Biosynthesis of short-chain-length-polyhydroxyalkanoates during the dual-nutrient-limited zone by Ralstonia eutropha

Biosynthesis of PHAs by Raltonia eutropha during the dual nutrient-limitation-zone was investigated with mixed organic acids as carbon sources and (NH₄)₂SO₄ as nitrogen source. Two different methods of maintaining the dual-nutrient-limitation zone were adopted by feeding mixed acids and (NH₄)₂SO₄ at determined rates into the fermentation cultures which were initially free of carbon sources (method A) or nitrogen sources (method B). The results indicate that, firstly, with the increase of the width of the dual-nutrient-limitation zone, the yield of short-chain-length-polyhydroxyalkanoates also increases and it suggests that most of the short-chain-length-polyhydroxyalkanoates were biosynthesized during the dual-nutrient-limitation zone. Secondly, in contrast with the dual-nutrient-limitation method of limiting the nitrogen source first (method B), the dual-nutrient-limitation method of limiting the carbon source first (method A) was more favourable for the production of short-chain-length-polyhydroxyalkanoates, and the maximum production of short-chain-length-polyhydroxyalkanoates of these two methods are 3.72 and 2.55 g/l, respectively.     

Nitrogen regulation of amino acid catabolism in Neurospora crassa

Neurospora crassa can utilize numerous compounds including certain amino acids as a sole nitrogen source. Mutants of the nit-2 locus, a regulatory gene which is postulated to mediate nitrogen catabolite repression, are deficient in the ability to utilize several amino acids as well as other nitrogen sources used by wild type. Various enzymes involved in amino acid catabolism were found to be regulated in distinct ways. Arginase, ornithine transaminase, and pyrroline-5-carboxylate dehydrogenase are all inducible enzymes but are not subject to nitrogen catabolite repression. By contrast, proline oxidase and the amino acid transport system(s) are controlled by nitrogen repression and their synthesis is increased markedly when nitrogen source is limiting. Unlike wild type, the nit-2 mutant cannot derepress amino acid transport, although proline oxidase is regulated in a normal fashion.This work was supported by Grant R01 GM-23367 from the National Institutes of Health. T. J. F. was supported by an NIH Predoctoral Traineeship in Developmental Biology; G. A. M. is supported by NIH Career Development Award GM-00052.     

Carbon assimilation and extracellular antigens of some yeast-like fungi

Many yeast-like fungi assimilated n-hexadecane, butylamine and putrescine as sole carbon sources. Methanol was not assimilated. This points to a physiological similarity to endomycetous, hydrocarbon-utilizing yeasts. Stephanoascus ciferrii assimilated uric acid, adenine and allantoin as sole source of carbon and nitrogen. All strains of Geotrichum candidum and many other yeast-like fungi assimilated acetoin and butan-2,3-diol. Assimilation tests for adenine, uric acid, allantoin, acetoin and butan-2,3-diol were found to be suitable for taxonomic purposes.Extracellular antigens immunologically related to those produced by Geotrichum candidum were detected in the cell-free culture liquids of several yeast-like fungi. The extracellular antigen excreted by Stephanoascus ciferrii was species-specific.     

Mutations affecting extracellular protease production in the filamentous fungusAspergillus nidulans

The extracellular proteases ofAspergillus nidulans are known to be regulated by carbon, nitrogen and sulphur metabolite repression. In this study, a mutant with reduced levels of extracellular protease was isolated by screening for loss of halo production on milk plates. Genetic analysis of the mutant showed that it contains a single, recessive mutation, in a gene which we have designatedxprE, located on chromosome VI. ThexprE1 mutation affected the production of extracellular proteases in response to carbon, nitrogen and, to a lesser extent, sulphur limitation. Three reversion mutations,xprF1, xprF2 andxprG1, which suppressxprE1, were characterised. BothxprF andxprG map to chromosome VII but the two genes are unlinked. ThexprF1, xprF2 andxprG1 mutants showed high levels of milk-clearing activity on medium containing milk as a carbon source but reduced growth on a number of nitrogen sources. Evidence is presented that thexprE1 andxprG1 mutations alter expression of more than one protease and affect levels of alkaline protease gene mRNA.     

Effects of Carbon and Nitrogen Sources on the Levels of Several NADP- and NAD-Linked Dehydrogenase Activities of Hydrocarbon-utilizable Candida Yeasts

The variation of activities of several NADP-linked and NAD-linked dehydrogenases were studied during the aerobic growth of two species of hydrocarbon-utilizable Candida yeasts on different carbon and nitrogen sources. The level of NADP-linked isocitrate dehydrogenase in C. tropicalis and C. lipolytica growing on acetate was significantly higher than that in the yeasts growing on glucose. The glucose-grown cells of C. tropicalis showed a high activity of glucose-6-phosphate dehydrogenase as compared with the acetate-grown cells, while the enzyme level in C. lipolytica was low regardless of carbon sources used. The cells of both yeasts growing on n-alkane and oleic acid contained relatively low activity of NADP-linked isocitrate dehydrogenase. Presence of ion in the acetate medium increased the level of NADP-linked isocitrate dehydrogenase activity. These results suggest that different types of NADPH-generating systems operate alternatively in these yeasts depending upon carbon and nitrogen sources.     

Utilization of hexamethylenetetramine (urotropine) by bacteria and yeasts

A slow growing bacterial population able to utilize hexamethylelenetetramine (urotropine) as sole source of carbon, nitrogen and energy was isolated from soil. From this crude enrichment culture two bacteria were isolated and identified as Brevundimonas diminuta and a Phyllobacterium sp. by sequencing of 16S ribosomal DNA. These bacteria also grew on urotropine but at a lower rate than the enrichment culture. Addition of glucose to the latter resulted in growth of some yeasts that overgrew the bacteria. Assimilation of urotropine as sole nitrogen source is very common among yeasts, 46 out of 60 species tested showed this characteristic.     

Expression of the <Emphasis Type="Italic">UGA4</Emphasis> gene encoding the δ-aminolevulinic and γ-aminobutyric acids permease in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> is controlled by amino acid-sensing systems

In yeasts, several sensing systems localized to the plasma membrane which transduce information regarding the availability and quality of nitrogen and carbon sources and work in parallel with the intracellular nutrient-sensing systems, regulate the expression and activity of proteins involved in nutrient uptake and utilization. The aim of this work was to establish whether the cellular signals triggered by amino acids modify the expression of the UGA4 gene which encodes the δ-aminolevulinic (ALA) and γ-aminobutyric (GABA) acids permease. In the present paper, we demonstrate that extracellular amino acids regulate UGA4 expression and that this effect seems to be mediated by the amino acid sensor complex SPS (SSY1, PTR3, SSY5).     

Mutations affecting regulation of the anabolic argF and the catabolic aru genes in Pseudomonas aeruginosa PAO

Summary The nucleotide sequence required for a fully functional promoter and operator of the Pseudomonas aeruginosa argF gene (argF _po), the arginine-repressible gene for anabolic ornithine carbamoyltransferase, was defined within a 160 by region. The streptomycin (Sm) resistance genes strAB of plasmid RSF1010 were fused to argF _po. This construct in P. aeruginosa strain PAO conferred resistance to Sm. Mutants of strain PAO were selected which were resistant to Sm in the presence of arginine due to constitutive expression of argF _po —strAB. These mutants were designated argR. They were unable to grow or grew poorly on arginine or ornithine as the sole carbon and nitrogen source. This growth defect (Aru^–/Oru^– phenotype) was correlated with a reduced level of N-succinylornithine aminotransferase, an enzyme participating in the major aerobic pathway for arginine and ornithine catabolism in this organism. The argR mutants were classified into four groups by transduction analysis and three argR mutations were mapped on the PAO chromosome. argR9901 and argR9902 were co-transducible with car-9 (at 1 min) and thus close to the oru-310 locus; argR9906 was localized in the oruI (=aru) gene cluster (67 min). Some aru mutants, which have been isolated previously and which produce very low amounts of all enzymes in the arginine succinyltransferase pathway, were unable to repress the argF gene in an arginine medium. Thus, P. aeruginosa PAO appears to have multiple genes that are involved in the regulation of both the anabolic argF and the catabolic aru genes.Abbreviations Arg^– arginine auxotrophy - Aru arginine utilization - Oru ornithine utilization     

Expression of the nitrate transporter nrt2 gene from the symbiotic basidiomycete Hebeloma cylindrosporum is affected by host plant and carbon sources

Although the function of the extramatrical mycelium of ectomycorrhizal fungi is considered essential for the acquisition of nitrogen by forest trees, gene regulation in this fungal compartment is poorly characterized. In this study, the expression of the nitrate transporter gene nrt2 from the ectomycorrhizal basidiomycete Hebeloma cylindrosporum was shown to be regulated by plant host and carbon sources. In the presence of a low fructose concentration, nrt2 expression could not be detected in the free-living mycelium but was high in the extramatrical symbiotic mycelium associated to the host plant Pinus pinaster. In the absence of nitrogen or in the presence of nitrate, high sugar concentrations in the medium were able to enhance nrt2 expression. Nevertheless, in the presence of high fructose concentration, high ammonium concentration still completely repressed nrt2 expression indicating that the nitrogen repression overrides sugar stimulation. This is the first report revealing an effect of host plant and of carbon sources on the expression of a fungal nitrate transporter-encoding gene.     

Nutrition of three species of microsporum

Summary The growth ofMicrosporum Cookei, M. distortum, andM. nanum was compared on solid media containing 23 different carbon sources and 25 different nitrogen sources.M. nanum grew well only on media containing ribose, xylose, levulose, or erythritol as the carbon source.M. distortum andM. Cookei were found to utilize a far greater variety of carbon sources. Both grew well on dextrose, levulose, galactose, mannose, ribose, arabinose, rhamnose, sucrose, cellobiose, lactose, trehalose, melibiose, melezitose, raffinose, dextrin, and mannitol; in additionM. Cookei grew well on xylose, maltose, and erythritol. Aspartic acid, arginine, and citrulline were favorable sources of nitrogen for all three species. M. nanum also grew well on alanine, ornithine, histidine, and proline;M. distortum on glycine, serine, asparagine, glutamic acid, leucine, and cysteine; andM. Cookei on asparagine, tyrosine, ornithine, histidine, leucine, isoleucine, and proline.WithM. Cookei andM. distortum grown in liquid media in shake culture, mannitol was the best carbon source and tyrosine the best of the nitrogen sources tested. Nutritional differences exist among these three species,M. Audouini, M. canis, andM. gypseum.From a thesis, under the direction of Dr.Frederick T. Wolf, submitted in partial fulfillment of the requirements for the degree Master of Arts, August 1961.     

Altered control of glutamate dehydrogenases in ornithine utilization mutants of Pseudomonas aeruginosa

Two classes of ornithine-nonutilizing (oru) mutants of Pseudomonas aeruginosa PAO were investigated. Strains carrying the oru-310 mutation were entirely unable to grow on l-ornithine as the only carbon and nitrogen source and were affected in the assimilation of a variety of nitrogen sources (e.g., amino acids, nitrate). The oru-310 mutation caused changes in the regulation of the catabolic NAD-dependent glutamate dehydrogenase; this enzyme was no longer inducible by glutamate but instead could be induced by ammonia. The oru-310 locus was cotransducible with car-9 and tolA in the 10 min region of the chromosome. An oru-314 mutant was severely handicapped in ornithine medium but could grow when a good carbon source was added; the mutant also showed pleiotropic growth effects related to nitrogen metabolism. The oru-314 mutation affected the regulation of the anabolic NADP-dependent glutamate dehydrogenase, which was no longer repressed by glutamate but showed normal derepression in the presence of ammonia. The oru-314 locus was mapped by transduction near met-9011 at 55 min. Both oru mutants could grow on l-glutamate, l-proline, or l-ornithine amended with 2-oxoglutarate, albeit slowly. We speculate that insufficient 2-oxoglutarate concentrations might account, at least in part, for the Oru^- phenotype of the mutants.     

Ornithine cycle in Nostoc PCC 73102. Arginase,OCT and arginine deiminase,and the effects of addition of external arginine,ornithine, or citrulline

Arginase, ornithine carbamoyl transferase (OCT) and arginine deiminase activities were found in cell-free extracts of Nostoc PCC 73102, a free-living cyanobacterium originally isolated from the cycad Macrozamia. Addition of either arginine, ornithine or citrulline to the growth medium induced significant changes in their in vitro activities. Moreover, growth in darkness, compared to in light, induced higher in vitro activities. The in vitro activities of arginase and arginine deiminase, two catabolic enzymes primarily involved in the breakdown of arginine, increased substantially by a combination of growth in darkness and addition of either arginine, or ornithine, to the growth medium. The most significant effects on the in vitro OCT activities where observed in cells grown with the addition of ornithine. Cells grown in darkness exhibited about 6% of the in vivo nitrogenase activity observed in cells grown in light. However, addition of external carbon (glucose and fructose) to cells grown in darkness resulted in in vivo nitrogenase activity levels similar to, or even higher than, cells grown in light. Growth with high in vivo nitrogenase activity or in darkness with the addition of external carbon, resulted in repressed levels of in vitro arginase and arginine deiminase activities. It is suggested that nitrogen starvation induces a mobilization of the stored nitrogen, internal release of the amino compound arginine, and an induction of two catabolic enzymes arginase and arginine deiminase. A similar and even more pronunced induction can be observed by addition of external arginine to the growth medium.     

The specific transport system for lysine is fully inhibited by ammonium in Penicillium chrysogenum: An ammonium-insensitive system allows uptake in carbon-starved cells

The regulation exerted by ammonium and other nitrogen sources on amino acid utilization was studied in swollen spores of Penicillium chrysogenum. Ammonium prevented the L-lysine, L-arginine and L-ornithine utilization by P. chrysogenum swollen spores seeded in complete media, but not in carbon-deficient media. Transport of L-[¹⁴C]lysine into spores incubated in presence of carbon and nitrogen sources was fully inhibited by ammonium ions (35 mM). However, in carbon-derepressed conditions (growth in absence of sugars, with amino acids as the sole carbon source) L-[¹⁴C]lysine transport was only partially inhibited. Competition experiments showed that L-lysine (1 mM) inhibits the utilization of L-arginine, and vice versa, L-arginine inhibits the L-lysine uptake. High concentrations of L-ornithine (100 mM) prevented the L-lysine and L-arginine utilization in P. chrysogenum swollen spores. In summary, ammonium seems to prevent the utilization of basic amino acids in P. chrysogenum spores by inhibiting the transport of these amino acids through their specific transport system(s), but not through the general amino acid transport system that is operative under carbon-derepression conditions.     

Effect of Carbon Source on Enzymes and Metabolites of Arginine Metabolism in Neurospora

The levels of enzymes and metabolites of arginine metabolism were determined in exponential cultures of Neurospora crassa grown on various carbon sources. The carbon sources decreased in effectiveness (as determined by generation times) in the following order: sucrose, acetate, glycerol, and ethanol. The basal and induced levels of the catabolic enzymes, arginase (EC 3.5.3.1) and ornithine transaminase (EC 2.6.1.13), were lower in mycelia grown on poor carbon sources. Arginase was more sensitive to variations in carbon source than was ornithine transaminase. Induction of both enzymes was sensitive to nitrogen metabolite control, but this sensitivity was reduced in mycelia grown on glycerol or ethanol. The pools of arginine and ornithine were reduced in mycelia grown in unsupplemented medium containing poor carbon sources, but the biosynthetic enzyme ornithine transcarbamylase (EC 2.1.3.3) was not derepressed. The arginine pools were similar, regardless of carbon source, in mycelia grown in arginine-supplemented medium. The ornithine pool was reduced by growth on poor carbon sources. The rate of arginine degradation was proportional to the level of arginase in both sucrose- and glycerol-grown mycelia. The distribution of arginine between cytosol and vesicles was only slightly altered by growth on glycerol instead of sucrose. The slightly smaller cytosolic arginine concentration did not appear to be sufficient to account for the alterations in basal and induced enzyme levels. The results suggest a possible carbon metabolite effect on the expression or turnover of a variety of genes for enzymes of arginine metabolism in Neurospora.     

N2-Succinylornithine in ornithine catabolism of Pseudomonas aeruginosa

Most Pseudomonas aeruginosa PAO mutants which were unable to utilize l-arginine as the sole carbon and nitrogen source (aru mutants) under aerobic conditions were also affected in l-ornithine utilization. These aru mutants were impaired in one or several enzymes involved in the conversion of N²-succinylornithine to glutamate and succinate, indicating that the latter steps of the arginine succinyltransferase pathway can be used for ornithine catabolism. Addition of aminooxyacetate, an inhibitor of the N²-succinylornithine 5-aminotransferase, to resting cells of P. aeruginosa in ornithine medium led to the accumulation of N²-succinylornithine. In crude extracts of P. aeruginosa an ornithine succinyltransferase (l-ornithine:succinyl-CoA N²-succinyltransferase) activity could be detected. An aru mutant having reduced arginine succinyltransferase activity also had correspondingly low levels of ornithine succinyltransferase. Thus, in P. aeruginosa, these two activities might be due to the same enzyme, which initiates aerobic arginine and ornithine catabolism.Abbreviations OAT ornithine 5-aminotransferase - SOAT N²-succinylornithine 5-aminotransferase - Oru ornithine utilization - Aru arginine utilization     

Effect of Different Carbon and Nitrogen Sources on Clostridium argentinense Toxigenicity in Coculture withPseudomonas mendocina

The effect of different carbon and nitrogen sources on the production of toxin by Clostridium argentinense was examined. The toxin production by C. argentinense in coculture with Pseudomonas mendocina increased in all the cases in relation to that produced by monocultures independent of the nature of the source. Using dextrin as carbon source C. argentinense produced the highest levels of toxin both in monocultures (300 LD₅₀/mL) and in cocultures with P. mendocina (5000 LD₅₀/mL). Experiments run in a microfermenter showed that the slow growth of cocultures associated with the assimilation of dextrin and the pH and Eh profiles favoured the production of toxin. Of the nitrogen sources assayed, corn steep liquor sustained the highest levels of toxin in both monocultures and cocultures with 3 and 2.8 fold increases with respect to that obtained using proteose peptone. The toxin production by C. argentinense cultures and C. argentinense–P. mendocina cocultures was highly dependent on the nature of the carbon and nitrogen sources used in the culture media. Growth of C. argentinense on substrates slowly assimilated stimulated the production of toxin.     

Mutants of Neurospora crassa Deficient in Ornithine-δ-Transaminase

The arg-12(s) mutation of Neurospora causes a partial block in the ornithine transcarbamylase (OTC) reaction. Strains carrying this mutation will use endogenous ornithine, but not exogenous ornithine, as a precursor of arginine. Certain strains carrying arg-12(s) may be used for direct selection of variants able to use exogenous ornithine as an arginine precursor. Among eight such derivatives, six lacked the catabolic enzyme ornithine transaminase (OTA). All six mutations were alleles of a single gene, designated ota, on linkage group III. No mutation affected arginase, the first enzyme in the catabolic pathway with OTA. Strains carrying ota mutations alone are vigorous prototrophs, but, unlike wild-type Neurospora, fail to use ornithine efficiently as a sole nitrogen source. The selection method by which ota mutants arose suggests that OTA is severely competitive with OTC for exogeneous ornithine.     

Biotransformation of albendazole by Cunninghamella blakesleeana: effect of carbon and nitrogen source

A filamentous fungus Cunninghamella blakesleeana was screened for its ability to biotransform the anthelmintic drug albendazole. The fungus produced three metabolites in the presence of the carbon and nitrogen sources studied. The transformation was identified by HPLC and the structures of the transformation products were assigned by LC-MS-MS analysis and on the basis of previous reports. The results indicated that the fungus metabolized albendazole into albendazole sulfoxide (M1), albendazole sulfone (M2) and an N-methyl metabolite of albendazole sulfoxide (M3). The effect of carbon and nitrogen source on the biotransformation of albendazole was significant. Among the carbon and nitrogen sources studied, fructose and urea respectively produced maximum extent of biotransformation in terms of substrate depletion. Among the carbon sources studied, maltose produced a higher percentage of M1 whereas M2 and M3 were produced to their maximum extent in presence of d-fructose in terms of metabolite per unit quantity of biomass. In the case of nitrogen sources, ammonium acetate, calcium nitrate and barium nitrate produced maximum percentage of M1, M2 and M3 respectively. The results reveal that the carbon and nitrogen source significantly influence the microbial transformation both qualitatively and quantitatively.     

碳氮源对花榈木胚性愈伤组织诱导、发育及有机物积累的影响

为探讨花榈木体胚发生过程中不同碳氮源处理对胚性愈伤组织诱导、发育和有机物积累的影响,并筛选出有利于花榈木体胚发生的碳氮源,优化体胚发生体系,该研究以成熟胚为外植体,通过单因素试验分析3种碳源、4种蔗糖浓度和6种氮源处理下胚性愈伤组织诱导、发育和有机物积累的差异。结果表明：(1)蔗糖中胚性愈伤组织诱导率显著高于葡萄糖和麦芽糖,但其体胚诱导率、体胚分化率、胚性愈伤组织可溶性糖、淀粉和可溶性蛋白含量差异不显著。(2)随着蔗糖浓度的升高,胚性愈伤组织、体细胞胚(体胚)诱导率、体胚分化率、胚性愈伤组织重量和可溶性蛋白含量呈先升高后降低的趋势,均以添加30 g·L^-1蔗糖最高,而胚性愈伤组织可溶性糖和淀粉含量呈增加的趋势。(3)在6种氮源处理中,胚性愈伤组织诱导率以添加500 mg·L^-1谷氨酰胺的处理最高,体胚诱导率则以添加谷氨酰胺和水解酪蛋白的处理较高,但不同氮源处理间体胚分化率无差异;添加有机氮源的处理其胚性愈伤组织可溶性蛋白含量显著高于无氮源处理。总之,不同的碳氮源通过影响花榈木胚性愈伤组织的诱导、发育和有机物的积累,从而影响其体胚诱导率,但对体...     

Molecular analysis of inorganic nitrogen assimilation in yeasts

Assimilation of nitrate and various other inorganic nitrogen compounds by different yeasts was investigated. Nitrate, nitrite, hydroxylamine, hydrazine, ammonium sulphate, urea and L-asparagine were tested as sole sources of nitrogen for the growth of Candida albicans, C. pelliculosa, Debaryomyces hansenii, Saccharomyces cerevisiae, C. tropicalis, and C. utilis. Ammonium sulphate and L-asparagine supported the growth of all the yeasts tested except D. hansenii while hydroxylamine and hydrazine failed to support the growth of any. Nitrate and nitrite were assimilated only by C. utilis. Nitrate utilization by C. utilis was also accompanied by the enzymatic activities of NAD(P)H: nitrate oxidoreductase (EC 1.6.6.2) and NAD(P)H: nitrite oxidoreductase (EC 1.6.6.4), but not reduced methyl viologen-or FAD-nitrate oxidoreductases (EC 1.7.99.4). It is demonstrated here that nitrate and nitrite reductase activities are responsible for the ability of C. utilis to assimilate primary nitrogen.