Purification and characterization of a methylene urea-hydrolyzing enzyme from Rhizobium radiobacter (Agrobacterium tumefaciens)

Slow-release fertilizers are gaining acceptance to increase fertilizer use efficiency and reduce environmental impact. The release of nitrogen from methylene urea, a common slow release N fertilizer, is controlled by microbial decomposition. An enzyme hydrolyzing slow-release nitrogen fertilizer, methylene urea, was purified from Rhizobium radiobacter (Agrobacterium tumefaciens) to homogeneity using a four-step purification procedure with an overall yield of 3%. The active enzyme has a molecular mass of approximately 180 kDa determined by size exclusion chromatography, and the SDS page of the purified protein indicated three subunits of different sizes (62, 34 and 32 kDa). The N-terminal amino acid sequence of the 62 kDa fragment indicates identity with urease subunits from Mycobacterium tuberculosis (73%) and Helicobacter pylori (71%). However, for the internal amino acid sequences of the 62 kDa fragment no matches with known proteins were found. Some internal peptides in the smaller subunits (32 and 34 kDa) are homologous to urease subunits and unknown proteins in Agrobacterium tumefaciens. Based on the kinetic properties, substrate selectivity, and inhibition characteristics, the novel enzyme (MUase) is an intracellular enzyme complex with urease activity. The enzymatic mechanism of methylene urea breakdown was studied using a novel LC-MS method for MU analysis, which indicates that all cold-water soluble nitrogen forms of methylene urea are subjected to hydrolysis, and the hydrolysis proceeds via methylurea, urea and other yet unidentified hydrolysis-products, suggesting that the isolated enzyme complex performs a multistep hydrolysis. The microbiological and molecular data is useful in determining the soil factors affecting the efficacy of methylene urea as a slow release fertilizer in agricultural production systems.     

Purification and characterization of hydrolase with chitinase and chitosanase activity from commercial stem bromelain

A hydrolase with chitinase and chitosanase activity was purified from commercial stem bromelain through sequential steps of SP-Sepharose ion-exchange adsorption, HiLoad Superdex 75 gel filtration, HiLoad Q Sepharose ion-exchange chromatography, and Superdex 75 HR gel filtration. The purified hydrolase was homogeneous, as examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme exhibited chitinase activity for hydrolysis of glycol chitin and 4-methylumbelliferyl beta-D-N,N',N' '-triacetylchitotrioside [4-MU-beta-(GlcNAc)(3)] and chitosanase activity for chitosan hydrolysis. For glycol chitin hydrolysis, the enzyme had an optimal pH of 4, an optimal temperature of 60 degrees C, and a K(m) of 0.2 mg/mL. For the 4-MU-beta-(GlcNAc)(3) hydrolysis, the enzyme had an optimal pH of 4 and an optimal temperature of 50 degrees C. For the chitosan hydrolysis, the enzyme had an optimal pH of 3, an optimal temperature of 50 degrees C, and a K(m) of 0.88 mg/mL. For hydrolysis of chitosans with various N-acetyl contents, the enzyme degraded 30-80% deacetylated chitosan most effectively. The enzyme split chitin or chitosan in an endo-manner. The molecular mass of the enzyme estimated by gel filtration was 31.4 kDa, and the isoelectric point estimated by isoelectric focusing electrophoresis was 5.9. Heavy metal ions of Hg(2+) and Ag(+), p-hydroxymercuribenzoic acid, and N-bromosuccinimide significantly inhibited the enzyme activity.     

Soil-borne strain IC14 of Serratia plymuthica with multiple mechanisms of antifungal activity provides biocontrol of Botrytis cinerea and Sclerotinia sclerotiorum diseases

Plant-associated strain IC14 of the Gram-negative bacterium Serratia plymuthica isolated from soil around melon roots was shown to suppress a wide range of phytopathogenic fungi in vitro. Foliar application of strain IC14 protected cucumber against Botrytis cinerea gray mold and Sclerotinia sclerotiorum white mold diseases of leaves under greenhouse conditions, reducing disease incidence by 76 and 84%, respectively. The strain possessed chitinolytic and proteolytic activities, produced the antibiotic pyrrolnitrin [3-chloro-4-(2′-nitro-3′-chlorophenyl)pyrrole] and siderophores, and secreted the plant growth hormone indole-3-acetic acid. An endochitinase with an apparent molecular mass of 58 kDa, was estimated to be the main secreted chitinolytic enzyme. Two mutants, one with increased chitinolytic activity and the second deficient in chitinolytic activity, were obtained by miniTn5-insertion mutagenesis. Neither mutant differed appreciably from the parental strain in the production of other antifungal compounds or in suppression of B. cinerea and S. sclerotiorum on plates or in the greenhouse, suggesting that chitinolytic activity is less essential for biocontrol of these pathogens by strain IC14. The obtained results present novel information concerning the potential of the soil-borne S. plymuthica strains as biocontrol agents of foliar diseases caused by plant pathogenic fungi.     

Phosphotriesterases of flavobacterium sp

An enzyme system having phosphotriesterase activity was partially purified from Flavobacterium sp. by means of gel filtration and preparative gel isotachophoresis. Flavobacterium phosphotriesterase showed maximum activity between pH 8–10 and was unaffected by the presence of metal ions. Non-ionic detergents were potent and irreversible inhibitors of activity. Inhibition was also observed with mercurial thiol reagents and cysteine, although in the latter case inhibition could be reversed by oxidation in air or with K₃Fe(CN)₆. Activity was restricted towards substrates having electron withdrawing aromatic or heterocyclic leaving group such as parathion, paraoxon, diazinon and their analogues. Substrate analogues having the weakly electrophilic 4-aminophenyl group were not hydrolysed and in some cases acted as competitive inhibitors. Product inhibition by 4-nitrophenol (but not by the phosphorus containing moiety) was observed.     

Biological control of the root-knot nematode Meloidogyne javanica by Trichoderma harzianum

The filamentous fungi Trichoderma spp. is currently developed as biocontrol agents against many plant pathogens. Recent studies have shown that these fungi are able to infect nematode eggs and juveniles. In this research, biological control of root-knot nematode (Meloidogyne javanica) by Trichoderma harzianum BI was investigated in greenhouse and laboratory experiments. Results showed that different concentrations (10²–10⁸ spores/ml) of T. harzianum BI decreased nematode infection and other parameters significantly, compared to control. T. harzianum BI was able to penetrate nematode egg mass matrix and significantly decreased nematode egg hatching level. Specific activities of resistance-related enzymes, namely peroxidase (POX), polyphenol oxidase (PPO) and phenylalanine ammonia lyase (PAL) increased significantly in T. harzianum BI inoculated plants. Maximum activities of POX, PPO and PAL were observed at the 5, 5 and 6 days after inoculation, respectively. Chitinase activity was also increased in culture filtrates of T. harzianum BI grown on wheat bran moistened with salt solution supplemented with colloidal chitin or nematode eggs. Maximum activity of chitinase was recorded at the 4 days after inoculation, in media supplemented with colloidal chitin (1.15 U/min per ml) and nematode eggs (0.85 U/min per ml). Results suggested that direct parasitism of eggs through the increase in extracellular chitinase activity, which would be indicator of eggs infection capability, and inducing plant defense mechanisms leading to systemic resistance are two main suppression mechanisms used by T. harzianum BI against nematode.     

Production of lignocellulose-degrading enzymes and degradation of leaf litter by saprotrophic basidiomycetes isolated from a Quercus petraea forest

Due to the production of lignocellulose-degrading enzymes, saprotrophic basidiomycetes can significantly contribute to the turnover of soil organic matter. The production of lignin- and polysaccharide-degrading enzymes and changes of the chemical composition of litter were studied with three isolates from a Quercus petraea forest. These isolates were capable of fresh litter degradation and were identified as Gymnopus sp., Hypholoma fasciculare and Rhodocollybia butyracea. Within 12 weeks of incubation, H. fasciculare decomposed 23%, R. butyracea 32% and Gymnopus sp. 38% of the substrate dry mass. All fungi produced laccase and Mn-peroxidase (MnP) and none of them produced lignin peroxidase or other Mn-independent peroxidases. There was a clear distinction in the enzyme production pattern between R. butyracea or H. fasciculare compared to Gymnopus sp. The two former species caused the fastest mass loss during the initial phase of litter degradation, accompanied by the temporary production of laccase (and MnP in H. fasciculare) and also high production of hydrolytic enzymes that later decreased. In contrast, Gymnopus sp. showed a stable rate of litter mass loss over the whole incubation period with a later onset of ligninolytic enzyme production and a longer lasting production of both lignin and cellulose-degrading enzymes. The activity of endo-cleaving polysaccharide hydrolases in this fungus was relatively low but it produced the most cellobiose hydrolase. All fungi decreased the C/N ratio of the litter from 24 to 15-19 and Gymnopus sp. also caused a substantial decrease in the lignin content. Analytical pyrolysis mass spectrometry of litter decomposed by this fungus showed changes in the litter composition similar to those caused by white-rot fungi during wood decay. These changes were less pronounced in the case of H. fasciculare and R. butyracea. All fungi also changed the mean masses of humic acid and fulvic acid fractions isolated from degraded litter. The humic acid fraction after degradation by all three fungi contained more lignin and less carbohydrates. Compared to the decomposition by saprotrophic basidiomycetes, litter degradation in situ on the site of fungal isolation resulted in the relative enrichment of lignin and differences in lignin composition revealed by analytical pyrolysis. It can most probably be explained by the participation of non-basidiomycetous fungi and bacteria during natural litter decomposition.     

Physiological aspects of N2-fixation by a Spirillum from Digitaria roots

Studies of the physiology of the Spirillum lipoferum recognized as the major organism responsible for N₂-fixation in the roots of Digitaria decumbens cv transvala were performed in order to improve the methods of culture and help to explain the physiology of this N₂-fixing grass bacterial association.Methods for isolation, purification and N₂-fixation assays are described. Acetylene concentrations used for N₂-ase activity measurements should be at least 12%. the Vmax of cultures in the log phase being at a pC₂H₂ of 0.12 atm and the apparent K_m 0.022 atm. Optimal temperatures for N₂-dependent growth are between 32 and 40°C. and little N₂-fixation is observed below 24°C. At 42°C the N₂-ase is inactivated. When cultures grown at 28 or 36°C are transferred to lower temperatures nitrogenase activity declines rapidly. One hour after transfer to 17°C activity is about half that before transfer and is maintained at this level for at least 8 h. After transfer to 10°C activity ceases after 1 h. Growth is very pH dependent, optimal growth on N₂ occurring only between pH 6.8 and 7.8. Nitrogen fixation below pH 5.5 and above 8.0 is less than one-quarter of the optimal. No N₂-fixation occurs in the absence of O₂ and maximal N₂-dependent growth is reached at 1.5% O₂ in the gas mixture bubbled through liquid cultures.In contrast to previous reports, several sugars including glucose can be used by the Spirillum for N₂-fixation, but only when small amounts of starter nitrogen or organic acids are added to the medium. Efficiencies of N₂-fixation on malate and glucose are similar and about 60% of that of cells incorporating NH⁺₄-N. Efficiency of NO^-₃ incorporation is 74% of that of NH⁺₄-N grown cultures. High observed efficiencies (52 mg N₂ fixed g^?1 malate or glucose) are attributed to carbon limited growth at optimum or O₂ limited conditions, both facilitated by slow diffusion rates through the semi-solid agar medium used.     

Biological control of damping-off caused by Rhizoctonia solani using chitinase-producing Paenibacillus illinoisensis KJA-424

A bacterium having strong chitinolytic activity was isolated from a coastal soil in Korea and identified as Paenibacillus illinoisensis KJA-424 on the basis of the nucleotide sequence of a 16S rRNA gene. By activity staining after SDS-PAGE, three major chitinase bands with chitinolytic activity, approximate molecular weight of 63, 54 and 38 kDa were detected. On co-culture Rhizoctonia solani with KJA-424, abnormal swelling and deformation of R. solani hyphae were observed, where the release of N-acetyl-d-glucosamine was detected. The bacterium suppressed the symptom of damping-off cucumber seedlings caused by R. solani, in greenhouse trial.     

Parasitism of sclerotia of Sclerotium rolfsii by trichoderma harzianum

The ability of Trichoderma harzianum isolate 203 to attack the soil-borne plant pathogen Sclerotium rolfsii is apparently connected with the production by the isolates of chitinase and β-(1,3)-glucanase inside the attacked sclerotia during parasitism.SEM and TEM micrographs show that the mycoparasite degraded walls of sclerotial cells and the attacked cells lost their cytoplasmic contents. It is assumed that T. harzianum utilizes sclerotial cell contents thus enabling it to sporulate intensively on the sclerotial surface and inside the digested cells.     

Pretreatment and Enzymatic Hydrolysis of Sorghum Bran

Sorghum bran has potential to serve as a low‐cost feedstock for production of fuel ethanol. Sorghum bran from a decortication process (10%) was used for this study. The approximate chemical composition of sorghum bran was 30% starch, 18% hemicellulose, 11% cellulose, 11% protein, 10% crude fat, and 3% ash. The objective of this research was to evaluate the effectiveness of selected pretreatment methods such as hot water, starch degradation, dilute acid hydrolysis, and combination of those methods on enzymatic hydrolysis of sorghum bran. Methods for pretreatment and enzymatic hydrolysis of sorghum bran involved hot water treatment (10% solid, w/v) at 130°C for 20 min, acid hydrolysis (H₂SO₄), starch degradation, and enzymatic hydrolysis (60 hr, 50°C, 0.9%, v/v) with commercial cellulase and hemicellulose enzymes. Total sugar yield by using enzymatic hydrolysis alone was 9%, obtained from 60 hr of enzyme hydrolysis. Hot water treatment facilitated and increased access of the enzymes to hemicellulose and cellulose, improving total sugar yield up to 34%. Using a combination of starch degradation, optimum hot water treatment, and optimum enzymatic hydrolysis resulted in maximum total sugar yield of up to 75%.     

A method for linking in situ activities of hydrolytic enzymes to associated organisms in forest soils

A root window-based, enzyme-imprinted, membrane system has been modified to enable visualization of the activities of hydrolytic enzymes (acid phosphatase, aminopeptidase, chitinase, and β-glucosidase) in situ in forest soils. The approach can be used to correlate the distribution of enzyme activity with visible features such as roots, mycorrhizas, or mycelial mats. In addition, it enables accurate spatial soil sampling for analysis of microbial communities associated with enzyme activities. The substrates are colorimetric conjugates of napthol, where color develops instantly in the field, or fluorimetric conjugates of 4-methylumbelliferone, whose fluorescent products are detected by a gel-documenting system. The method will allow important questions about the relationship between taxonomic and functional diversity of soil microorganisms to be addressed and identification of enzyme activity hot-spots in soil.     

Degradation of ioxynil by a soil fungus, Fusarium solani

A fungus, Fusarium solani, isolated from the soil, degraded ioxynil (3,5-diiodo-4-hydroxyben-zonitrile) in pure culture into at least eight products. Five products were detected in the organic fractions extracted from a culture grown in [¹⁴C]cyano-labeled ioxynil. Three additional products were separated by ion-exchange chromatography of the acidified aqueous phase. Cultures grown in the presence of [¹⁴C]ring-labeled ioxynil produced the same products in the organic extract and four to five products in the aqueous phase. The cyano-carbon of ioxynil was released as CO₂ at a faster rate than that of ring-carbons and was released after the initial ring cleavage. Two of the metabolites were identified as 3,5-diiodo-4-hydroxybenzamide and 3,5-diiodo-4-hydroxybenzoic acid.     

Microbial enzyme activities in leaf litter, humus and mineral soil layers of European forests

The rationale of the study was to investigate microbial activity in different soil horizons in European forests. Hence, activities of chitinase and cellulase, microbial biomass carbon (C_mic) and basal respiration were measured in litter, fragmentation, humus and mineral soil layers collected several times from various beech and spruce forests. Sites were selected to form a gradient in N availability. Analyses were also performed on beech litter from a litterbag transplant experiment. Furthermore, microbiological parameters were measured in horizons of beech and spruce chronosequence sites with different stand age in order to investigate the influence of forest rotation, and hence changes in soil organic matter (SOM) dynamics, on microbial activity. Finally in horizons of one beech forest, the seasonal variation of selected microbiological parameters was measured more intensively. β-Glucosaminidase and cellobiohydrolase activities were measured using fluorogenic 4-methylumbelliferyl substrates to estimate chitinase and cellulase activities, respectively. On a spatial scale, chitinase and cellulase activities, C_mic determined by substrate induced respiration, and basal respiration ranged from 144 to 1924 and 6-177 nmol 4-MU g⁻¹ org-C h⁻¹, 8-48 mg C g⁻¹ org-C and 11-149 μg CO₂-C g⁻¹ org-C h⁻¹, respectively; in general values were significantly lower in layers of humus and mineral soil than of litter. Chitinase activity, C_mic and basal respiration from humus and mineral soil layers, together, correlated positively, while none correlated with cellulase activity. Similarly in the litter layer, no correlations were found between the microbiological parameters. On a seasonal scale, a time lag between a burst in basal respiration rate and activities of both enzymes were observed. In general, activities of cellulase and chitinase, C_mic and basal respiration, did not change with stand age, except in the humus layer in the spruce chronosequence, where C_mic decreased with stand age. In the litter layer, cellulase activity was significantly and positively related to the C:N ratio, while only a tendency for chitinase activity was shown, indicating that enzyme activities decreased with increasing N availability. In accordance, the enzyme activities and C_mic decreased significantly with increasing chronic N deposition in the humus layer, while basal respiration only tended to decrease with increasing N deposition. In contrast, enzyme activities in beech litter from litterbags after 2 years of incubation were generally higher at sites with higher N deposition. The results show different layer-specific responses of enzyme activities to changes in N availability, indicating different impacts of N availability on decomposition of SOM and stage of litter decomposition.     

Soil enzyme activity in an old-growth northern hardwood forest: Interactions between soil environment,ectomycorrhizal fungi and plant distribution

Plants and soil microbes produce extracellular enzymes (EE) that catalyze the hydrolysis of nitrogen (N) and phosphorus (P) containing compounds in soil and other enzymes involved in degradation of lignin and cellulose. We explored whether soil enzyme activity involved in carbon (C), N and P cycling were correlated with plant distribution, soil chemical conditions and the identity of fungi colonizing tree roots in an old growth forest remnant. Terminal restriction fragment length polymorphism (TRFLP) was used to determine the presence of root fungi and standard fluorometric analysis was used to determine soil enzyme activities. Soil enzymes were consistently positively correlated with soil C and N, but not CN ratio. Soil P was also correlated with enzyme activity during both June and September sampling. We saw no significant relationships between herbaceous plant cover and enzyme activity in June, but there were significant positive correlations between α-glucosidase and herbaceous plant coverage in September. We also found that some enzymes were significantly correlated with the identity of fungi colonizing tree roots separated from the soil cores. Chitinase and β-glucosidase were positively correlated with the genera Russula and Piloderma while chitinase was negatively correlated with Amanita and Entoloma. In addition, phosphatase was positively correlated with Russula, Meliniomyces and Solenopezia. Our results suggest that enzyme activity in old growth forest soils are affected by a variety of environmental factors, and that herbaceous plants and some root fungi may be associated with sites of elevated or decreased decomposition potential and nutrient cycling.     

Non-pathogenic Fusarium strains protect the seedlings of Lepidium sativum from Pythium ultimum

Two root-colonizing Fusarium strains, Ls-F-in-4-1 and Rs-F-in-11, isolated from roots of Brassicaceae plants, induced the resistance in Lepidium sativum seedlings against Pythium ultimum. These strains caused an increase in the content of benzyl isothiocyanate, and of its precursor glucotropaeolin, in the roots of the host plants. The increased isothiocyanate content is one of the factors contributing to the resistance of L. sativum against P. ultimum. To be transformed into the fungitoxic compound benzyl isothiocyanate, glucotropaeolin has to be hydrolyzed by myrosinase, which can be produced either by plants or microorganisms. The Fusarium strain Ls-F-in-4-1 has a myrosinase activity but the strain Rs-F-in-11 has not. These results suggest that both strains are able to trigger the metabolic pathway leading to benzyl isothiocyanate production in the plant. In the case of the myrosinase-negative strain Rs-F-in-11, hydrolyzation into isothiocyanate is only due to the myrosinase activity of the plant, and in the other case, the myrosinase produced by the strain Ls-F-in-11 also would contribute to the production of isothiocyanate. This paper reports a new mode of action of non-pathogenic Fusarium strains in controlling P. ultimum.     

Effects of elevated CO2 concentration on rhizodeposition from Lolium perenne grown on soil exposed to 9 years of CO2 enrichment

The effects of enriched CO₂ atmosphere on partitioning of recently assimilated carbon were investigated in a plant-soil-microorganism system in which Lolium perenne seedlings were planted into cores inserted into the resident soil within a sward that had been treated with elevated CO₂ for 9 consecutive years, under two N fertilisation levels (Swiss FACE experiment). The planted cores were excavated from the ambient (35 Pa pCO₂) and enriched (60 Pa pCO₂) rings at two dates, in spring and autumn, during the growing season. The cores were brought back to the laboratory for ¹⁴C labelling of shoots in order to trace the transfer of recently assimilated C both within the plant and to the soil and microbial biomass. At the spring sampling, high N supply stimulated shoot and total dry matter production. Consistently, high N enhanced the allocation of recently fixed C to shoots, and reduced it to belowground compartments. Elevated CO₂ had no consequences for DM or the pattern of C allocation. At the autumn sampling, at high N plot, yield of L. perenne was stimulated by elevated CO₂. Consistently, ¹⁴C was preferentially allocated aboveground and, consequently belowground recent C allocation was depressed and rhizodeposition reduced. At both experimental periods, total soil C content was similar in all treatments, providing no evidence for soil carbon sequestration in the Swiss Free Air CO₂ Enrichment experiment (FACE) after 9 years of enrichment. Recently assimilated C and soil C were mineralised faster in soils from enriched rings, suggesting a CO₂-induced shift in the microbial biomass characteristics (structure, diversity, activity) and/or in the quality of the root-released organic compounds.     

Seasonality of soil biological properties in a poplar plantation growing under elevated atmospheric CO2

Microorganisms are the regulators of decomposition processes occurring in soil, they also constitute a labile fraction of potentially available N. Microbial mineralization and nutrient cycling could be affected through altered plant inputs at elevated CO_2. An understanding of microbial biomass and microbial activity in response to belowground processes induced by elevated CO₂ is thus crucial in order to predict the long-term response of ecosystems to climatic changes. Microbial biomass, microbial respiration, inorganic N, extractable P and six enzymatic activities related to C, N, P and S cycling (β-glucosidase, cellulase, chitinase, protease, acid phosphatase and arylsulphatase) were investigated in soils of a poplar plantation exposed to elevated CO_2. Clones of Populus alba, Populus nigra and Populus x euramericana were grown in six 314 m² plots treated either with atmospheric (control) or enriched (550 μmol mol⁻¹ CO₂) CO₂ concentration with FACE technology (free-air CO₂ enrichment). Chemical and biochemical parameters were monitored throughout a year in soil samples collected at five sampling dates starting from Autumn 2000 to Autumn 2001.The aim of the present work was: (1) to determine if CO₂ enrichment induces modifications to soil microbial pool size and metabolism, (2) to test how the seasonal fluctuations of soil biochemical properties and CO₂ level interact, (3) to evaluate if microbial nutrient acquisition activity is changed under elevated CO₂.CO₂ enrichment significantly affected soil nutrient content and three enzyme activities: acid phosphatase, chitinase and arylsulphatase, indicators of nutrient acquisition activity. Microbial biomass increased by a 16% under elevated CO₂. All soil biochemical properties were significantly affected by the temporal variability and the interaction between time and CO₂ level significantly influenced β-glucosidase activity and microbial respiration. Data on arylsulphatase and chitinase activity suggest a possible shift of microbial population in favour of fungi induced by the FACE treatment.     

土壤中几丁质酶的研究进展

几丁质酶是影响土壤中氮矿化的一种重要的酶,其分解几丁质控制着氮循环的关键步骤。本文对土壤中几丁质酶的分类、来源、分布、影响因素、催化动力学特性及测定方法进行了综合评述,以期为我国开展土壤几丁质酶的相关研究提供理论参考。     

Autolysis of chlamydospores of Fusarium solani f. sp. cucurbitae in chitin and laminarin amended soils

Lysis of chlamydospores of Fusarium solani f. sp. cucurbitae is enhanced both in chitin and in laminarin amended soils. In soil amended with both chitin and laminarin, lysis of chlamydospores resembles that in control soil. Addition of chitin and laminarin to soil stimulates the growth of both bacteria and actinomycetes. In soils amended with chitin and with both chitin and laminarin, a chitinolytic microflora is stimulated. Penetration of chlamydospore cells by soil microorganisms has not been observed using the electron microscope. The mechanism of lysis of chlamydospores in soil is discussed.     

The production of N-nitrosoiminodiacetate from nitrilotriacetate and nitrate by microorganisms growing in mixed culture

Mixed populations of microorganisms growing in a recycling soil percolator produced N-nitrosoiminodiacetate (NIDA) after 30 days growth using sodium nitrilotriacetate (NTA), a detergent additive, and NaNO₃ as C and N source respectively. Control experiments using autoclaved soil or substituting either acetate (C source) or NH⁺₄ (N source) did not show any N-nitroso derivative production. Five distinct strains of microorganisms were isolated which could grow on an NTA/NO^?₃ minimal medium but none produced NIDA in pure culture. Mixed cultures of two strains of Pseudomonas produced NIDA after 4 days percolation, using glass beads as a support. Cell-free extracts of the mixed cultures synthesized NIDA after 30 min incubation at 30°C with an activity of 27 μmoles NIDA produced/mg protein h^?1. Physical separation of the two organisms and the cell-free extracts by a dialysis membrane in a chamber determined that only one of the two organisms was responsible for the synthesis of NIDA, but the production was dependent upon the presence of the other organism. The possible relationship between the two organisms is discussed.