Metabolism of quaternary carbon compounds: 2,2-dimethylheptane and tertbutylbenzene.

Two Achromobacter strains capable of utilizing 2,2-dimethylheptane or tertbutylbenzene as the sole carbon and energy source were isolated from waste-water. Pivalic acid was found in the cultures of Achromobacter A1 containing 2,2-dimethylheptane. From cultures of Achromobacter A2 in the presence of tertbutylbenzene, a diol was isolated and identified as 2,3-dihydro-2,3-dihydroxytertbutylbenzene. Evidence for meta cleavage of the aromatic ring and for accumulation of pivalic acid in the cultures was also obtained. A metabolic pathway for tertbutylbenzene is suggested.     

Degradation of 3-phenylbutyric acid by Pseudomonas sp.

Pseudomonas sp. isolated by selective culture with 3-phenylbutyrate (3-PB) as the sole carbon source metabolized the compound through two different pathways by initial oxidation of the benzene ring and by initial oxidation of the side chain. During early exponential growth, a catechol substance identified as 3-(2,3-dihydroxyphenyl)butyrate (2,3-DHPB) and its meta-cleavage product 2-hydroxy-7-methyl-6-oxononadioic-2,4-dienoic acid were produced. These products disappeared during late exponential growth, and considerable amounts of 2,3-DHPB reacted to form brownish polymeric substances. The catechol intermediate 2,3-DHPB could not be isolated, but cell-free extracts were able only to oxidize 3-(2,3-dihydroxyphenyl)propionate of all dihydroxy aromatic acids tested. Moreover, a reaction product caused by dehydration of 2,3-DHPB on silica gel was isolated and identified by spectral analysis as (--)-8-hydroxy-4-methyl-3,4-dihydrocoumarin. 3-Phenylpropionate and a hydroxycinnamate were found in supernatants of cultures grown on 3-PB; phenylacetate and benzoate were found in supernatants of cultures grown on 3-phenylpropionate; and phenylacetate was found in cultures grown on cinnamate. Cells grown on 3-PB rapidly oxidized 3-phenylpropionate, cinnamate, catechol, and 3-(2,3-dihydroxyphenyl)propionate, whereas 2-phenylpropionate, 2,3-dihydroxycinnamate, benzoate, phenylacetate, and salicylate were oxidized at much slower rates. Phenylsuccinate was not utilized for growth nor was it oxidized by washed cell suspensions grown on 3-PB. However, dual axenic cultures of Pseudomonas acidovorans and Klebsiella pneumoniae, which could not grow on phenylsuccinate alone, could grow syntrophically and produced the same metabolites found during catabolism of 3-PB by Pseudomonas sp. Washed cell suspensions of dual axenic cultures also immediately oxidized phenylsuccinate, 3-phenylpropionate, cinnamate, phenylacetate, and benzoate.     

Catabolism of phenylpropionic acid and its 3-hydroxy derivative by Escherichia coli

A number of laboratory strains and clinical isolates of Escherichia coli utilized several aromatic acids as sole sources of carbon for growth. E. coli K-12 used separate reactions to convert 3-phenylpropionic and 3-(3-hydroxyphenyl)propionic acids into 3-(2,3-dihydroxyphenyl)propionic acid which, after meta-fission of the benzene nucleus, gave succinate, pyruvate, and acetaldehyde as products. Enzyme assays and respirometry showed that all enzymes of this branched pathway were inducible and that syntheses of enzymes required to convert the two initial growth substrates into 3-(2,3-dihydroxyphenyl)propionate are under separate control. E. coli K-12 also grew with 3-hydroxycinnamic acid as sole source of carbon; the ability of cells to oxidize cinnamic and 3-phenylpropionic acids, and hydroxylated derivatives, was investigated. The lactone of 4-hydroxy-2-ketovaleric acid was isolated from enzymatic reaction mixtures and its properties, including optical activity, were recorded.     

Microbial oxidation of dimethylnaphthalene isomers.

Three bacterial strains, identified as Alcaligenes sp. strain D-59 and Pseudomonas sp. strains D-87 and D-186, capable of growing on 2,6-dimethylnaphthalene (2,6-DMN) as the sole source of carbon and energy were isolated from soil samples. 2,6-Naphthalene dicarboxylic acid was formed in the culture broths of these three strains grown on 2,6-DMN. In addition, 2-hydroxymethyl-6-methylnaphthalene and 6-methylnaphthalene-2-carboxylic acid were detected in the culture broth of strain D-87. Strain D-87 grew well on 1,2-, 1,3-, 1,4-, 1,5-, 2,3-, and 2,7-DMN as the sole source of carbon and energy and accumulated 2-methylnaphthalene-3-carboxylic acid and 2,3-naphthalene dicarboxylic acid from 2,3-DMN, 4-methylnaphthalene-1-carboxylic acid from 1,4-DMN, and 7-methylnaphthalene-2-carboxylic acid from 2,7-DMN.     

Utilization of capsaicin and vanillylamine as growth substrates by Capsicum (hot pepper)-associated bacteria

Capsaicin contributes to the organoleptic attributes of hot peppers. Here, we show that capsaicin is utilized as a growth nutrient by certain bacteria. Enrichment cultures utilizing capsaicin were successfully initiated using Capsicum-derived plant material or leaves of tomato (a related Solanaceae) as inocula. No other sources of inoculum examined yielded positive enrichments. Of 25 isolates obtained from enrichments: all utilized 8-methylnonanoic acid; nine were found capable of degrading capsaicin as sole carbon and energy source; 11 were found capable of utilizing vanillylamine; but only two strains could use either of these latter two compounds as sole nitrogen source. Phylogenetic analysis of capsaicin degraders revealed them to be strains of Variovorax and Ralstonia, whereas the vanillylamine degraders were strains of Pseudomonas and Variovorax. Neither of the two strains isolated from one enrichment culture originally inoculated with dried pepper fruit was capable of using capsaicin as sole carbon and nitrogen source. However, good growth was achieved under such conditions when the two isolates, a strain of Variovorax paradoxusThat degraded capsaicin when provided with ammonium, and a vanillylamine degrading strain of Pseudomonas putida, were cultured together. A cross-feeding of capsaicin-derived carbon and nitrogen between members of pepper-associated consortia is proposed.     

A novel isoindoline,porritoxin sulfonic acid,from Alternaria porri and the structure-phytotoxicity correlation of its related compounds

Novel zinniol-related compound 3, named porritoxin sulfonic acid, with an isoindoline skeleton was isolated from the culture liquid of Alternaria porri. The structure was determined to be 2-(2"-sulfoethyl)-4-methoxy-5-methyl-6-(3'-methyl-2'-butenyloxy)-2,3-dihydro-1H-isoindol-1-one. The phytotoxic activities of three isoindolines (1-3) were evaluated in a seedling-growth assay against stone leek and lettuce.     

Studies on the biodegradation of nonionic surfactants applied in the polyester fiber industry. I. Activated sludge bacteria degrading the surfactants.

The paper presents characteristics of 76 strains of bacteria capable of utilizing nonionic surfactants Cirrasol FP, Cirrasol SF 200 and Cirrasol TCS as the source of carbon. The strains were isolated from two activated sludges adapted to the purification of wastes containing the above compounds at concentration 150--200 mg/l. The isolated strains belonged to the genera: Achromobacter, Alcaligenes, Arthrobacter, Flavobacterium, Mycobacterium, Nocardia, Pseudomonas and Xanthomonas. With load 0.11 mg surfactant/mg d.w./day bacteria belonging to Alcaligenes were dominating. With load 0.18--0.31 mg surfactant/mg d.w./day microorganisms were dominated by Pseudomonas. The highest intensity of degradation of the studied surfactant was shown by species: Alcaligenes viscolactis, Nocardia blackwellii and Pseudomonas rathonis.     

Isolation and characterization of a fluoranthene-utilizing strain of Pseudomonas paucimobilis.

A soil bacterium capable of utilizing fluoranthene as the sole source of carbon and energy for growth was purified from a seven-member bacterial community previously isolated from a creosote waste site for its ability to degrade polycyclic aromatic hydrocarbons. By standard bacteriological methods, this bacterium was characterized taxonomically as a strain of Pseudomonas paucimobilis and was designated strain EPA505. Utilization of fluoranthene by strain EPA 505 was demonstrated by increase in bacterial biomass, decrease in aqueous fluoranthene concentration, and transient formation of transformation products in liquid cultures where fluoranthene was supplied as the sole carbon source. Resting cells grown in complex medium showed activity toward anthraquinone, benzo[b]fluorene, biphenyl, chrysene, and pyrene as demonstrated by the disappearance of parent compounds or changes in their UV absorption spectra. Fluoranthene-grown resting cells were active against these compound as well as 2,3-dimethylnaphthalene, anthracene, fluoranthene, fluorene, naphthalene, and phenanthrene. These studies demonstrate that organic compounds not previously reported to serve as growth substrates can be utilized by axenic cultures of microorganisms. Such organisms may possess novel degradative systems that are active toward other compounds whose biological degradation has been limited because of inherent structural considerations or because of low aqueous solubility.     

Isolation and characterization of a fluoranthene-utilizing strain of Pseudomonas paucimobilis

A soil bacterium capable of utilizing fluoranthene as the sole source of carbon and energy for growth was purified from a seven-member bacterial community previously isolated from a creosote waste site for its ability to degrade polycyclic aromatic hydrocarbons. By standard bacteriological methods, this bacterium was characterized taxonomically as a strain of Pseudomonas paucimobilis and was designated strain EPA505. Utilization of fluoranthene by strain EPA 505 was demonstrated by increase in bacterial biomass, decrease in aqueous fluoranthene concentration, and transient formation of transformation products in liquid cultures where fluoranthene was supplied as the sole carbon source. Resting cells grown in complex medium showed activity toward anthraquinone, benzo[b]fluorene, biphenyl, chrysene, and pyrene as demonstrated by the disappearance of parent compounds or changes in their UV absorption spectra. Fluoranthene-grown resting cells were active against these compound as well as 2,3-dimethylnaphthalene, anthracene, fluoranthene, fluorene, naphthalene, and phenanthrene. These studies demonstrate that organic compounds not previously reported to serve as growth substrates can be utilized by axenic cultures of microorganisms. Such organisms may possess novel degradative systems that are active toward other compounds whose biological degradation has been limited because of inherent structural considerations or because of low aqueous solubility.     

Degradation of phenanthrene and anthracene by Nocardia otitidiscaviarum strain TSH1, a moderately thermophilic bacterium

Aims:  The metabolism of phenanthrene and anthracene by a moderate thermophilic Nocardia otitidiscaviarum strain TSH1 was examined.
Methods and Results:  When strain TSH1 was grown in the presence of anthracene, four metabolites were identified as 1,2-dihydroxy-1,2-dihydroanthracene, 3-(2-carboxyvinyl)naphthalene-2-carboxylic acid, 2,3-dihydroxynaphthalene and benzoic acid using gas chromatography-mass spectrometry (GC-MS), reverse phase-high performance liquid chromatography (RP-HPLC) and thin-layer chromatography (TLC). Degradation studies with phenanthrene revealed 2,2'-diphenic acid, phthalic acid, 4-hydroxyphenylacetic acid, o -hydroxyphenylacetic acid, benzoic acid, a phenanthrene dihydrodiol, 4-[1-hydroxy(2-naphthyl)]-2-oxobut-3-enoic acid and 1-hydroxy-2-naphthoic acid (1H2NA), as detectable metabolites.
Conclusions:  Strain TSH1 initiates phenanthrene degradation via dioxygenation at the C-3 and C-4 or at C-9 and C-10 ring positions. Ortho -cleavage of the 9,10-diol leads to formation of 2,2'-diphenic acid. The 3,4-diol ring is cleaved to form 1H2NA which can subsequently be degraded through o -phthalic acid pathway. Benzoate does not fit in the previously published pathways from mesophiles. Anthracene metabolism seems to start with a dioxygenation at the 1 and 2 positions and ortho -cleavage of the resulting diol. The pathway proceeds probably through 2,3-dicarboxynaphthalene and 2,3-dihydroxynaphthalene. Degradation of 2,3-dihydroxynaphthalene to benzoate and transformation of the later to catechol is a possible route for the further degradation of anthracene.
Significance and Impact of the Study:  For the first time, metabolism of phenanthrene and anthracene in a thermophilic Nocardia strain was investigated.     

Agrobactin, a siderophore from Agrobacterium tumefaciens.

A siderophore (microbial iron transport compound) was isolated from low iron cultures of Agrobacterium tumefaciens B6. The substance was characterized as a threonyl peptide of spermidine acylated with 3 residues of 2,3-dihydroxybenzoic acid, the carbonyl group of 1 residue of the latter participating in an oxazoline ring with the beta-hydroxyl of the threonine moiety. The compound, N-[3-(2,3-dihydroxybenzamido)propyl]-N-[4-(2,3-dihydroxybenzamido)butyl]-2-(2,3-dihydroxyphenyl)-trans-5-methyl-oxazoline-4-carboxamide, was given the trivial name agrobactin. Exposure to acid opened the oxazoline ring to afford agrobactin A. Ferric agrobactin A and agrobactin A itself, but not agrobactin or its ferric complex, had some capacity to feed iron to enterobactin-deficient strains of Escherichia coli and Salmonella typhimurium. Agrobactin was produced by A. tumefaciens in response to iron deficiency and was able to reverse the iron starvation in this organism precipitated by the presence of a ferric complexing agent not utilized by the cells.     

Bacterial metabolism of carbofuran.

Fifteen bacteria capable of degrading carbofuran (2,3-dihydro-2,2-dimethyl-7-benzofuranyl methylcarbamate) were isolated from soil samples with a history of pesticide application. All isolates were gram negative and were oxidase- and catalase-positive rods; they occurred singly or as short chains. All of the identified isolates belonged to one of two genera, Pseudomonas and Flavobacterium. They were separated into three groups based on their mode of utilization of carbofuran. Six isolates were placed in group I; these isolates utilized carbofuran as a sole source of nitrogen. Seven isolates were placed in group II; these isolates utilized the pesticide as a sole source of carbon. Isolates of both groups I and II hydrolyzed carbofuran to carbofuran phenol. Two isolates, designated group III, also utilized carbofuran as a sole source of carbon. They degraded the pesticide more rapidly, however, so up to 40% of [14C]carbofuran was lost as 14CO2 in 1 h. The results suggest that these isolates degrade carbofuran by utilizing an oxidative pathway.     

Bacterial metabolism of carbofuran

Fifteen bacteria capable of degrading carbofuran (2,3-dihydro-2,2-dimethyl-7-benzofuranyl methylcarbamate) were isolated from soil samples with a history of pesticide application. All isolates were gram negative and were oxidase- and catalase-positive rods; they occurred singly or as short chains. All of the identified isolates belonged to one of two genera, Pseudomonas and Flavobacterium. They were separated into three groups based on their mode of utilization of carbofuran. Six isolates were placed in group I; these isolates utilized carbofuran as a sole source of nitrogen. Seven isolates were placed in group II; these isolates utilized the pesticide as a sole source of carbon. Isolates of both groups I and II hydrolyzed carbofuran to carbofuran phenol. Two isolates, designated group III, also utilized carbofuran as a sole source of carbon. They degraded the pesticide more rapidly, however, so up to 40% of [14C]carbofuran was lost as 14CO2 in 1 h. The results suggest that these isolates degrade carbofuran by utilizing an oxidative pathway.     

Antibacterial bromophenols from the marine red alga Rhodomela confervoides

Two bromophenols, together with three known compounds, were isolated from the methanolic extract of the marine alga, Rhodomela confervoides. By means of MS and NMR spectroscopic analyses, they were identified as 3-bromo-4-[2,3-dibromo-4,5-dihydroxyphenyl] methyl-5-(hydroxymethyl) 1,2-benzenediol (1) and 3-bromo-4-[2,3-dibromo-4,5-dihydroxyphenyl] methyl-5- (ethoxymethyl) 1,2-benzenediol (2). Three known compounds were also isolated, namely 3-bromo-4-[2,3-dibromo-4,5-dihydroxyphenyl] methyl-5-(methoxymethyl) 1,2-benzenediol (3), 4,4'- methylenebis [5,6-dibromo-1,2-benzenediol] (4) and bis (2,3-dibromo-4,5-dihydroxybenzyl) ether (5). Compound 5 was the most active against five strains of bacteria with the MIC less than 70 microg/ml, while compounds 2, 3 and 4 exhibited moderate activity.     

Enantioselective Metabolism of Chiral 3-Phenylbutyric Acid, an Intermediate of Linear Alkylbenzene Degradation, by Rhodococcus rhodochrous PB1

Rhodococcus rhodochrous PB1 was isolated from compost soil by selective culture with racemic 3-phenylbutyric acid as the sole carbon and energy source. Growth experiments with the single pure enantiomers as well as with the racemate showed that only one of the two enantiomers, (R)-3-phenylbutyric acid, supported growth of strain PB1. Nevertheless, (S)-3-phenylbutyric acid was cometabolically transformed to, presumably, (S)-3-(2,3-dihydroxyphenyl)butyric acid (the absolute configuration at the C-3 atom is not known yet) by (R)-3-phenylbutyric acid-grown cells of strain PB1, as shown by (sup1)H nuclear magnetic resonance spectroscopy of the partially purified compound and gas chromatography-mass spectrometry analysis of the trimethylsilyl derivative. Oxygen uptake rates suggest that either 3-phenylpropionic acid or cinnamic acid (trans-3-phenyl-2-propenoic acid) is the substrate for aromatic ring hydroxylation. This view is substantiated by the fact that 3-(2,3-dihydroxyphenyl)propionic acid was a substrate for meta cleavage in cell extracts of (R)-3-phenylbutyric acid-grown cells of strain PB1. Gas chromatography-mass spectrometry analysis of trimethylsilane-treated ethyl acetate extracts of incubation mixtures showed that both the meta-cleavage product, 2-hydroxy-6-oxo-2,4-nonadiene-1,9-dicarboxylic acid, and succinate, a hydrolysis product thereof, were formed during such incubations.     

Production of 4,5-dihydro-4,5-dihydroxyphthalate from phthalate by a mutant strain of Pseudomonas testosteroni M4-1

Summary Pseudomonas testosteroni M4-1, capable of using phthalate as the sole carbon and energy source, was isolated. Tn5 mutagenesis using pSUP2021 yielded mutant strains of M4-1 that are defective in phthalate metabolism and produce a dihydrodiol compound. The dihydrodiol compound produced by mutant strain M4-122 was isolated and identified as 4,5-dihydro-4,5-dihydroxyphthalate (DDP) by elementary analysis, mass analysis and nuclear magnetic resonance. Various conditions to increase the yield of DDP from phthalate were examined for mutant strain M4-122. With resting cells 6 g DDP/1 were produced. The additional of ethanol to the resting-cell reaction mixture enhanced DDP production and 10 g DDP/1 was produced from 8.3 g/1 of phthalate. Offprint requests to: T. Omori     

Isolation and characterization of Escherichia coli mutants defective for phenylpropionate degradation

Mutants of Escherichia coli defective in catabolism of 3-phenylpropionate, 3-(3-hydroxyphenyl)propionate, or both were isolated after mutagenesis with ethylmethane sulfonate. Nine phenotypically distinct classes of mutants were identified, including strains lacking each of the first five enzyme activities for the degradation of these compounds and mutants pleiotropically negative for some of these activities. Characterization of these mutants was greatly facilitated by the use of indicator media in which accumulation of 3-(2,3-dihydroxyphenyl)propionate or 2-hydroxy-6-ketononadienedioic acid led to the formation of dark red or bright yellow colors, respectively, in the medium. Assays with wild-type and mutant strains indicated that 3-phenylpropionate (or its dihydrodiol), but none of the hydroxylated derivatives tested, induced the synthesis of enzymes for its conversion to 3-(2,3-dihydroxyphenyl)propionate. The remaining enzymes were induced by the 2- or 3-hydroxy or 2,3-dihydroxy derivatives of 3-phenylpropionate, with the 2-hydroxy compound acting as an apparent gratuitous inducer. Metabolism to nonaromatic intermediates appeared to be unnecessary for full induction of any pathway enzyme. One unusual class of mutants, in which 2-keto-4-pentenoate hydratase appeared to be uninducible, indicated a level of control not previously shown in meta-fission catabolic pathways.     

Biodegradation of chrysene by the bacterial strains isolated from oily sludge

The biodegradation studies were conducted to test the ability of the bacterial strains (Chry2 and Chry3) isolated from the oily sludge obtained from Gujarat refinery, India, for utilization of chrysene in the liquid medium. Biodegradation of the compound was confirmed using gas chromatography and the percent degradation was calculated to be 15.0 and 17% by Chry2 and Chry3, respectively. The biodegradation results were supported by increase in viable cell count and dry biomass, in the presence of chrysene as the sole carbon source. Both the cultures produced biosurfactant which was indicated by the reduction in surface tension of the growth medium. Presence of catechol 2, 3-dioxygenase gene in Chry3 indicated its potential for degradation of PAHs through meta cleavage degradation pathway. Both the strains were found to possess catechol 1,2-dioxygenase and catechol 2,3-dioxygenase enzyme activities. Based on morphological and biochemical tests, the cultures were tentatively identified as Bacillus sp. (Chry2) and Pseudomonas sp. (Chry3).     

Microbial Transformation of Terpenoids. I. Indentification of Metabolites Produced by a Pseudomonad from Citronellal and Citral

A bacterium capable of utilizing citronellal or citral as the sole source of carbon and energy has been isolated from soil by the enrichment culture technique. It metabolizes citronellal to citronellic acid (65%), citronellol (0.6%), dihydrocitronellol (0.6%), menthol (0.75%), and 3,7-dimethyl-1,7-octane diol (1.7%). The metabolites of citral were geranic acid (62%), 6-methyl-5-heptanoic acid (0.5%), 3-methyl-2-butenoic acid (1%), and 1-hydroxy-3, 7-dimethyl-6-octen-2-one (0.75%).     

Microbial degradation of hydrocarbons. Catabolism of 1-phenylalkanes by Nocardia salmonicolor

1. Nocardia salmonicolor grew on a variety of alkanes, 1-phenylalkanes and 1-cyclo-hexylalkanes as sole carbon and energy sources. 2. Growth on 1-phenyldodecane in batch culture was diauxic. Isocitrate lyase activity was induced during lag phase, reaching a maximum activity in the first growth phase, during which both the aromatic ring and the side chain were degraded. However, 4-phenylbutyrate, 4-phenylbut-3-enoate, 4-phenylbut-2-enoate, 3-phenylpropionate, cinnamate and phenylacetate accumulated in the growth medium. These compounds disappeared at the onset of diauxic lag and four hydroxylated compounds accumulated; one was 4-(o-hydroxyphenyl)but-3-enoate and another was identified as 4-(o-hydroxyphenyl)butyrate. These compounds were utilized during the second growth phase. 3. Washed 1-phenyldodecane-grown cells oxidized acetate, cinnamate, 3,4-dihydroxyphenylacetate, homogentisate, o-, m- and p-hydroxyphenylacetate, phenylacetate, and 4-phenylbutyrate rapidly without lag. 4. Extracts of such cells rapidly oxidized homogentisate,3,4-dihydroxyphenylacetate, catechol and protocatechuate. 5. The organism grew readily on 4-phenylbutyrate, phenylacetate, o-hydroxyphenylacetate, homogentisate and 3,4-dihydroxyphenylacetate as sole carbon energy sources, but growth was slow on cinnamate and 4-phenylbut-3-enoate. 6. When cinnamate and phenylacetate were sole carbon sources for growth, phenylacetate and o-hydroxyphenylacetate respectively were detected in culture supernatants. 4-Phenylbut-3-enoate and 4-phenylbutyrate both yielded a mixture of cinnamate and phenylacetate. 7. It is proposed that 1-phenyldodecane is catabolized by ω-oxidation of the terminal methyl group, side-chain β-oxidation to 4-phenylbutyrate, both β- and α-oxidation to phenylacetic acid, hydroxylation to homogentisate via o-hydroxyphenylacetate and ring cleavage to maleylacetoacetate. Catabolism via 3,4-dihydroxyphenylacetate may also occur. 8. Growth on 1-phenylnonane was also diauxic and cinnamic acid, phenylpropionic acid, benzoic acid and hydroxyphenylpentanoic acid accumulated in the medium. Respirometric data and ring-cleavage enzyme activities showed similar patterns to those obtained after growth on 1-phenyldodecane. The results suggest that the main catabolic routes for 1-phenyldodecane and 1-phenylnonane may converge at cinnamate. 9. Possible reasons for diauxie are discussed.