Degradation of Plasticizer Di-n-butylphthalate by Delftia sp. TBKNP-05

Bacterial strain Delftia sp. TBKNP-05, isolated by para-hydroxybenzoate enrichment technique, is capable of degrading di-n-butylphthalate (DBP) as a sole source of carbon and energy. Analysis of intermediates by thin-layer chromatography and high-performance liquid chromatography indicated the presence of monobutylphthalate (MBP), phthalate (PA), and protocatechuate (PCA). The washed cells grown on DBP and PA showed appreciable oxidation of DBP, MBP, PA, and PCA. The enzyme activities in cell-free extracts of Delftia sp. TBKNP-05 exhibited the presence of DBP esterase, MBP esterase, PA-dioxygenase, and PCA 4,5-dioxygenase. The PCA is metabolized by meta-cleavage pathway, leading to further mineralization of the compound in this bacterium.     

Degradation of a Plasticizer, di-n-Butylphthalate by Delftia sp. TBKNP-05

A bacterial strain Delftia sp. TBKNP-05 isolated by para-hydroxybenzoate enrichment technique is capable of degrading di-n-butylphthalate (DBP) as a sole source of carbon and energy. Analysis of intermediates by thin layer chromatography and high performance liquid chromatography indicated the presence of monobutylphthalate (MBP), phthalate (PA), and protocatechuate (PCA). The washed cells grown on DBP and PA showed appreciable oxidation of DBP, MBP, PA, and PCA. The enzyme activities in cell free extracts of Delftia sp. TBKNP-05 exhibited the presence of DBP esterase, MBP esterase, PA-dioxygenase, and protocatechuate 4, 5-dioxygenase. The protocatechuate is metabolized by a meta-cleavage pathway leading to further mineralization of the compound in this bacterium.     

Bacterial degradation of benzalphthalide

APseudomonas sp., isolated by an enrichment culture technique, grew on benzalphthalide at up to 1 g/l as sole carbon source. Cells oxidized both benzalphthalide ando-phthalate at enhanced rates compared with glucose-grown cells, but catechol, gentisate and protocatechuate were oxidized slowly and equally by benzalphthalide-and glucose-grown cells.     

Genetic and biochemical characterization of a 4-hydroxybenzoate hydroxylase from <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Corynebacterium glutamicum uses 4-hydroxybenzoic acid (4HBA) as sole carbon source for growth. Previous studies showed that 4HBA was taken up into cells via PcaK, and the aromatic ring was cleaved via protocatechuate 3,4-dioxygenase. In this study, the gene pobA _Cg (ncgl1032) involved in the conversion of 4HBA into 3,4-dihydroxybenzoate (protocatechuate) was identified, and the gene product PobA _Cg was characterized as a 4HBA 3-hydroxylase, which is a homodimer of PobA_Cg. The pobA _Cg is physically associated with pcaK and formed a putative operon, but the two genes were located distantly to the pca cluster, which encode other enzymes for 4HBA/protocatechuate degradation. This new 4HBA 3-hydroxylase is unique in that it prefers NADPH to NADH as a cosubstrate, although its sequence is similar to other 4HBA 3-hydroxylases that prefer NADH as a cosubstrate. Sited-directed mutagenesis on putative NADPH-binding sites, D38 and T42, further improved its affinity to NADPH as well as its catalytic efficiency.     

Utilization of phthalate esters by micrococci

Several strains of Micrococcus have been isolated by enrichment with one of several phthalate esters as sole carbon source. They have been separated into four groups by their esterase content and nutritional characteristics. The catabolic potential for phthalate utilization found in these strains provides further support for designation of the four groups. Pathways for phthalate utilization by 4,5-dihydroxyphthalate and/or 3,4-dihydroxyphthalate and protocatechuate and/or 2,3-dihydroxybenzoate are outlined, which suggests that micrococci possess substantial potential for the catabolism of aromatic compounds.     

Phthalate and 4-hydroxyphthalate metabolism in Pseudomonas testosteroni: purification and properties of 4,5-dihydroxyphthalate decarboxylase.

Phthalate is degraded through 4,5-dihydroxyphthalate and protocatechuate in Pseudomonas testosteroni NH1000. The ezyme 4,5-dihydroxyphthalate decarboxylase, catalyzing the conversion of 4,5-dihydroxyphthalate to protocatechuate and carbon dioxide, was purified approximately 130-fold from phthalate-induced cells of a protocatechuate 4,5-dioxygenase-deficient mutant of P. testosteroni. The most purified preparation showed a single protein band on sodium dodecyl sulfate-acrylamide disc gel electrophoresis with a molecular weight of 38,000. The apparent molecular weight of the native enzyme determined by Sephadex G-200 column chromatography was 150,000. Among the substrate analogs tested, only 4-hydroxyphthalate served as a substrate, which was decarboxylated to form m-hydroxybenzoate. The apparent Km values for 4,5-dihydroxyphthalate and 4-hydroxyphthalate were estimated to be 10.5 micrometer and 1.25 mM, respectively, and the Vmax for the former was 10 times larger than that for the latter. Whereas the wild-type strain could utilize 4-hydroxyphthalate as a sole source of carbon, none of the following could grow with the compound: 4,5-dihydroxyphthalate decarboxylase-deficient, m-hydroxybenzoate-nondegradable, and protocatechuate 4,5-dioxygenase-deficient mutants. Since one-step revertants of these mutants could utilize 4-hydroxyphthalate, the compound appears to be metabolized through m-hydroxybenzoate and protocatechuate in P. testosteroni NH1000.     

DL-4-hydroxyphenylglycine catabolism in Pseudomonas putida MW27

Thirteen bacteria were isolated on D-4-hydroxyphenylglycine as sole carbon and energy source. Seven strains transaminated only the D-enantiomer while the other six isolates transaminated both enantiomers of 4-hydroxyphenylglycine. One of the six strains utilizing both enantiomers was characterized as a Pseudomonas putida. This strain, MW27, employed two enantioselective transaminases, to catalyze the initial step in the metabolism of DL-4-hydroxyphenylglycine. The product of the transamination, 4-hydroxyphenylglyoxylate, was further metabolized via 4-hydroxybenzaldehyde and 4-hydroxybenzoate to protocatechuate. Preliminary results indicate that both transaminases are co-ordinately synthesized together with the 4-hydroxyphenylglyoxylate decarboxylase and the NADP⁺-dependent 4-hydroxybenzaldehyde dehydrogenase.     

Phthalate and 4-hydroxyphthalate metabolism in Pseudomonas testosteroni: purification and properties of 4,5-dihydroxyphthalate decarboxylase.

Phthalate is degraded through 4,5-dihydroxyphthalate and protocatechuate in Pseudomonas testosteroni NH1000. The ezyme 4,5-dihydroxyphthalate decarboxylase, catalyzing the conversion of 4,5-dihydroxyphthalate to protocatechuate and carbon dioxide, was purified approximately 130-fold from phthalate-induced cells of a protocatechuate 4,5-dioxygenase-deficient mutant of P. testosteroni. The most purified preparation showed a single protein band on sodium dodecyl sulfate-acrylamide disc gel electrophoresis with a molecular weight of 38,000. The apparent molecular weight of the native enzyme determined by Sephadex G-200 column chromatography was 150,000. Among the substrate analogs tested, only 4-hydroxyphthalate served as a substrate, which was decarboxylated to form m-hydroxybenzoate. The apparent Km values for 4,5-dihydroxyphthalate and 4-hydroxyphthalate were estimated to be 10.5 micrometer and 1.25 mM, respectively, and the Vmax for the former was 10 times larger than that for the latter. Whereas the wild-type strain could utilize 4-hydroxyphthalate as a sole source of carbon, none of the following could grow with the compound: 4,5-dihydroxyphthalate decarboxylase-deficient, m-hydroxybenzoate-nondegradable, and protocatechuate 4,5-dioxygenase-deficient mutants. Since one-step revertants of these mutants could utilize 4-hydroxyphthalate, the compound appears to be metabolized through m-hydroxybenzoate and protocatechuate in P. testosteroni NH1000.     

Characterization of <Emphasis Type="Italic">Rhodococcus wratislaviensis</Emphasis> strain J3 that degrades 4-nitrocatechol and other nitroaromatic compounds

The bacterial strain J3 was isolated from soil by selective enrichment on mineral medium containing 4-nitrocatechol as the sole carbon and energy source. This strain was identified as Rhodococcus wratislaviensis on the basis of morphology, biochemical, physiological and chemotaxonomic characterization and complete sequencing of the 16S rDNA gene. The isolated bacterium could utilize 4-nitrocatechol, 3-nitrophenol and 5-nitroguaiacol as sole carbon and energy sources. Stoichiometric release of nitrites was measured during degradation of 4-nitrocatechol both in growing cultures and for stationary phase cells. The J3 strain was unable to degrade 4-nitroguaiacol, 2-nitrophenol, 4-nitrophenol, 2,4-dinitrobenzoic acid, 4,5-dimethoxy-2-nitrobenzoic acid and 2,3-difluoro-6-nitrophenol. The J3 strain is deposited in the Czech Collection of Microorganisms as CCM 4930.     

Degradation of 4-aminobenzenesulfonate by a two-species bacterial coculture

The mutualistic interactions in a 4-aminobenzenesulfonate (sulfanilate) degrading mixed bacterial culture were studied. This coculture consisted of Hydrogenophaga palleronii strain S1 and Agrobacterium radiobacter strain S2. In this coculture only strain S1 desaminated sulfanilate to catechol-4-sulfonate, which did not accumulate in the medium but served as growth substrate for strain S2. During growth in batch culture with sulfanilate as sole source of carbon, energy, nitrogen and sulfur, the relative cell numbers (colony forming units) of both strains were almost constant. None of the strains reached a cell number which was more than threefold higher than the cell number of the second strain. A mineral medium with sulfanilate was inoculated with different relative cell numbers of both strains (relative number of colony forming units S1:S2 2200:1 to 1:500). In all cases, growth was found and the proportion of both strains moved towards an about equal value of about 3:1 (strain S1:strain S2). In contrast to the coculture, strain S1 did not grow in a mineral medium in axenic culture with 4-aminobenzenesulfonate or any other simple organic compound tested. A sterile culture supernatant from strain S2 enabled strain S1 to grow with 4-aminobenzenesulfonate. The same growth promoting effect was found after the addition of a combination of 4-aminobenzoate, biotin and vitamin B₁₂. Strain S1 grew with 4-aminobenzenesulfonate plus the three vitamins with about the same growth rate as the mixed culture in a mineral medium. When (resting) cells of strain S1 were incubated in a pure mineral medium with sulfanilate, up to 30% of the oxidized sulfanilate accumulated as catechol-4-sulfonate in the culture medium. In contrast, only minor amounts of catechol-4-sulfonate accumulated when strain S1 was grown with 4ABS in the presence of the vitamins.Abbreviations 4ABS 4-aminobenzenesulfonate - CFU colony forming units - 4CS catechol-4-sulfonate - 4HB 4-hydroxybenzoate     

Biodegradation of 4-nitroanisole by two Rhodococcus spp.

Two Rhodococcus strains, R. opacus strain AS2 and R. erythropolis strain AS3, that were able to use 4-nitroanisole as the sole source of carbon and energy, were isolated from environmental samples. The first step of the degradation involved the O-demethylation of 4-nitroanisole to 4-nitrophenol which accumulated transiently in the medium during growth. Oxygen uptake experiments indicated the transformation of 4-nitrophenol to 4-nitrocatechol and 1,2,4-trihydroxybenzene prior to ring cleavage and then subsequent mineralization. The nitro group was removed as nitrite, which accumulated in the medium in stoichiometric amounts. In R. opacus strain AS2 small amounts of hydroquinone were produced by a side reaction, but were not further degraded.     

Utilization of homoserine lactone as a sole source of carbon and energy by soil Arthrobacter and Burkholderia species

Homoserine lactone (HSL) is a ubiquitous product of metabolism. It is generated by all known biota during the editing of certain mischarged aminoacyl-tRNA reactions, and is also released as a product of quorum signal degradation by bacterial species expressing acyl-HSL acylases. Little is known about its environmental fate over long or short periods of time. The mammalian enzyme paraoxonase, which has no known homologs in bacteria, has been reported to degrade HSL via a lactonase mechanism. Certain strains of Variovorax and Arthrobacter utilize HSL as a sole source of nitrogen, but not as a sole source of carbon or energy. In this study, the enrichment and isolation of four strains of soil bacteria capable of utilizing HSL as a carbon and energy source are described. Phylogenetic analysis of these isolates indicates that three are distinct members of the genus Arthrobacter, whereas the fourth clusters within the non-clinical Burkholderia. The optimal pH for growth of the isolates ranged from 6.0 to 6.5, at which their HSL-dependent doubling times ranged from 1.4 to 4 h. The biodegradation of HSL by these 4 isolates far outpaced its chemical decay. HSL degradation by soil bacteria has implications for the consortial mineralization of acyl-homoserine lactones by bacteria associated with quorum sensing populations.     

Utilization of catechin byRhizobium sp.

Rhizobium sp. isolated fromLeucaena leucocephala utilized catechin as sole carbon source. Optimum growth occurred at 2 to 5 mM. From replacement cultures containing catechin, phloroglucinolcarboxylic acid, phloroglucinol, resorcinol, protocatechuate, catechol and hydroxyquinol were separated by chromatography. Rothera's test confirmed theortho ring fission of the end products of catechin.     

Biodegradation of Cypermethrin by <Emphasis Type="Italic">Micrococcus</Emphasis> sp. strain CPN 1

A bacterium capable of utilizing pyrethroid pesticide cypermethrin as sole source of carbon was isolated from soil and identified as a Micrococcus sp. The organism also utilized fenvalerate, deltamethrin, perimethrin, 3-phenoxybenzoate, phenol, protocatechuate and catechol as growth substrates. The organism degraded cypermethrin by hydrolysis of ester linkage to yield 3-phenoxybenzoate, leading to loss of its insecticidal activity. 3-Phenoxybenzoate was further metabolized by diphenyl ether cleavage to yield protocatechuate and phenol as evidenced by isolation and identification of metabolites and enzyme activities in the cell-free extracts. Protocatechuate and phenol were oxidized by ortho-cleavage pathway. Thus, the organism was versatile in detoxification and complete mineralization of pyrethroid cypermethrin     

Biodegradation of 4-nitrotoluene by Pseudomonas sp. strain 4NT.

A strain of Pseudomonas spp. was isolated from nitrobenzene-contaminated soil on 4-nitrotoluene as the sole source of carbon, nitrogen, and energy. The organism also grew on 4-nitrobenzaldehyde, and 4-nitrobenzoate. 4-Nitrobenzoate and ammonia were detected in the culture fluid of glucose-grown cells after induction with 4-nitrotoluene. Washed suspensions of 4-nitrotoluene- or 4-nitrobenzoate-grown cells oxidized 4-nitrotoluene, 4-nitrobenzaldehyde, 4-nitrobenzyl alcohol, and protocatechuate. Extracts from induced cells contained 4-nitrobenzaldehyde dehydrogenase, 4-nitrobenzyl alcohol dehydrogenase, and protocatechuate 4,5-dioxygenase activities. Under anaerobic conditions, cell extracts converted 4-nitrobenzoate or 4-hydroxylaminobenzoate to protocatechuate. Conversion of 4-nitrobenzoate to protocatechuate required NADPH. These results indicate that 4-nitrotoluene was degraded by an initial oxidation of the methyl group to form 4-nitrobenzyl alcohol, which was converted to 4-nitrobenzoate via 4-nitrobenzaldehyde. The 4-nitrobenzoate was reduced to 4-hydroxylaminobenzoate, which was converted to protocatechuate. A protocatechuate 4,5-dioxygenase catalyzed meta-ring fission of the protocatechuate. The detection of 4-nitrobenzaldehyde and 4-nitrobenzyl alcohol dehydrogenase and 4-nitrotoluene oxygenase activities in 4-nitrobenzoate-grown cells suggests that 4-nitrobenzoate is an inducer of the 4-nitrotoluene degradative pathway.     

Biodegradation of 3-Nitrobenzoate by <Emphasis Type="Italic">Bacillus flexus</Emphasis> strain XJU-4

Bacillus flexus strain XJU-4 utilized 3-nitrobenzoate at 12 mM as a sole source of carbon and energy. This strain also utilized 4-nitrobenzoate, 2-nitrotoluene and nitrobenzene as growth substrates. The optimum conditions for degradation of 3-nitrobenzoate by the organism were found to be at pH 7.0 and temperature 30°C. Metabolite analysis, growth and enzymatic studies have revealed that the organism degraded 3-nitrobenzoate by oxidative mechanism through protocatechuate with the release of nitrite. The cells grown on 3-nitrobenzoate utilized protocatechuate but not 3-hydroxybenzoate, 3-aminobenzoate, 4-hydroxy-3-nitrobenzoate and 4-nitrocatechol. The cell-free extract of Bacillus flexus strain XJU-4 grown on 3-nitrobenzoate contained the activity of protocatechuate 2,3-dioxygenase, which suggest that protocatechuate was further degraded by a novel 2,3-dioxygenative meta-cleavage pathway.     

Degradation of monohydroxylated benzoates by strain KUFI-6N of the yeast-like fungus Exophiala jeanselmei

Strain KUFI-6N of Exophiala jeanselmei, a cyclohexanol-utilizing yeast-like fungus, was found to grow on 3 isomers of hydroxybenzoate that functioned as the sole carbon sources. Distinct and highly specific hydroxylases converted p- and m-hydroxybenzoate to protocatechuate and o-hydroxybenzoate to catechol.     

Biodegradation of propoxur by Pseudomonas species

A bacterium capable of degrading propoxur (2-isopropoxyphenyl-N-methylcarbamate) was isolated from soil by enrichment cultures and was identified as a Pseudomonas species. The organism grew on propoxur at 2 g/l as sole source of carbon and nitrogen, and accumulated 2-isopropoxyphenol as metabolite in the culture medium. The cell free extract of Pseudomonas sp. grown on propoxur contained the activity of propoxur hydrolase. The results suggest that the organism degraded propoxur by hydrolysis to yield 2-isopropoxyphenol and methylamine, which was further utilized as carbon source.     

Aerobic and anaerobic degradation of furan-3-carboxylate by Paracoccus denitrificans strain MK33

An organism identified as Paracoccus denitrificans was isolated from an enrichment culture with furan-3-carboxylate as its sole source of carbon and energy. The organism degraded furan-3-carboxylate under aerobic and — in the presence of nitrate - under anaerobic conditions. The aerobic degradation was initiated by an oxygenase reaction to form probably 2-hydroxy-3-furoate. Under anaerobic conditions the first intermediate was 3-furoyl-CoA which was reduced by NADPH to probably 4,5-dihydro-furoyl-CoA. Succinic semialdehyde was an intermediate in both aerobic and anaerobic mechanism of furan-3-carboxylate.Abbreviations F-3-C furan-3-carboxylate - 3-FCoA 3-furoyl-CoA     

Utilization of aromatic compounds as carbon and energy sources during growth and N2-fixation by free-living nitrogen fixing bacteria

Six species of free-living nitrogen fixing bacteria, Azomonas agilis, Azospirillum brasilense, Azospirillum lipoferum, Azotobacter chroococcum, Azotobacter vinelandii, and Beijerinckia mobilis, were surveyed for their ability to grow and fix N₂ using aromatic compounds as sole carbon and energy source. All six species grew and expressed nitrogenase activity on benzoate, catechol, 4-hydroxybenzoate, naphthalene, protocatechuate, and 4-toluate. In many cases, growth rates on one or more aromatic compounds were comparable to or greater than those on the non-aromatic substrates routinely used for cultivation of the organisms. Specific activity of nitrogenase in extracts of aromatic-grown cells often exceeded that in cells grown on non-aromatic substrates. All six species growing on substrates typically converted to catechol expressed inducible catechol 1,2-dioxygenase and/or catechol 2,3-dioxygenase. When grown on substrates typically converted to protocatechuate, inducible protocatechuate 3,4-dioxygenase and/or protocatechuate 4,5-dioxygenase was expressed. A. chroococcum expressed only ortho cleavage dioxygenases during growth on naphthalene and 4-toluate and only meta cleavage dioxygenases on the other aromatics. B. mobilis expressed only ortho cleavage dioxygenases. The other four species examined expressed both ortho and meta cleavage enzymes.A preliminary account of this work was presented at the 91st General Meeting of the American Society for Microbiology, Dallas, TX, 1991