Phage mediated horizontal transfer of the sopE1 gene increases enteropathogenicity of Salmonella enterica serotype Typhimurium for calves

Anaerobic fungi are an important component of the cellulolytic ruminal microflora. Ammonia alone as N source supports growth, but amino acid mixtures are stimulatory. In order to evaluate the extent of de novo synthesis of individual amino acids in Piromyces communis and Neocallimastix frontalis, isotope enrichment in amino acids was determined during growth on (15)NH(4)Cl in different media. Most cell N (0.78 and 0.63 for P. communis and N. frontalis, respectively) and amino acid N (0.73 and 0.59) continued to be formed de novo from ammonia when 1 g l(-1) trypticase was added to the medium; this concentration approximates the peak concentration of peptides in the rumen after feeding. Higher peptide/amino acid concentrations decreased de novo synthesis. Lysine was exceptional, in that its synthesis decreased much more than other amino acids when Trypticase or amino acids were added to the medium, suggesting that lysine synthesis might limit fungal growth in the rumen.     

Production of specific site probes of tRNA structure by enrichment with carbon 13 at particular locations.

Escherichia coli C6 rel met cys was cultured in a stringently defined minimal medium containing 13C-enriched metabolites in order to (1) achieve maximal 13C isotopic enrichment of tRNA; and (2) produce site specific but natural, non-perturbing NMR probes of tRNA structure and function. Growth conditions were manipulated to achieve optimal culture growth concomitant with maximal in vivo incorporation of various 13C-enriched nucleic acid precursors, including L-[methyl-13C] methionine, [2-(13)C] adenine, and [2-(13)C] uracil. Effective blockage of purine biosynthesis de novo was accomplished with the addition of the antimetabolite 6-mercaptopurine to the growth medium. Transfer RNAs specifically 13C-enriched in all methyl groups (57 atom %), C2 of adenine (60 atom %), and C2 of uracil (82 atom %) and C2 of cytosine (73 atom %) have been produced.     

Analysis of docosahexaenoic acid biosynthesis in Crypthecodinium cohnii by 13C labelling and desaturase inhibitor experiments

The lipids of the heterotrophic microalga Crypthecodinium cohnii contain the omega-3 polyunsaturated fatty acid (PUFA) and docosahexaenoic acid (22:6) to a level of over 30%. The pathway of 22:6 synthesis in C. cohnii is unknown. The ability of C. cohnii to use 13C-labelled externally supplied precursor molecules for 22:6 biosynthesis was tested by 13C NMR analysis. Furthermore, the presence of desaturases (typical for aerobic PUFA synthesis) was studied by the addition of specific desaturase inhibitors in the growth medium. The addition of 1-(13)C acetate or 1-(13)C butyrate in the growth medium resulted in 22:6 with only the odd carbon atoms enriched. Apparently, two-carbon units were used as building blocks for 22:6 synthesis and butyrate was first split into two-carbon units prior to incorporation in 22:6. When 1-(13)C oleic acid was added to the growth medium, 1-(13)C oleic acid was incorporated into the lipids of C. cohnii but was not used as a precursor for the synthesis of 22:6. Specific desaturase inhibitors (norflurazon and propyl gallate) inhibited lipid accumulation in C. cohnii. The fatty acid profile, however, was not altered. In contrast, in the arachidonic acid-producing fungus, Mortierella alpina, these inhibitors not only decreased the lipid content but also altered the fatty acid profile. Our results can be explained by the presence of three tightly regulated separate systems for the fatty acid production by C. cohnii, namely for (1). the biosynthesis of saturated fatty acids, (2). the conversion of saturated fatty acids to monounsaturated fatty acids and (3). the de novo synthesis of 22:6 with desaturases involved.     

Characterization of central carbon metabolism of Streptococcus pneumoniae by isotopologue profiling

The metabolism of Streptococcus pneumoniae was studied by isotopologue profiling after bacterial cultivation in chemically defined medium supplemented with [U-(13)C(6)]- or [1,2-(13)C(2)]glucose. GC/MS analysis of protein-derived amino acids showed lack of (13)C label in amino acids that were also essential for pneumococcal growth. Ala, Ser, Asp, and Thr displayed high (13)C enrichments, whereas Phe, Tyr, and Gly were only slightly labeled. The analysis of the labeling patterns showed formation of triose phosphate and pyruvate via the Embden-Meyerhof-Parnas pathway. The labeling patterns of Asp and Thr suggested formation of oxaloacetate exclusively via the phosphoenolpyruvate carboxylase reaction. Apparently, α-ketoglutarate was generated from unlabeled glutamate via the aspartate transaminase reaction. A fraction of Phe and Tyr obtained label via the chorismate route from erythrose 4-phosphate, generated via the pentose phosphate pathway, and phosphoenolpyruvate. Strikingly, the data revealed no significant flux from phosphoglycerate to Ser and Gly but showed formation of Ser via the reverse reaction, namely by hydroxymethylation of Gly. The essential Gly was acquired from the medium, and the biosynthesis pathway was confirmed in experiments using [U-(13)C(2)]glycine as a tracer. The hydroxymethyl group in Ser originated from formate, which was generated by the pyruvate formate-lyase. Highly similar isotopologue profiles were observed in corresponding experiments with pneumococcal mutants deficient in PavA, CodY, and glucose-6-phosphate dehydrogenase pointing to the robustness of the core metabolic network used by these facultative pathogenic bacteria. In conclusion, this study demonstrates the dual utilization of carbohydrates and amino acids under in vitro conditions and identifies the unconventional de novo biosynthesis of serine by pneumococci.     

13C nuclear magnetic resonance studies of Pseudomonas putida fatty acid metabolic routes involved in poly(3-hydroxyalkanoate) synthesis.

The formation of poly(3-hydroxyalkanoates) (PHAs) in Pseudomonas putida KT2442 from various carbon sources was studied by 13C nuclear magnetic resonance spectroscopy, gas chromatography, and gas chromatography-mass spectroscopy. By using [1-13C]decanoate, the relation between beta-oxidation and PHA formation was confirmed. The labeling pattern in PHAs synthesized from [1-13C]acetate corresponded to the formation of PHAs via de novo fatty acid biosynthesis. Studies with specific inhibitors of the fatty acid metabolic pathways demonstrated that beta-oxidation and de novo fatty acid biosynthesis function independently in PHA formation. Analysis of PHAs derived from [1-13C]hexanoate showed that both fatty acid metabolic routes can function simultaneously in the synthesis of PHA. Furthermore, evidence is presented that during growth on medium-chain-length fatty acids, PHA precursors can be generated by elongation of these fatty acids with an acetyl coenzyme A molecule, presumably by a reverse action of 3-ketothiolase.     

De Novo Synthesis of Amino Acids by the Ruminal Bacteria Prevotella bryantii B14, Selenomonas ruminantium HD4, and Streptococcus bovis ES1

The influence of peptides and amino acids on ammonia assimilation and de novo synthesis of amino acids by three predominant noncellulolytic species of ruminal bacteria, Prevotella bryantii B₁4, Selenomonas ruminantium HD4, and Streptococcus bovis ES1, was determined by growing these bacteria in media containing ¹⁵NH₄Cl and various additions of pancreatic hydrolysates of casein (peptides) or amino acids. The proportion of cell N and amino acids formed de novo decreased as the concentration of peptides increased. At high concentrations of peptides (10 and 30 g/liter), the incorporation of ammonia accounted for less than 0.16 of bacterial amino acid N and less than 0.30 of total N. At 1 g/liter, which is more similar to peptide concentrations found in the rumen, 0.68, 0.87, and 0.46 of bacterial amino acid N and 0.83, 0.89, and 0.64 of total N were derived from ammonia by P. bryantii, S. ruminantium, and S. bovis, respectively. Concentration-dependent responses were also obtained with amino acids. No individual amino acid was exhausted in any incubation medium. For cultures of P. bryantii, peptides were incorporated and stimulated growth more effectively than amino acids, while cultures of the other species showed no preference for peptides or amino acids. Apparent growth yields increased by between 8 and 57%, depending on the species, when 1 g of peptides or amino acids per liter was added to the medium. Proline synthesis was greatly decreased when peptides or amino acids were added to the medium, while glutamate and aspartate were enriched to a greater extent than other amino acids under all conditions. Thus, the proportion of bacterial protein formed de novo in noncellulolytic ruminal bacteria varies according to species and the form and identity of the amino acid and in a concentration-dependent manner.     

Factors to consider in using [U-C]palmitate for analysis of sphingolipid biosynthesis by tandem mass spectrometry

This study describes the use of a stable-isotope labeled precursor ([U-13C]palmitate) to analyze de novo sphingolipid biosynthesis by tandem mass spectrometry. It also describes factors to consider in interpreting the data, including the isotope's location (13C appears in three isotopomers and isotopologues: [M + 16] for the sphingoid base or N-acyl fatty acid, and [M + 32] for both); the isotopic enrichment of palmitoyl-CoA; and its elongation, desaturation, and incorporation into N-acyl-sphingolipids. For HEK293 cells incubated with 0.1 mM [U-13C]palmitic acid, ～60% of the total palmitoyl-CoA was 13C-labeled by 3 h (which was near isotopic equilibrium); with this correction, the rates of de novo biosynthesis of C16:0-ceramide, C16:0-monohexosylceramide, and C16:0-sphingomyelins were 62 ± 3, 13 ± 2, and 60 ± 11 pmol/h per mg protein, respectively, which are consistent with an estimated rate of appearance of C16:0-ceramide using exponential growth modeling (119 ± 11 pmol/h per mg protein). Including estimates for the very long-chain fatty acyl-CoAs, the overall rate of sphingolipid biosynthesis can be estimated to be at least ～1.6-fold higher. Thus, consideration of these factors gives a more accurate picture of de novo sphingolipid biosynthesis than has been possible to-date, while acknowledging that there are inherent limitations to such approximations.     

Decreased purine synthesis during amino acid starvation of human lymphoblasts

Normal human lymphoblasts starved for each of several essential, but not essential, amino acids had decreased DNA and RNA synthesis but no change in free intracellular purine nucleotides. The rates of purine nucleotide synthesis via the de novo and salvage pathways were measured by incorporating [14C]formate and [14C]hypoxanthine labels, respectively, into lymphoblasts starved for an amino acid or treated with a protein synthesis inhibitor. After 3 h of starvation, purine synthesis via the de novo pathway decreased 90% and via the salvage pathway decreased 60%. Cycloheximide and puromycin each reduced de novo synthesis by 96% and salvage synthesis by 72%. The decrease in purine synthesis de novo after removal of the amino acid was of first order kinetics and was fully and rapidly reversed by reconstitution with the amino acid. The synthesis of alpha-N-formylglycinamide ribonucleotide declined 97% after amino acid starvation; the synthesis of purines from 5-aminoimidazole-4-carboxamide riboside decreased 41%. The synthesis of guanylates decreased more than the synthesis of adenylates during amino acid starvation.     

Constrained de novo sequencing of conotoxins

De novo peptide sequencing by mass spectrometry (MS) can determine the amino acid sequence of an unknown peptide without reference to a protein database. MS-based de novo sequencing assumes special importance in focused studies of families of biologically active peptides and proteins, such as hormones, toxins, and antibodies, for which amino acid sequences may be difficult to obtain through genomic methods. These protein families often exhibit sequence homology or characteristic amino acid content; yet, current de novo sequencing approaches do not take advantage of this prior knowledge and, hence, search an unnecessarily large space of possible sequences. Here, we describe an algorithm for de novo sequencing that incorporates sequence constraints into the core graph algorithm and thereby reduces the search space by many orders of magnitude. We demonstrate our algorithm in a study of cysteine-rich toxins from two cone snail species (Conus textile and Conus stercusmuscarum) and report 13 de novo and about 60 total toxins.     

A defined medium for rumen bacteria and identification of strains impaired in de novo biosynthesis of certain amino acids

A completely defined growth medium has been developed to determine the nitrogen requirements for several species of ruminal bacteria, and has revealed two strains which are impaired in de novo biosynthesis of certain amino acids. Using NH₄Cl as a sole nitrogen source, the medium supported growth of Butyrivibrio, Selenomonas, Prevotella and Streptococcus species. One strain of B. fibrisolvens (E14) and one strain of P. ruminicola (GA33) did not grow in the presence of NH₄Cl until the medium was supplemented with amino acids or peptides. For B. fibrisolvens strain E14, methionine was identified as the specific growth-limiting amino acid although methionine alone did not support growth in the absence of NH₄Cl. For P. ruminicola strain GA33, any individual amino acid other than methionine or cysteine could supplement the medium and support growth. Enzyme assays confirmed a lack of NADH and NADPH-dependent glutamate dehydrogenase (GDH) activities in this strain.     

Intracellular Conditions Required for Initiation of Solvent Production by Clostridium acetobutylicum

We investigated the intracellular physiological conditions associated with the induction of butanol-producing enzymes in Clostridium acetobutylicum. During the acidogenic phase of growth, the internal pH decreased in parallel with the decrease in the external pH, but the internal pH did not go below 5.5 throughout batch growth. Butanol was found to dissipate the proton motive force of fermenting C. acetobutylicum cells by decreasing the transmembrane pH gradient, whereas the membrane potential was affected only slightly. In growing cells, the switch from acid to solvent production occurred when the internal undissociated butyric acid concentration reached 13 mM and the total intracellular undissociated acid concentration (acetic plus butyric acids) was at least 40 to 45 mM. Similar values were obtained when cultures were supplemented with 50 mM butyric acid initially or when a phosphate-buffered medium was used instead of an acetate-buffered medium. To measure the induction of the enzymes involved in solvent synthesis, we determined the rates of conversion of butyrate to butanol in growing cells. The rate of butanol formation reached a maximum in the mid-solvent phase, when the butanol concentration was 50 mM. Although more solvent accumulated later, de novo enzyme synthesis decreased and then ceased.     

Role of the mouse visceral yolk sac in nutrition: inhibition by a somatomedin inhibitor

A low molecular weight somatomedin inhibitory serum fraction (SI), obtained from streptozotocin-induced diabetic rats, causes morphological abnormalities and growth reduction in mouse embryos grown in whole embryo culture (WEC). These abnormalities are thought to be caused, at least in part, by a failure of the visceral yolk sac (VYS) to properly degrade proteins, a process that normally provides the conceptus with amino acids and peptides for de novo protein synthesis (histiotrophic nutrition). To test this hypothesis, embryos exposed to the SI were provided with a mixture of ten essential amino acids (supplemented group) in an attempt to circumvent SI-induced VYS dysfunction. Results showed that 82.4% (14/17) of embryos in the amino acid-supplemented group exhibited improved growth and development compared to those embryos exposed to medium containing the SI alone (unsupplemented group). Supplemented embryos showed greater expansion of the brain regions, improved visceral arch development, and increased protein content compared to nonsupplemented SI-treated embryos. However, these parameters were still reduced compared to controls. VYSs from both the unsupplemented and amino acid-supplemented groups were identical with respect to alterations in morphology and increased protein content compared to VYSs from conceptuses cultured in control medium (with or without amino acid supplementation). The improvement in embryonic growth and development due to amino acid supplementation in spite of VYS abnormalities supports the hypothesis that nutritional deprivation is one aspect of SI-induced teratogenesis.     

Selective utilization of exogenous amino acids by Dehalococcoides ethenogenes strain 195 and its effects on growth and dechlorination activity

Bacteria of the genus Dehalococcoides are important members of bioremediation communities because of their ability to detoxify chloroethenes to the benign end product ethene. Genome-enabled studies conducted with Dehalococcoides ethenogenes 195 have revealed that two ATP-binding cassette (ABC)-type amino acid transporters are expressed during its exponential growth stages. In light of previous findings that Casamino Acids enhanced its dechlorination activity, we hypothesized that strain 195 is capable of importing amino acids from its environment to facilitate dechlorination and growth. To test this hypothesis, we applied isotopomer-based dilution analysis with (13)C-labeled acetate to differentiate the amino acids that were taken up by strain 195 from those synthesized de novo and to determine the physiological changes caused by the significantly incorporated amino acids. Our results showed that glutamate/glutamine and aspartate/asparagine were almost exclusively synthesized by strain 195, even when provided in excess in the medium. In contrast, phenylalanine, isoleucine, leucine, and methionine were identified as the four most highly incorporated amino acids, at levels >30% of respective proteinogenic amino acids. When either phenylalanine or all four highly incorporated amino acids were added to the defined mineral medium, the growth rates, dechlorination activities, and yields of strain 195 were enhanced to levels similar to those observed with supplementation with 20 amino acids. However, genes for the putative ABC-type amino acids transporters and phenylalanine biosynthesis exhibited insignificant regulation in response to the imported amino acids. This study also demonstrates that using isotopomer-based metabolite analysis can be an efficient strategy for optimizing nutritional conditions for slow-growing microorganisms.     

Dynamics of a de novo designed three-helix bundle protein studied by 15N, 13C, and 2H NMR relaxation methods

Understanding how the amino acid sequence of a polypeptide chain specifies a unique, functional three-dimensional structure remains an important goal, especially in the context of the emerging discipline of de novo protein design. Alpha3D is a single chain protein of 73 amino acids resulting from a de novo design effort. Previous solution nuclear magnetic resonance studies of alpha3D confirm that the protein adopts the designed structure of a three-helix bundle. Furthermore, alpha3D has been previously shown to possess all of the major thermodynamic and structural characteristics of natural proteins, though it shares no sequence homology to any protein sequence in the database. In this work, the backbone and side-chain dynamics of alpha3D were investigated using 15N, 13C, and 2H nuclear magnetic resonance relaxation methods with the aim of assessing the character of the internal motions of this native-like protein of de novo design. At the backbone level, both 15N and 13C(alpha) relaxation studies indicate highly restrictive motion on the picosecond to nanosecond time scale in the alpha-helical regions of alpha3D, with increasing mobility at the ends of the alpha-helices and in the two loop regions. This is largely consistent with what is seen in proteins of natural origin. Overall, the view provided by both 2H and 13C methyl relaxation methods suggest that the side chains of alpha3D are more dynamic compared to natural proteins. Regions of relative flexibility bound clusters of rigid methyl-bearing side-chain groups that are interspersed with aromatic and beta-branched amino acids. The time scale of motions associated with methyl-bearing side chains of alpha3D are significantly longer than that seen in natural proteins. These results indicate that the strategies underlying the design of alpha3D have largely, but not completely, captured both the structural and dynamic character of natural proteins.     

Thyroid hormone responsive protein Spot14 enhances catalysis of fatty acid synthase in lactating mammary epithelium

Thyroid hormone responsive protein Spot 14 has been consistently associated with de novo fatty acid synthesis activity in multiple tissues, including the lactating mammary gland, which synthesizes large quantities of medium chain fatty acids (MCFAs) exclusively via FASN. However, the molecular function of Spot14 remains undefined during lactation. Spot14-null mice produce milk deficient in total triglyceride and de novo MCFA that does not sustain optimal neonatal growth. The lactation defect was rescued by provision of a high fat diet to the lactating dam. Transgenic mice overexpressing Spot14 in mammary epithelium produced total milk fat equivalent to controls, but with significantly greater MCFA. Spot14-null dams have no diminution of metabolic gene expression, enzyme protein levels, or intermediate metabolites that accounts for impaired de novo MCFA. When [¹³C] fatty acid products were quantified in vitro using crude cytosolic lysates, native FASN activity was 1.6-fold greater in control relative to Spot14-null lysates, and add back of Spot14 partially restored activity. Recombinant FASN catalysis increased 1.4-fold and C = 14:0 yield was enhanced 4-fold in vitro following addition of Spot14. These findings implicate Spot14 as a direct protein enhancer of FASN catalysis in the mammary gland during lactation when maximal MCFA production is needed.     

Incorporation of labelled leucine (H3 Leu.)into proteins secreted by pituitary cells in culture

Human hypophyseal tissues from spontaneous abortions or hypophysectomized adults were cultured with tritiated leucine to determine whether secreted growth hormone (GH) was biosynthesized de novo during culture. Cultures were grown on chicken plasma clots, in Leighton tubes in Eagle's medium with 10 mcCi leucine and 10% calf serum, without antibiotics, in air. Labeled GH was measured by double antibody precipitation, membrane ultrafiltration and scintillation, and total GH by radioimmunoassay. Total radioactivity was measured by filtering the medium on Sephadex G100 with .13 M borate buffer pH 8.4 to separate labeled proteins and free leucine. There were 2 small early peaks of labeled protein containing 3-5% of the total radioactivity. Total GH formed 4 peaks, the largest in a late peak of low specific activity. The labeled GH appeared in several regions, the greatest in early peaks of high specific activity. The results showed definite neosynthesis of labeled GH. There seemed to be several types of GH molecules, some newly formed and intensely labeled and others either preexisting or made from older amino acid pools.     

Probing in vivo metabolism by stable isotope labeling of storage lipids and proteins in developing Brassica napus embryos

Developing embryos of Brassica napus accumulate both triacylglycerols and proteins as major storage reserves. To evaluate metabolic fluxes during embryo development, we have established conditions for stable isotope labeling of cultured embryos under steady-state conditions. Sucrose supplied via the endosperm is considered to be the main carbon and energy source for seed metabolism. However, in addition to 220 to 270 mM carbohydrates (sucrose, glucose, and fructose), analysis of endosperm liquid revealed up to 70 mM amino acids as well as 6 to 15 mM malic acid. Therefore, a labeling approach with multiple carbon sources is a precondition to quantitatively reflect fluxes of central carbon metabolism in developing embryos. Mid-cotyledon stage B. napus embryos were dissected from plants and cultured for 15 d on a complex liquid medium containing (13)C-labeled carbohydrates. The (13)C enrichment of fatty acids and amino acids (after hydrolysis of the seed proteins) was determined by gas chromatography/mass spectrometry. Analysis of (13)C isotope isomers of labeled fatty acids and plastid-derived amino acids indicated that direct glycolysis provides at least 90% of precursors of plastid acetyl-coenzyme A (CoA). Unlabeled amino acids, when added to the growth medium, did not reduce incorporation of (13)C label into plastid-formed fatty acids, but substantially diluted (13)C label in seed protein. Approximately 30% of carbon in seed protein was derived from exogenous amino acids and as a consequence, the use of amino acids as a carbon source may have significant influence on the total carbon and energy balance in seed metabolism. (13)C label in the terminal acetate units of C(20) and C(22) fatty acids that derive from cytosolic acetyl-CoA was also significantly diluted by unlabeled amino acids. We conclude that cytosolic acetyl-CoA has a more complex biogenetic origin than plastidic acetyl-CoA. Malic acid in the growth medium did not dilute (13)C label incorporation into fatty acids or proteins and can be ruled out as a source of carbon for the major storage components of B. napus embryos.     

The de novo and salvage pathways of GDP-mannose biosynthesis are both sufficient for the growth of bloodstream-form Trypanosoma brucei

The sugar nucleotide GDP-mannose is essential for Trypanosoma brucei. Phosphomannose isomerase occupies a key position on the de novo pathway to GDP-mannose from glucose, just before intersection with the salvage pathway from free mannose. We identified the parasite phosphomannose isomerase gene, confirmed that it encodes phosphomannose isomerase activity and localized the endogenous enzyme to the glycosome. We also created a bloodstream-form conditional null mutant of phosphomannose isomerase to assess the relative roles of the de novo and salvage pathways of GDP-mannose biosynthesis. Phosphomannose isomerase was found to be essential for parasite growth. However, supplementation of the medium with low concentrations of mannose, including that found in human plasma, relieved this dependence. Therefore, we do not consider phosphomannose isomerase to be a viable drug target. We further established culture conditions where we can control glucose and mannose concentrations and perform steady-state [U-(13) C]-D-glucose labelling. Analysis of the isotopic sugar composition of the parasites variant surface glycoprotein synthesized in cells incubated in 5 mM [U-(13) C]-D-glucose in the presence and absence of unlabelled mannose showed that, under physiological conditions, about 80% of GDP-mannose synthesis comes from the de novo pathway and 20% from the salvage pathway.     

Two biosynthetic pathways for aromatic amino acids in the archaeon Methanococcus maripaludis

Methanococcus maripaludis is a strictly anaerobic, methane-producing archaeon. Aromatic amino acids (AroAAs) are biosynthesized in this autotroph either by the de novo pathway, with chorismate as an intermediate, or by the incorporation of exogenous aryl acids via indolepyruvate oxidoreductase (IOR). In order to evaluate the roles of these pathways, the gene that encodes the third step in the de novo pathway, 3-dehydroquinate dehydratase (DHQ), was deleted. This mutant required all three AroAAs for growth, and no DHQ activity was detectible in cell extracts, compared to 6.0 +/- 0.2 mU mg(-1) in the wild-type extract. The growth requirement for the AroAAs could be fulfilled by the corresponding aryl acids phenylacetate, indoleacetate, and p-hydroxyphenylacetate. The specific incorporation of phenylacetate into phenylalanine by the IOR pathway was demonstrated in vivo by labeling with [1-(13)C]phenylacetate. M. maripaludis has two IOR homologs. A deletion mutant for one of these homologs contained 76, 74, and 42% lower activity for phenylpyruvate, p-hydoxyphenylpyruvate, and indolepyruvate oxidation, respectively, than the wild type. Growth of this mutant in minimal medium was inhibited by the aryl acids, but the AroAAs partially restored growth. Genetic complementation of the IOR mutant also restored much of the wild-type phenotype. Thus, aryl acids appear to regulate the expression or activity of the de novo pathway. The aryl acids did not significantly inhibit the activity of the biosynthetic enzymes chorismate mutase, prephenate dehydratase, and prephenate dehydrogenase in cell extracts, so the inhibition of growth was probably not due to an effect on these enzymes.     

Metabolic network analysis of Bacillus clausii on minimal and semirich medium using (13)C-labeled glucose

Using (13)C-labeled glucose fed to the facultative alkalophilic Bacillus clausii producing the alkaline serine protease Savinase, the intracellular fluxes were quantified in continuous cultivation and in batch cultivation on a minimal medium. The flux through the pentose phosphate pathway was found to increase with increasing specific growth rate but at a much lower level than previously reported for Bacillus subtilis. Two futile cycles in the pyruvate metabolism were included in the metabolic network. A substantial flux in the futile cycle involving malic enzyme was estimated, whereas only a very small or zero flux through PEP carboxykinase was estimated, indicating that the latter enzyme was not active during growth on glucose. The uptake of the amino acids in a semirich medium containing 15 of the 20 amino acids normally present in proteins was estimated using fully labeled glucose in batch cultivations. It was found that leucine, isoleucine, and phenylalanine were taken up from the medium and not synthesized de novo from glucose. In contrast, serine and threonine were completely synthesized from other metabolites and not taken up from the medium. Valine, proline, and lysine were partly taken up from the medium and partly synthesized from glucose. The metabolic network analysis was extended to include analysis of growth on the semirich medium containing amino acids, and the metabolic flux distribution on this medium was estimated and compared with growth on minimal medium.