The utilization of the mixtures of monosaccharides by some mucorales

Summary The utilization of mixtures of monosaccharides byBlakeslea trispora Thaxter,Choanephora circinans (Naganish &Kawakami)Hesseltine &Benjamin,Gilbertella persicaria var.indica Mehrotra &Mehrotra andHelicostylum piriforme Bainier was studied. The effect of sorbose on the utilization of other sugars present in the mixtures was also studied. It was found that all the mixtures of sugars in combination with asparagine or ammonium chloride were valueless for all the organisms exceptHelicostylum piriforme. Growth ofHelicostylum piriforme on the mixtures with asparagine as the nitrogen source was better than on the mixtures with ammonium chloride as the source of nitrogen. Asparagine being a favourable source counteractecd sorbose inhibition, while ammonium chloride failed to do so. On the other hand, both of the nitrogen sources failed to counteract sorbose inhibition in the rest of the organisms. None of the organisms could finish sorbose and rhamnose from any of the mixtures within the specified period.     

Utilization of nitrate,nitrite and ammonium by Chlamydomonas reinhardii

In phototrophically grown Chlamydomonas cells, ammonium strongly inhibited the utilization of nitrate or nitrite. Under darkness, or in the presence of an uncoupler or inhibitor of the non-cyclic photosynthetic electron flow, the utilization of nitrate, nitrite or ammonium was suppressed. l-Methionine-d,l-sulfoximine (MSX) or azaserine, which blocks the assimilation of ammonium, inhibited the consumption of nitrate, but not nitrite, by the cells. Ammonium produced an immediate inhibition of the permease for nitrate in Chlamydomonas growing with nitrate, while ammonium-grown cells lacked this permease. The synthesis of nitrate-reductase activity was dependent on an active permease. In N-starved Chlamydomonas cells, previously treated with MSX, the permease for nitrate was insensitive to inhibition by ammonium, and a significant amount of nitrate reductase was synthetized. These cells photoproduce ammonium by reducing nitrate. Nitrogen-repleted cells, treated with MSX, actively photoproduced ammonium by reducing nitrite, but not nitrate.Abbreviations DCMU N-(3,4-dichlorophenyl)N,N-di-methyl-urea - PCCP Carbonylcyanid-p-trifluoromethoxy-phenylhydrazone - Mops 2-(N-morpholino)propanesulfonic acid - MSX l-Methionine-d,l-sulfoximine     

Organic and inorganic nitrogen source effects on the metabolism of Clostridium acetobytulicum

Summary The effects of organic and inorganic nitrogen combinations on cell growth, solvent production and nitrogen utilization by Clostridium acetobutylicum ATCC 824 was studied in batch fermentations. Fermentations in media with 10 mM glutamic acid, as the organic nitrogen source, and 0 mM to 10 mM ammonium chloride, as the inorganic nitrogen source had a solvent yield of 0.8 to 1.08 mmol solvent/mmol glucose used, with a slow fermentation rate (2 mmol solvent/l h^-1). When media contained 20 mM or 30 mM glutamic acid as well as 2.5 to 7.5 mM ammonium chloride the fermentation rate increased (5.5 mmol/l h^-1) while the solvent yield remained constant (0.86 to 0.96 mmol solvent/mmol glucose used). Total solvent production was higher in media containing 20 mM or 30 mM glutamic acid than with 10 mM glutamic acid.     

l-Stereoselective amino acid amidase with broad substrate specificity from Brevundimonas diminuta: characterization of a new member of the leucine aminopeptidase family

Brevundimonas diminuta TPU 5720 produces an amidase acting l-stereoselectively on phenylalaninamide. The enzyme (LaaA_Bd) was purified to electrophoretic homogeneity by ammonium sulfate fractionation and four steps of column chromatography. The final preparation gave a single band on SDS-PAGE with a molecular weight of ≈53,000. The native molecular weight of the enzyme was about 288,000 based on gel filtration chromatography, suggesting that the enzyme is active as a homohexamer. It had maximal activity at 50°C and pH 7.5. LaaA_Bd lost its activity almost completely on dialysis against potassium phosphate buffer (pH 7.0), and the amidase activity was largely restored by the addition of Co²⁺ ions. The enzyme was, however, inactivated in the presence of ethylenediaminetetraacetic acid even in the presence of Co²⁺, suggesting that LaaA_Bd is a Co²⁺-dependent enzyme. LaaA_Bd had hydrolyzing activity toward a broad range of l-amino acid amides including l-phenylalaninamide, l-glutaminamide, l-leucinamide, l-methioninamide, l-argininamide, and l-2-aminobutyric acid amide. Using information on the N-terminal amino acid sequence of the enzyme, the gene encoding LaaA_Bd was cloned from the chromosomal DNA of the strain and sequenced. Analysis of 4,446 bp of the cloned DNA revealed the presence of seven open-reading frames (ORFs), one of which (laaA _Bd ) encodes the amidase. LaaA_Bd is composed of 491 amino acid residues (calculated molecular weight 51,127), and the deduced amino acid sequence exhibits significant similarity to that of ORFs encoding hypothetical cytosol aminopeptidases found in the genomes of Caulobacter crescentus, Bradyrhizobium japonicum, Rhodopseudomonas palustris, Mesorhizobium loti, and Agrobacterium tumefaciens, and leucine aminopeptidases, PepA, from Rickettsia prowazekii, Pseudomonas putida ATCC 12633, and Escherichia coli K-12. The laaA _Bd gene modified in the nucleotide sequence upstream from its start codon was overexpressed in an E. coli transformant. The activity of the recombinant LaaA_Bd in cell-free extracts of the E. coli transformant was 25.9 units mg⁻¹ with l-phenylalaninamide as substrate, which was 50 times higher than that of B. diminuta TPU 5720.     

Purification and properties of glutamine synthetase from Nocardia asteroides

Glutamine synthetase (GS, EC 6.3.1.2) from Nocardia asteroides was purified to homogeneity by ammonium sulfate precipitation, Sephadex G-150, and DEAE-Sepharose chromatography. The native molecular weight of the purified enzyme was determined to be 720 kDa. SDS-PAGE analysis of the purified preparation revealed a single band corresponding to 59 kDa, indicating the possible presence of 12 identical subunits. The divalent cations Mn^2- and Mg²⁺ were found to be essential for optimal transferase and biosynthetic activity, respectively. The optimal pH and temperature for both activities of the enzyme were found to be 7.2 and 50°C. Amino acids such as l-alanine, glycine, and aspartate inhibited the GS activity. The K _m values for the substrates of the biosynthetic reaction ATP, glutamate, and ammonium chloride were found to be 400 m, 7.7mm, and 200 m, respectively. Addition of ammonium chloride to the nitrogen-limited culture resulted in a decrease of GS transferase and biosynthetic activities. Phosphodiesterase treatment of the extract from ammonia-shocked cultures showed an increase in GS transferase activity. The results indicate the possible regulation of GS by covalent modification.     

Short-term ammonium inhibition of nitrate utilization by Anacystis nidulans and other cyanobacteria

Ammonium at low concentrations caused a rapid and effective inhibition of nitrate utilization in the light by the cyanobacterium Anacystis nidulans without affecting the cellular level of nitrate reductase activity. The inhibition was reversible, and the ability of the cells to utilize nitrate was restored immediately after ammonium had been exhausted. The inhibitory effect was dependent on consumption by the cells of the added ammonium which was rapidly incorporated into amino acids. In the presence of L-methionine-d,l-sulfoximine (MSX) or azaserine, inhibitors of the glutamine synthetase-glutamate synthase pathway, ammonium did not exhibit any inhibitory effect on nitrate utilization. Ammonium assimilation, rather than ammonium itself, seems to regulate nitrate utilization in A. nidulans. Short-term inhibition by ammonium of nitrate utilization and its prevention by MSX were also demonstrated in the filamentous cyanobacteria Anabaena and Nostoc.Abbreviations MSX L-Methionine-d-l-sulfoximine     

NADH-dependent reduction of d-proline in Clostridium sticklandii. Reconstitution from three fractions containing NADH dehydrogenase,d-proline reductase,and a third protein factor

The enzyme system from Clostridium sticklandii catalyzing the NADH-dependent reduction of d-proline was co-purified by chromatography on DEAE-cellulose at pH 8.2 and ammonium sulfate fractionation, and resolved into fractions containing three different protein components, NADH dehydrogenase, d-proline reductase and a third protein factor, by chromatography on DEAE-cellulose at pH 7.0. Upon recombination of the fractions containing the three different protein components, the NADH-dependent reduction of d-proline was successfully reconstituted. The NADH dehydrogenase fractions oxidized NADH in the presence of artificial electron acceptors, and were inhibited by p-hydroxymercuriphenylsulfonate (50% at 80 nM). They contained 3–4 different enzyme bands as revealed by polyacrylamide-gel electropherograms stained with the NADH-dependent reduction of 2,3,5-triphenyltetrazolium chloride. d-Proline reduction was also coupled to a leuco-methylene blue-generating system containing d-glucose and glucose-oxidase (EC 1.1.3.4). Circumstantial evidence indicated that, among the clostridial proteins, only d-proline reductase and the third protein factor were needed for this reaction.Non-standard abbreviations TTC 2,3,5-triphenyltetrazolium chloride     

Regulation of α-aminoadipyl-cysteinyl-valine,isopenicillin N synthetase,isopenicillin N isomerase and deacetoxycephalosporin C synthetase by nitrogen sources in Streptomyces lactamdurans

Summary Ammonium and asparagine produced a concentration-dependent reduction of cephamycin C biosynthesis by Streptomyces lactamdurans. Addition of ammonium salts at 1 mM concentration reduced cephamycin biosynthesis by resting cells of S. lactamdurans, whereas concentrations of asparagine above 10 mM were required to get the same effect. High ammonium concentrations decreased glutamine synthetase activity in cell extracts of S. lactamdurans in parallel to the reduction of antibiotic biosynthesis. Ammonium supplementation decreased the pool of glutamic acid and glutamine whereas the intracellular content of ammonium, alanine, and phosphoserine increased significantly. The pool of the tripeptide (l--aminoadipyl)-l-cysteinyl-d-valine, an intermediate in cephamycin biosynthesis, was greatly reduced in ammonium-supplemented cultures. Isopenicillin N synthetase, that converts the tripeptide (l--aminoadipyl)-l-cysteinyl-d-valine to isopenicillin N, isopenicillin N isomerase (that isomerises isopenicillin N to penicillin N) and deacetoxycephalosporin C synthetase (converting penicillin N into deacetoxycephalosporin C) were also reduced in ammonium-supplemented cultures. However, the activities of these enzymes were not inhibited in vitro by 40 mM ammonium, suggesting that the enzymes were repressed but not inhibited by ammonium in vivo.     

Nitrogen requirements of certain isolates of alternaria tenuis auct

Three isolates ofA. tenuis isolated from the diseased leaves ofMangifera indica l. Musa paradisiaca l. andPsidium guajava l. were investigated. They were grown on different sources of nitrogen viz., potassium nitrate, sodium nitrate, calcium nitrate, ammonium nitrate, sodium nitrite, ammonium sulphate, ammonium chloride, glycine, DL-valine, L-glutamic acid, urea, thiourea, L-asparagine and peptone. They were also grown on the medium lacking nitrogen. A wide variation was observed in the growth and reproduction of the different isolates. The growth of all of them was good on potassium nitrate, calcium nitrate, glycine, DL-valine, L-glutamic acid, L-asparagine and peptone but the sporulation was satisfactory on calcium nitrate only. Sodium nitrite supported moderate growth of banana leaf isolate whereas there was no growth of the other two isolates. None of the organisms could grow on the medium lacking nitrogen as well as on thiourea. The results obtained with the isolates under study have been compared with those of earlier investigators and it has been clearly established that the different isolates ofA. tenuis could show marked differences in their nitrogen requirements.     

Ionic and non-ionic compounds in the germination of spores of bacillus megaterium texas

Summary Germination requirements of suspensions of spores of Bacillus megaterium, Texas strain, an l-alanine-inosine type, have been examined employing a decrease in optical density as the criterion of germination. In deionized water, l-alanine and inosine were devoid of germinative powers. They were effective only in conjunction with any one of a large variety of salts. Data are given for germination by the monovalent and divalent alkali metal chlorides. The potassium halides were germinative; potassium fluoride was the best. Salts of organic acids, including fatty acids and polycarboxylic acids, were germinative. The need for inosine could be bypassed by various salts, e.g., ammonium propionate or salts of dipicolinic acid. Also, l-alanine was replaceable by a variety of amino acids, provided suitable ions were present. In the presence of magnesium chloride, sodium dipicolinate could substitute for either inosine or l-alanine, but not both. Salts of n-hexylamine and n-heptylamine bypassed the need for both l-alanine and inosine. A primary role for ions in germination is proposed and a secondary, augmentative action is attributed to l-alanine and inosine.     

Nitrogen and sulphur requirements of Colletotrichum inamdarii Lal

Summary Nitrogen and sulphur requirements ofColletotrichum inamdarii Lal isolated from the leaves ofCarissa carandas L. have been studied. DL-serine, L-asparagine and L-phenylalanine have been found to be of good nitrogen source followed by potassium nitrate, calcium nitrate, magnesium nitrate, DL-alanine, ammonium nitrate, glutamic acid, ammonium sulphate, DL-valine, aspartic acid, ammonium chloride, ammonium hydrogencarbonate, L-histidine and potassium nitrite. There was no growth in the absence of nitrogen.Sporulation was excellent on calcium nitrate and sodium nitrate, Very good on DL-serine, potassium nitrate, and magnesium nitrate. Good on L-asparagine, L-phenylalanine and ammonium oxalate. Fair on DL-alanine, DL-leucine, ammonium sulphate, DL-valine, ammonium chloride and L-histidine whereas poor on glutamic acid, aspartic acid, ammonium tartarate and ammonium nitrate. Few spores were observed on ammonium hydrogencarbonate but potassium nitrite did not show any sporulation.Amongst the sulphur compounds sodium bisulphate gave the best growth and good sporulation, followed by sodium thiosulphate, magnesium sulphate, ammonium sulphate and potassium sulphate. Thiourea gave negligible growth whereas it failed to grow on zinc sulphate and potassium persulphate.     

Modulation of different forms of glutamine synthetases inRhizobium phaseoli and their possible role in nitrogen fixation

The effect of a number of compounds structurally related to glutamic acid and other nitrogenous compounds on the composition of three forms of glutamine synthetase (GS) inRhizobium phaseoli has been examined in detail. Amino acids like glutamic acid, glutamine, and a fixed source of nitrogen like ammonium chloride did not alter the relative glutamine synthetase composition.l-Methioninedl-sulfoximine (MSX), a glutamate analogue, significantly repressed the synthesis of GSIII to a greater extent.,N-oxalyl,-diaminopropionic acid (ODAP), another glutamate analogue, selectively stimulated the synthesis of GSII, and the effect of ODAP on GSII synthesis was greatly enhanced in the presence of ethylenediamine or ammonium chloride. Ethylenediamine itself caused a predominant synthesis of GSIII.-Cyanoalanine-grownR. phaseoli did not synthesize GSI. The synthesis of the three different glutamine synthetases can thus be differentially modulated.     

Biosynthesis of 5-(p-aminophenyl)-1,2,3,4-tetrahydroxypentane by methanogenic bacteria

In an attempt to establish the nature of the ammonium-assimilation products which mediate the inhibition by ammonium of nitrate uptake in cyanobacteria, the effect of different amino acids on nitrate utilization by intact Anacystis nidulans cells has been assayed. To exclude an indirect inhibition of nitrate uptake through the ammonium which the amino acids might release, the cells were pretreated with l-methionine-d,l-sulfoximine (MSX), a potent inactivator of glutamine synthetase. Under these conditions, several l-amino acids, but not the corresponding d-isomers, affected nitrate utilization to a variable extent, causing inhibitions ranging between 20 and 80% when added at 20 mM concentration.For most of the inhibitory amino acids, including l-isoleucine, l-leucine and l-valine, a correlation was found between their ability to act as amino group donors to -ketoglutarate, in reactions catalyzed by A. nidulans cell-free extracts, and their inhibitory effect on nitrate utilization. l-Glutamine, l-asparagine and glycine, being effective inhibitors of nitrate utilization, were poor substrates for the transaminating activity to -ketoglutarate, however. The possible role of the latter amino acids as mediators in the ammonium-promoted inhibition of nitrate uptake is discussed.Abbreviations MSX l-methionine-d,l-sulfoximine - MTA-5 mixed alkyltrimethylammonium bromide - Mops morpholinopropane sulfonic acid     

Production of l-glutamic acid by aBacillus sP

A strain ofBacillus cereus var.mycoides isolated from Burdwan soil producesl-glutamate in the medium. The strain is able to grow and produce in a synthetic medium but supplementation with casamino acid or yeast extract improves the yield. Maintenance of pH of the fermentation medium near neutrality prolongs the active growth period and improves the yield. Glucose and ammonium nitrate were found to be most suitable carbon and nitrogen sources, respectively. Cane sugar molasses (as a substitute for glucose) significantly stimulated the growth but glutamate production was less. Various B vitamins stimulate the growth and glutamate yield. The yield of glutamate under optimal condition is 5.2 g/l.     

Isolation and characterization of a marine methanogenic bacterium from the biofilm of a shiphull in Los Angeles harbor

A marine mesophilic, irregular coccoid methanogen, which shows close resemblance toMethanococcus sp., was isolated from the biofilm of shiphulls docked in Los Angeles harbor. Hydrogen plus carbon dioxide or formate served as substrates for methanogenesis in a mineral salt medium. The isolate did not use acetate and methanol as sole source of carbon and energy. The organism had an optimal pH range of 6.8–7.0 and a temperature optimum of 37°C. Elevated levels of sodium chloride were required for optimum growth. Optimum levels of total sulfide and magnesium chloride for growth were 1.0mm and 10mm respectively. The isolate used ammonia as nitrogen source. The concentration of 30mm ammonium chloride supported maximum growth of the isolate.     

Transport of l(-)malic acid and other dicarboxylic acids in the yeast Candida sphaerica

Summary dl-Malic acid grown cells of Candida sphaerica (anamorph of Kluyveromyces marxianus) formed a saturable transport system that mediated accumulative transport of l(-)malic acid with the following kinetic parameters at pH 5.0: V _max, 0.44 nmol l(-)malate·s^-1 per milligram dry weight; K _m ,0.1 mM l(-)malate. Initial uptake of the acid was accompanied by disappearance of extracellular protons, the rates of which followed Michaelis-Menten kinetics as a function of the acid concentration. Variation with extracellular pH of the K _m values, calculated either as the concentrations of anions or of undissociated acid, pointed to anions as the transported form. Furthermore, accumulated free acid suffered rapid efflux after the addition of the protonophore carbonylcyanide-M-chlorophenyl-hydrazone (CCCP). These results suggested that the transport system was a dicarboxylate-proton symporter. The system was inducible and was subject to glucose repression. Succinic, fumaric, -ketoglutaric, oxaloacetic and d-malic acid, but not maleic, malonic, oxalic nor l(+)-tartaric acid, apparently used the same transport system since they acted as competitive inhibitors of l(-)malic acid transport and induced proton movements that followed Michaelis-Menten kinetics. Experiments with glucose-repressed cells showed that undissociated dicarboxylic acid (measured with labelled succinic acid) entered the cells slowly by simple diffusion. The permeability of the cells for undissociated acid increased exponentially with pH, the diffusion constant increasing 100-fold between pH 3.5 and 6.0.     

Engineering of an <Emphasis Type="SmallCaps">l</Emphasis>-arabinose metabolic pathway in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Corynebacterium glutamicum was metabolically engineered to broaden its substrate utilization range to include the pentose sugar l-arabinose, a product of the degradation of lignocellulosic biomass. The resultant CRA1 recombinant strain expressed the Escherichia coli genes araA, araB, and araD encoding l-arabinose isomerase, l-ribulokinase, and l-ribulose-5-phosphate 4-epimerase, respectively, under the control of a constitutive promoter. Unlike the wild-type strain, CRA1 was able to grow on mineral salts medium containing l-arabinose as the sole carbon and energy source. The three cloned genes were expressed to the same levels whether cells were cultured in the presence of d-glucose or l-arabinose. Under oxygen deprivation and with l-arabinose as the sole carbon and energy source, strain CRA1 carbon flow was redirected to produce up to 40, 37, and 11%, respectively, of the theoretical yields of succinic, lactic, and acetic acids. Using a sugar mixture containing 5% d-glucose and 1% l-arabinose under oxygen deprivation, CRA1 cells metabolized l-arabinose at a constant rate, resulting in combined organic acids yield based on the amount of sugar mixture consumed after d-glucose depletion (83%) that was comparable to that before d-glucose depletion (89%). Strain CRA1 is, therefore, able to utilize l-arabinose as a substrate for organic acid production even in the presence of d-glucose.     

Extracellular deamination of l-amino acids by Chlamydomonas reinhardtii cells

When grown in the light and in a Tris-acetate phosphate medium, cells of Chlamydomonas reinhardtii Dang. can use the following l-amino acids as a sole nitrogen source: asparagine, glutamine, arginine, lysine, alanine, valine, leucine, isoleucine, serine, methionine, histidine, and phenylalanine, whereas, in the absence of acetate, the cells only used l-arginine. The utilization system in the acetate medium consisted of an extracellular deaminating activity induced by l-amino acids; it took between 10 to 30 h before the system appeared in cells previously grown with ammonium. This deaminase activity was nonspecific, required an organic carbon source for its de-novo synthesis, and was sensitive to high ammonium concentration and light deprivation.Abbreviations HPLC high-performance liquid chromatography - TAP Tris-acetate-phosphate This work was supported by a grant of the CAICYT, Spain. The secretarial assistance of C. Santos and I. Molina is gratefully acknowledged.To whom correspondence should be addressed.     

Studies on Aspergilli XVI. Effect of pH,temperature, and carbon and nitrogen interaction

Summary The effect of pH, temperature, and carbon and nitrogen interaction on the growth and sporulation ofAspergillus nidulans (Eidam)Wint.,A. rugulosus Thom &Raper,A. variecolor (Berk. &Br.)Thom &Raper andA. quadrilineatus was studied. All the moulds could grow on a wide range of pH (2.0 to 12.0) but the growth was poor on too acid and too alkaline media. Best growth ofA. rugulosus, A. quadrilineatus, andA. violaceus was seen at pH 6.5 and that ofA. nidulans andA. variecolor at pH 7.0. In general maximum production of perithecia was recorded between pH 6.0 and 8.0.All the above species ofAspergillus under study could grow between a temperature range of 10° C–48° C, but the growth was poor at 10° C and 48° C. The present moulds showed good growth at 20° C, 25°C, and 30° C. At 40° CA. nidulans andA. rugulosus showed moderate growth while the rest of the Aspergilli attained good growth. Temperatures between 20° C–30° C favoured excellent perithecial production.In general, little improvement in growth was noted on media containing good carbon and nitrogen sources. Malic acid was found to be useless when supplied singly. But, poor growth was recorded when supplied in combination with amino acids, amide, and peptone. This was due to the fact that these N sources also supplied carbon for their metabolism.     

Futile cycling of ammonium ions via the high affinity potassium uptake system (Kdp) of Escherichia coli

Escherichia coli Frag1 was grown under various nutrient limitations in chemostat culture at a fixed temperature, dilution rate and pH both in the presence and the absence of a high concentration of ammonium ions by using either ammonium chloride or dl-alanine as the sole nitrogen source. The presence of high concentrations of ammonium ions in the extracellular fluids of potassium-limited cultures of E. coli Frag1 caused an increase of the specific rate of oxygen consumption of these cultures. In contrast, under phosphate-, sulphate- or magnesium-limited growth conditions no such increase could be observed. The presence of high concentrations of ammonium ions in potassium-limited cultures of E. coli Frag5, a mutant strain of E. coli Frag1 which lacks the high affinity potassium uptake system (Kdp), did not increase the specific rate of oxygen consumption.These results indicate that ammonium ions, very similar to potassium ions both in charge and size, are transported via the K dp leading to a futile cycle of ammonium ions and ammonia molecules (plus protons) across the cytoplasmic membrane. Both the uptake of ammonium ions and the extrusion of protons would increase the energy requirement of the cells and therefore increase their specific rate of oxygen consumption. The involvement of a (methyl)ammonium transport system in this futile cycle could be excluded.