Biosynthesis of α-Ketoglutarate by the Reductive Carboxylation of Succinate in Bacteroides ruminicola

Experiments with growing cells and with cell-free extracts of Bacteroides ruminicola indicate that this anaerobic bacterium can synthesize alpha-ketoglutarate by a reductive carboxylation of succinate. When the organism was grown in medium containing succinate-1,4-(14)C, most of the radioactivity in cells was in the protein fraction and most of the (14)C in protein was in the glutamic acid family of amino acids (glutamate, proline, and arginine). When unlabeled succinate was added to culture medium containing glucose-U-(14)C, incorporation of radioactivity into the glutamic acid family of amino acids was greatly reduced. This supports the concept that succinate is an intermediate in synthesis of alpha-ketoglutarate. Cell-free extracts of the organism incubated with succinate-1,4-(14)C incorporated (14)C into amino acids and most of this was found in glutamate. The cofactors which stimulate glutamate synthesis from succinate by extracts from these cells appear to be similar to the factors that have been demonstrated with extracts from photosynthetic bacteria. The position of label in glutamate synthesized from succinate-1,4-(14)C, the probable absence of isocitric dehydrogenase, and studies with labeled citrate and with inhibitors of citric acid cycle enzymes support the concept of a reductive carboxylation of succinate as the only, or at least a major, mechanism for synthesis of alpha-ketoglutarate in this organism. This appears to be the first evidence for a net synthesis of alpha-ketoglutarate by this reaction in a nonphotosynthetic heterotrophic organism.     

Microbial oxidation of dibenzothiophene by the thermophilic organism Sulfolobus acidocaldarius

The refractory organic sulfur compound dibenzothiophene (DBT) has been oxidized by the thermophilic, sulfur oxidizing organism Sulfolobus acidocaldarius. Sulfate ions were released into the medium as the oxidation product. The kinetics of this oxidation have been investigated on the basis of sulfate released as a result of oxidation. Dibenzothiophene was found to be inhibitory to the organisms for initial concentrations over 500 mg/L. The organism may prove to be capable of oxidizing thiophene compounds present in oil refinery wastewater, coal, and crude oil.     

Evidence for the Existence of an Aerobic Pathway for Synthesis of Monounsaturated Fatty Acids by Alcaligenes faecalis

Studies on the composition of total fatty acids of Alcaligenes faecalis harvested at different growth phases have been carried out. Ability of the organism to desaturate palmitic and stearic acid has also been tested. The organism contained palmitic (16:0), stearic (18:0), palmitoleic (16:1), cis-vaccenic (18:1), cyclopropane (17: big dn tri, open and 19: big dn tri, open), and three hydroxy acids. Increase in cyclopropane acids and corresponding decrease in monounsaturated acids in direct proportion to the age of the culture were observed, whereas other fatty acids remained relatively unaltered. A growing culture of the organism was found to desaturate [1-(14)C]palmitic acid supplied in the medium to hexadecanoic acid. Resting cells desaturated [1-(14)C]palmitic and [1-(14)C]stearic acid giving rise to about 50% of (14)C in the COOH group of corresponding monounsaturated fatty acids.     

Some aspects of the regulation of the production of extracellular proteolytic enzymes by a marine bacterium

A highly proteolytic Gram-negative, rod-shaped bacterium was isolated from the gills of fresh plaice and the effect of culture conditions on the production of proteolytic enzymes was investigated. When the organism, strain SA 1, was grown in the presence of complex mixtures of proteins and amino acids, both endopeptidase and aminopeptidase activity was demonstrated in the cell-free culture medium. However, synthesis of these enzymes was not observed when the organism was grown in a mineral medium with lactate or succinate as the only carbon and energy source. Synthesis of both endopeptidase and aminopeptidase was induced by the presence of amino acids in the medium. Of the amino acids tested, l-phenylalanine was found to be the best single inducer for the production of endopeptidase. When in addition one or more different amino acids were added, endopeptidase production was found to increase with increasing complexity of the mixture, up to a maximum which was obtained with five different amino acids. Production of the aminopeptidase was optimal when l-glutamic acid was used as a single inducer. For this enzyme the amount of enzyme activity released in the medium decreased with increasing complexity of the amino acid mixture. Endopeptidase as well as aminopeptidase activity was found to accumulate in the medium at the end of the logarithmic growth phase, when the culture was no longer growing exponentially. When the stationary phase was reached, enzyme production stopped. Production of both enzymes was immediately halted upon addition of chloramphenicol and was found to be repressed by glucose and lactate. These results suggest that synthesis of proteolytic extracellular enzymes by the organism studied is controlled by an efficient regulatory mechanism, in which growth rate is an important parameter.     

Microbial origin of phenylcarboxylic acids in the human body

In previous studies we demonstrated increased amounts of phenylcarboxylic acids (PCA) in serum of patients with sepsis. This observation prompted the present study of the ability of the human microbiome bacteria to produce PCA in vitro. PCA were detected in culture media by gas chromatography-mass spectrometry. Increased amounts of phenyllactic and p-hydroxyphenyllactic acids were produced by Klebsiella pneumonia, Escherichia coli, and Staphylococcus aureus. Certain strict anaerobes (bifidobacteria, lactobacteria, eubacteria) have also been found to actively produce these PCA, but these bacteria are not etiologically linked to sepsis. Thus our results demonstrate the ability of sepsis-related bacteria to produce PCA and provide experimental support for the theory that the accumulation of PCA in the blood of patients with sepsis results from microbial degradation of phenylalanine and tyrosine.     

A metabolic study to decipher amino acid catabolism-directed biofuel synthesis in Acetoanaerobium sticklandii DSM 519

Acetoanaerobium sticklandii DSM 519 is a hyper-ammonia-producing anaerobe. It has the ability to produce organic solvents and acids from protein catabolism through Stickland reactions and specialized pathways. Nevertheless, its protein catabolism-directed biofuel production has not yet been understood. The present study aimed to decipher such growth-associated metabolic potential of this organism at different growth phases using metabolic profiling. A seed culture of this organism was grown separately in metabolic assay media supplemented with gelatin and or a mixture of amino acids. The extracellular metabolites produced by this organism were qualitatively analyzed by gas chromatography–mass spectrometry platform. The residual amino acids after protein degradation and amino acids assimilation were identified and quantitatively measured by high-performance liquid chromatography (HPLC). Organic solvents and acids produced by this organism were detected and the quantity of them determined with HPLC. Metabolic profiling data confirmed the presence of amino acid catabolic products including tyramine, cadaverine, methylamine, and putrescine in fermented broth. It also found products including short-chain fatty acids and organic solvents of the Stickland reactions. It reported that amino acids were more appropriate for its growth yield compared to gelatin. Results of quantitative analysis of amino acids indicated that many amino acids either from gelatin or amino acid mixture were catabolised at a log-growth phase. Glycine and proline were poorly consumed in all growth phases. This study revealed that apart from Stickland reactions, a specialized system was established in A. sticklandii for protein catabolism-directed biofuel production. Acetone–butanol–ethanol (ABE), acetic acid, and butyric acid were the most important biofuel components produced by this organism. The production of these components was achieved much more on gelatin than amino acids. Thus, A. sticklandii is suggested herein as a potential organism to produce butyric acid along with ABE from protein-based wastes (gelatin) in bio-energy sectors.

     

Biological removal of pyritic sulfur from coal by the thermophilic organism Sulfolobus acidocaldarius

More than 90% of initial pyritic sulfur was removed from bituminous coal samples (containing 2.1% pyritic sulfur) using the thermophilic organism Sulfolobus acidocaldarius. Microbial desulfurization rate was improved nearly ten fold by adjusting the N/P and N/Mg ratios in the nutrient medium. Environmental conditions were optimized. The optimal values of temperature and pH were 70 degrees C and 1.5, respectively. The influence of certain process variables (such as coal pulp density, particle size, and initial cell number density) on the rate of pyritic sulfur removal were determined. A pulp density of 20%, particle size of D (p) < 48 mum, and an initial cell number density of 10(12) cells/g pyrite in coal were found to be optimal. The carbon dioxide enriched air did not improve the rate of pyritic sulfur removal compared to pure air at 10% pulp density of coal samples containing 2.1% pyritic sulfur. The kinetics of microbial leaching of pyritic sulfur from coal was investigated. The rate of leaching was found to be first order with respect to pyritic sulfur concentration in the reaction medium.     

Growth of obligate autotrophic bacteria on glucose in a continuous flow-through apparatus

Nitrosomonas europaea, Nitrobacter agilis, Thiobacillus denitrificans, T. neapolitanus, and T. thioparus (all obligate autotrophic bacteria) have been grown in dialysis culture, on glucose salts media, in the absence of their specific inorganic energy source. Metabolic products for N. agilis grown on nitrite salts medium were identified as keto acids. Pyruvic acid inhibited this organism at 5 x 10(-5)m. Keto acids were not inhibitory for the thiobacilli grown on thiosulfate medium. However, when T. denitrificans was grown on glucose with dialysis, addition of 5 x 10(-4)m pyruvate inhibited growth. Thus, it appears pyruvate may be inhibitory for other autotrophs, as has been reported for T. thiooxidans.     

THE BIOGENESIS OF MITOCHONDRIA : III. The Lipid Composition of Aerobically and Anaerobically Grown Saccharomyces cerevisiae as Related to the Membrane Systems of the Cells

The growth conditions known to influence the occurrence of mitochondrial profiles and other cell membrane systems in anaerobic cells of S. cerevisiae have been examined, and the effect of the several growth media on the lipid composition of the organism has been determined. The anaerobic cell type containing neither detectable mitochondrial profiles nor the large cell vacuole may be obtained by the culture of the organism on growth-limiting levels of the lipids, ergosterol, and unsaturated fatty acids. Under these conditions, the organism has a high content of short-chain saturated fatty acids (10:0, 12:0), phosphatidyl choline, and squalene, compared with aerobically grown cells, and it is especially low in phosphatidyl ethanolamine and the glycerol phosphatides (phosphatidyl glycerol + cardiolipin). The high levels of unsaturated fatty acids normally found in the phospholipids of the aerobic cells are largely replaced by the short-chain saturated acids, even though the phospholipid fraction contains virtually all of the small amounts of unsaturated fatty acid present in the anaerobic cells. Such anaerobic cells may contain as little as 0.12 mg of ergosterol per g dry weight of cells while the aerobic cells contain about 6 mg of ergosterol per g dry weight. Anaerobic cell types containing mitochondrial profiles can be obtained by the culture of the organism in the presence of excess quantities of ergosterol and unsaturated fatty acids. Such cells have increased levels of total phospholipid, ergosterol, and unsaturated fatty acids, although these compounds do not reach the levels found in aerobic cells. The level of ergosterol in anaerobic cells is markedly influenced by the nature of the carbohydrate in the medium; those cells grown on galactose media supplemented with ergosterol and unsaturated fatty acids have well defined mitochondrial profiles and an ergosterol content (2 mg per g dry weight of cells) three times that of equivalent glucose-grown cells which have poorly defined organelle profiles. Anaerobic cells which are low in ergosterol synthesize increased amounts of squalene.     

Microbial investigations on the suffocation disease of rice in Taiwan

Summary Previous experiments indicate that denitrifiers appeared to be extremely explosive in the rhizosphere of the diseased rice plant. This result encouraged us to clarify the relationships between the development of the disease and the organism.A basal medium of mineral nutrients plus asparagine nitrate with vitamins and amino acids proved to be more effective not only for the growth but also for producing pigments under aerobic conditions. Yeast extract instead of vitamins and amino acids was a better medium for anaerobic cultivation. The organism was identified by physiological tests asPseudomonas putida. The disease symptoms of red-brownish blast spots spreading from tip toward base of the leaf and black rotten root with putrid odor can be reproduced by inoculation of the organism. This suggests that some phytotoxin might be produced by the organism toxic to the cytochrome system of the plant, inhibiting the photosynthetic process as symbolized by necrosis. This also suggests that the organism would use sulfate from ammonium sulfate fertilizer applied in the absence of nitrate as H₂ acceptor for the metabolic process resulting in hydrogen sulfide, which reduced the oxygenizing ability of the rice root leading to iron toxicity and potassium deficiency. It is evident that the organisms are mainly responsible for the suffocation disease of rice in poorly drained paddy soil.     

Inactivation of rat liver HMG-CoA reductase phosphatases by polycarboxylic acids

Incubation of the four purified HMG-CoA reductase phosphatases with the sodium salts of eleven polycarboxylic acids at concentrations of 40 mM, inactivated the enzymes to different degrees depending on the structure of the carboxylic acids. Maleate, malonate, oxalate, citrate, and hydroxymethylglutarate produced full inactivation at the concentration tested. When the four phosphatases were incubated with these acids, a concentration-dependent inactivation was observed. Fumarate, the trans isomer of maleate, produced little inactivation of the four phosphatases. Mevalonate did not inactivate at all. A relationship between those concentrations of acid that produced a 50% inactivation and the logarithm of the stability constant of Mg2+ or Mn2+ salts of polycarboxylic acids was observed. When reductase phosphatases were incubated with mixtures of polycarboxylic sodium salts and Mg2+ or Mn2+, an increase in the molar ratio divalent cation/carboxylic acid determined an increase in the four reductase phosphatase activities. The inactivating effect of citrate was on the phosphatases (high and low forms) and not on the substrates (HMG-CoA reductase, phosphorylase, and glycogen synthase). Reactivation of the citrate-inactivated phosphatases by Mn2+ and Mg2+ depended on the phosphorylated substrates, Mn2+ being the better activator. It is concluded that HMG-CoA reductase phosphatases are metalloenzymes.     

Repression of Acetyl-Coenzyme A Carboxylase by Unsaturated Fatty Acids: Relationship to Coenzyme Repression

It has been reported that the level of d-biotin in the growth medium of Lactobacillus plantarum regulates the synthesis of apoacetyl-coenzyme A (CoA) carboxylase; high levels cause repression, and deficient levels effect derepression. In this study, evidence has been obtained which suggests that coenzyme repression by biotin is an indirect effect; i.e., biotin regulates the synthesis of unsaturated fatty acids which are the true repressors of the acetyl-CoA carboxylase. This was observed in an experiment in which long-chain unsaturated fatty acids were added to media containing deficient, sufficient, or excess levels of d-biotin. In every case, independently of the biotin concentration for growth, the unsaturated fatty acids caused a severe repression of the carboxylase. Saturated fatty acids were without effect. The level of oleic acid required to give maximal repression was 50 mug/ml. The free fatty acids had no adverse effect on the activity of the cell-free extracts nor on the permeation of d-biotin into the cell. Saturated and unsaturated fatty acids decreased the rate of holocarboxylase formation from d-biotin and the apoacetyl-CoA carboxylase in the extracts. It is concluded that there are at least three mechanisms that control the acetyl-CoA carboxylase in this organism: (i) indirect coenzyme repression by d-biotin, (ii) repression by unsaturated fatty acids, and (iii) regulation of the activity of the holocarboxylase synthetase by both saturated and unsaturated fatty acids.     

Growth characteristics of a pseudomonad which utilizes pyridoxine or pyridoxamine as a carbon source

Cultural characteristics of Pseudomonas MA-1, which has been extensively used as a source of enzymes that degrade vitamin B(6), confirm its classification as a pseudomonad. The organism requires biotin for growth; any one of several amino acids is also required for growth when pyridoxine (but not when glucose) serves as the energy source. Growth of Pseudomonas MA-1 is inhibited by one or more unidentified substances formed when pyridoxine is autoclaved with the medium. Modified cultural conditions are described which permit the large-scale growth of the organism with pyridoxine as carbon source.     

Function of Growth Factors for Rumen Microorganisms I. Nutritional Characteristics of Selenomonas ruminantium

Nutritional characteristics of Selenomonas ruminantium var. lactilytica isolated from a sheep rumen were studied. The organism required for growth the addition of a clarified rumen fluid to a Trypticase-yeast extract medium with either lactate or glucose as an energy source. The requirement for rumen fluid was found to be satisfied by volatile fatty acids in glucose media and by biotin in lactate media. Straight-chain saturated fatty acids with C(3) to C(10) carbon skeleton had been found to be effective. Among them, n-valerate was most effective at the lowest concentration. An abnormal morphology was observed with n-valerate-deficient glucose media. n-Valerate was essential in glucose media, and it was stimulatory in lactate media. Fermentation products from glucose were lactate, propionate, and acetate, and fermentation products from lactate were propionate and acetate. When cells were grown in a glucose medium containing n-valerate-C(14), the label was present in cell fractions. Almost all of the activity was found in lipid materials.     

Production of L-serine by the methanol utilizing bacterium,Pseudomonas 3ab

Summary A bacterium capable of growth on methanol and some organic acids as sole source of carbon and energy has been isolated and designated Pseudomonas 3ab. This facultative methylotrophic organism apparently utilizes the serine pathway of formaldehyde fixation.When methanol was used as the sole carbon source for growth, L-serine production by Pseudomonas 3ab occurred upon the addition of glycine and methanol at the end of the exponential growth phase. The maximum yield of L-serine (4.7 g/l) was obtained when 20 g/l glycine and 8 g/l methanol were added and the pH of the culture medium was changed to 8.5.Although Pseudomonas 3ab is unable to grow on L-serine or glycine, it is very active in decomposing these amino acids. The degradation of L-serine and glycine has been shown to be pH-dependent with a minimum at pH 8.5–9.0.     

Comparative metabolomics reveals the mechanism of avermectin production enhancement by <Emphasis Type="Italic">S</Emphasis>-adenosylmethionine

It was found that S-adenosylmethionine (SAM) could effectively improve avermectin titer with 30–60 μg/mL addition to FH medium. To clearly elucidate the mechanism of SAM on intracellular metabolites of Streptomyces avermitilis, a GC–MS-based comparative metabolomics approach was carried out. First, 230 intracellular metabolites were identified and 14 of them remarkably influenced avermectin biosynthesis were discriminative biomarkers between non-SAM groups and SAM-treated groups by principal components analysis (PCA) and partial least squares (PLS). Based on further key metabolic pathway analyses, these biomarkers, such as glucose, oxaloacetic acid, fatty acids (in soybean oil), threonine, valine, and leucine, were identified as potentially beneficial precursors and added in medium. Compared with single-precursor feeding, the combined feeding of the precursors and SAM markedly increased the avermectin titer. The co-feeding approach not only directly verified our hypothesis on the mechanism of SAM by comparative metabolomics, but also provided a novel strategy to increase avermectin production.     

Effect of castration on purine biosynthesis in the rat liver and kidney

With the present study, the effects of testosterone deficiency on the biosynthesis of purine nucleotides and nucleic acids in rat liver and kidney has been investigated. 20% homogenates were prepared in 1 N HCl04, centrifuged and the nucleic acid were extracted from the precipitate with 0.7 N PCA (15 min at 90 degrees C): the acid-soluble purines were precipitated with 1 M AgNO3 from the supernatant of the centrifuged homogenate, then extracted with 1 M HC1. An increase of the specific activity of the liver (A, +49%; G, +47%) and of the kidney (A, +54%; G, +34%) has been observed (P less than 0.05), while no variation of the specific activity of nucleic acids was evident after castration.     

Differential amino acid utilization by Chlamydia psittaci (strain guinea pig inclusion conjunctivitis) and its regulatory effect on chlamydial growth

The effect of omission of individual amino acids from growth medium on the multiplication of Chlamydia psittaci (strain guinea pig inclusion conjunctivitis) in cycloheximide-treated McCoy cells has been examined. Marked differences were observed in the amounts of particular amino acids required for normal chlamydial multiplication: omission of either leucine, phenylalanine or valine completely inhibited multiplication, whereas absence of any one of another 10 amino acids had no effect on numbers of cells infected. Threshold concentrations of 80, 80 and approx. 8 nmol ml-1 for leucine, valine and phenylalanine, respectively, were needed for normal chlamydial multiplication. These requirements could not be related either to unusually high content in the whole organism, to degradation in the medium, or, from studies with leucine, to deficient association of leucine with host cells. Leucine deprivation at late stages of the developmental cycle also appeared to regulate multiplication. Possible mechanisms responsible for these effects are discussed.