Transport of methylamine by Pseudomonas sp. MA.

Pseudomonas sp. MA grows on methylamines as a sole source of carbon, nitrogen, and energy. The transport of methylamine into the organism was investigated. It was found that this organism possesses an inducible transport system for methylamine having the following physical parameters: pH optimum, 7.2; temperature optimum, 30 to 35 degrees C; Km, 1 to 30 mM; Vmax, 90 to 120 nmol/min per mg (dry weight) of cells. Methylamine uptake was curtailed by azide, cyanide, and carbonyl cyanide-m-chlorophenylhydrazone; osmotic shock treatment reduced the uptake by 50%. The uptake was not effectively inhibited by ammonium ion, amino acids, or amides, but was competitively inhibited by short-chain alkylamines. Cells grown on succinate-ammonium chloride did not possess the transport system, but it could be induced in such cells by methylamine in 20 h. Cells grown with methylamine as a sole nitrogen, but not carbon, source transported methylamine at a reduced rate.     

Uptake of methylamine and methanol by Pseudomonas sp. strain AM1.

The uptake of methylamine and of methanol by the facultative methylotroph Pseudomonas sp. strain AM1 was investigated. It was found that this organism possesses two uptake systems for methylamine. One of these operates when methylamine is the sole source of carbon, nitrogen, and energy. It has a Km of 1.33 X 10(-4) M and a Vmax of 67 nmol/min per mg of cells (dry weight). The other system, found when methylamine is the sole nitrogen source only, has a Km of 1.2 X 10(-5) M and a Vmax of 8.9 nmol/min per mg of cells (dry weight). Both uptake systems were severely inhibited by azide, cyanide, carbonyl cyanide-m-chlorophenyl hydrazone, and N-ethylmaleimide, but only the high-affinity system was inhibited by ammonium ions with a Ki of 7.7 mM. Both systems were susceptible to osmotic shock treatment, competitively inhibited by ethylamine, and unaffected by most amino acids. Methanol uptake showed a Km of 4.8 microM and a Vmax of 60.6 nmol/min per mg of cells (dry weight) and was not inhibited by osmotic shock treatment. Azide, cyanide, and N-ethylmaleimide curtailed uptake, but carbonyl cyanide-m-chlorophenyl hydrazone merely reduced the rate of uptake. A methanol dehydrogenase mutant, M15A, was unable to take up methanol. It is proposed that methanol diffuses into the cell where it is rapidly oxidized by methanol dehydrogenase.     

Regulation of methanol and methylamine dehydrogenases in Methylophilus methylotrophus

Abstract Methylophilus methylotrophus can use methylamine as sole source of carbon and nitrogen. Measurements of the specific activity of methylamine dehydrogenase (MNDH) in bacteria grown in batch or chemostat culture showed that MNDH was induced by methylamine and repressed when methanol or NH₄⁺ were provided as alternative carbon or nitrogen sources. The degree of repression varied with the growth conditions. Methanol dehydrogenase (MDH) was present in bacteria growtn on methylamine as sole carbon source, but the specific activity was low compared with that in bacteria grown on medium containing methanol, indicating that this enzyme is induced by methanol.     

Methylamine metabolism in Hansenula polymorpha: an in vivo 13C and 31P nuclear magnetic resonance study.

Methylamine uptake, oxidation, and assimilation were studied in Hansenula polymorpha, a methylotrophic yeast. The constitutive ammonia transport system was shown to be effective at accumulating methylamine within cells cultured with methylamine or ammonia as a nitrogen source. [13C]methylamine oxidation rates were measured in vivo in methylamine-adapted cells by 13C nuclear magnetic resonance and were found to be lower than its uptake rate into the cells. The 13C label of methylamine was found exclusively in trehalose and glycerol, and [13C]formaldehyde was also extensively assimilated, indicating the presence of an assimilation pathway for the methylamine carbon. In vivo 31P nuclear magnetic resonance analysis showed major differences in the endogenous polyphosphate levels and mean chain length during adaptation of the cells from ammonia to methylamine, indicating that methylamine accumulated in the vacuole in the same manner as basic amino acids and purines. [13C]glucose metabolism was drastically altered during adaptation of the cells from ammonia to methylamine as a nitrogen source. The total rate of glucose utilization and the rate of ethanol production fell. Direct trehalose synthesis from glucose increased, indicating a switch from carbon utilization for growth to that for storage. The rate of methylamine oxidation was sufficient to support a much higher flow of carbon into central biosynthetic pathways. These results suggest that this reduction in biosynthetic carbon flow, rather than nitrogen availability, was the main factor responsible for reducing the growth rate of the yeast when ammonia was replaced by methylamine as the nitrogen source.     

Expression of a Rhizopus oryzae lipase in Pichia pastoris under control of the nitrogen source-regulated formaldehyde dehydrogenase promoter

A Rhizopus oryzae lipase gene has been expressed in Pichia pastoris as a reporter using the formaldehyde dehydrogenase 1 promoter (PFLD1) of this organism, which has been reported to be strongly and independently induced by either methanol as sole carbon source or methylamine as sole nitrogen source. Levels of lipase expressed and secreted under the control of the PFLD1 at different induction conditions have been compared to those obtained with the commonly used alcohol oxidase 1 promoter (PAOX1) in small (shake flask) and 1l bioreactor batch cultures. PFLD1-controlled heterologous gene expression was strongly repressed by excess of either glycerol or glucose-but not sorbitol-during growth using methylamine both as sole nitrogen source and inducing substrate. Co-induction of PFLD1 with methanol and methylamine resulted in a synergistic effect on extracellular lipase expression levels. In all tested conditions, the substitution of ammonium for methylamine as carbon source provoked a clear decrease in the specific growth rate and yield of biomass per gram of carbon source. Overall, this study demonstrates that the PFLD1 promoter is at least as efficient as the PAOX1 for extracellular expression of heterologous proteins in P. pastoris bioreactor cultures and provides a first basis for the further design of methanol-free high cell density fed-batch cultivation strategies for controlled overproduction of foreign proteins in P. pastoris.     

Production of volatile nitrogenous compounds from the degradation of streptomycin by Pseudomonas maltophilia

Ammonia, methylamine, and pyridine were detected in broth filtrates of a streptomycin-degrading strain of Pseudomonas maltophilia during growth on streptomycin as a sole carbon and nitrogen source. Ammonia and methylamine, quantitatively measured by conversion to chromophores with picryl sulfonic acid, were found to accumulate in broth, whereas pyridine concentration increased in the early stages of streptomycin degradation and then decreased as the degradation of the antibiotic neared completion. Exogenous pyridine was metabolized by washed-cell suspensions. Use of N-streptomycin-methyl-(14)C showed that the methylamine arose from the N-l-glucosamine-methyl moiety of streptomycin. Methylamine was an end product and was not further metabolized by cells.     

The effect of pH on the selection of carbaryl-degrading bacteria from garden soil

At an alkaline pH and in an aqueous solution carbaryl hydrolyses to form 1-naphthol, methylamine and carbon dioxide, but it is much more stable at an acid pH. Soil perfusion column experiments indicated that the rate of carbaryl degradation at pH 6.0 to 7.0 was limited by the rate of chemical hydrolysis. Bacterial communities of at least 12 and 14 members were selected in continuous cultures using carbaryl as the sole carbon and nitrogen source at pH 6.0. These communities were supported by the slow formation of hydrolysis products and a carbaryl-degrading bacterium was not selected after > 2000 h. A bacterial community of at least eight members was selected using equimolar 1-naphthol and methylamine as its sole carbon and nitrogen source. In contrast, after a lag of between 10 and 50 days, soil perfusion column and continuous culture enrichments at pH 5.2 and 5.0, respectively, led to the selection of a Pseudomonas sp. which could utilize carbaryl as its sole carbon and nitrogen source.     

The effect of pH on the selection of carbaryl-degrading bacteria from garden soil

At an alkaline pH and in an aqueous solution carbaryl hydrolyses to form 1-naphthol, methylamine and carbon dioxide, but it is much more stable at an acid pH. Soil perfusion column experiments indicated that the rate of carbaryl degradation at pH 6.0 to 7.0 was limited by the rate of chemical hydrolysis. Bacterial communities of at least 12 and 14 members were selected in continuous cultures using carbaryl as the sole carbon and nitrogen source at pH 6.0. These communities were supported by the slow formation of hydrolysis products and a carbaryl-degrading bacterium was not selected after greater than 2000 h. A bacterial community of at least eight members was selected using equimolar 1-naphthol and methylamine as its sole carbon and nitrogen source. In contrast, after a lag of between 10 and 50 days, soil perfusion column and continuous culture enrichments at pH 5.2 and 5.0, respectively, led to the selection of a Pseudomonas sp. which could utilize carbaryl as its sole carbon and nitrogen source.     

Methylamine and ammonia transport in Saccharomyces cerevisiae.

Methylamine (methylammonium ion) entered Saccharomyces cerevisiae X2180-A by means of a specific active transport system. Methylamine uptake was pH dependent (maximum rate between pH 6.0 and 6.5) and temperature dependent (increasing up to 35 C) and required the presence of a fermentable or oxidizable energy source in the growth medium. At 23 C the vmax for methylamine transport was similar 17 nmol/min per mg of cells (dry weight) and the apparent Km was 220 muM. The transport system exhibited maximal activity in ammonia-grown cells and was repressed 60 to 70 percent when glutamine or asparagine was added to the growth medium. There was no significant derepression of the transport system during nitrogen starvation. Ammonia (ammonium ion) was a strong competitive inhibitor of methylamine uptake, whereas other amines inhibited to a much lesser extent. Mutants selected on the basis of their reduced ability to transport methylamine (Mea-R) simultaneously exhibited a decreased ability to transport ammonia.     

Hydrogenosomes in known species of rumen entodiniomorphid protozoa

Abstract Yeasts of the genus Kluyveromyces grew very slowly on methylamine as sole nitrogen source. Methylamine oxidase activity in cell-free extracts was very low. Under conditions known to separate methylamine oxidase from benzylamine oxidase in other yeast genera, only a single enzyme was detected in Kluyveromyces lactis . This enzyme could oxidize benzylamine, n -butylamine and (very poorly) methylamine. The enzyme lost no activity on heating at 45°C and had a high affinity and V _max for benzylamine and 1-aminoalkanes of long-chain length, with a very low affinity and V _max for methylamine. It is concluded that growth of K. lactis on methylamine involves only benzylamine oxidase, and that a methylamine oxidase of the type found in other yeasts does not occur.     

Biodegradation of Carbaryl by a Micrococcus Species

A bacterium capable of utilizing carbaryl as sole source of carbon was isolated from garden soil and identified as a Micrococcus species. The organism also utilized carbofuran, naphthalene, 1-naphthol, and several other aromatic compounds as growth substrates. The organism degraded carbaryl by hydrolysis to yield 1-naphthol and methylamine. 1-Naphthol was further metabolized via salicylate by a gentisate pathway, as evidenced by oxygen uptake and enzymatic studies. Received: 27 November 2000 / Accepted: 29 December 2000     

Causes of conductance change in yeast cultures.

The conductance change due to growth of Saccharomyces cerevisiae Y112, Zygosaccharomyces bailii M and Rhodotorula rubra NCYC 63 in culture media containing glucose, tartrate pH buffer and ammonium ions as sole nitrogen source was compared with that in a medium containing L-asparagine as sole nitrogen source. Decreases in conductance were observed in glucose-ammonium cultures of all three yeasts while little change occurred in cultures with L-asparagine as sole nitrogen source. This supports the hypothesis that the metabolic activity primarily responsible for conductance change in yeast cultures is the uptake of charged ammonium ions as nitrogen source and the reaction of protons with pH buffer compounds. Rhodotorula rubra cultures with L-asparagine as sole carbon source caused large increases in conductance with growth. Chemical analyses of culture filtrates showed that this increase in conductance was due to use of L-asparagine as carbon source and the excretion of nitrogen surplus to biosynthetic needs as ammonium. In addition, the production of aspartate, acetate and bicarbonate contributed to the increase in conductance.     

Causes of conductance change in yeast cultures

The conductance change due to growth of Saccharomyces cerevisiae Y112, Zygosaccharomyces bailii M and Rhodotorula rabra NCYC 63 in culture media containing glucose, tartrate pH buffer and ammonium ions as sole nitrogen source was compared with that in a medium containing L-asparagine as sole nitrogen source. Decreases in conductance were observed in glucose-ammonium cultures of all three yeasts while little change occurred in cultures with L-asparagine as sole nitrogen source. This supports the hypothesis that the metabolic activity primarily responsible for conductance change in yeast cultures is the uptake of charged ammonium ions as nitrogen source and the reaction of protons with pH buffer compounds.
Rhodotorula rubra cultures with L-asparagine as sole carbon source caused large increases in conductance with growth. Chemical analyses of culture filtrates showed that this increase in conductance was due to use of L-asparagine as carbon source and the excretion of nitrogen surplus to biosynthetic needs as ammonium. In addition, the production of aspartate, acetate and bicarbonate contributed to the increase in conductance.     

Regulation of methylammonium transport inParacoccus denitrificans

Depending on the growth conditionsParacoccus denitrificans synthesizes two different carriers mediating uptake of methylamine. When used as a nitrogen source, methylamine is transported via a NH ₄ ⁺ carrier, and its transport is inhibited by NH ₄ ⁺ but not by ethylamine. When used as a carbon source, methylamine is transported by a specific alkylamine carrier, and its transport is inhibited by ethylamine but not by NH ₄ ⁺ . The NH ₄ ⁺ carrier is under nitrogen control, the alkylamine carrier under carbon control.Abbreviations MA Methylamine - FCCP p-trifluormethoxycarbonylcyanide-phenylhydrazone     

Control of benzylamine oxidase activity in Kluyveromyces fragilis grown on primary amines as nitrogen source

Abstract After growth on a mixture of ammonium and either methylamine or n -butylamine as nitrogen sources, benzylamine oxidase activity in yeasts from a number of different genera was found to be repressed to a lesser extent by ammonium than was methylamine oxidase. Catalase activity was better repressed by ammonium with methylamine as the nitrogen source than with n -butylamine. During growth of Kluyveromyces fragilis on equimolar mixtures of ammonium and an amine as nitrogen sources, benzylamine oxidase synthesis began during the period of exclusive growth on ammonium, and a period of simultaneous use of both nitrogen sources was observed just before the ammonium was exhausted. Addition of ammonium to cultures growing on n -butylamine as nitrogen source had no immediate repressive effect on benzylamine oxidase or catalase synthesis. However, growth on limiting ammonium in the absence of amines did give rise to low levels of amine oxidase and derepression of catalase activity. It is concluded that benzylamine oxidase in yeasts is induced strongly by amines as well as being less strongly repressed by ammonium than methylamine oxidase.     

Biogenesis and turnover of peroxisomes involved in the concurrent oxidation of methanol and methylamine in Hansenula polymorpha

Growth of Hansenula polymorpha in shake flasks and chemostat cultures in the presence of methanol as the sole source of carbon and methylamine as the sole source of nitrogen was associated with the development of peroxisomes in the cells. The organelles were involved in the concurrent oxidation of these two compounds, since they contained both alcohol oxidase and amine oxidase, which are key enzymes in methanol and methylamine metabolism, respectively. In addition catalase was present. Peroxisomes with a completely crystalline substructure were observed in methanol-limited chemostat-grown cells. Amine oxidase probably formed an integral part of these crystalloids, whereas catalase was present in a freely diffusable form. Transfer of cells, grown in a methanol-limited chemostat in the presence of methylamine into glucose/ammonium sulphate media resulted in the loss of both alcohol oxidase and amine oxidase activity from the cells. This process was associated with degradation of the crystalline peroxisomes. However, when cells were transferred into glucose/methylamine media, amine oxidase activity only declined during 2 h after the transfer and thereafter increased again. This subsequent rise in amine oxidase activity was associated with the development of new peroxisomes in the cells in which degradation of the crystalline peroxisomes, originally present, continued. These newly formed organelles probably originated from peroxisomes which had not been affected by degradation. When in the methanollimited chemostat methylamine was replaced by ammonium sulphate, repression of the synthesis of amine oxidase was observed. However, inactivation of this enzyme or degradation of peroxisomes was not detected. The decrease of amine oxidase activity in the culture was accounted for by dilution of enzyme as a result of growth and washout.     

NITROGEN-LIMITED GROWTH RESPONSE OF RUMINAL BACTERIUM SELENOMONAS RUMINANTIUM STRAIN D TO METHYLAMINE ADDITION IN A MINIMAL MEDIUM

Methylamine is often found as a naturally-occurring metabolite in the rumen of sheep and cattle fed a variety of diets. The objective of this study was to examine the effect of methylamine on nitrogen-limited growth of Selenomonas ruminantium strain D grown on a nitrogen-free basal medium that would not support bacterial growth without the addition of a nitrogen source (ammonia, urea, glutamine, cysteine, or glutamate). In this medium S. ruminantium growth rates were significantly faster (P < 0.05) with 11 mM ammonia-nitrogen than any other nitrogen source and were slowest on glutamate-nitrogen (1 and 11 mM). Maximum optical density was greater (P < 0.05) for all nitrogen sources when the respective nitrogen source was increased from 1 mM to 11 mM nitrogen. Addition of 10 mM methylamine significantly decreased (P < 0.05) maximum optical density compared to the respective nitrogen source and concentration without added methylamine for cells grown on 1 mM glutamine-, 1 mM cysteine-, 11 mM ammonia-, 11 mM glutamine-, 11 mM cysteine- or 11 mM glutamate-nitrogen. It appears that ruminal methylamine could decrease extent of growth of ruminal selenomonads depending on the nitrogen source(s) available for nitrogen assimilation.     

Isolation and Characterization of a Mutant of Arthrobacter sp. Strain GLP-1 Which Utilizes the Herbicide Glyphosate as Its Sole Source of Phosphorus and Nitrogen

Arthrobacter sp. strain GLP-1, grown on glucose as a carbon source, utilizes the herbicide glyphosate [N-(phosphonomethyl)glycine] as its sole source of phosphorus as well as its sole source of nitrogen. The mutant strain GLP-1/Nit-1 utilizes glyphosate as its sole source of nitrogen as well. In strain GLP-1, P_i was a potent competitive inhibitor of glyphosate uptake (K_i, 24 μM), while the affinity of P_i for the uptake system of strain GLP-1/Nit-1 was reduced by 2 orders of magnitude (K_i, 2.3 mM). It is concluded that the inability of strain GLP-1 to utilize glyphosate as a source of nitrogen is due to the stringent control of glyphosate uptake by excess phosphate released during the degradation of the herbicide.