Pan-genome analysis identifies intersecting roles for Pseudomonas specialized metabolites in potato pathogen inhibition.

Agricultural soil harbors a diverse microbiome that can form beneficial relationships with plants, including the inhibition of plant pathogens. Pseudomonas spp. are one of the most abundant bacterial genera in the soil and rhizosphere and play important roles in promoting plant health. However, the genetic determinants of this beneficial activity are only partially understood. Here, we genetically and phenotypically characterize the Pseudomonas fluorescens population in a commercial potato field, where we identify strong correlations between specialized metabolite biosynthesis and antagonism of the potato pathogens Streptomyces scabies and Phytophthora infestans. Genetic and chemical analyses identified hydrogen cyanide and cyclic lipopeptides as key specialized metabolites associated with S. scabies inhibition, which was supported by in planta biocontrol experiments. We show that a single potato field contains a hugely diverse and dynamic population of Pseudomonas bacteria, whose capacity to produce specialized metabolites is shaped both by plant colonization and defined environmental inputs.

Potato scab and blight are two major diseases which can cause heavy crop losses. They are caused, respectively, by the bacterium Streptomyces scabies and an oomycete (a fungus-like organism) known as Phytophthora infestans. Fighting these disease-causing microorganisms can involve crop management techniques ­ for example, ensuring that a field is well irrigated helps to keep S. scabies at bay. Harnessing biological control agents can also offer ways to control disease while respecting the environment. Biocontrol bacteria, such as Pseudomonas, can produce compounds that keep S. scabies and P. infestans in check. However, the identity of these molecules and how irrigation can influence Pseudomonas population remains unknown. To examine these questions, Pacheco-Moreno et al. sampled and isolated hundreds of Pseudomonas strains from a commercial potato field, closely examining the genomes of 69 of these. Comparing the genetic information of strains based on whether they could control the growth of S. scabies revealed that compounds known as cyclic lipopeptides are key to controlling the growth of S. scabies and P. infestans. Whether the field was irrigated also had a large impact on the strains forming the Pseudomonas population. Working out how Pseudomonas bacteria block disease could speed up the search for biological control agents. The approach developed by Pacheco-Moreno et al. could help to predict which strains might be most effective based on their genetic features. Similar experiments could also work for other combinations of plants and diseases.

Plant growth promoting Pseudomonas aeruginosa from Valeriana wallichii displays antagonistic potential against three phytopathogenic fungi.

The soil nature and characterstics are directly related to the micro-organisms present, bio-mineralization process, plant type and thus having harmonius and interdependent relationships. Soil bacteria having antagonistic activity against phytopathogens, play an important role in root growth, overall plant growth and also their composition depends upon the plant species. Population explosion across globe has resulted in indiscriminate use of chemical fertilizers, fungicides and pesticides, thus posing serious risk to plant productivity and soil flora. Plant growth promoting rhizobacteria (PGPRs) are considered safer than chemical fertilizers as they are eco-friendly and sustain longer after colonization in rhizospheric soil. PGPRs are preferred as a green choice and acts as a superior biocontrol agents against phytopathogens. In the present study, a potential rhizobacteria, Pseudomonas aeruginosa (isolate-2) was isolated from the rhizosphere of a medicinal plant, Valeriana wallichi. The bacterial isolate exhibited qualitative tests for plant growth promoting determinatives. It was also subjected to in-vitro biocontrol activity against potential phytopathogens viz. Alternaria alternata, Aspergillus flavus and F. oxysporum. The antagonistic efficacy against F. oxysporum was 56.2% followed by Alternaria alternata to be 51.02%. The maximum inhibition of radial growth of F. oxysporum was 69.2%, Alternaria alternata (46.4%) and Aspergillus flavus (15%). The Pseudomonas aeruginosa exhibited plant growth promotion rhizobacterial activity which can be expoited as biofertilizers. This study deals with microbial revitalization strategy and offers promising solution as a biocontrol agent to enhance crop yield. Further, PGPRs research using the interdisciplinary approaches like biotechnology, nanotechnology etc. will unravel the molecular mechanisms which may be helpful for maximizing its potential in sustainable agriculture.

Investigating the formulation of alginate- gelatin encapsulated Pseudomonas fluorescens (VUPF5 and T17-4 strains) for controlling Fusarium solani on potato.

Nanotechnology is one of the most fascinating sciences with a great potential to improve many agricultural products. Use of nanoparticles in plant disease management is a novel area which may prove very effective in future. Use of nanomaterials and biocompatible compounds in nano-encapsulation of antagonist bacteria is an important step in enhancing the efficiency of these agents in adverse environmental conditions. Two strains of Pseudomonas fluorescens (VUPF5 and T17-4) were used for alginate-gelatin nanocomposite beads with different concentrations of gelatin. The moisture content, swelling, and releasing of encapsulated viable bacteria was investigated in vitro and in vivo conditions. The results of FT-IR and X-ray diffraction analysis revealed that when gelatin was added into sodium alginate, electrostatic interaction occurred. The swelling and moisture content of nanocomposite beads grew with gelatin enhancement. The maximum encapsulation efficiency at the gelatin concentration of 1.5% in VUPF5 and T17-4 was 91.23% and 87.23%, respectively. Further, the greenhouse experiment showed that inoculation of potato with bacterial strains and nanocomposite beads of these strains reduced disease incidence. The encapsulation method described in this study can be effectively used to protect the plant probiotic bacteria inoculum from harmful conditions of the soil for its successful establishment in the rhizosphere.

Pharmacological treatment of inhalation injury after nuclear or radiological incidents: The Chinese and German approach.

Inhalation injury is often associated with burns and significantly increases morbidity and mortality. The main toxic components of fire smoke are carbon monoxide, hydrogen cyanide, and irritants. In the case of an incident at a nuclear power plant or recycling facility associated with fire, smoke may also contain radioactive material. Medical treatments may vary in different countries, and in this paper, we discuss the similarities and differences in the treatments between China and Germany. Carbon monoxide poisoning is treated by 100% oxygen administration and, if available, hyperbaric oxygenation in China as well as in Germany. In addition, antidotes binding the cyanide ions and relieving the respiratory chain are important. Methemoglobin-forming agents (e.g., nitrites, dimethylaminophenol) or hydroxocobalamin (Vitamin B12) are options. The metabolic elimination of cyanide may be enhanced by sodium thiosulfate. In China, sodium nitrite with sodium thiosulfate is the most common combination. The use of dimethylaminophenol instead of sodium nitrite is typical for Germany, and hydroxocobalamin is considered the antidote of choice if available in cases of cyanide intoxications by fire smoke inhalation as it does not further reduce oxygen transport capacity. Systematic prophylactic use of corticosteroids to prevent toxic pulmonary edema is not recommended in China or Germany. Stable iodine is indicated in the case of radioiodine exposure and must be administered within several hours to be effective. The decorporation of metal radionuclides is possible with Ca (DTPA) or Prussian blue that should be given as soon as possible. These medications are used in both countries, but it seems that Ca (DTPA) is administered at lower dosages in China. Although the details of the treatment of inhalation injury and radionuclide(s) decorporation may vary, the general therapeutic strategy is very similar in China and Germany.

Platinum and rhenium extraction from a spent refinery catalyst using Bacillus megaterium as a cyanogenic bacterium: statistical modeling and process optimization.

The present study evaluated the potential of Bacillus megaterium as a cyanogenic bacterium to produce cyanide for solubilization of platinum and rhenium from a spent refinery catalyst. Response surface methodology was applied to study the effects and interaction between two main effective parameters including initial glycine concentration and pulp density. Maximum Pt and Re recovery was obtained 15.7% and 98%, respectively, under optimum conditions of 12.8 g/l initial glycine concentration and 4% (w/v) pulp density after 7 days. Increasing the free cyanide concentration to 3.6 mg/l, varying the pH from 6.7 to 9, and increasing the dissolved oxygen from 2 to 5mg/l demonstrated the growth characteristics of B. megaterium during bioleaching process. The modified shrinking core model was used to determine the rate limiting step of the process. It was found that diffusion through the product layer is the rate controlling step.

Age versus stage: does ontogeny modify the effect of phosphorus and arbuscular mycorrhizas on above- and below-ground defence in forage sorghum?

Arbuscular mycorrhizas (AM) can increase plant acquisition of P and N. No published studies have investigated the impact of P and AM on the allocation of N to the plant defence, cyanogenic glucosides. We investigated the effects of soil P and AM on cyanogenic glucoside (dhurrin) concentration in roots and shoots of two forage sorghum lines differing in cyanogenic potential (HCNp). Two harvest times allowed plants grown at high and low P to be compared at the same age and the same size, to take account of known ontogenetic changes in shoot HCNp. P responses were dependent on ontogeny and tissue type. At the same age, P-limited plants were smaller and had higher shoot HCNp but lower root HCNp. Ontogenetically controlled comparisons showed a P effect of lesser magnitude, and that there was also an increase in the allocation of N to dhurrin in shoots of P-limited plants. Colonization by AM had little effect on shoot HCNp, but increased root HCNp and the allocation of N to dhurrin in roots. Divergent responses of roots and shoots to P, AM and with ontogeny demonstrate the importance of broadening the predominantly foliar focus of plant defence studies/theory, and of ontogenetically controlled comparisons.

Protozoa Drive the Dynamics of Culturable Biocontrol Bacterial Communities.

Some soil bacteria protect plants against soil-borne diseases by producing toxic secondary metabolites. Such beneficial biocontrol bacteria can be used in agricultural systems as alternative to agrochemicals. The broad spectrum toxins responsible for plant protection also inhibit predation by protozoa and nematodes, the main consumers of bacteria in soil. Therefore, predation pressure may favour biocontrol bacteria and contribute to plant health. We analyzed the effect of Acanthamoeba castellanii on semi-natural soil bacterial communities in a microcosm experiment. We determined the frequency of culturable bacteria carrying genes responsible for the production of the antifungal compounds 2,4-diacetylphloroglucinol (DAPG), pyrrolnitrin (PRN) and hydrogen cyanide (HCN) in presence and absence of A. castellanii. We then measured if amoebae affected soil suppressiveness in a bioassay with sugar beet seedlings confronted to the fungal pathogen Rhizoctonia solani. Amoebae increased the frequency of both DAPG and HCN positive bacteria in later plant growth phases (2 and 3 weeks), as well as the average number of biocontrol genes per bacterium. The abundance of DAPG positive bacteria correlated with disease suppression, suggesting that their promotion by amoebae may enhance soil health. However, the net effect of amoebae on soil suppressiveness was neutral to slightly negative, possibly because amoebae slow down the establishment of biocontrol bacteria on the recently emerged seedlings used in the assay. The results indicate that microfaunal predators foster biocontrol bacterial communities. Understanding interactions between biocontrol bacteria and their predators may thus help developing environmentally friendly management practices of agricultural systems.

Pesticide tolerant and phosphorus solubilizing Pseudomonas sp. strain SGRAJ09 isolated from pesticides treated Achillea clavennae rhizosphere soil.

In this study, an attempt was made to identify an effective phosphate solubilizing bacteria from pesticide polluted field soil. Based on the formation of solubilization halo on Pikovskaya's agar, six isolates were selected and screened for pesticide tolerance and phosphate (P) solubilization ability through liquid assay. The results showed that only one strain (SGRAJ09) obtained from Achillea clavennae was found to tolerate maximum level of the pesticides tested and it was phylogenetically identified as Pseudomonas sp. It possessed a wide range of pesticide tolerance, ranging from 117 µg mL(-1) for alphamethrin to 2,600 µg mL(-1) for endosulfan. The available P concentrations increased with the maximum and double the maximum dose of monocrotophos and imidacloprid, respectively. On subjected to FT-IR and HPLC analysis, the presence of organic acids functional group in the culture broth and the production of gluconic acid as dominant acid aiding the P solubilization were identified. On comparison with control broth, monocrotophos and imidacloprid added culture broth showed quantitatively high organic acids production. In addition to gluconic acid production, citric and acetic acids were also observed in the pesticide amended broth. Furthermore, the Pseudomonas sp. strain SGRAJ09 possessed all the plant growth promoting traits tested. In presence of monocrotophos and imidacloprid, its plant growth promoting activities were lower than that of the pesticides unamended treatment.

Effects of cyanogenic plants on fitness in two host strains of the fall armyworm (Spodoptera frugiperda).

The generalist moth, Spodoptera frugiperda (J. E. Smith) consists of two genetic subgroups (host strains) that differ in their distribution among host plant species. The corn strain prefers crop plants such as corn, sorghum, and cotton, while the rice strain is found in small grasses such as Cynodon spp. and rice. Little is known about the physiological factors that drive this host preference. Here, we report a feeding study with natural host plants and an artificial diet containing cyanide. We found that corn, two Cynodon spp. (bermudagrass C. dactylon (L.) Persoon, 'NuMex Sahara', and stargrass C. nlemfuensis var. nlemfuensis Vanderyst, 'Florona'), and a hybrid between bermudagrass and stargrass, 'Tifton 85', exhibited differences in the concentration of the cyanogenic precursors or cyanogenic potential (HCNp) and the release of hydrogen cyanide per unit time or cyanogenic capacity (HCNc). Corn plants released low levels of hydrogen cyanide, while stargrass had greater HCNp/HCNc than bermudagrass and 'Tifton 85'. Feeding studies showed that corn strain larvae experienced higher mortality than the rice strain when fed stargrass or artificial diet supplemented with cyanide. Also, corn strain larvae excreted higher levels of cyanogenic compounds than the rice strain when fed Cynodon spp. These differences in excretion suggest potential disparities in cyanide metabolism between the two strains. We hypothesize that differences in the susceptibility to cyanide levels in various host plants could play a role in driving strain divergence and what appears to be the incipient speciation of this moth.

Pro-inflammatory activity in rats of thiocyanate, a metabolite of the hydrocyanic acid inhaled from tobacco smoke.

OBJECTIVE: To seek a mechanism linking tobacco smoking with the increased incidence and severity of rheumatoid arthritis, deduced from many retrospective surveys, by studying arthritis/fibrosis development in rats. METHODS: Rats (>300) received low levels of sodium/potassium thiocyanate (10 or 25 mmol/l) in their drinking water to raise their blood thiocyanate levels, mimicking the elevated levels of blood, salivary and urinary thiocyanate found in smokers. RESULTS: Thiocyanate supplements increased the severity of experimental arthritis induced by tailbase injection of (1) Freund's complete adjuvants (mycobacteria plus various adjuvant-active oils), (2) collagen type-II with Freund's incomplete adjuvant (no mycobacteria), (3) the synthetic lipid amine, avridine in an oil and (4) the natural hydrocarbons squalene (C(30)H(50)) and pristane (C(19)H(40)). This pro-arthritic effect was independent of sex, rat strain or changing diet and housing facilities. Thiocyanate supplements also amplified the acute/persisting inflammatory responses to paw injections of pristane, zymosan and microcrystalline hydroxyapatite. Iodide salts also mimicked some of these effects of thiocyanate. CONCLUSION: Thiocyanate, a detoxication product of HCN present in tobacco smoke, increased (or even induced) inflammatory responses to several agents causing arthritis or fibrotic inflammation in rats. It, therefore, can act as a co-arthritigen, or 'virulence factor' and could be a therapeutic target to reduce arthritis expression and morbidity.

Chromium reduction, plant growth-promoting potentials, and metal solubilizatrion by Bacillus sp. isolated from alluvial soil.

The plant growth-promoting potentials, production of siderophore and solubilization of insoluble phosphorus (P) and zinc and lead by the chromium (vi) -reducing Bacillus species, PSB 1, PSB 7, and PSB 10, was assessed both in the presence and absence of chromium under in vitro conditions. The Bacillus strains tolerated chromium up to the concentration of 500 (PSB1), 400 (PSB7), and 550 microg ml(-1) (PSB10), respectively, on nutrient agar plates. Bacillus sp. PSB 10 reduced Cr (vi) by 87% at pH 7, which was followed by Bacillus sp. PSB 1 (83%) and PSB 7 (74%) in nutrient broth after 120 h of incubation. A concentration of 50 microg ml(-1) of Cr (vi) was completely reduced by Bacillus sp. PSB 1 and PSB 10 (after 100 h) and PSB 7 (after 120 h). The Bacillus strains PSB 1, PSB 7, and PSB 10 produced 19.3, 17.7, and 17.4 microg ml(-1) of indole acetic acid, respectively, in luria bertani broth at 100 microg ml(-1) of tryptophan, which consistently decreased with an increase in chromium concentration. The Bacillus strains were positive for siderophore, HCN, and ammonia both in the absence and presence of chromium. The Bacillus strains solubilized 375 (PSB 1), 340 (PSB 7), and 379 (PSB 10) microg ml(-1) P, respectively, in Pikovskaya broth devoid of chromium. In contrast, chromium at 150 microg ml(-1) reduced the amount of P solubilized by 17 (PSB 1), 15 (PSB 7), and 9% (PSB 10) compared to control. The tested bacterial strains solubilized a considerable amount of zinc and lead in nutrient broth both in the absence and presence of chromium. Generally, the chromium reduction and the plant growth-promoting potentials of chromium-reducing Bacillus were strongly correlated at the tested concentration of chromium. The present observations demonstrated that the chromium-reducing, metal-solubilizing, and plant growth-promoting potentials of the Bacillus strains PSB1, PSB 7, and PSB10 were not adversely affected by the chromium application and, hence, may be applied for raising the productivity of crops under metal-contaminated soils.

Glass distilling collector applied for HCN recovery from submerged culture broth and fruiting body of Pleurotus eryngii for identification and quantification.

Detection and surveillance of food commodities containing cyanide is a crucial issue of food safety. In this study, five strains of Pleurotus eryngii (P. eryngii) were grown in submerged culture of yeast malt broth (YMB) with the suspected production of HCN. A safety-warranted U-bent glass distilling collector with three enlarged bulbs on each arm was designed to recover the broth vapor. When AgNO(3) solution was used as an absorbent to interact with the vapor, a white precipitate was formed. The precipitate was isolated and identified as AgCN by FT-Raman spectroscopic analysis. When the absorbent was substituted by KOH, after evaporation to dryness, dissolved in D(2)O, and followed by (13)C-NMR analysis, a KCN spectrum was achieved. Formation of AgCN and KCN confirmed HCN production in the broth by P. eryngii. When a sodium picrate solution (1.4%) was used as an absorbent and various authentic KCN solutions were applied for distillation and followed by absorbance determination at 510 nm, a linear dose-dependent relationship was obtained and the procedure was applied for HCN quantification of the marketed P. eryngii mushrooms (fruiting body). As estimated, 67.3% of the products contained HCN less than 1.0 mg/kg, 17.3% between 1.0 and 2.0 mg/kg, and 15.4% higher than 2.0 mg/kg. When the mushrooms were sliced and cooked in water at 95 degrees C for 6 min, 89.1% of the original HCN was lost. When the P. eryngii strains were respectively grown by submerged cultivation in YMB or YMB supplemented with 2.5% glycine for 16 days, HCN content was slightly higher in the latter than in the former for each strain.

Characterization of a new Pseudomonas aeruginosa strain NJ-15 as a potential biocontrol agent.

Phylogenetic characterization of soil isolate NJ-15, based on sequence homology of a partial 746-bp fragment of 16SrDNA amplicon, with the ribosomal database sequences (http://www.msu.edu/RDP/cgis/phylip.cgi), validated the strain as Pseudomonas aeruginosa. The strain NJ-15 produced a substantial amount of indole acetic acid (IAA) in tryptophan-supplemented medium. Besides, the strain also exhibited significant production of both the siderophore and hydrogen cyanide (HCN) on chrome azurol S and King's B media, respectively. The data revealed lower HCN production under iron-limiting conditions vis-à-vis higher HCN release with iron stimulation. Significant growth inhibition of phytopathogenic fungi occurred in the order as Fusarium oxysporum > Trichoderma herizum > Alternaria alternata > Macrophomina phasiolina upon incubation with strain NJ-15 cells. Thus, the secondary metabolites producing new Pseudomonas aeruginosa strain NJ-15 exhibited innate potential of plant growth promotion and biocontrol activities in vitro.

Characterisation of a cyanide hydratase gene in the phytopathogenic fungus Leptosphaeria maculans.

A gene encoding a cyanide hydratase was cloned from an aggressive isolate of Leptosphaeria maculans, the fungus which causes blackleg disease of oilseed Brassica spp. This enzyme catalyses the breakdown of hydrogen cyanide to a less toxic compound, formamide. The predicted amino acid sequence of cyanide hydratase in L. maculans is 77% and 82% identical to cyanide hydratases from two other ascomycetes, Gloeocercospora sorghi and Fusarium lateritium, respectively. The gene is present as a single copy in the L. maculans genome, in both aggressive and non-aggressive isolates, although there is a restriction fragment length polymorphism between these two isolate groups for this gene. The cyanide hydratase promoter contains four putative target sites for GATA transcription factors, proteins that regulate nitrogen metabolism and other processes. Transcription of cyanide hydratase in an aggressive L. maculans isolate is induced strongly by potassium cyanide. Transcription of the gene is detectable in cotyledons of Brassica juncea and B. napus during infection. L. maculans can utilise the reaction product, formamide, as a sole source of nitrogen.

Crystallization and preliminary crystallographic analysis of the cyanogenic beta-glucosidase from the white clover Trifolium repens L.

The cyanogenic beta-glucosidase from Trifolium repens (white clover) has been crystallized, in a form suitable for X-ray analysis, from ammonium sulphate solutions. The crystals, which diffract to 3.0 A, are tetragonal, space group P4(3)2(1)2 or its enantiomorph P4(1)2(1)2. The cell dimensions are a = b = 69.92 A, c = 248.38 A.

Digestibility of boiled and oven-dried cassava in infants and small children.

Boiled fresh cassava or oven-dried cassava flour provided 50% of the energy and less than 10% of the protein in diets of convalescent malnourished infants; casein was added to complete 8% of energy as protein, and vegetable oil (soybean-cottonseed) was added to make 20% of energy as fat. Despite high wet weights of feces (172 +/- 42 and 214 +/- 41 g/d), their dry weights (20 +/- 3 and 22 +/- 2 g/d) and their energy (6.9 +/- 0.7 and 7.6 +/- 0.5% of intake) and nitrogen (17 +/- 3 and 20 +/- 3% of intake) contents were low, and their fat content (4.4 +/- 1.3 and 5.2 +/- 1.2% of intake) was very low. Cassava is a surprisingly effective source of energy which interferes little or not at all with digestion of added protein and fat in weaning diets. For its safe use, it is important that home or industrial processing almost completely eliminate its potential hydrocyanic acid content, and that a good quality protein supplement be consumed regularly in nutritionally adequate amounts.

Laetrile toxicity studies in dogs.

Dogs were fed laetrile and fresh, sweet almonds under various conditions. The doses of laetrile were similar to those prescribed for patients with cancer and ranged on a basis of gram to square meter from an equivalent of the oral dose for man to five times this dose. Six of the ten dogs died of cyanide poisoning. One dog recovered, and three dogs, at the time of sacrifice, demonstrated various levels of neurologic impairment, ranging from difficulty in walking to coma. These studies demonstrate that oral laetrile is highly toxic when taken with some common table foods. We predict that there will be an increased incidence of cyanide poisoning in man as laetrile becomes more readily available.

Cyanide formation from histidine in Chlorella. A general reaction of aromatic amino acids catalyzed by amino acid oxidase systems.

The formation of HCN from D-histidine in Chlorella vulgaris extracts is shown to be due to the combined action of a soluble protein and a particulate component. Either horse-radish peroxidase (EC 1.11.1.7) or a metal ion with redox properties can be substituted for the particulate component. Ions of manganese and vanadium are especially effective, as are o-phenanthroline complexes of iron. Cobalt ions are less active. The D-amino acid oxidase (EC 1.4.3.3) from kidney and the L-amino acid oxidase (EC 1.4.3.2) from snake venom likewise cause HCN production from histidine when supplemented with the particulate preparation from Chlorella or with peroxidase or with a redox metal ion. The stereospecificity of the amino acid oxidase determines which of the two stereoisomers of histidine is active as an HCN precursor. Though histidine is the best substrate for HCN production, other naturally occurring aromatic amino acids (viz. tyrosine, phenylalanine and tryptophan) can also serve as HCN precursors with these enzyme systems. The relative effectiveness of each substrate varies with the amino acid oxidase enzyme and with the supplement. With respect to this latter property, the particulate preparation from Chlorella behaves more like a metal ion than like peroxidase.

A D-amino acid oxidase from Chlorella vulgaris.

A procedure has been developed for the partial purification from Chlorella vulgaris of an enzyme which catalyzes the formation of HCN from D-histidine when supplemented with peroxidase of a metal with redox properties. Some properties of the enzyme are described. Evidence is presented that the catalytic activity for HCN formation is associated with a capacity for catalyzing the oxidation of a wide variety of D-amino acids. With D-leucine, the best substrate for O2 consumption, 1 mol of ammonia is formed for half a mol of O2 consumed in the presence of catalase. An inactive apoenzyme can be obtained by acid ammonium sulfate precipitation, and reactivated by added FAD. On the basis of these criteria, the Chlorella enzyme can be classified as a D-amino acid oxidase (EC 1.4.3.3). Kidney D-amino acid oxidase and snake venom L-amino acid oxidase, which likewise form HCN from histidine on supplementation with peroxidase, have been compared with the Chlorella D-amino acid oxidase. The capacity of these enzymes for causing HCN formation from histidine is about proportional to their ability to catalyze the oxidation of histidine.