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osmo-sensing by n- and c-terminal extensions of the glycine betaine uptake system betp of corynebacterium glutamicum.the major uptake carrier for the compatible solute glycine betaine in corynebacterium glutamicum is the secondary transport system betp. it is effectively regulated by the external osmolality both on the level of expression and of activity. betp carries highly charged domains both at the n and at the c terminus. we investigated the role of these extensions in the regulatory response to hyperosmotic stress. mutants of the betp gene coding for proteins with truncated n- and c-terminal extensions w ...19989446558
nadph oxidase system as a superoxide-generating cyanide-resistant pathway in the respiratory chain of corynebacterium glutamicum.the respiratory chain of corynebacterium glutamicum was investigated, especially with respect to a cyanide-resistant respiratory chain bypass oxidase. the membranes of c. glutamicum had nadh, succinate, lactate, and nadph oxidase activities, and menaquinone, and cytochromes a 598, b 562(558), and c 550 as respiratory components. the nadh, succinate, lactate, and nadph oxidase systems, all of which were more cyanide-resistant than n,n,n',n'-tetramethyl-p-phenylene diamine oxidase activity (cytoch ...199827385451
glutamate overproduction in corynebacterium glutamicum triggered by a decrease in the level of a complex comprising dtsr and a biotin-containing subunit.glutamate overproduction in corynebacterium glutamicum is induced by tween 40, biotin-limitation, or sublethal amounts of penicillin. disruption of the dtsr gene, which encodes a putative component of a biotin-containing enzyme complex involved in fatty acid synthesis, causes constitutive overproduction of glutamate. we report here that overexpression of dtsr inhibits the induction of glutamate overproduction. in contrast, the level of dtsr in the wild type strain was found to decrease in the pr ...199927380236
hyperproduction of tryptophan by corynebacterium glutamicum with the modified pentose phosphate pathway.a classically derived tryptophan-producing corynebacterium glutamicum strain was recently significantly improved both by plasmid-mediated amplification of the genes for the rate-limiting enzymes in the terminal pathways and by construction of a plasmid stabilization system so that it produced more tryptophan. this engineered strain, ky9218 carrying pkw9901, produced 50 g of tryptophan per liter from sucrose after 80 h in fed-batch cultivation without antibiotic pressure. analysis of carbon balan ...199910347033
a special reactor design for investigations of mixing time effects in a scaled-down industrial l-lysine fed-batch fermentation processa specially designed model reactor based on a 42-l laboratory fermentor was equipped with six stirrers (rushton turbines) and five cylindrical disks. in this model reactor, the mixing time, theta(90), turned out to be 13 times longer compared with the 42-l standard laboratory fermentor fitted with two rushton turbines and four wall-fixed longitudinal baffles. to prove the suitability of the model reactor for scaledown studies of mixing-time-dependent processes, parallel exponential fed-batch cul ...199910404240
cloning of the argf gene encoding the ornithine carbamoyltransferase from corynebacterium glutamicum.the argf gene encoding ornithine carbamoyl-transferase (otcase; ec2.1.3.3) has been cloned from corynebacterium glutamicum by transforming the escherichia coli arginine auxotroph with the genomic dna library. the cloned dna also complements the e. coli argg mutant, suggesting a clustered organization of the genes in the genome. we have determined the dna sequence of the minimal fragment complementing the e. coli argf mutant. the coding region of the cloned gene is 957 nucleotides long with a ded ...199910420995
ribonucleotide reductase (rnr) of corynebacterium glutamicum atcc 13032--genetic characterization of a second class iv enzyme.ribonucleotide reductases (rnrs) encoded by nrd (nucleotide reduction) genes are unique enzymes providing the dna precursors in all living organisms and several viruses. the designation of four classes of rnrs reflects their use of diverse metallo-cofactors. using oligonucleotide primers derived from conserved domains of the primary structure of known nrda and nrde proteins, an internal 938 bp fragment of the nrde gene was amplified from genomic dna of corynebacterium glutamicum. with this pcr p ...199910439398
crystallization and preliminary x-ray diffraction studies of monomeric isocitrate dehydrogenase from corynebacterium glutamicum.a monomeric isocitrate dehydrogenase has been crystallized for the first time. this enzyme catalyzes the conversion of isocitrate to oxalosuccinate and subsequently to alpha-ketoglutarate and co(2); the coenzyme nadp(+) is reduced to nadph during the reaction. polyethylene glycol 2000 monomethyl ether was used to crystallize the enzyme in space group c2 with unit-cell parameters a = 137.1, b = 54.6, c = 126.4 a, beta = 108.2 degrees. the very small crystal (0. 05 x 0.20 x 0.05 mm) diffracted to ...199910489453
establishment of a gene transfer system for rhodococcus opacus pd630 based on electroporation and its application for recombinant biosynthesis of poly(3-hydroxyalkanoic acids).a gene transfer system for rhodococcus opacus pd630 based on electroporation was established and optimized employing the escherichia coli-rhodococcus shuttle vectors pnc9501 and pnc9503 as well as the e. coli-corynebacterium glutamicum shuttle vector pjc1 as suitable cloning vectors for r. opacus pd630, resulting in transformation efficiencies up to 1.5 x 10(5) cfus/microgram plasmid dna. applying the optimized electroporation protocol to the pnc9501-derivatives pak68 and pak71 harboring the ent ...199910570798
a method for the determination of pyruvate carboxylase activity during the glutamic acid fermentation with corynebacterium glutamicum.a discontinuous lactate dehydrogenase coupled assay is described for the evaluation of the pyruvate carboxylase activity (pc, ec 6.4.1.1) in a glutamate overproducing strain of corynebacterium glutamicum. after an initial permeabilisation period of the cells, the method consisted of the fluorometric determination of the remaining pyruvate level after transformation into oxaloacetate by the endogenous pc. the assay was demonstrated to be powerful and enabled the determination of the c. glutamicum ...199910579510
characterization of activity and expression of isocitrate lyase in mycobacterium avium and mycobacterium tuberculosis.analysis by two-dimensional gel electrophoresis revealed that mycobacterium avium expresses several proteins unique to an intracellular infection. one abundant protein with an apparent molecular mass of 50 kda was isolated, and the n-terminal sequence was determined. it matches a sequence in the m. tuberculosis database (sanger) with similarity to the enzyme isocitrate lyase of both corynebacterium glutamicum and rhodococcus fascians. only marginal similarity was observed between this open readi ...199910572116
isolation of the muri gene from brevibacterium lactofermentum atcc 13869 encoding d-glutamate racemase.the muri gene encoding d-glutamate racemase plays an important role in the biosynthesis of d-glutamic acid, an essential component of cell wall peptidoglycan of almost all eubacteria. a dna fragment that could rescue the auxotrophy of d-glutamic acid in the escherichia coli muri mutant strain wm335 was isolated from brevibacterium lactofermentum atcc 13869 belonging to the coryneform bacteria. dna sequencing reveals that it encodes a protein of 284 amino acid residues, which shows a high level o ...199910386367
the corynebacterium glutamicum insertion sequence iscg2 prefers conserved target sequences located adjacent to genes involved in aspartate and glutamate metabolism.an is element, termed iscg2, was identified in the chromosome of corynebacterium glutamicum atcc 13032. after screening a cosmid library of the c. glutamicum atcc 13032 genome, six copies of iscg2 including their flanking regions were sequenced and analyzed. iscg2 is 1636 bp in length and has 26-bp imperfect inverted repeats flanked by 3-bp direct repeats. by comparisons with other is elements, iscg2 was classified as a member of the is30 family of insertion sequences. the six copies of iscg2 we ...199910589846
class 1 integrons, gene cassettes, mobility, and epidemiology.integrons are genetic elements that, although unable to move themselves, contain gene cassettes that can be mobilized to other integrons or to secondary sites in the bacterial genome. the majority of approximately 60 known gene cassettes encode resistance to antibiotics. recently, a number of gene cassettes encoding extended-spectrum beta-lactamases or carbapenemases have been described. up to at least five cassettes may be present in an integron, which leads to multiresistance. frequently, more ...199910614949
expression of the escherichia coli catabolic threonine dehydratase in corynebacterium glutamicum and its effect on isoleucine production.the catabolic or biodegradative threonine dehydratase (e.c. 4.2.1. 16) of escherichia coli is an isoleucine feedback-resistant enzyme that catalyzes the degradation of threonine to alpha-ketobutyrate, the first reaction of the isoleucine pathway. we cloned and expressed this enzyme in corynebacterium glutamicum. we found that while the native threonine dehydratase of c. glutamicum was totally inhibited by 15 mm isoleucine, the heterologous catabolic threonine dehydratase expressed in the same st ...199910388709
[l-lysine production by corynebacterium glutamicum non growing cells].the production of l-lysine by corynebacterium glutamicum (gigo) non growing cells was studied. a system of fermentation was developed where it was possible to separate the physiologic states of growth from those of production of l-lysine. the biomass propagation step carried out in batch cultures with yeast extract and glucose substrate showed cellular growth without production of l-lysine. the production of l-lysine was achieved when the cells were collected and incubated in the mineral glucose ...199910974714
conversion of glutamate to glutamine by permeabilized corynebacterium glutamicum.glutamate was converted to glutamine by corynebacterium glutamicum permeabilized by ethanol (10%). atp was supplied to the reaction by dried saccharomyces cerevisiae or regenerated by acetyl kinase. high glutamine synthetase activity in c. glutamicum was induced by cultivation of the microorganism in media containing glutamate as the sole source of nitrogen.199910997130
nitrogen regulation in corynebacterium glutamicum: isolation of genes involved and biochemical characterization of corresponding proteins.the regulation of nitrogen assimilation was investigated in the gram-positive actinomycete corynebacterium glutamicum. biochemical studies and site-directed mutagenesis revealed that glutamine synthetase activity is regulated via adenylylation in this organism. the genes encoding the central signal transduction protein ph (glnb) and the primary nitrogen sensor uridylyltransferase (glnd) were isolated and sequenced. additionally, genes putatively involved in the degradation of ornithine (ocd) and ...199910227160
positively regulated expression of the escherichia coli arabad promoter in corynebacterium glutamicum.in corynebacterium glutamicum the promoter of the arabad escherichia coli gene is positively regulated by both arabinose and the arac gene product, as it is the case in the natural host. if the l-arabinose inducer and an active arac gene are present, significant amounts of arabad promoter expression take place in the absence of the e. coli crp protein. these results show that the c. glutamicum rna polymerase is activated by the e. coli positive regulator of transcription arac.199910234830
cloning and analysis of the arob gene encoding dehydroquinate synthase from corynebacterium glutamicum.the arob gene encoding dehydroquinate synthase of corynebacterium glutamicum has been cloned by complementation of an aro auxotrophic mutant of escherichia coli with the genomic dna library. the recombinant plasmid contained a 1.4-kb fragment that complemented the escherichia coli dehydroquinate-synthase-deficient mutant. the nucleotide sequences of the subcloned dna has been determined. the sequences contain an open reading frame of 360 codons, from which a protein with a molecular mass of abou ...199910907426
response of the central metabolism in corynebacterium glutamicum to the use of an nadh-dependent glutamate dehydrogenase.the extensive use of 13c enrichments in precursor metabolites for flux quantification does not rely on nadph stoichiometries and can therefore be used to quantify reducing power fluxes. as an application of this concept, the nadph fluxes were quantified in an l-lysine producer of corynebacterium glutamicum grown into metabolic and isotopic steady state with [1-13c]glucose. in this case, where the organism's nadph-dependent glutamate dehydrogenase consumes reducing power, the nadph flux generated ...199910935753
metabolic engineering from a cybernetic perspective: aspartate family of amino acids.using the modular cybernetic framework developed by varner and ramkrishna (varner and ramkrishna; 1998a, b) a cybernetic model is formulated that describes the time evolution of the aspartate family of amino acids in corynebacterium lactofermentum atcc 21799. the network model formulation is employed in the role of a diagnostic tool for the overproduction of threonine. more precisely, having determined a parameter set that describes the time evolution of a base strain (lysine producer), the mode ...199910935757
importance of phosphoenolpyruvate carboxylase of corynebacterium glutamicum during the temperature triggered glutamic acid fermentation.to give clues about the respective importance of phosphoenol-pyruvate carboxylase (pepc) and pyruvate carboxylase (pc) in corynebacterium glutamicum metabolism during a temperature triggered glutamic acid fermentation, pepc activity was genetically amplified and pc activity was suppressed by biotin limitation in the culture medium. in absence of pc activity, glutamate production was dramatically reduced whereas lactate excretion was strongly increased. whereas pepc amplification in excess of bio ...199910937826
metabolic analysis of glutamate production by corynebacterium glutamicum.the dynamic behavior of the metabolism of corynebacterium glutamicum during l-glutamic acid fermentation, was evaluated by quantitative analysis of the evolution of intracellular metabolites and key enzyme concentrations. glutamate production was induced by an increase of the temperature and a final concentration of 80 g/l was attained. during the production phase, various other compounds, notably lactate, trehalose, and dha were secreted to the medium. intracellular metabolites analysis showed ...199910937937
glutamate excretion as a major kinetic bottleneck for the thermally triggered production of glutamic acid by corynebacterium glutamicum.the study was aimed at evaluating the extent of flux control exercised by the amino acid excretion step on the glutamate production flux in c. glutamicum 2262 strain that is induced for glutamate excretion by an upward temperature shift. cells initially induced to excrete glutamate were cultivated at different controlled temperatures between 33 and 40 degrees c, and changes in glutamate excretion flux and intracellular concentration were determined in response to increased culture temperature. t ...199910937940
the lyse superfamily: topology of the lysine exporter lyse of corynebacterium glutamicum, a paradyme for a novel superfamily of transmembrane solute translocators.in corynebacterium glutamicum the lyse carrier protein exhibits the unique function of exporting l-lysine. we here analyze the membrane topology of lyse, a protein of 236 amino acyl residues, using phoa- and lacz-fusions. the amino-terminal end of lyse is located in the cytoplasm whereas the carboxy-terminal end is found in the periplasm. although 6 hydrophobic domains were identified based on hydropathy analyses, only five transmembrane spanning helices appear to be present. the additional hydr ...199910943564
molecular analysis of the corynebacterium glutamicum transketolase gene.transketolase is important in production of the aromatic amino acids in corynebacterium glutamicum. the complete nucleotide sequence of the c. glutamicum transketolase gene has been identified. the dna-derived protein sequence is highly similar to the transketolase of mycobacterium tuberculosis, taxonomically related to c. glutamicum. the alignment of the n-terminus regions between both transketolases showed ttg to be the most probable start codon. potential ribosomal binding and promoter region ...199910586507
a heat shock following electroporation induces highly efficient transformation of corynebacterium glutamicum with xenogeneic plasmid dna.an improved method for the electrotransformation of wild-type corynebacterium glutamicum (atcc 13032) is described. the two crucial alterations to previously developed methods are: cultivation of cells used for electrotransformation at 18 degrees c instead of 30 degrees c, and application of a heat shock immediately following electrotransformation. cells cultivated at sub optimal temperature have a 100-fold improved transformation efficiency (10(8) cfu micrograms-1) for syngeneic dna (dna isolat ...199910570802
analysis of the corynebacterium glutamicum dapa promoter.deletion and mutational analysis of the promoter p-dapa from corynebacterium glutamicum was performed to identify regions and particular nucleotides important for its function. an extended -10 region and a stretch of six t's at positions -55 to -50 were found to be the most important elements in the promoter function. the results of mutational analysis of p-dapa are consistent with the conclusions of statistical computer-aided analysis of 44 c. glutamicum promoter sequences.199910498736
sequence analysis and expression of the aspartokinase and aspartate semialdehyde dehydrogenase operon from rifamycin sv-producing amycolatopsis mediterranei.a approximately 4.8 kb kpni fragment, from the upstream region of the methylmalonyl-coa mutase gene (mutab) of rifamycin sv-producing amycolatopsis mediterranei, was cloned and partially sequenced. codon preference analysis showed three complete orfs. orf2 is internal to orf1, and encodes a polypeptide corresponding to 172 amino acids, whereas orf1 encodes a polypeptide of 421 amino acids. they were identified as the encoding genes of aspartokinase alpha- and beta-subunits by comparing the amino ...199910521665
analysis of corynebacterium glutamicum methionine biosynthetic pathway: isolation and analysis of metb encoding cystathionine gamma-synthase.the metb gene encoding cystathionine y-synthase, the second enzyme of methionine biosynthetic pathway, was isolated from a psl109-based corynebacterium glutamicum gene library via complementation of an escherichia coli metb mutant. a dna-sequence analysis of the cloned dna identified an open-reading frame of 1161 bp which encodes a protein with the molecular weight of 41,655 comprising of 386 amino acids. the putative protein product showed good amino acid-sequence homology to its counterpart in ...199910420990
characterization of a facultatively psychrophilic bacterium, vibrio rumoiensis sp. nov., that exhibits high catalase activitya novel facultatively psychrophilic bacterium, strain s-1, which exhibits extraordinarily high catalase activity was isolated from the drain pool of a fish product processing plant that uses h2o2 as a bleaching and microbicidal agent. the catalase activity of the isolate was 1 or 2 orders of magnitude higher than those of corynebacterium glutamicum, staphylococcus aureus, pseudomonas fluorescens, and five other species tested in this study. the strain seemed to possess only one kind of catalase, ...19999872761
expression of the corynebacterium glutamicum pand gene encoding l-aspartate-alpha-decarboxylase leads to pantothenate overproduction in escherichia coli.the corynebacterium glutamicum pand gene was identified by functional complementation of an escherichia coli pand mutant strain. sequence analysis revealed that the coding region of pand comprises 411 bp and specifies a protein of 136 amino acid residues with a deduced molecular mass of 14.1 kda. a defined c. glutamicum pand mutant completely lacked l-aspartate-alpha-decarboxylase activity and exhibited beta-alanine auxotrophy. the c. glutamicum pand (pandc. g.) as well as the e. coli pand (pand ...199910103247
purification and stabilization of a monomeric isocitrate dehydrogenase from corynebacterium glutamicum.monomeric isocitrate dehydrogenase was expressed in corynebacterium glutamicum cells harboring pek-icdes1, a plasmid carrying the gene for the enzyme. two- to three-fold higher expression levels of the recombinant enzyme were observed in such cells when grown in fermentors, compared to those grown in shaker incubators. the enzyme was purified to homogeneity by ammonium sulfate fractionation, sephadex g-150 gel filtration, fplc mono q anion-exchange chromatography, and affinity gel chromatography ...199910092494
site-specific integration of corynephage phi16: construction of an integration vector.phi16, a temperate phage induced from corynebacterium glutamicum atcc 21792, lysogenizes its host via site-specific recombination. the phage attachment site, attp, was located to a 6.5 kb bamhi fragment of the phi16 genome. this fragment also contained phi16 integrative functions. the minimal phage dna fragment required for integration was defined. this 1630 bp region contained a large open reading frame, int, encoding a protein of 416 amino acids with similarity in its carboxyl-terminal domain ...199910217487
structure and organization of the rrnd operon of 'brevibacterium lactofermentum': analysis of the 16s rrna gene.five rrna operons (rrn) were found by hybridization in the genome of 'brevibacterium lactofermentum' atcc 13869 and corynebacterium glutamicum atcc 13032. 'b. lactofermentum' dsm 20412 differed from the other corynebacteria tested in showing six hybridizing bamhi bands. two of the rrn operons (rrnd and rrne) were located in a single cosmid. sequencing of the rrnd operon showed that it contains a complete 16s rrna-23s rna-5s rrna gene cluster. phylogenetic studies using the complete 16s rrna sequ ...199910220171
the tetab genes of the corynebacterium striatum r-plasmid ptp10 encode an abc transporter and confer tetracycline, oxytetracycline and oxacillin resistance in corynebacterium glutamicum.the tetracycline resistance region of the 50-kb r-plasmid ptp10 from the clinical isolate corynebacterium striatum m82b was analyzed in corynebacterium glutamicum atcc 13032 and confined to a 4.4-kb sphi-sa/i dna fragment. nucleotide sequence analysis revealed two open reading frames, termed teta and tetb, specifying proteins of 513 and 528 amino acids, respectively. the deduced amino acid sequences of tetab displayed similarity to atp-binding cassette transporters including strv and strw of str ...199910220896
flux through the tetrahydrodipicolinate succinylase pathway is dispensable for l-lysine production in corynebacterium glutamicum.the n-succinyl-ll-diaminopimelate desuccinylase gene (dape) in the four-step succinylase branch of the l-lysine biosynthetic pathway of corynebacterium glutamicum was disrupted via marker-exchange mutagenesis to create a mutant strain that uses only the one-step meso-diaminopimelate dehydrogenase branch to overproduce lysine. this mutant strain grew and utilized glucose from minimal medium at the same rate as the parental strain. in addition, the dape- strain produced lysine at the same rate as ...199910222581
d-pantothenate synthesis in corynebacterium glutamicum and use of panbc and genes encoding l-valine synthesis for d-pantothenate overproduction.d-pantothenate is synthesized via four enzymes from ketoisovalerate, which is an intermediate of branched-chain amino acid synthesis. we quantified three of these enzyme activities in corynebacterium glutamicum and determined specific activities ranging from 0.00014 to 0.001 micromol/min mg (protein)-1. the genes encoding the ketopantoatehydroxymethyl transferase and the pantothenate synthetase were cloned, sequenced, and functionally characterized. these studies suggest that panbc constitutes a ...199910223988
identification and disruption of betl, a secondary glycine betaine transport system linked to the salt tolerance of listeria monocytogenes lo28.the trimethylammonium compound glycine betaine (n,n, n-trimethylglycine) can be accumulated to high intracellular concentrations, conferring enhanced osmo- and cryotolerance upon listeria monocytogenes. we report the identification of betl, a gene encoding a glycine betaine uptake system in l. monocytogenes, isolated by functional complementation of the betaine uptake mutant escherichia coli mkh13. the betl gene is preceded by a consensus sigmab-dependent promoter and is predicted to encode a 55 ...199910224004
cloning of the transketolase gene and the effect of its dosage on aromatic amino acid production in corynebacterium glutamicum.transketolase is a key enzyme of the non-oxidative pentose phosphate pathway. the effect of its overexpression on aromatic amino acid production was investigated in corynebacterium glutamicum, a typical amino-acid-producing organism. for this purpose, the transketolase gene of the organism was cloned on the basis of its ability to complement a c. glutamicum transketolase mutant with pleiotropically shikimic-acid-requiring, ribose- and gluconic-acid-negative phenotype. the gene was shown by delet ...199910091326
cloning, sequence analysis, expression and inactivation of the corynebacterium glutamicum pta-ack operon encoding phosphotransacetylase and acetate kinase.the corynebacterium glutamicum ack and pta genes encoding the acetate-activating enzymes acetate kinase and phosphotransacetylase were isolated, subcloned on a plasmid and re-introduced into corynebacterium glutamicum. relative to the wild-type, the recombinant strains showed about tenfold higher specific activities of both enzymes. sequence analysis of a 3657 bp dna fragment revealed that the ack and pta genes are contiguous in the corynebacterial chromosome, with pta upstream and the last nucl ...199910075432
in vivo fluxes in the ammonium-assimilatory pathways in corynebacterium glutamicum studied by 15n nuclear magnetic resonanceglutamate dehydrogenase (gdh) and glutamine synthetase (gs)-glutamine 2-oxoglutarate-aminotransferase (gogat) represent the two main pathways of ammonium assimilation in corynebacterium glutamicum. in this study, the ammonium assimilating fluxes in vivo in the wild-type atcc 13032 strain and its gdh mutant were quantitated in continuous cultures. to do this, the incorporation of 15n label from [15n]ammonium in glutamate and glutamine was monitored with a time resolution of about 10 min with in v ...199910049869
analysis of the integration functions of phi304l: an integrase module among corynephages.plasmid p12929 was shown to integrate into the chromosome of corynebacterium glutamicum rm3 and bl15. the minimal integrating fragment was subsequently defined. the arms flanking the integrated plasmid (attl and attr) were identified, allowing for the determination of the attp and the attb attachment sites. the attb site is located at the 3' end of an orf presenting 62-78% identity with l19 ribosomal proteins. integration in the attb site does not result in the inactivation of this gene because ...199910049830
identification of mechanosensitive ion channels in the cytoplasmic membrane of corynebacterium glutamicum.patch-clamp experiments performed on membrane fragments of corynebacterium glutamicum fused into giant liposomes revealed the presence of two different stretch-activated conductances, 600 to 700 ps and 1,200 to 1,400 ps in 0.1 m kcl, that exhibited the same characteristics in terms of kinetics, ion selectivity, and voltage dependence.199910049402
accumulation of anthranilic acid and n-glucosylanthranilic acid by a corynebacterium glutamicum mutant resistant to dl-serine hydroxamate.during a study on the effect of dl-serine hydroxamate on corynebacterium glutamicum (jcm1318, a wild strain), a mutant resistant to the drug, strain to3002, was isolated. this mutant accumulated five ehrlich's reagent positive fluorescent substances in the culture medium. two major and one minor fluorescent products were isolated by preparative high-performance liquid chromatography following charcoal column chromatography from the culture supernatant. one major product was identified as anthran ...199912501374
redirection of carbon flux to lysine in a recombinant of corynebacterium lactofermentum atcc 21799 by limited supply of pantothenate.to increase carbon flux to lysine, minimized production of amino acids that are biosynthetically related to lysine, for example, isoleucine and valine, is required. by limiting the supply of pantothenate, the precursor of coenzyme a, the carbon flux was redirected from isoleucine and valine to lysine in the recombinant of corynebacterium lactofermentum atcc 21799 containing the plasmid pgc77. the pgc77 contains hom(dr), thrb, and ilva encoding feedback-deregulated homoserine dehydrogenase, homos ...199916232592
development of a kinetic model for l-lysine biosynthesis in corynebacterium glutamicum and its application to metabolic control analysis.a mathematical model describing intracellular lysine synthesis by corynebacterium glutamicum in batch fermentation was developed. the model is based on material balance equations of the key metabolites, and includes mechanistically based, experimentally matched rate equations for individual enzymes. from the measurements of the levels of intra- and extracellular metabolites during cultivation, the kinetic parameters in the model were identified through the decomposition of the network of reactio ...199916232634
metabolic control analysis for lysine synthesis using corynebacterium glutamicum and experimental verification.metabolic control analysis was carried out for the lysine biosynthetic pathway in corynebacterium glutamicum atcc 21253 using mechanism-based kinetic models developed for enzymatic reactions for this pathway. the rate-limiting steps during lysine production were determined with the aid of flux control coefficients, which indicated that the lysine synthesis flux is governed mainly by both aspartokinase and permease activity in the export system. analyses indicated that an increase in the activity ...200016232840
attenuation control of ilvbnc in corynebacterium glutamicum: evidence of leader peptide formation without the presence of a ribosome binding site.the ilvbnc operon of corynebacterium glutamicum encodes acetohydroxy acid synthase and isomero-reductase, which are key enzymes of l-isoleucine, l-valine and l-leucine syntheses. in this study we identified the transcript initiation site of ilvbnc operon 292 nucleotides in front of the first structural gene, and detected the formation of a short transcript from the leader region in addition to the full-length transcript of the operon. this identifies the control of ilvbnc transcription by an att ...200016232899
growth rate influences reductive biodegradation of the organophosphorus pesticide demeton by corynebacterium glutamicum.the organophosphorous pesticide, demeton-s-methyl was transformed by corynebacterium glutamicum in co-metabolism with more readily degradable substrates. glucose, acetate and fructose were tested as growth substrates, and the highest demeton-s-methyl biotransformation average rate (0.78 mg l(-1) h(-1)) and maximum instantaneous rate (1.4 mg l(-1) h(-1)) were achieved on fructose. this higher efficiency seems to be linked to the atypical behavior of c. glutamicum grown on fructose, characterized ...200011587440
pulsed high-field gradient in vivo nmr spectroscopy to measure diffusional water permeability in corynebacterium glutamicum.pulsed high-field gradient in vivo nmr spectroscopy was used to measure diffusional water permeability in cell suspensions of the gram-positive bacterium corynebacterium glutamicum. two different regions of h2o mobility were detected. one was characterized by the apparent coefficient of self-diffusion, d(1 app) = (4.6-12.7)x10(-8) cm(2) s(-1), depending on the observation time t. the other region was characterized by d(2) = 1.4x10(-5) cm(2) s(-1). the value of d(2) was similar to the diffusion c ...200010683237
reductive cleavage of demeton-s-methyl by corynebacterium glutamicum in cometabolism on more readily metabolizable substrates.corynebacterium glutamicum is able to biotransform demeton-s-methyl, an organophosphorus compound, during cometabolism with more readily metabolizable substrates. among the cosubstrates used, fructose is the growth substrate that is most favorable for demeton-s-methyl biotransformation. the reaction mechanism of demeton-s-methyl biotransformation involves reductive cleavage of an s-c bond, which leads to accumulation of dimethyl thiophosphate in the culture medium.200010698792
metabolic flux distributions in corynebacterium glutamicum during growth and lysine overproduction. reprinted from biotechnology and bioengineering, vol. 41, pp 633-646 (1993).the two main contributions of this article are the solidification of corynebacterium glutamicum biochemistry guided by bioreaction network analysis, and the determination of basal metabolic flux distributions during growth and lysine synthesis. employed methodology makes use of stoichiometrically based mass balances to determine flux distributions in the c. glutamicum metabolic network. presented are a brief description of the methodology, a thorough literature review of glutamic acid bacteria b ...200010699864
characterization of the in vivo acceptors of the mycoloyl residues transferred by the corynebacterial ps1 and the related mycobacterial antigens 85.mycolic acids, long-chain (c70-c90) alpha-alkyl, beta-hydroxy fatty acids, are characteristic cell envelope components of mycobacteria; similar but shorter-chain substances occur in corynebacteria and related taxa. these compounds apparently play an important role in the physiology of these bacteria. the deduced n-terminal region of ps1, one of the two major secreted proteins of corynebacterium glutamicum encoded by the csp1 gene, is similar to the antigens 85 complex of mycobacterium tuberculos ...200010712685
two-dimensional electrophoretic analysis of corynebacterium glutamicum membrane fraction and surface proteins.an improved protocol for the two-dimensional analysis of proteins of the corynebacterium glutamicum cytoplasmic membrane fraction is described. by use of increased 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (chaps) concentrations (2-4%) and an optimized electrophoresis protocol, horizontal streaking of proteins of the cytoplasmic membrane fraction was almost completely avoided. more important, in contrast to a previously published method, both a sample tray and ipg-phor isoelectri ...200010726773
the 51,409-bp r-plasmid ptp10 from the multiresistant clinical isolate corynebacterium striatum m82b is composed of dna segments initially identified in soil bacteria and in plant, animal, and human pathogens.the 51,409-bp dna sequence of the multiresistance plasmid ptp10 from the gram-positive opportunistic human pathogen corynebacterium striatum m82b has been determined. fully automated genome interpretation led to the identification of 47 orfs. analysis of the genetic organization of ptp10 suggests that the plasmid is composed of eight dna segments, the boundaries of which are represented by transposons and insertion sequences. the dna segments of ptp10 are highly similar to (1) a plasmid-encoded ...200010732668
a mutation in the corynebacterium glutamicum ltsa gene causes susceptibility to lysozyme, temperature-sensitive growth, and l-glutamate production.the corynebacterium glutamicum mutant ky9714, originally isolated as a lysozyme-sensitive mutant, does not grow at 37 degrees c. complementation tests and dna sequencing analysis revealed that a mutation in a single gene of 1,920 bp, ltsa (lysozyme and temperature sensitive), was responsible for its lysozyme sensitivity and temperature sensitivity. the ltsa gene encodes a protein homologous to the glutamine-dependent asparagine synthetases of various organisms, but it could not rescue the aspara ...200010781535
quantitative determination of metabolic fluxes during coutilization of two carbon sources: comparative analyses with corynebacterium glutamicum during growth on acetate and/or glucose.growth of corynebacterium glutamicum on mixtures of the carbon sources glucose and acetate is shown to be distinct from growth on either substrate alone. the organism showed nondiauxic growth on media containing acetate-glucose mixtures and simultaneously metabolized these substrates. compared to those for growth on acetate or glucose alone, the consumption rates of the individual substrates were reduced during acetate-glucose cometabolism, resulting in similar total carbon consumption rates for ...200010809686
another unusual type of citric acid cycle enzyme in helicobacter pylori: the malate:quinone oxidoreductase.the only enzyme of the citric acid cycle for which no open reading frame (orf) was found in the helicobacter pylori genome is the nad-dependent malate dehydrogenase. here, it is shown that in this organism the oxidation of malate to oxaloacetate is catalyzed by a malate:quinone oxidoreductase (mqo). this flavin adenine dinucleotide-dependent membrane-associated enzyme donates electrons to quinones of the electron transfer chain. similar to succinate dehydrogenase, it is part of both the electron ...200010809701
response to nitrogen starvation in corynebacterium glutamicum.proteins strongly synthesized in corynebacterium glutamicum during nitrogen restriction were examined by two-dimensional gel electrophoresis and microsequencing. two main groups of enzymes were identified beside miscellaneous proteins, enzymes involved (i) in protein synthesis, and (ii) in carbon metabolism. biochemical measurements revealed an increase of oxygen consumption during nitrogen starvation, indicating an enhanced energy demand of the cells. by northern hybridizations, an increased tr ...200010828405
kinetic properties of the glucose-6-phosphate and 6-phosphogluconate dehydrogenases from corynebacterium glutamicum and their application for predicting pentose phosphate pathway flux in vivo.the glucose-6-phosphate (glc6p) and 6-phosphogluconate (6pg) dehydrogenases of the amino-acid-producing bacterium corynebacterium glutamicum were purified to homogeneity and kinetically characterized. the glc6p dehydrogenase was a heteromultimeric complex, which consists of zwf and opca subunits. the product inhibition pattern of the glc6p dehydrogenase was consistent with an ordered bi-bi mechanism. the 6pg dehydrogenase was found to operate according to a theorell-chance ordered bi-ter mechani ...200010848959
microbiological investigation on black crusts from open-air stone monuments of bologna (italy).samples of black crusts taken from open-air stone monuments in bologna dating back to between the twelfth and the nineteenth century were considered. the microbiological test procedures isolated from a high number of chemoorganotrophic bacteria, each of the samples although it was expected to find chemo-lithotrophic (specially sulfur-oxidizing and nitrifying) bacteria on account of the great amounts of sulphur, carbon, nitrogen dioxides and ammonia in the air. sporogenous and asporogenous specie ...200010872690
cloning of the malic enzyme gene from corynebacterium glutamicum and role of the enzyme in lactate metabolism.malic enzyme is one of at least five enzymes, known to be present in corynebacterium glutamicum, capable of carboxylation and decarboxylation reactions coupling glycolysis and the tricarboxylic acid cycle. to date, no information is available concerning the physiological role of the malic enzyme in this bacterium. the male gene from c. glutamicum has been cloned and sequenced. the protein encoded by this gene has been purified to homogeneity, and the biochemical properties have been established. ...200010877795
characterization of the ribosomal rrnd operon of the cephamycin-producer 'nocardia lactamdurans' shows that this actinomycete belongs to the genus amycolatopsis.the cephamycin producer strain 'nocardia lactamdurans' contains four ribosomal rna (rrn) operons. one of them (rrnd) was cloned from a dna library in the bifunctional cosmid pjar4. a 2229 bp region of rrnd has been sequenced. the 'n. lactamdurans' rrnd operon maintains the canonical order 5'-16s-23s-5s-3'. four of the consensus gürtler-stanisch sequences were found in the 16s rrna gene and a fifth one in the sequenced 5' region of the 23s rrna gene. the anti shine-dalgarno sequence of 'n. lactam ...200010879974
menaquinol oxidase activity and primary structure of cytochrome bd from the amino-acid fermenting bacterium corynebacterium glutamicum.cytochrome d was spectroscopically detected in membrane fractions of the amino-acid-fermenting, high-g+c gram-positive bacterium corynebacterium glutamicum. inhibition of nadh oxidase activity in the membranes by cyanide suggested that the main terminal respiratory oxidase during the stationary phase was a type of cytochrome bd. cytochrome bd-type quinol oxidase, purified from the membranes, was composed of two subunits. its reduced form showed absorption peaks at 627, 595, and 560 nm, which wer ...200010896219
characterization of glk, a gene coding for glucose kinase of corynebacterium glutamicum.the glk gene from corynebacterium glutamicum was isolated by complementation using escherichia coli zsc113 (ptsg ptsm glk). we sequenced a total of 3072 bp containing the 969-bp open reading frame encoding glucose kinase (glk). the glk gene has a deduced molecular mass of 34.2 kda and contains a typical atp binding site. comparison with protein sequences revealed homologies to glk from streptomyces coelicolor (43%) and bacillus megaterium (35%). the glk gene in c. glutamicum was inactivated on t ...200010913707
pyruvate carboxylase from mycobacterium smegmatis: stabilization, rapid purification, molecular and biochemical characterization and regulation of the cellular level.this is the first report on the purification and characterization of an anaplerotic enzyme from a mycobacterium. the anaplerotic reactions play important roles in the biochemical differentiation of mycobacteria into non-replicating stages. we have purified and characterized a pyruvate carboxylase (pyc) from mycobacterium smegmatis and cloned and sequenced its gene. we have developed a very rapid and efficient purification protocol that provided pyc with very high specific activities (up to 150 u ...200010913817
urease of corynebacterium glutamicum: organization of corresponding genes and investigation of activity.the corynebacterium glutamicum genes encoding urease were isolated and sequenced. while urea, ureb and urec are encoding structural subunits of urease, uree, uref, ureg and ured are encoding accessory proteins. as deduced from dna sequence analyses, the ure genes are transcriptionally coupled, this was proven by rt-pcr at least for ureabc. gene disruption experiments revealed that both structural (urec) and accessory proteins (ured) are indispensable for urease activity and growth on urea. ureas ...200010930756
rapid cloning of metk encoding methionine adenosyltransferase from corynebacterium glutamicum by screening a genomic library on a high density colony-array.the genes sam1 and sam2 encoding the two different methionine adenosyltransferases (ec 2.5.1.6) in saccharomyces cerevisiae were used as templates to generate specific dna-probes. this heterologous mixture of dna-probes was hybridized under low stringency hybridization conditions to a corynebacterium glutamicum colony-array representing the complete genome. subsequently, one genomic fragment was isolated which contained the c. glutamicum methionine adenosyltransferase gene metk (1.224 kb). when ...200011094286
diversity of l-methionine catabolism pathways in cheese-ripening bacteria.enzymatic activities that could be involved in methanethiol generation in five cheese-ripening bacteria were assayed, and the major sulfur compounds produced were identified. l-methionine and alpha-keto-gamma-methyl-thio-butyric acid demethiolating activities were detected in whole cells and cell extracts (cfes) of all the bacteria tested. no l-methionine deaminase activity could be detected in any of the ripening bacteria and l-methionine aminotransferase was detected in cfes of brevibacterium ...200011097940
the three-dimensional structure of the ternary complex of corynebacterium glutamicum diaminopimelate dehydrogenase-nadph-l-2-amino-6-methylene-pimelate.the three-dimensional (3d) structure of corynebacterium glutamicum diaminopimelate d-dehydrogenase in a ternary complex with nadph and l-2-amino-6-methylene-pimelate has been solved and refined to a resolution of 2.1 a. l-2-amino-6-methylene-pimelate was recently synthesized and shown to be a potent competitive inhibitor (5 microm) vs. meso-diaminopimelate of the bacillus sphaericus dehydrogenase (sutherland et al., 1999). diaminopimelate dehydrogenase catalyzes the reversible nadp+ -dependent o ...200011106178
comparative functional characterization in vitro of heptosyltransferase i (waac) and ii (waaf) from escherichia coli.heptosyltransferase ii, encoded by the waaf gene of escherichia coli, is a glycosyltransferase involved in the synthesis of the inner core region of lipopolysaccharide. the gene was subcloned from plasmid pwsb33 [brabetz, w., müller-loennies, s., holst, o. & brade, h. (1997) eur. j. biochem. 247, 716-724] into a shuttle vector for the expression in the gram-positive host corynebacterium glutamicum. the in vitro activity of the enzyme was investigated in comparison to that of heptosyltransferase ...200011054112
pathway analysis and metabolic engineering in corynebacterium glutamicum.the gram-positive bacterium corynebacterium glutamicum is used for the industrial production of amino acids, e.g. of l-glutamate and l-lysine. during the last 15 years, genetic engineering and amplification of genes have become fascinating methods for studying metabolic pathways in greater detail and for the construction of strains with the desired genotypes. in order to obtain a better understanding of the central metabolism and to quantify the in vivo fluxes in c. glutamicum, the [13c]-labelli ...200011076021
functions of the membrane-associated and cytoplasmic malate dehydrogenases in the citric acid cycle of corynebacterium glutamicum.like many other bacteria, corynebacterium glutamicum possesses two types of l-malate dehydrogenase, a membrane-associated malate:quinone oxidoreductase (mqo; ec 1.1.99.16) and a cytoplasmic malate dehydrogenase (mdh; ec 1.1.1.37) the regulation of mdh and of the three membrane-associated dehydrogenases mqo, succinate dehydrogenase (sdh), and nadh dehydrogenase was investigated. mqo, mdh, and sdh activities are regulated coordinately in response to the carbon and energy source for growth. compare ...200011092846
comparative study of the effects of peptidoglycan monomer and structurally related adamantyltripeptides on humoral immune response to ovalbumin in the mouse.peptidoglycan monomer, glcnac-murnac-l-ala-d-isogln-mesodap(omega nh2)-d-ala-d-ala (pgm) originating from brevibacterium divaricatum and synthetic adamantyltripeptides, diastereoisomers of d,l-(adamant-2-yl)-gly-l-ala-d-isogln (adtp1 and adtp2) exhibit immunomodulating activity. an experimental model in the mouse has been established with suboptimal amounts of ovalbumin (ova) as the immunogen, and parallel testing of adjuvant activity of these three immunomodulators was carried out in balb/c, c5 ...200010649625
a brevibacterium lactofermentum 16s rrna gene used as target site for homologous recombination.genes for rrna are highly conserved and present in multiple copies in most prokaryotic organisms increasing the number of theoretical sites for homologous recombination. they might be targets for integration events between unrelated microorganisms providing that an efficient genetic transfer is present. we have used a plasmid containing a portion of the 16s rrna gene from the rrnd operon of brevibacterium lactofermentum to transform the same strain resulting in non-essential inactivation of vari ...200010754248
in vivo quantification of parallel and bidirectional fluxes in the anaplerosis of corynebacterium glutamicum.the c(3)-c(4) metabolite interconversion at the anaplerotic node in many microorganisms involves a complex set of reactions. c(3) carboxylation to oxaloacetate can originate from phosphoenolpyruvate and pyruvate, and at the same time multiple c(4)-decarboxylating enzymes may be present. the functions of such parallel reactions are not yet fully understood. using a (13)c nmr-based strategy, we here quantify the individual fluxes at the anaplerotic node of corynebacterium glutamicum, which is an e ...200010946002
3-deoxy-d-manno-oct-2-ulosonic acid (kdo) transferase (waaa) and kdo kinase (kdka) of haemophilus influenzae are both required to complement a waaa knockout mutation of escherichia coli.the lipopolysaccharide (lps) of the deep rough mutant haemophilus influenzae i69 consists of lipid a and a single 3-deoxy-d-manno-oct-2-ulosonic acid (kdo) residue substituted with one phosphate at position 4 or 5 (helander, i. m., lindner, b., brade, h., altmann, k., lindberg, a. a., rietschel, e. t., and zähringer, u. (1988) eur. j. biochem. 177, 483-492). the waaa gene encoding the essential lps-specific kdo transferase was cloned from this strain, and its nucleotide sequence was identical to ...200010952982
amtr, a global repressor in the nitrogen regulation system of corynebacterium glutamicum.the uptake and assimilation of nitrogen sources is effectively regulated in bacteria. in the gram-negative enterobacterium escherichia coli, the ntrb/c two-component system is responsible for the activation of transcription of different enzymes and transporters, depending on the nitrogen status of the cell. in this study, we investigated regulation of ammonium uptake in corynebacterium glutamicum, a gram-positive soil bacterium closely related to mycobacterium tuberculosis. as shown by northern ...200010972815
a highly specific monomeric isocitrate dehydrogenase from corynebacterium glutamicum.the monomeric isocitrate dehydrogenase (idh) of corynebacterium glutamicum is compared to the topologically distinct dimeric idh of escherichia coli. both idhs have evolved to efficiently catalyze identical reactions with similar ph optimum as well as striking specificity toward nadp and isocitrate. however, the monomeric idh is 10-fold more active (calculated as kcat/km.isocitrate/km.nadp) and 7-fold more nadp-specific than the dimeric enzyme, favoring nadp over nad by a factor of 50,000. such ...200011185559
osmosensor and osmoregulator properties of the betaine carrier betp from corynebacterium glutamicum in proteoliposomes.the secondary glycine betaine uptake system betp of corynebacterium glutamicum was purified from escherichia coli membranes in strep-tagged form after heterologous expression of the betp gene and was reconstituted in e. coli lipids. betp retained its kinetic properties (v(max) and k(m) for betaine and na(+)) as compared with intact cells. the influence of driving forces (na(+) gradient and/or electrical potential) on betaine uptake was quantified in proteoliposomes. betp was effectively regulate ...200010625602
cloning of the histidine biosynthetic genes from corynebacterium glutamicum: organization and analysis of the hisg and hise genes.the physically linked hisg and hise genes, encoding for atp-phosphoribosyltransferase and phosphoribosyl-atp-pyrophosphohydrolase were isolated from the corynebacterium glutamicum gene library by complementation of escherichia coli histidine auxotrophs. they are two of the nine genes that participate in the histidine biosynthetic pathway. molecular genetics and sequencing analysis of the cloned 9-kb insert dna showed that it carries the hisg and hise genes. in combining this result with our prev ...200011006846
structure of the urease operon of corynebacterium glutamicum.urease activity of corynebacterium glutamicum results in a rapid ph increase upon addition of urea to the growth medium. the urease operon c. glutamicum was isolated of and sequenced. seven open reading frames were identified; urea, ureb, and urec were homologues of other bacterial urease structural genes, uree, uref, ureg, and ured exhibited homology to urease accessory genes. disruption of urec prevented the utilization of urea as a nitrogen source by c. glutamicum. urease activity was induced ...200011328647
functions of the membrane-associated and cytoplasmic malate dehydrogenases in the citric acid cycle of escherichia coli.oxidation of malate to oxaloacetate in escherichia coli can be catalyzed by two enzymes: the well-known nad-dependent malate dehydrogenase (mdh; ec 1.1.1.37) and the membrane-associated malate:quinone-oxidoreductase (mqo; ec 1.1.99.16), encoded by the gene mqo (previously called yojh). expression of the mqo gene and, consequently, mqo activity are regulated by carbon and energy source for growth. in batch cultures, mqo activity was highest during exponential growth and decreased sharply after on ...200011092847
tetz, a new tetracycline resistance determinant discovered in gram-positive bacteria, shows high homology to gram-negative regulated efflux systems.the complete nucleotide sequence of the tetracycline resistance plasmid pag1 from the gram-positive soil bacterium corynebacterium glutamicum 22243 (formerly corynebacterium melassecola 22243) was determined. the r-plasmid has a size of 19,751 bp and contains at least 18 complete open reading frames. the resistance determinant of pag1 revealed homology to gram-negative tetracycline efflux and repressor systems of tet classes a through j. the highest levels of amino acid sequence similarity were ...200011078655
modeling and experimental design for metabolic flux analysis of lysine-producing corynebacteria by mass spectrometry.experimental design of (13)c-tracer studies for metabolic flux analysis with mass spectrometric determination of labeling patterns was performed for the central metabolism of corynebacterium glutamicum comprising various flux scenarios. ratio measurement of mass isotopomer pools of corynebacterium products lysine, alanine, and trehalose is sufficient to quantify the flux partitioning ratios (i) between glycolysis and pentose phosphate pathways (phi(ppp)), (ii) between the split pathways in the l ...200111289793
characterization of the phosphoenolpyruvate carboxykinase gene from corynebacterium glutamicum and significance of the enzyme for growth and amino acid production.corynebacterium glutamicum possesses phosphoenolpyruvate (pep) carboxykinase, oxaloacetate decarboxylase and malic enzyme, all three in principle being able to catalyze the first step in gluconeogenesis. to investigate the role of pep carboxykinase for growth and amino acid production, the respective pck gene was isolated, characterized and used for construction and analysis of mutants and overexpressing strains. sequence analysis of the pck gene predicts a polypeptide of 610 amino acids showing ...200111565516
development and validation of corynebacterium dna microarrays.we have developed dna microarray techniques for studying corynebacterium glutamicum. a set of 52 c. glutamicum genes encoding enzymes from primary metabolism was amplified by pcr and printed in triplicate onto glass slides. total rna was extracted from cells harvested during the exponential-growth and lysine production phases of a c. glutamicum fermentation. fluorescently labeled cdnas were prepared by reverse transcription using random hexamer primers and hybridized to the microarrays. to estab ...200111319117
h+-atpase defect in corynebacterium glutamicum abolishes glutamic acid production with enhancement of glucose consumption rate.a mutant of corynebacterim glutamicum ('brevibacterium flayum') atcc14067 with a reduced h+-atpase activity, f172-8, was obtained as a spontaneous neomycin-resistant mutant. the atpase activity of strain f172-8 was reduced to about 25% of that of the parental strain. strain f172-8 was cultured in a glutamic-acid fermentation medium containing 100 g/l of glucose using ajar fermentor. it was found that glucose consumption per cell during the exponential phase was higher by 70% in the mutant than i ...200111762601
a corynebacterium glutamicum mutant with a defined deletion within the rplk gene is impaired in (p)ppgpp accumulation upon amino acid starvation.the rplk gene of corynebacterium glutamicum atcc13032 comprises 438 nucleotides and encodes a protein of 145 amino acids with a molecular mass of 15.3 kda. the amino acid sequence revealed extensive similarities to the large ribosomal subunit protein l11 from several gram-positive and gram-negative bacteria. the c. glutamicum rplk gene is located downstream of sece, representing part of the protein export apparatus, and of nusg, encoding a transcription antiterminator protein. the rplk gene is f ...200111238976
application of maldi-tof ms to lysine-producing corynebacterium glutamicum: a novel approach for metabolic flux analysis.in the present work, a novel comprehensive approach of (13)c-tracer studies with labeling measurements by maldi-tof ms, and metabolite balancing was developed to elucidate key fluxes in the central metabolism of lysine producing corynebacterium glutamicum during batch culture. maldi-tof ms methods established allow the direct quantification of labeling patterns of low molecular mass corynebacterium products from 1 microl of diluted culture supernatant. a mathematical model of the central coryneb ...200111298764
single and double overexpression of c(4)-cycle genes had differential effects on the pattern of endogenous enzymes, attenuation of photorespiration and on contents of uv protectants in transgenic potato and tobacco plants.to improve the efficiency of co(2) fixation in c(3) photosynthesis, c(4)-cycle genes were overexpressed in potato and tobacco plants either individually or in combination. overexpression of the phosphoenolpyruvate carboxylase (pepc) gene (ppc) from corynebacterium glutamicum (cppc) or from potato (stppc, deprived of the phosphorylation site) in potato resulted in a 3-6-fold induction of endogenous cytosolic nadp malic enzyme (me) and an increase in the activities of nad-me (3-fold), nadp isocitr ...200111520867
organization and transcriptional analysis of a six-gene cluster around the rplk-rpla operon of corynebacterium glutamicum encoding the ribosomal proteins l11 and l1.a cluster of six genes, trna(trp)-sece-nusg-rplk-rpla-pkwr, was cloned and sequenced from a corynebacterium glutamicum cosmid library and shown to be contiguous in the c. glutamicum genome. these genes encode a tryptophanyl trna, the protein translocase component sece, the antiterminator protein nusg, and the ribosomal proteins l11 and l1 in addition to pkwr, a putative regulatory protein of the laci-galr family. s1 nuclease mapping analysis revealed that nusg and rplk are expressed as separate ...200111319098
pyruvate carboxylase is a major bottleneck for glutamate and lysine production by corynebacterium glutamicum.corynebacterium glutamicum possesses both phosphoenolpyruvate carboxylase (pepcx) and pyruvate carboxylase (pcx) as anaplerotic enzymes for growth on carbohydrates. to analyze the significance of pcx for the amino acid production by this organism, the wild-type pyc gene, encoding pcx, was used for the construction of defined pyc-inactive and pyc-overexpressing strains and the glutamate, lysine and threonine production capabilities of these recombinant strains of c. glutamicum were tested in comp ...200111321586
identification of essential cysteine residues in 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase from corynebacterium glutamicum.to ascertain the functional role of cysteine residue in 3-deoxy-d-arabino-heptulosonate-7-phosphate (dahp) synthase from corynebacterium glutamicum, site-directed mutagenesis was performed to change each of the three residues to serine. plasmids were constructed for high-level overproduction and one-step purification of histidine-tagged dahp synthase. analysis of the purified wild-type and mutant enzymes by sds-polyacrylamide gel electrophoresis showed an apparent protein band with a molecular m ...200111381336
regulation of aromatic amino acid biosynthesis in gamma-proteobacteria.computational comparative techniques were applied to analysis of the aromatic amino acid regulons in gamma-proteobacteria. this resulted in characterization of the trpr and tyrr regulons in the genomes of yersinia pestis, haemophilus influenzae, vibrio cholerae and other bacteria and identification of new members of the phhr regulon in the genome of pseudomonas aeruginosa. candidate attenuators were constructed for all studied genomes, including the trpba operon of the very distantly related bac ...200111545272
purification and characterization of l-2,3-butanediol dehydrogenase of brevibacterium saccharolyticum c-1012 expressed in escherichia coli.the l-2,3-butanediol dehydrogenase produced in e. coli jm109/plbd2-ctc was purified by 5 steps. the molecular mass of this enzyme was estimated at 110 kda and the subunit was measured to be 30 kda. the l-bdh had some differences from the bdhs from other sources in substrate specificity, pi value, ph stability, effects of divalent cations, and organic acids.200111577733
corynebacterium glutamicum: a dissection of the pts.the high-gc gram-positive actinomycete corynebacterium glutamicum is commercially exploited as a producer of amino acids that are used as animal feed additives and flavor enhancers. despite its beneficial role, carbon metabolism and its possible influence on amino acid metabolism is poorly understood. we have addressed this issue by analyzing the phosphotransferase system (pts), which in many bacteria controls the flux of nutrients and therefore regulates carbon metabolism. the general pts phosp ...200111361073
l-glutamic acid production in a novel three phase fluidized bed reactor using coimmobilized bio-catalyst.the production of l-glutamic acid has been studied using coimmobilized whole cells of pseudomonas reptilivora and micrococcus glutamicus in a three phase fluidized bed bioreactor with selected production medium enriched with glucose. the effect of initial substrate concentration, temperature, ph and aeration rate on the yield of glutamic acid has been investigated. it has been found that the acid production increases exponentially with substrate concentration and mass transfer co-efficient varie ...200111347434
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