Metabolic regulation in the facultative methylotroph Arthrobacter P1. Growth on primary amines as carbon and energy sources

During growth of the facultative methylotroph Arthrobacter P1 on methylamine or ethylamine both substrates are metabolized initially in an identical fashion, via the respective aldehydes. The regulatory mechanisms governing the synthesis and activities of enzymes involved in amine and aldehyde utilization were studied in substrate transition experiments. Transfer of ethylamine-grown cells into a medium with methylamine resulted in immediate exeretion of low levels of formaldehyde (max. 0.5 mM) and formate. In the reverse experiment, transfer of methylaminegrown cells into a medium with ethylamine, excretion of much higher levels of acetaldehyde (max. 3.5 mM) occurred. These different levels of aldehyde accumulation were also observed in studies with mutants of Arthrobacter P1 blocked in the synthesis of hexulose phosphate synthase or acetaldehyde dehydrogenase. In wild type Arthrobacter P1, aldehyde production resulted in rapid induction of the synthesis of enzymes involved in their degradation but also in temporary inhibition of further amine utilization and growth. The latter aetivities only resumed at normal rates after the disappearance of the aldehydes from the cultures. Acetaldehyde utilization resulted in intermittent excretion of ethanol and acetate, whereas formaldehyde utilization resulted in further accumulation of formate.During growth of Arthrobacter P1 in the presence of methylamine accumulation of toxic levels of formaldehyde is prevented because of the rapid synthesis of hexulose phosphate synthase to high activities and, in transient state situations, by feedback inhibition of formaldehyde on the activities of the methylamine transport system and amine oxidase.Abbreviations DTNB 5,5-dithiobis-(2-nitrobenzoate) - HPS hexulosephosphate synthase - MS mineral salts - RuMP ribulose monophosphate     

Isolation and characterization of mutants of the facultative methylotroph Arthrobacter P1 blocked in one-carbon metabolism

Among methylamine and/or ethylamine minus mutants of Arthrobacter P1 four different classes were identified, which were blocked either in the methylamine transport system, amine oxidase, hexulose phosphate synthase or acetaldehyde dehydrogenase. The results indicated that a common primary amine oxidase is involved in the metabolism of methylamine and ethylamine. Growth on ethylamine, however, was not dependent on the presence of the methylamine transport system. In mutants lacking amine oxidase, methylamine was unable to induce the synthesis of hexulose phosphate synthase, thus confirming the view that the actual inducer for the latter enzyme is not methylamine, but its oxidation product formaldehyde. Contrary to expectation, when the formaldehyde fixing enzyme hexulose phosphate synthase was deleted (mutant Art 11), accumulation of formaldehyde during growth on choline or on glucose plus methylamine as a nitrogen source did not occur. Evidence was obtained to indicate that under these conditions formaldehyde may be oxidized to carbon dioxide via formate, a sequence in which peroxidative reactions mediated by catalase are involved. In addition, a specific NAD-dependent formaldehyde dehydrogenase was detected in choline-grown cells of wild type Arthrobacter P1 and strain Art 11. This enzyme, however, does not play a role in methylamine or formaldehyde metabolism, apparently because these compounds do not induce its synthesis.Abbreviations RuMP ribulose monophosphate - HPS hexulose phosphate synthase - HPI hexulose phosphate isomerase     

Uptake of methylamine via an inducible,energy-dependent transport system in the facultative methylotroph Arthrobacter P1

Cytoplasmic membrane vesicles were prepared by a lysozyme-salt treatment from Arthrobacter P1 grown on methylamine as the carbon and energy source. In the presence of an ascorbate-phenazine methosulphate electron donor system, these vesicles accumulated methylamine in unmodified form by an inducible transport system. This system has a high affinity for methylamine (K_app=20–25 M). The effect of the ionophores valinomycin and nigericin combined with membrane potential () and pH-gradient (pH) measurements demonstrated that methylamine uptake is electrogenic and driven by the . Optimal activity is observed at pH 6.5 and 30°C. Methylamine uptake was not affected by the presence of ammonium ions but was inhibited by the primary amines ethylamine (competitively), propylamine, butylamine and benzylamine. In addition, formaldehyde and acetate, at a concentration of 1 mM, inhibited methylamine uptake almost completely. These compounds were shown to be non-competitive inhibitors. A strong inhibition observed in the presence of plumbagin could be relieved by addition of dithiothreitol. This indicates that the oxidation-reduction state of, probably, carrier dithiol-disulfide-groups is an important factor in methylamine translocation in Arthrobacter P1.     

Arthrobacter P 1, a fast growing versatile methylotroph with amine oxidase as a key enzyme in the metabolism of methylated amines

A facultative methylotrophic bacterium was isolated from enrichment cultures containing methylamine as the sole carbon source. It was tentatively identified as an Arthrobacter species. Extracts of cells grown on methylamine or ethylamine contained high levels of amine oxidase (E.C. 1.4.3.) activity. Glucose- or choline-grown cells lacked this enzyme. Oxidation of primary amines by the enzyme resulted in the formation of H₂O₂; as a consequence high levels of catalase were present in methylamine-and ethylamine-grown cells. The significance of catalase in vivo was demonstrated by addition of 20 mM aminotriazole (a catalase inhibitor) to exponentially growing cells. This completely blocked growth on methylamine whereas growth on glucose was hardly affected. Cytochemical studies showed that methylamine-dependent H₂O₂ production mainly occurred on invaginations of the cytoplasmic membrane. Assimilation of formaldehyde which is generated during methylamine oxidation was by the FBP variant of the RuMP cycle of formaldehyde fixation. The absence of NAD-dependent formaldehyde and formate dehydrogenases indicated the operation of a non-linear oxidation sequence for formal-dehyde via hexulose phosphate synthase. Enzyme profiles of the organism grown on various substrates suggested that the synthesis of amine oxidase, catalase and the enzymes of the RuMP cycle is not under coordinate control.     

Regulation and function of transaldolase isoenzymes involved in sugar and one-carbon metabolism in the ribulose monophosphate cycle methylotroph Arthrobacter P1

In the facultative methylotroph Arthrobacter P1 the enzyme transaldolase plays an important role in both the pentose phosphate pathway and in the ribulose monophosphate cycle of formaldehyde fixation.Among gluconate-negative mutants of Arthrobacter P1 strains occurred which also were unable to grow on xylose because they had lost the ability to synthesize transaldolase. Furthermore, this loss of transaldolase activity resulted in decreased growth rates on a number of other heterotrophic substrates. Contrary to expectation, these mutants still grew on methylamine and were even able to use gluconate as a carbon source at normal rates provided methylamine was supplied as a nitrogen source. Under these conditions high levels of transaldolase were observed.Partial purification of the transaldolases synthesized by gluconate-grown cells of wild type Arthrobacter P1 and methylamine-grown cells of one of these mutants, strain Art 98, revealed the presence of transaldolase isoenzymes. These enzymes displayed similar kinetics but were very different in heat sensitivity. Functionally these isoenzymes are apparently very similar but their synthesis is regulated differently. One of the enzymes is synthesized constitutively whereas the other is specifically induced during growth on C₁ compounds. Strain Art 98 has lost the ability to synthesize the constitutive transaldolase. It is postulated that the C₁-induced transaldolase serves to ensure a sufficiently high rate of regeneration of ribulose-5-phosphate during growth on C₁ compounds.Abbreviations RuMP ribulose monophosphate - DEAE diethylaminoethyl     

Regulation of methylamine and formaldehyde metabolism in Arthrobacter P1

The regulation of methylamine and formaldehyde metabolism in Arthrobacter P1 was investigated in carbonlimited continuous cultures. To avoid toxic effects of higher formaldehyde concentrations, formaldehyde-limited cultures were established in smooth substrate transitions from choline-limitation. Evidence was obtained that the synthesis of enzymes involved in the conversion of methylamine into formaldehyde and in formaldehyde fixation is induced sequentially in this organism. Compared to growth with methylamine the molar growth yield on formaldehyde was approximately 30% higher. This difference is mainly due to the expenditure of energy for the uptake of methylamine from the medium.The addition of a pulse of a heterotrophic substrate, glucose or acetate, to C₁ substrate-limited continuous cultures resulted in relief of carbon limitation and transient synthesis of increasing amounts of cell material. Concomitantly, a significant decrease in the specific activities of hexulose phosphate synthase was observed. However, the total activity of hexulose phosphate synthase in these cultures remained clearly in excess of that required to fix the formaldehyde that became available in time. The observed strong decrease in the specific activities of this RuMP cycle enzyme strongly suggests that its synthesis is controlled via catabolite repression exerted by the metabolism of heterotrophic substrates.Abbreviations HPS 3-Hexulose-6-phosphate synthase - HPI 3-hexulose-6-phosphate isomerase - RuMP ribulose monophosphate     

Purification and characterization of an NAD+-linked formaldehyde dehydrogenase from the facultative RuMP cycle methylotrophArthrobacter P1

WhenArthrobacter P₁ is grown on choline, betaine, dimethylglycine or sarcosine, an NAD⁺-dependent formaldehyde dehydrogenase is induced. This formaldehyde dehydrogenase has been purified using ammonium sulphate fractionation, anion exchange- and hydrophobic interaction chromatography. The molecular mass of the native enzyme was 115 kDa±10 kDa. Gel electrophoresis in the presence of sodium dodecyl sulphate indicated that the molecular mass of the subunit was 56 kDa±3 kDa, which is consistent with a dimeric enzyme structure. After ammonium sulphate fractionation the partially purified enzyme required the addition of a reducing reagent in the assay mixture for maximum activity. The enzyme was highly specific for its substrates and the K_m values were 0.10 and 0.80 mM for formaldehyde and NAD⁺, respectively. The enzyme was heat-stable at 50° C for at least 10 min and showed a broad pH optimum of 8.1 to 8.5. The addition of some metal-binding compounds and thiol reagents inhibited the enzyme activity.Abbreviation RuMP Ribulose monophosphate     

Mapping of some genes involved in C-1 metabolism in the facultative methylotroph Methylobacterium sp strain AM1 (Pseudomonas AM1)

R68.45 mediated mobilisation of the chromosome of Methylobacterium sp strain AM1 has been investigated. High frequencies of cotransfer of four genes required for C-1 metabolism with the genes coding for streptomycin, phosphonomycin and cycloserine resistance were demonstrated. A preliminary map of this region has been constructed on the basis of the results of three and four factor crosses showing that not all the C-1 genes are contiguous.Abbreviations Str streptomycin - Pho phosphonomycin - Cyc cycloserine - Tc tetracycline - Km kanamycin - Cb carbenicillin - Ade adenine - Thi thiamine - Met methionine     

Oxidation of amines by yeasts grown on 1-aminoalkanes or putrescine as the sole source of carbon,nitrogen and energy

The maximum growth rate of Trichosporon cutaneum CBS 8111 in chemostat cultures was 0.185 h^-1 on ethylamine and 0.21 h^-1 on butylamine, that of Candida famata CBS 8109 was 0.32 h^-1 on putrescine.The amine oxidation pattern of the ascomycetous strains studied, viz. Candida famata CBS 8109, Stephanoascus ciferrii CBS 4856 and Trichosporon adeninovorans CBS 8244 was independent of the amine that had been used as the growth substrate. It resembled that of benzylamine/putrescine oxidase found in other ascomycetous yeasts. However, differences in pH optimum and substrate specificity were observed between the amine-oxidizing systems of these three species.The amine oxidation pattern of cell-free extracts of Trichosporon cutaneum CBS 8111 varied with the amine that was used as growth substrate. The enzyme system produced by Cryptococcus laurentii CBS 7140 failed to oxidize isobutylamine and benzylamine, and showed a high pH optimum.The synthesis of amine oxidase in the four yeast strains studied was not repressed by ammonium chloride and was weakly repressed by glucose but was strongly repressed if both compounds were present in the growth medium.     

Regulation of methylotrophic and heterotrophic metabolism in Arthrobacter P1. Growth on mixtures of methylamine and acetate in batch and continuous cultures

Incubations of Arthrobacter P1 in batch culture in media with mixtures of acetate and methylamine resulted in sequential utilization of the two carbon substrates, but not in diauxic growth. Irrespective of the way cells were pregrown, acetate was the preferred substrate and subsequent studies showed that this is due to the fact that acetate is a strong inhibitor of the methylamine transport system and amine oxidase in Arthrobacter P1. An analysis of enzyme activities in cell-free extracts showed that synthesis of amine oxidase occurred already in the first growth phase with acetate, whereas rapid synthesis of hexulose phosphate synthase was only observed once methylamine utilization started. It is therefore concluded that in Arthrobacter P1 the synthesis of the enzymes specific for methylamine oxidation is not regulated co-ordinately with those involved in formaldehyde fixation, but induced sequentially by methylamine and formaldehyde, respectively.During growth of Arthrobacter P1 on the same mixture in carbon- and energy source-limited continuous cultures both substrates were used simultaneously and completely at dilution rates below the _max on either of these substrates. Addition of methylamine, in concentrations as low as 0.5 mM, to the medium reservoir of an acetate-limited continuous culture (D=0.10 h^-1) already resulted in synthesis of both amine oxidase and hexulose phosphate synthase. In the reverse experiment, addition of acetate to the medium reservoir of a methylamine-limited continuous culture (D=0.10 h^-1), acetate was initially only used as an energy source. Synthesis of the glyoxylate cycle enzymes, however, did occur at acetate concentration in the feed above 7.5–10 mM. This indicates that at acetate concentrations below 10 mM the metabolism of the C₁ substrate methylamine is able to cause a complete repression of the synthesis of the enzymes involved in carbon assimilation from the C₂ substrate acetate.Abbreviations HPS Hexulose phosphate synthase - MS mineral salts - RuMP ribulose monophosphate     

Phenylalanine and tyrosine metabolism in the facultative methylotroph Nocardia sp. 239

Nocardia sp. 239 is able to use l-tyrosine and both d- and l-phenylalanine as carbon-, energy- and nitrogen sources for growth. The catabolism of these compounds is by way of (4-hydroxy)phenylpyruvate and (4-hydroxy)-phenylacetate as intermediates and the pathways merge at the level of homogentisate. The conversion of the amino acids into (4-hydroxy)phenylpyruvate is catalyzed by an inducible NAD-dependent phenylalanine dehydrogenase and l-tyrosine aminotransferase, respectively. Incubation of the organism in media with l-phenylalanine plus phenyl-pyruvate resulted in diauxic growth, with phenylpyruvate used first. Phenylalanine dehydrogenase activity cold only be detected after depletion of phenylpyruvate, in the ensuing second growth phase on l-phenylalanine. During growth on phenylalanine plus methanol, low levels of phenylalanine dehydrogenase were detected and this resulted in simultaneous utilization of the two substrates. Following diepoxyoctane treatment, mutants of Nocardia sp. 239 affected in phenylalanine and phenylpyruvate degradation were isolated. Double mutants blocked in both phenylalanine dehydrogenase and phenylpyruvate decarboxylase completely failed to catabolize phenylalanine. The absence of these enzymes did not affect growth on tyrosine.Abbreviations RuMP ribulose monophosphate - EMS ethylmethanesulphonate - NTG N-methyl-N-nitro-N-nitrosoguanidine     

Regulation of ammonia (NH3-N) metabolism in the marine pulmonate,onchidium verruculatum. I. effects of a neuroendocrine centre on tissue ammonia levels

Effect of ganglionic extirpations and replacement on the ammonia levels in the blood and various tissues of the marine pulmonate, O. verruculatum was studied. Three hrs after extirpation of the pleurovisceral (PV) complex only and not any other ganglia, revealed a significant (P < 0.001) decline in the blood and foot ammonia levels and significant (P < 0.001) increment in the hepatopancreatic ammonia level. However, no change was observed in the mantle and nephridial ammonia levels. Replacement of the PV complex, either via implantation of intact ganglia or boiled and unboiled homogenate administration (1 gn/animal) into the ganglionectomised group, restored ammonia levels in the blood and foot and showed significant (P < 0.005) decrease in hepatopancreatic ammonia levels. The results are discussed in the light of hormonal regulation of the nitrogen metabolism.     

Interrelationships between carbohydrate metabolism and nitrogen assimilation in cultured plant cells

In sycamore cells grown on nitrate as opposed to glutamate there is a higher pentose phosphate pathway carbon flux relative to glycolysis in the early stages of cell growth when nitrate assimilation is most active. The high pentose phosphate pathway activity compared with glycolysis in nitrate grown cells is accompanied by enhanced levels of hexokinase, pyruvate kinase, glucose-6-phosphate de-hydrogenase, 6-phosphogluconate dehydrogenase and transketolase. There is no significant increase in activity of the solely glycolytic enzyme, phosphofructokinase. It is suggested that the increased pentose phosphate pathway activity in nitrate grown cells is correlated with a demand by nitrite assimilation for NADPH.II=Jessup and Fowler, 1976 b     

Glucose catabolism by Thiobacillus A2 grown in chemostat culture under carbon or nitrogen limitation

Thiobacillus A2 was grown in glucose- or ammonium-limited chemostats and relative contributions of the Embden-Meyerhof (EM), Entner-Doudoroff (ED) and pentose phosphate (PP) pathways to glucose catabolism estimated by ¹⁴C-glucose radiorespirometry. In fast growing strain GFI, the EM pathway predominated (41–79%) under all growth conditions with the PP pathway contributing 18–30%. The ED pathway was apparently absent under some conditions of glucose limitation. In contrast, wild type Thiobacillus A2 exhibited predominance of the EM pathway (43–48%) under ammonium-limitation but apparent predominance of the PP pathway (43–55%) under glucose-limitation, although all three pathways were calculated to operate. Under some conditions of glucose limitation the EM pathway was possibly considerably depressed. No clear pattern of response of the three pathways to altered environmental conditions could be deduced, although marked change in pathway activities were obviously induced. Growth yield was apparently unaffected by variation in pathways. The problems of interpreting such complex radiorespirometric data are discussed.Abbreviations EM Embden-Meyerhof - ED Entner-Doudoroff - KDPG 2-keto-3-deoxy-6-phosphogluconate - 6-PG 6-phosphogluconate - PK phosphoketolase - PP pentose phosphate     

Suitability of hydroxyapatite and iron phosphate as P sources for Lupinus albus grown in nutrient solution

In the framework of efforts to introduce Tuber melanosporum as a cultivated crop to Israel, spores of the truffle, obtained from fruit-bodies procured in Italy and France, were used to inoculate oak seedlings and hazel suckers. Typical T. melanosporum mycorrhizas were observed 3 months after inoculation on roots of both plant species. One- to two-year-old mycorrhizal seedlings were outplanted at a number of experimental sites and irrigated regularly. Two sites characterized by alkaline soil but differing in soil composition and climatic conditions were chosen for the present study. DNA of ascocarps used for inoculation, DNA of re-isolated cultures and fungal DNA taken from tree mycorrhizas 4 years after outplanting were compared with T. melanosporum reference cultures by molecular methods. All T. melanosporum profiles proved to be identical except for one belonging to a reference culture, which exhibited an unusual HinfI ITS-RFLP pattern. A single base substitution, responsible for the different HinfI restriction site, distinguished the ITS region of this culture from a published T. melanosporum ITS sequence. ITS restriction polymorphism analyses determined that roots of all potted plants tested and many 4-year-old trees from the two experimental plots (irrespective of soil and climatic differences) were colonized by T. melanosporum.     

Growth and mycelial strand production of Rigidoporus lignosus with various nitrogen and carbon sources

Growth and differentiation of mycelial strands in Rigidoporus lignosus have been shown to depend on suitable combinations of the pH of the media and the nature of the nitrogen and carbon sources. Amino acids as sole nitrogen sources gave rise to vegetative mycelium. At pH 4.5, growth and mycelial strand differentiation required asparagine, as the fungus failed to grow in the absence of this amino acid. However, at pH 6, differentiation of strands occurred appreciably in asparagine-deficient media, suggesting a close balance between pH and amino acid requirements. Ammonium was required for strand differentiation, while nitrate, as a sole nitrogen source, maintained the fungus undifferentiated. Of the carbohydrates tested, only glucose, fructose and mannose supported strand differentiation. Starch was found to be particularly effective in promoting growth of vegetative mycelium. Strand differentiation required more specific conditions than growth of the vegetative mycelium.     

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.     

The differential effects of TCA-cycle acids on the growth of plant cells cultured in liquid media containing various nitrogen sources

Alfalfa (Medicago sativa L. cv. Canadian No. 1), tobacco (Nicotiana tabacum L. var. humilis) and wheat (triticum monococcum L.) cells were grown in a defined, liquid medium containing either ammonium sulfate, L-glutamine or potassium nitrate as the sole nitrogen source, and the effects of the tricarboxylic-acid (TCA) intermediates, citrate and -ketoglutarate (5, 10, 15 mM), on the growth (dry-weight increase) of these cells was observed. The three cell suspension cultures exhibited a different growth response to the TCA-cycle intermediate supplied, depending upon the concentration of the additive and the nitrogen source. Citrate (5 mM) greatly enhanced growth of alfalfa and wheat cells in an ammonium-based medium but was less effective at higher concentrations, and in the case of alfalfa cells markedly inhibited growth. Tobacco cell growth was inhibited by all citrate concentrations tested. In contrast, all concentrations of -ketoglutarate used stimulated the growth of all three cell cultures in an ammonium-based medium. Alfalfa and wheat cells grown in an L-glutamine-based medium were influenced by citrate in a manner similar to that in ammonium-based medium. The growth of tobacco cells was slightly enhanced by 5 mM citrate but inhibited by higher concentrations. -Ketoglutarate, at all concentrations tested, was stimulatory to the growth of the cells of all three species in a glutamine-based medium, except for alfalfa cells which were inhibited at 15 mM. Both TCA-cycle acids inhibited the growth of alfalfa and tobacco cells grown on a nitrate-based medium whereas the growth of wheat cells was almost unaffected.     

Urease formation in purple sulfur bacteria (Chromatiaceae) grown on various nitrogen sources

Batch cultures of Thiocapsa roseopersicina strain 6311, Thiocystis violacea strain 2311 and Chromatium vinosum strain 1611, grown anaerobically in the light on sulfide with urea, ammonia, N₂ or casein hydrolysate as nitrogen source exhibited urease activity, while Chromatium vinosum strain D neither showed any degradation of urea nor urease activity on any of the nitrogen sources tested.In T. violacea and C. vinosum strain 1611 urease was little affected by the nitrogen source and seemed to be constitutive. In T. roseopersicina, however, the enzyme was repressed by ammonia (although a low basal level of activity remained) and, to a lesser degree, induced by urea: The presense of urea stimulated a temporary increase in urease activity in the early exponential growth phase. The highest activities, however, were found after growth on N₂, and especially on 0.1% casein hydrolysate (in the absence or after exhaustion of external ammonia), but not before the stationary growth phase was reached. Derepressed urease synthesis required an efficient external source of nitrogen.In cultures of T. roseopersicina urease activity showed a periodic oscillation which depended on the repeated feeding with sulfide and subsequent variation in the sulfur content of the cells. The possible reasons of this oscillation are discussed.     

Changes of cell size distribution during the batch culture of Arthrobacter strain PI/1-95

The size distributions of an arthrobacter, approximately 1 m in diameter, were analysed using a Coulter® Multisizer II instrument thereby making it possible to distinguish between the different stages in the morphological cycle. The results indicated that at the beginning of exponential phase a shift occurred from large to smaller cells, the cell size distributions in both categories were asymmetric, skewed towards higher values than the means. During the course of the batch culture the cells in the larger class decreased in size and the two classes coalesced. The size distribution of the smaller class exhibited an additional shift towards smaller cells. After which neither the number of classes nor the size distribution changed. It was concluded that the Coulter® Multisizer II instrument provides a feasible method to extract information not only about mean cell size but also about cell size distributions.