Oxidative decomposition of malonic acid as basis for the action of manganese peroxidase in the absence of hydrogen peroxide.

Manganese peroxidase (MnP) from the ligninolytic basidiomycetes Phlebia radiata and Nematoloma frowardii was found to decompose malonate oxidatively in the absence of H2O2 in a reaction system consisting of the enzyme, sodium malonate and MnCl2. The enzymatic oxidation resulted in a substantial decrease in malonate concentration and the formation of CO2, oxalate, glyoxylate and formate. Simultaneously with the decomposition of malonate, Mn(II) was oxidized to Mn(III) leading to high transient concentrations of the latter. MnP action in the absence of H2O2 started slowly after a lag period of 3 h. The lag period was considerably shortened after a single addition of Mn(III). Superoxide dismutase and catalase inhibited the enzymatic reaction partly, ascorbate completely. ESR studies demonstrated the formation of a carbon-centered radical during the course of the reaction. We propose that the latter generates peroxides that can be used by MnP to oxidize Mn(II) to Mn(III).

Production of manganese peroxidase and organic acids and mineralization of 14C-labelled lignin (14C-DHP) during solid-state fermentation of wheat straw with the white rot fungus nematoloma frowardii

The basidiomycetous fungus Nematoloma frowardii produced manganese peroxidase (MnP) as the predominant ligninolytic enzyme during solid-state fermentation (SSF) of wheat straw. The purified enzyme had a molecular mass of 50 kDa and an isoelectric point of 3.2. In addition to MnP, low levels of laccase and lignin peroxidase were detected. Synthetic 14C-ring-labelled lignin (14C-DHP) was efficiently degraded during SSF. Approximately 75% of the initial radioactivity was released as 14CO2, while only 6% was associated with the residual straw material, including the well-developed fungal biomass. On the basis of this finding we concluded that at least partial extracellular mineralization of lignin may have occurred. This conclusion was supported by the fact that we detected high levels of organic acids in the fermented straw (the maximum concentrations in the water phases of the straw cultures were 45 mM malate, 3.5 mM fumarate, and 10 mM oxalate), which rendered MnP effective and therefore made partial direct mineralization of lignin possible. Experiments performed in a cell-free system, which simulated the conditions in the straw cultures, revealed that MnP in fact converted part of the 14C-DHP to 14CO2 (which accounted for up to 8% of the initial radioactivity added) and 14C-labelled water-soluble products (which accounted for 43% of the initial radioactivity) in the presence of natural levels of organic acids (30 mM malate, 5 mM fumarate).

Involvement of cytochrome P-450 in the 15alpha-hydroxylation of 13-ethyl-gon-4-ene-3,17-dione by Penicillium raistrickii.

The 15alpha-hydroxylation of 13-ethyl-gon-4-ene-3,17-dione (GD) with different subcellular fractions of Penicillium raistrickii i 477 was investigated. Cytochrome P-450 was shown to be involved in this reaction. The steroid transformation was inhibited by carbon monoxide, metyrapone, p-CMB, iodoacetamide, N-methylmaleimide and several metal ions. The 15alpha-hydroxylase was observed to be dependent on nicotinamide-adenine dinucleotide phosphate (NADPH) replaceable by NaIO4, and the activity was enhanced by a NADPH-regenerating system, indicating the involvement of the NADPH-cytochrome c (P-450) reductase. This was further confirmed by the inhibition of the hydroxylase activity in the presence of cytochrome c. No effect was observed in the presence of azide and antimycin A. Solubilized microsomes gave an absorption maximum at 453 nm in carbon monoxide difference spectrum, and showed a Type-I GD-binding spectrum typically for cytochrome P-450 interaction with substrate. First results about the inducibility of the enzymes involved in the 15alpha-hydroxylation of GD are shown.

Degradation of polycyclic aromatic hydrocarbons by manganese peroxidase of Nematoloma frowardii.

The degradation of polycyclic aromatic hydrocarbons by a manganese peroxidase crude preparation of Nematoloma forwardii was demonstrated for a mixture of eight different polycyclic aromatic hydrocarbons, and the five individual polycyclic aromatic hydrocarbons phenanthrene, anthracene, pyrene, fluoranthene, and benzo[alpha]pyrene. Oxidation of polycyclic aromatic hydrocarbons was enhanced by the addition of glutathione, a mediator substance, able to form reactive thiyl radicals. Glutathione-mediated manganese peroxidase (1.96 U ml(-1)) was capable of mineralizing [14C]pyrene (7.3%),[14C]anthracene (4.7%), [14C]benzo[alpha]pyrene (4.0%), [14C]benz(alpha)anthracene 2.9%), and [14C]phenanthrene (2.5%) in a period of 168 h. This is the first description of direct enzymatic mineralization of polycyclic aromatic hydrocarbons by manganese peroxidase, and indicates their important role in the oxidation of polycyclic aromatic hydrocarbons by wood-decaying fungi.

Degradation of lignite (low-rank coal) by ligninolytic basidiomycetes and their manganese peroxidase system

Ligninolytic basidiomycetes (wood and leaf-litter-decaying fungi) have the ability to degrade low-rank coal (lignite). Extracellular manganese peroxidase is the crucial enzyme in the depolymerization process of both coal-derived humic substances and native coal. The depolymerization of coal by Mn peroxidase is catalysed via chelated Mn(III) acting as a diffusible mediator with a high redox potential and can be enhanced in the presence of additional mediating agents (e.g. glutathione). The depolymerization process results in the formation of a complex mixture of lower-molecular-mass fulvic-acid-like compounds. Experiments using a synthetic 14C-labeled humic acid demonstrated that the Mn peroxidase-catalyzed depolymerization of humic substances was accompanied by a substantial release of carbon dioxide (17%-50% of the initially added radio-activity was released as 14CO2). Mn peroxidase was found to be a highly stable enzyme that remained active for several weeks under reaction conditions in a liquid reaction mixture and even persisted in sterile and native soil from an opencast mining area for some days.

Effects of ryegrass on biodegradation of hydrocarbons in soil.

The effects of growing ryegrass (Lolium perenne L.) on the biodegradation of hydrocarbons was studied in laboratory scale soil columns. Degradation of hydrocarbons as well as bacterial numbers, soil respiration rates and soil dehydrogenase activities were determined. In the rhizosphere soil system, aliphatic hydrocarbons disappeared faster than in unvegetated columns. Abiotic loss by evaporation was of minor significance. Elimination of pollutants was accompanied by an increase in microbial numbers and activities. The microbial plate counts and soil respiration rates were substantially higher in the rhizosphere than in the bulk soil. The results indicate that biodegradation of hydrocarbons in the rhizosphere is stimulated by plant roots.

Nature and significance of microbial cometabolism of xenobiotics.

Microbial cometabolism, i.e. "transformation of a non-growth substrate in the obligate presence of a growth substrate or another transformable compound" (Dalton and Stirling 1982) is a whole-cell phenomenon physiologically based on coupling of different catabolic pathways at the cellular level. It is frequently observed in transformation of xenobiotic non-growth substrates by individual microbial species. Transformation processes of this type are usually mediated by appropriate non-specific enzymes of the peripheric cellular metabolism able to modify a variety of substances other than their natural substrates. The precise mechanisms of coupling between metabolism of xenobiotic non-growth substrates and of particular additional carbon substrates may be different depending on the substrates and the microbial species involved. However, experimental data indicate that the primary function of the respective additional carbon substrates is to supply either energy, cofactors or metabolites for the different cellular events involved in the transformation process (e.g. uptake of the xenobiotic non-growth substrate, functioning of appropriate degradative enzymes of the peripheric cellular metabolism). Cometabolism of xenobiotics involves nothing special or novel from the standpoint of biochemistry. On the contrary, there are numerous examples where the turnover of particular natural compounds by certain aerobic or anaerobic microorganisms is essentially based on coupling of different catabolic pathways at the cellular level by transfer of hydrogen (i.e. reducing power) and/or energy between two or more enzymatic reactions. Synthetic chemicals which resist total degradation by individual microbial species may undergo mineralization due to complementary catabolic sequences mediated by certain multispecies microbial associations with cometabolic transformations being the initial steps. Although taking place in certain natural habitats (e.g. rhizospheres, sewage), microbial cometabolism of xenobiotics in natural ecosystems occurs with slow rates since the respective cometabolizing populations are generally small and will not increase in number or biomass in response to the introduced chemicals. However, under conditions of axenic microbial cultures, high concentrations of biomass, and appropriate substrate mixtures cometabolism of synthetic chemicals may be a useful technique of considerable practical importance to accumulate biochemical products at high yields. In addition, cometabolic capabilities of wild-type microorganisms may serve as a tool for the construction of microbial strains with a new degradative potential for recalcitrant xenobiotic compounds.

Phaseolotoxin production by Pseudomonas syringae pv. phaseolicola: the influence of temperature.

Phaseolotoxin (N-sulphodiaminophosphinyl-ornithyl-alanyl-homoarginine) is a phytotoxic secondary metabolite produced by Pseudomonas syringae pv. phaseolicola. The production of the phytotoxin is strongly regulated by temperature. The optimal temperature for phaseolotoxin production is 18 degrees C. Temperatures in the range between 18 degrees C and 30 degrees C inhibit the production of phaseolotoxin in an increasing manner. By temperature shift experiments and the inhibition of protein synthesis by chloramphenicol it was demonstrated, that the synthesis of enzymes related to toxin synthesis and not the regulation of the activity of the responsible enzyme system is affected by higher temperatures.

[Physiology of aniline catabolism by achromobacter Ir2]. / Physiologie des Anilinabbaus durch Achromobacter Ir 2

A bacterium was isolated from soil which utilizes aniline as sole source of carbon and nitrogen. It was identified as Achromobacter sp. The cells grow at concentrations in the range of 0.5 to 1.25 mg aniline/ml with a growth rate of 0.3 h-1. Substrate inhibition was observed at concentrations higher than 1.5 mg/ml, 3.0 mg/ml completely inhibit the growth. The yield coefficient was 0.63. Aniline was degraded with an activity of 200 microng/mg cell dry weight/hour. Aniline was assimilated and completely degraded. The remaining nitrogen was quantitatively detected as ammonia. The enzyme system involved in aniline degradation was induced by aniline but not repressed by succinate and ammonia.

[Repression of urease biosynthesis in Neurospora crassa by ammonium ions].

The regulation of the synthesis of the enzyme urease (urea amido hydrolase E.C. 3.5.1.5.) in Neurospora crassa was investigated. The biosynthesis of urease is repressed by ammonium ions. Under ammonium excess conditions the specific activity of urease decreases from 0.980 to 0.180 mumoles NH3/min/mg protein. By addition of cycloheximide it was shown that ammonia influences the synthesis of this enzyme. Enzyme induction by the substrate could be excluded. Even under the conditions of highest repression a specific activity of urease of 0.180 mumoles NH3/min/mg protein was measured. Possible causes of this constitutive enzyme level are discussed.

[Studies on the variability of the phaseolotoxin production by Pseudomonas syringae pv. phaseolicola]. / Untersuchungen zur Variabilität der Phaseolotoxinbildung bei Pseudomonas syringae pv. phaseolicola.

Isolation of bacteria from a field of bean plants (Phaseolus vulgaris L.) with conspicuous symptoms of halo blight disease resulted in 123 bacterial strains from which 57 were identified as Pseudomonas syringae pv. phaseolicola . At 18 degrees C the phaseolotoxin production of the isolated strains differs widely in submerse culture. Only few strains produce high amounts of phaseolotoxin being comparable with those of the reference strain 1321, while most of the strains show a low capability of phaseolotoxin production. Furthermore, we proved the stability of phaseolotoxin production of 29 strains. After one year about 50% of the strains were extremely reduced in their capability of phaseolotoxin production. We found that the reason for this reduction of toxin production is the appearance of Tox- segregants within a Tox+ clone. At 24 degrees C all strains (with one exception) show considerable lower amounts of toxin production or none at all: maximally 30% of the toxin amounts synthesized at 18 degrees C were produced. At 28 degrees C none of the isolated strains produce phaseolotoxin . The present data allow the conclusion to be drawn that in natural environments there exists a wide spread regarding the amount and stability of the phaseolotoxin production.

Identification and population dynamics of bacteria in leaf spots of soybean.

The qualitative and quantitative composition of bacterial flora occurring inside the leaf spots of field grown soybeans was studied during the growing seasons (June to October) of 1989 and 1990. As a rule these leaf spots (necrotic lesions with chlorotic haloes) were caused by Pseudomonas syringae pv. glycinea. This pathogenic bacterium was predominantly found during the whole season in the symptomatic leaf tissue. Other species, mainly Erwinia herbicola, were also found in the same habitat. The population sizes of P. s. pv. glycinea increased from the beginning of symptom occurrence until July, stabilized until September, and then decreased a little. In general, the size of saprophytic populations was orders of magnitude lower than that of the pathogenic populations. The number of different bacterial genera per sample increased up to four genera per leaf spot by the end of the season. No significant influence of the occurring saprophytes on the population dynamics of the pathogen in planta could be observed.

Phenol and cresol metabolism in Bacillus pumilus isolated from contaminated groundwater.

From an aquifier contaminated with phenolic compounds seven bacterial strains able to grow on phenol and several mono- and disubstituted alkylphenols as sole source of carbon and energy were isolated. Five isolates belong to the genus Pseudomonas, two to the genus Bacillus. The isolate most active in utilization of the applied xenobiotics was identified as Bacillus pumilus and used for the investigation of the degradation pathways in liquid cultures. Cells of this strain precultured on phenol were able to utilize para-cresol as sole carbon source via the oxidation of the methylsubstituent and intradiol ring cleavage of the resulting protocatechuic acid, whereas an intradiol ring fission of the intermediate 4-methylcatechol led to 4-methylmuconolactone as dead end-product. Cells precultured on meta- and ortho-cresol were able to utilize the respective compounds as sole carbon sources via 3-methylcatechol, which induced the following extradiol ring fission pathway. Cells precultured on phenol were able to cooxidize meta- as well as ortho-cresol to 3-methylcatechol, which was cleaved via an intradiol ring fission, finally leading to the dead end-product 2-methylmuconolactone.

Cometabolic degradation of o-cresol and 2,6-dimethylphenol by Penicillium frequentans Bi 7/2.

o-Cresol induced glucose-grown resting mycelia of Penicillium frequentans Bi 7/2 (ATCC-number: 96048) immediately oxidized o-cresol and other phenols. After precultivation on glucose and phenol degradation started after a lag-phase of 24 hours. Metabolites of o-cresol metabolism were methylhydroquinone, methyl-p-benzoquinone, 2-methyl-5-hydroxyhydroquinone and 2-methyl-5-hydroxy-p-benzoquinone. The initial reaction is probably catalyzed by a NADPH dependent hydroxylase which is specific for o-cresol. The metabolism of 2,6-dimethylphenol (2,6-xylenol) occurred via 2,6-dimethylhydroquinone, 2,6-dimethyl-p-benzoquinone, 2,6-dimethyl-3-hydroxyhydroquinone, 2,6-dimethyl-3-hydroxy-p-benzoquinone and 3-methyl-2-hydroxybenzoic acid.

Unspecific degradation of halogenated phenols by the soil fungus Penicillium frequentans Bi 7/2.

Resting phenol-grown mycelia of the fungus Penicillium frequentans strain Bi 7/2 were shown to be capable of metabolizing various monohalogenated phenols as well as 3,4-dichlorophenol. 2,4.dichlorophenol could be metabolized in the presence of phenol as cosubstrate. In the first degradation step the halogenated phenols were oxidized to the corresponding halocatechols. Halocatechols substituted in para-position (4-halocatechols) were further degraded under formation of 4-carboxymethylenbut-2-en-4-olide. A partial dehalogenation took place splitting the ring system. 3-Halocatechols were cleaved to 2-halomuconic acids as dead end metabolites without a dehalogenation step. Dichlorophenols were only transformed to the corresponding catechols. In addition 3,5-dichloro-catechol was O-methylated to give two isomers of dichloroguiacol. The halogenated catechols with the exception of 4-fluorocatechol partly polymerized oxidatively in the culture fluid to form insoluble dark-brown products. The degradation of halophenols are due to the action of unspecific intracellular enzymes responsible for phenol catabolism (phenol hydroxylase, catechol-1,2-dioxygenase, muconate cycloisomerase I).

Regulation of phenol degradation in Pseudomonas putida.

In order to characterize the ability of Pseudomonas putida (TREVISAN 1889) MIGULA 1895 strain H to degrade various mono- and diphenolic aromatic compounds, respiratory activities towards phenol, catechol, and the cresol isomers were determined. The following rates of oxygen uptake (QO2) were obtained with resting phenol-grown cells: phenol -- 229, o-cresol -- 231, m-cresol -- 43, p-cresol -- 200, catechol -- 262. All these compounds were oxidized by a two-phase-kinetics, the first phase is characterized by a higher oxidation rate than the second. The oxidation of phenol as well as of p-cresol was found to be substrate-inhibited at concentrations above 0.25 mM. A Ki-value of 100 mg/l was calculated for phenol oxidation. The phenol-degrading enzyme system is induced, probably coordinately, by phenol and the cresol isomers. In strain H the degradation of phenol is carried out simultaneously with the assimilation of natural carbohydrates like glucose and sodium pyruvate. Aniline as well as sodium benzoate, though not metabolized by strain H, cause a concentration-dependent inhibition of phenol degradation in resting phenol-grown cells of that strain. The mechanism of this inhibition is discussed.

Occurrence of antimicrobial activities of bacteria from soybean leaf spots.

Bacteria were isolated from leaf spots of field grown soybeans during two growing seasons. The leaf spots yielded up to 4 different species and a total population size of about 10(7)-10(8) bacteria/cm2. The majority of the 192 isolates belonged to the species Pseudomonas syringae pv. glycinea (55%), causing leaf spots of bacterial blight on soybeans, and Erwinia herbicola (22%). The remaining isolates included bacteria from other genera, but occurred occasionally. The determination of biological activity of the isolates demonstrated that a high percentage of strains from the group Erwinia/Enterobacter produced biological active substances against Escherichia coli (69%) and against Chlorella pyrenoidosa (88%). The majority of P. syringae pv. glycinea strains failed to do so. None of the isolates affected the growth of Geotrichum candidum. The E. herbicola strains showed clear antagonistic properties against a wide range of isolated bacteria. Four E. herbicola strains inhibited the growth of nearly all other E. herbicola isolates and 6 other strains were active against most of the P. syringae pv. glycinea isolates. However, antagonistic interactions among strains isolated from a distinct leaf spot were very rarely.

Degradation of aniline and monochloroanilines by Rhodococcus sp. An 117 and a pseudomonad: a comparative study.

Two newly isolated aniline-degrading bacterial strains were characterized with regard to their enzyme systems responsible for aniline catabolism. One of them identified as a Rhodococcus sp. metabolized aniline exclusively via the beta-ketoadipate pathway by means of inducible enzymes. The aniline-degrading enzyme system of the second isolate, presumably a pseudomonad, was shown to consist of an inducible aniline-converting enzyme and constitutive meta-pathway enzymes. Both isolates failed to metabolize monochlorinated anilines in the absence of additional carbon sources. To explain this the ring-cleaving enzymes of both isolates were examined for their substrate specificities. Furthermore, the effect of 4-chlorocatechol on the enzymes catalyzing aniline conversion and catechol oxygenation was investigated.