtained for this plasmid isolated from transformed S. typhimurium and S. typhi were compared with those of pBR322 isolated from Escherichia coli K-12. In E. coli K-12, adenines at the sequence -GATC- and the second cytosines at - [Formula: see text] - are met hylated by enzymes coded for by the genes dam and dem, respectively. From comparison of the restriction patterns obtained, it is concluded that S. typhimurium and S. typhi contain genes responsible for deoxyribonucleic acid methylation equivalent to E. coli K-12 genes dam and dcm.     

Nalidixic Acid for Enrichment of Auxotrophs in Cultures of Salmonella typhimurium

Nalidixic acid kills only growing cells of Salmonella typhimurium and can be used to enrich for auxotrophs in populations where prototrophs predominate.     

Heterogeneity of the Stability of Messenger Ribonucleic Acid in Salmonella typhimurium

Cells of Salmonella typhimurium strain SL 282, deflagellated by mechanical shear, regenerated their flagella in the absence of tryptophan, an amino acid required for growth but not found in flagellin. Ribonucleic acid (RNA) synthesis was severely inhibited by tryptophan starvation. These findings suggested that the messenger RNA (mRNA) for flagellin might be stable. Actinomycin D was used to inhibit RNA synthesis in ethylenediaminetetraacetate-treated bacteria. The introduction of an F(lac) episome into strain SL 282 permitted the simultaneous study of the synthesis of flagellin, beta-galactosidase, and total protein. In the actinomycin-treated bacteria protein and beta-galactosidase syntheses were inhibited by 90%, whereas flagellin synthesis was unaffected. We conclude that the mRNA for flagellin synthesis is stable and that species of mRNA vary with respect to metabolic stability in S. typhimurium.     

Acid adaptation sensitizes Salmonella typhimurium to hypochlorous acid.

Acid adaptation of Salmonella typhimurium at a pH of 5.0 to 5.8 for one to two cell doublings resulted in marked sensitization of the pathogen to halogen-based sanitizers including chlorine (hypochlorous acid) and iodine. Acid-adapted S. typhimurium was more resistant to an anionic acid sanitizer than was its nonadapted counterpart. A nonselective plating medium of tryptose phosphate agar plus 1% pyruvate was used throughout the study to help recover chemically stressed cells. Mechanisms of HOCl-mediated inactivation of acid-adapted and nonadapted salmonellae were investigated. Hypochlorous acid oxidized a higher percentage of cell surface sulfhydryl groups in acid-adapted cells than in nonadapted cells, and sulfhydryl oxidation was correlated with cell inactivation. HOCl caused severe metabolic disruptions in acid-adapted and nonadapted S. typhimurium, such as respiratory loss and inability to restore the adenylate energy charge from a nutrient-starved state. Sensitization of S. typhimurium to hypochlorous acid by acid adaptation also involved increased permeability of the cell surface because nonadapted cells treated with EDTA became sensitized. The results of this study establish that acid-adapted S. typhimurium cells are highly sensitized to HOCl oxidation and that inactivation by HOCl involves changes in membrane permeability, inability to maintain or restore energy charge, and probably oxidation of essential cellular components. This study provides a basis for improved practical technologies to inactivate Salmonella and implies that acid pretreatment of food plant environments may increase the efficacy of halogen sanitizers.     

Biosynthesis and assembly of envelope lipoprotein in a glycerol-requiring mutant of Salmonella typhimurium.

A glycerol-requiring mutant of Salmonella typhimurium was used in a study of the biosynthesis and assembly of a structural lipoprotein in the cell envelope of gram-negative bacteria. Upon removal of glycerol from the growth medium, the biosynthesis of lipoprotein, as measured by radioactive arginine incorporation, was reduced by the same extent as that of other envelope proteins, the cumulative incorporation of arginine being 20% of that of the unstarved control cells. However, the incorporation of radioactive palmitate into lipoprotein was more severely curtailed after glycerol starvation, the cumulative rate of which was 8% of that observed in the unstarved cells. It was further observed that the lipoprotein synthesized in the glycerol-starved cells was more enriched in unmodified cysteine, which is known to be the N-terminal amino acid of lipoprotein, than that synthesized in the unstarved cells. We conclude that the synthesis of the apoprotein portion of Braun's lipoprotein proceeds independently of the attachment of diglyceride to the sulfhydryl group of the N-terminal cysteine and may, in fact, precede the incorporation of the diglyceride moiety.     

Deoxyribonucleic Acid Repair in a Highly Radiation-Resistant Strain of Salmonella typhimurium

Deoxyribonucleic acid repair was studied in gamma-irradiated wild-type Salmonella typhimurium and in a radiation-resistant derivative 20 times more resistant than wild type. After exposure to 20 or 50 krad, the wild-type strain (DB21) degraded 30 to 50% of its prelabeled DNA into acid-soluble fragments, whereas the radioresistant strain degraded less than 15% after 4 h of incubation. Post-irradiation synthesis of DNA in the wild-type strain DB21 was reduced after a dose of 20 krad and totally inhibited after exposure to 200 krad. With radiation-resistant strain, D21R6008, on the other hand, DNA synthesis was delayed after a dose of 200 krad but not inhibited. Doses of 20 and 200 krad produced a similar number of single-strand breaks in the DNA of both strains as determined by zone sedimentation analysis in alkaline sucrose gradients. The radiation-resistant strain D21R6008, on the other hand, DNA synthesis was strand breaks in its DNA and repairs these damages more rapidly than wild-type Salmonella.