Aspartate kinase-independent lysine synthesis in an extremely thermophilic bacterium, Thermus thermophilus: lysine is synthesized via alpha-aminoadipic acid not via diaminopimelic acid.
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An aspartate kinase-deficient mutant of Thermus thermophilus, AK001, was constructed. The mutant strain did not grow in a minimal medium, suggesting that T. thermophilus contains a single aspartate kinase. Growth of the mutant strain was restored by addition of both threonine and methionine, while addition of lysine had no detectable effect on growth. To further elucidate the lysine biosynthetic pathway in T. thermophilus, lysine auxotrophic mutants of T. thermophilus were obtained by chemical mutagenesis. For all lysine auxotrophic mutants, growth in a minimal medium was not restored by addition of diaminopimelic acid, whereas growth of two mutants was restored by addition of alpha-aminoadipic acid, a precursor of lysine in biosynthetic pathways of yeast and fungi. A BamHI fragment of 4.34 kb which complemented the lysine auxotrophy of a mutant was cloned. Determination of the nucleotide sequence suggested the presence of homoaconitate hydratase genes, termed hacA and hacB, which could encode large and small subunits of homoaconitate hydratase, in the cloned fragment. Disruption of the chromosomal copy of hacA yielded mutants showing lysine auxotrophy which was restored by addition of alpha-aminoadipic acid or alpha-ketoadipic acid. All of these results indicated that in T. thermophilus, lysine was not synthesized via the diaminopimelic acid pathway, believed to be common to all bacteria, but via a pathway using alpha-aminoadipic acid as a biosynthetic intermediate. (+info)
The site-specific integration of genetic elements may modulate thermostable protease production, a virulence factor in Dichelobacter nodosus, the causative agent of ovine footrot.
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The gram-negative anaerobe Dichelobacter nodosus is the causative agent of footrot in sheep. The authors have previously characterized two genetic elements, the intA (vap) and intB elements, which integrate into the genome of D. nodosus. In the virulent strain A198 there are two copies of the intA element. One copy is integrated into the 3' end of the tRNA-serGCU gene, close to the aspartokinase (askA) gene, and the second copy is integrated into the 3' end of the tRNA-serGGA gene, next to the polynucleotide phosphorylase (pnpA) gene. In this study, a new genetic element was identified in the benign strain C305, the intC element, integrated into the 3' end of the tRNA-serGCU gene, next to askA. The intC element was found in most D. nodosus strains, both benign and virulent, which were examined, and was integrated into tRNA-serGCU in most strains. Between the askA and tRNA-serGCU genes, a gene (designated glpA), was identified whose predicted protein product has very high amino acid identity with RsmA from the plant pathogen Erwinia carotovora. RsmA acts as a global repressor of pathogenicity in E. carotovora, by repressing the production of extracellular enzymes. In virulent strains of D. nodosus the intA element was found to be integrated next to pnpA, and either the intA or intC element was integrated next to glpA. By contrast, all but one of the benign strains had intB at one or both of these two positions, and the one exception had neither intA, intB nor intC at one position. The loss of the intC element from the virulent strain 1311 resulted in loss of thermostable protease activity, a virulence factor in D. nodosus. A model for virulence is proposed whereby integration of the intA and intC genetic elements modulates virulence by altering the expression of glpA, pnpA, tRNA-serGCU and tRNA-serGGA. (+info)
Organization of threonine biosynthesis genes from the obligate methylotroph Methylobacillus flagellatus.
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The genes encoding aspartate kinase (ask), homoserine dehydrogenase (hom), homoserine kinase (thrB) and threonine synthase (thrC) from the obligate methylotroph Methylobacillus flagellatus were cloned. In maxicells hom and thrC directed synthesis of 51 and 48 kDa polypeptides, respectively. The hom, thrB and thrC genes and adjacent DNA areas were sequenced. Of the threonine biosynthesis genes, only hom and thrC were tightly linked in the order hom-thrC. The gene for thymidylate synthase (thyA) followed thrC and the gene for aspartate aminotransferase (aspC) preceded hom. All four genes (aspC-hom-thrC-thyA) were transcribed in the same direction. mRNA analysis indicated that hom-thrC are apparently transcribed in one 7.5 kb transcript in M. flagellatus. Promoter analysis showed the presence of a functional promoter between aspC and hom. No functional promoter was found to be associated with the DNA stretch between hom and thrC. The thrB gene encoded an unusual type of homoserine kinase and was not linked to other threonine biosynthesis genes. (+info)
Effects of a feedback-resistant aspartokinase III gene on L-isoleucine production in Escherichia coli K-12.
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An L-isoleucine-overproducing recombinant strain of E. coli, TVD5, was also found to overproduce L-valine. The L-isoleucine productivity of TVD5 was markedly decreased by addition of L-lysine to the medium. Introduction of a gene encoding feedback-resistant aspartokinase III increased L-isoleucine productivity and decreased L-valine by-production. The resulting strain accumulated 12 g/l L-isoleucine from 40 g/l glucose, and suppression of L-isoleucine productivity by L-lysine was relieved. (+info)
Attractant regulation of the aspartate receptor-kinase complex: limited cooperative interactions between receptors and effects of the receptor modification state.
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The manner by which the bacterial chemotaxis system responds to a wide range of attractant concentrations remains incompletely understood. In principle, positive cooperativity between chemotaxis receptors could explain the ability of bacteria to respond to extremely low attractant concentrations. By utilizing an in vitro receptor-coupled kinase assay, the attractant-dependent response curve has been measured for the Salmonella typhimurium aspartate chemoreceptor. The attractant chosen, alpha-methyl aspartate, was originally used to quantitate high receptor sensitivity at low attractant concentrations by Segall, Block, and Berg [(1986) Proc. Natl. Acad. Sci. U.S.A. 83, 8987-8991]. The attractant response curve exhibits limited positive cooperativity, yielding a Hill coefficient of 1.7-2.4, and this Hill coefficient is relatively independent of both the receptor modification state and the mole ratio of CheA to receptor. These results disfavor models in which there are strong cooperative interactions between large numbers of receptor dimers in an extensive receptor array. Instead, the results are consistent with cooperative interactions between a small number of coupled receptor dimers. Because the in vitro receptor-coupled kinase assay utilizes higher than native receptor densities arising from overexpression, the observed positive cooperativity may overestimate that present in native receptor populations. Such positive cooperativity between dimers is fully compatible with the negative cooperativity previously observed between the two symmetric ligand binding sites within a single dimer. The attractant affinity of the aspartate receptor is found to depend on the modification state of its covalent adaptation sites. Increasing the the level of modification decreases the apparent attractant affinity at least 10-fold in the in vitro receptor-coupled kinase assay. This observation helps explain the ability of the chemotaxis pathway to respond to a broad range of attractant concentrations in vivo. (+info)
Evidence for direct interaction between enzyme I(Ntr) and aspartokinase to regulate bacterial oligopeptide transport.
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Bradyrhizobium japonicum transports oligopeptides and the heme precursor delta-aminolevulinic acid (ALA) by a common mechanism. Two Tn5-induced mutants disrupted in the lysC and ptsP genes were identified based on the inability to use prolyl-glycyl-glycine as a proline source and were defective in [(14)C]ALA uptake activity. lysC and ptsP were shown to be proximal genes in the B. japonicum genome. However, RNase protection and in trans complementation analysis showed that lysC and ptsP are transcribed separately, and that both genes are involved in oligopeptide transport. Aspartokinase, encoded by lysC, catalyzes the phosphorylation of aspartate for synthesis of three amino acids, but the lysC strain is not an amino acid auxotroph. The ptsP gene encodes Enzyme I(Ntr) (EI(Ntr)), a paralogue of Enzyme I of the phosphoenolpyruvate:sugar phosphotransferase (PTS) system. In vitro pull-down experiments indicated that purified recombinant aspartokinase and EI(Ntr) interact directly with each other. Expression of ptsP in trans from a multicopy plasmid complemented the lysC mutant, suggesting that aspartokinase normally affects Enzyme I(Ntr) in a manner that can be compensated for by increasing the copy number of the ptsP gene. ATP was not a phosphoryl donor to purified EI(Ntr), but it was phosphorylated by ATP in the presence of cell extracts. This phosphorylation was inhibited in the presence of aspartokinase. The findings demonstrate a role for a PTS protein in the transport of a non-sugar solute and suggest an unusual regulatory function for aspartokinase in regulating the phosphorylation state of EI(Ntr). (+info)
Aspartate kinase 2. A candidate gene of a quantitative trait locus influencing free amino acid content in maize endosperm.
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The maize (Zea mays) Oh545o2 inbred accumulates an exceptionally high level of free amino acids, especially lysine (Lys), threonine (Thr), methionine, and iso-leucine. In a cross between Oh545o2 and Oh51Ao2, we identified several quantitative trait loci linked with this phenotype. One of these is on the long arm of chromosome 2 and is linked with loci encoding aspartate (Asp) kinase 2 and Asp kinase (AK)-homoserine dehydrogenase (HSDH) 2. To investigate whether these enzymes can contribute to the high levels of Asp family amino acids, we measured their specific activity and feedback inhibition properties, as well as activities of several other key enzymes involved in Lys metabolism. We did not find a significant difference in total activity of dihydrodipicolinate synthase, HSDH, and Lys ketoglutarate reductase between these inbreds, and the feedback inhibition properties of HSDH and dihyrodipicolinate synthase by Lys and/or Thr were similar. The most significant difference we found between Oh545o2 and Oh51Ao2 is feedback inhibition of AK by Lys but not Thr. AK activity in Oh545o2 is less sensitive to Lys inhibition than that in Oh51Ao2, with a Lys I50 twice that of Oh51Ao2. AK activity in Oh545o2 endosperm is also higher than in Oh51Ao2 at 15 d after pollination, but not 20 d after pollination. The results indicate that the Lys-sensitive Asp kinase 2, rather than the Thr-sensitive AK-HSDH2, is the best candidate gene for the quantitative trait locus affecting free amino acid content in Oh545o2. (+info)
An integrated study of threonine-pathway enzyme kinetics in Escherichia coli.
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We have determined the kinetic parameters of the individual steps of the threonine pathway from aspartate in Escherichia coli under a single set of experimental conditions chosen to be physiologically relevant. Our aim was to summarize the kinetic behaviour of each enzyme in a single tractable equation that takes into account the effect of the products as competitive inhibitors of the substrates in the forward reaction and also, when appropriate (e.g. near-equilibrium reactions), as substrates of the reverse reactions. Co-operative feedback inhibition by threonine and lysine was also included as necessary. We derived the simplest rate equations that describe the salient features of the enzymes in the physiological range of metabolite concentrations in order to incorporate them ultimately into a complete model of the threonine pathway, able to predict quantitatively the behaviour of the pathway under natural or engineered conditions. (+info)