Oxidative metabolism of phenanthrene and anthracene by soil pseudomonads. The ring-fission mechanism

1. Phenanthrene is oxidatively metabolized by soil pseudomonads through trans-3,4-dihydro-3,4-dihydroxyphenanthrene to 3,4-dihydroxyphenanthrene, which then undergoes cleavage. 2. Some properties of the ring-fission product, cis-4-(1-hydroxynaphth-2-yl)-2-oxobut-3-enoic acid, are described. The Fe²⁺-dependent oxygenase therefore disrupts the bond between C-4 and the angular C of the phenanthrene nucleus. 3. An enzyme of the aldolase type converts the fission product into 1-hydroxy-2-naphthaldehyde (2-formyl-1-hydroxynaphthalene). An NAD-specific dehydrogenase is also present in the cell-free extract, which oxidizes the aldehyde to 1-hydroxy-2-naphthoic acid. This is then oxidatively decarboxylated to 1,2-dihydroxynaphthalene, thus allowing continuation of metabolism via the naphthalene pathway. 4. Anthracene is similarly metabolized, through 1,2-dihydro-1,2-dihydroxyanthracene to 1,2-dihydroxyanthracene, in which ring-fission occurs to give cis-4-(2-hydroxynaphth-3-yl)-2-oxobut-3-enoic acid. The position of cleavage is again at the bond between the angular C and C-1 of the anthracene nucleus. 5. Enzymes that convert the fission product through 2-hydroxy-3-naphthaldehyde into 2-hydroxy-3-naphthoic acid were demonstrated. The further metabolism of this acid is discussed. 6. The Fe²⁺-dependent oxygenase responsible for cleavage of all the o-dihydroxyphenol derivatives appears to be catechol 2,3-oxygenase, and is a constitutive enzyme in the Pseudomonas strains used.     

Oxidative metabolism of linoleic acid by human leukocytes

Upon incubation with human leukocytes, [1-14C] linoleic acid is almost exclusively transformed into 13-hydroxy-9Z, 11E-octadecadienoic acid (13-HODE) if the linoleic acid concentration is lower than 50 microM. Identification of 13-HODE was done by GLC-MS at the level of its methyl ester, trimethylsilyl ether and by comparison with authentic 13-HODE in two different HPLC systems. Analysis of the products by chiral phase HPLC shows that 13(S)-hydroxy-9Z, 11E-octadecadienoic acid is by far the major metabolite formed by human leukocytes. Comparison of reactions performed with intact or lyzed cells suggests that the formation of 13(S)-HODE by human leukocytes occurs in two steps, a dioxygenation catalyzed by a 15-lipoxygenase and a reduction of intermediate 13-HPODE by a glutathione-dependent peroxidase.     

Quorum-sensing regulation in soil pseudomonads

228 strains of soil and rhizosphere pseudomonads isolated in different geographic zones were screened, with the use of two tester systems, for the capacity to produce N-acyl-homoserine lactones (AHLs), which are autoinducers involved in quorum-sensing (QS) regulation. AHL production was found in 11.4% of the strains investigated. In five Pseudomonas chlororaphis strains shown to be active AHL producers and chosen for further study, PCR identified two QS systems that involved the phzI, phzR, csaI, and csaR genes; this finding suggests the conservative nature of these regulation systems in P. chloroaphis. Strain P. chlororaphis 449, chosen as a model object and studied in greater detail, produced three AHL species including N-butanoyl-homoserine lactone and N-hexanoyl-homoserine lactone. This strain produced three types of phenazine antibiotics, as well as siderophores and cyanide; it also exhibited antagonistic properties toward a wide spectrum of phytopathogenic fungi. The phzI and csaI genes, coding for synthases of AHLs of two types, were cloned and sequenced; mutants with knocked-out phzI and csaI genes were obtained. With the use of transposon mutagenesis and the gene substitution method, mutations were obtained in the global expression regulator genes gacS, coding for the GacA-GacS regulation system kinase, and rpoS, coding for the sigma S subunit of RNA polymerase. The effect of these mutations on the AHL synthesis and on the regulation of various metabolic processes in P. chlororaphis was studied.     

Quorum-sensing regulation in soil pseudomonads

228 strains of soil and rhizosphere pseudomonads isolated in different geographic zones were screened, with the use of two tester systems, for the capacity to produce N-acetyl-homoserine lactones (AHLs), which are autoinducers involved in quorum-sensing (QS) regulation. AHL production was found in 11.4% of the strains investigated. In five Pseudomonas chlororaphis strains shown to be active AHL producers and chosen for further study, PCR identified two QS systems that involved the phzI, phzR, csaI, and csaR genes; this finding suggests the conservative nature of these regulation systems in P. chlororaphis. Strain P. chlororaphis 449, chosen as a model object and studied in greater detail, produced three AHL species including N-butanoyl-homoserine lactone and N-hexanoyl-homoserine lactone. This strain produced three types of phenazine antibiotics, as well as siderophores and cyanide; it also exhibited antagonistic properties toward a wide spectrum of phytopathogenic fungi. The phzI and csaI genes, coding for synthases of AHLs of two types, were cloned and sequenced; mutants with knocked-out phzI and csal genes were obtained. With the use of transposon mutagenesis and the gene substitution method, mutations were obtained in the global expression regulator genes gacS, coding for the GacA-GacS regulation system kinase, and rpoS, coding for the sigma S subunit of RNA polymerase. The effect of these mutations on the AHL synthesis and on the regulation of various metabolic processes in P. chlororaphis was studied.     

The breakdown of phthalic acid esters by microorganisms

The distribution in environment of phthalic acid esters classified according to their inherent toxicity, teratogenic, mutagenic properties and cancerogenic activity into a group of priority pollutants of biosphere is described in this review. The role of microbial community in degradation of phthalate esters has been demonstrated. The effect of such environmental factors as pH of the medium, temperature, humidity, concentration of organic matter, the introduction of promising microbial strains in natural biocenoses has been examined. The pathways of phthalate ester metabolism have been analysed in representatives of active bacterial destructors belonging to genera Nocardia, Pseudomonas and Micrococcus. The decomposition rate of phthalate esters has been shown to be correlated with structure, concentration and culture conditions.     

Iron uptake and metabolism in pseudomonads

Pseudomonads are ubiquitous Gram-negative γ proteobacteria known for their extreme versatility and adaptability. Some are plant pathogens (Pseudomonas syringae) which have to survive on the surface of leaves while others can colonize the rhizosphere or survive in soil (Pseudomonas fluorescens, Pseudomonas putida), and one species, Pseudomonas entomophila, is an insect pathogen. The most investigated species, Pseudomonas aeruginosa, is known to be an opportunistic pathogen able to infect plants, nematodes, insects, and mammals, including humans. Like for other bacteria, iron is a key nutrient for pseudomonads. The fluorescent pseudomonads produce siderophores, the best known being the fluorescent high-affinity peptidic pyoverdines. Often diverse secondary siderophores of lower affinity are produced as well (pyochelin, pseudomonin, corrugatins and ornicorrugatins, yersiniabactin, and thioquinolobactin). Reflecting their large capacity of adaptation to changing environment and niche colonization, pseudomonads are able to obtain their iron from heme or from siderophores produced by other microorganisms (xenosiderophores) via the expression of outer membrane TonB-dependent receptors. As expected, iron uptake is exquisitely and hierarchically regulated in these bacteria. In this short review, the diversity of siderophores produced, receptors, and finally the way iron homeostasis is regulated in P. aeruginosa, P. syringae, P. putida, and P. fluorescens, will be presented and, when possible, put in relation with the lifestyle and the ecological niche.     

Degradation of 3-chlorobenzoate in soil by pseudomonads carrying biodegradative plasmids

Degradation of continuously added 3-chlorobenzoate (3-CB) was studied in samples of chernozem soil. Soil columns were inoculated withPseudomonas putida growing on 3-CB and carrying the biodegradation plasmid and withPseudomonas aeruginosa incapable of growth on 3-CB and carrying the inserted biodegradation plasmid pBS 2 determining ortho-cleavage of the aromatic ring. While the 3-CB degradation was observed in both inoculated variants, the native microflora of the soil under study was incapable to degrade 3-CB. Among pseudomonads isolated from inoculated soil at different stages of cultivation and growth on 3-CB, some had the taxonomic features ofP. putida as well as those differing in 1 –5 characteristics. The study of the activities of the enzymes cleaving the aromatic ring revealed the presence of pyrocatechol 1,2-dioxygenase in the isolated strains only, as estimated by means of benzoate and 3-CB as substrates.     

The regulation of naphthalene metabolism in pseudomonads

The activities of three enzymes specifically involved in naphthalene metabolism have been measured in $\text{Pseudomonas}$ NCIB 9816 after induction with salicylate or 2-hydroxybenzyl alcohol. The results indicate that naphthalene oxygenase, 1,2-dihydroxynaphthalene oxygenase and salicylaldehyde dehydrogenase are induced coordinately and it is suggested that all of the enzymes converting naphthalene to salicylate are regulated coordinately.     

Enhanced dimethyl phthalate biodegradation by accelerating phthalic acid di-oxygenation

The aerobic biodegradation of dimethyl phthalate (DMP) is initiated with two hydrolysis reactions that generate an intermediate, phthalic acid (PA), that is further biodegraded through a two-step di-oxygenation reaction. DMP biodegradation is inhibited when PA accumulates, but DMP’s biodegradation can be enhanced by adding an exogenous electron donor. We evaluated the effect of adding succinate, acetate, or formate as an exogenous electron donor. PA removal rates were increased by 15 and 30% for initial PA concentrations of 0.3 and 0.6 mM when 0.15 and 0.30 mM succinate, respectively, were added as exogenous electron donor. The same electron-equivalent additions of acetate and formate had the same acceleration impacts on PA removal. Consequently, the DMP-removal rate, even PA coexisting with DMP simultaneously, was accelerated by 37% by simultaneous addition of 0.3 mM succinate. Thus, lowering the accumulation of PA by addition of an electron increased the rate of DMP biodegradation.     

Competitive metabolism of naphthalene, methylnaphthalenes, and fluorene by phenanthrene-degrading pseudomonads.

Polynuclear aromatic hydrocarbons (PAHs) typically exist as complex mixtures in contaminated soils, yet little is known about the biodegradation of PAHs in mixtures. We have isolated two physiologically diverse bacteria, Pseudomonas stutzeri P-16 and P. saccharophila P-15, from a creosote-contaminated soil by enrichment on phenanthrene as the sole carbon source and studied their ability to metabolize several other two- and three-ring PAHs. Naphthalene, 1-methylnaphthalene, and 2-methylnaphthalene served as growth substrates for both organisms, while fluorene was only cometabolized. We also studied the effects of these compounds on initial rates of phenanthrene uptake in binary mixtures. Lineweaver-Burk analysis of kinetic measurements was used to demonstrate competitive inhibition of phenanthrene uptake by all four compounds, suggesting that multiple PAHs are being transformed by a common enzyme pathway in whole cells. Estimates of the inhibition coefficient, Ki, are reported for each compound. The occurrence of competitive metabolic processes in physiologically diverse organisms suggests that competitive metabolism may be a common phenomenon among PAH-degrading organisms.     

Naphthalene metabolism by pseudomonads: purification and properties of 1,2-dihydroxynaphthalene oxygenase.

1,2-Dihydroxynaphthalene oxygenase was purified from Pseudomonas putida NCIB 9816 grown on naphthalene as the sole source of carbon and energy. The enzyme had a subunit molecular weight of 19,000 and in a medium containing phosphate buffer, 1 mM mercaptoethanol, and 10% (vol/vol) ethanol had a native molecular weight greater than 275,000. The enzyme required Fe2+ for activity. It was inactivated slowly on standing, and inactivation was accelerated by dilution with aerated buffers and by H2O2. Bathophenanthroline sulfonate, o-phenanthroline, 8-hydroxyquinoline, and 2,2'-dipyridyl also inhibited the enzyme. The inactive enzyme was reactivated by anaerobic incubation with ferrous sulfate and ferrous ammonium sulfate. Thiol reagents and acetone, ethanol, or glycerol decreased the rate of loss of activity. The enzyme was most active with 1,2-dihydroxynaphthalene, for which the Km was 2.8 X 10(-4) M. 3-Methyl- and 4-methylcatechols were oxidized at 3 and 1.5%, respectively, of the rate of 1,2-dihydroxynaphthalene, and the Km for 3-methylcatechol was 1.5 X 10(-4) M. Purified 1,2-dihydroxynaphthalene oxygenase catalyzed the oxidation of 1,2-dihydroxynaphthalene, leading to the appearance of 2-hydroxychromene-2-carboxylic acid, but 3-methylcatechol was oxidized by this enzyme to 2-hydroxy-6-oxoheptadienoic acid. Thus, a product structurally analogous to 2-hydroxychromene-2-carboxylic acid was not observed when 3-methylcatechol was oxidized. This may indicate that 2-hydroxychromene-2-carboxylic acid results from cyclization of a ring fission product before release from the enzyme.     

Biodegradation of phthalic acid esters (PAEs) in soil bioaugmented with acclimated activated sludge

The degradation characteristics of four phthalic acid esters (PAEs), i.e. di-methyl phthalate (DMP), di-ethyl phthalate (DEP), di-n-butyl phthalate (DBP) and di-n-octyl phthalate (DOP) in the soil augmented with acclimated sludge was investigated in order to assess the efficacy of bioaugmentation as a strategy for remediating PAEs-contaminated soil and correlate the degradation rate of PAEs with their alkyl chain length. The results demonstrated that PAEs with shorter alkyl chain, that is, DMP and DEP could be degraded more quickly than DBP and DOP. The degradation of four PAEs in the soil conformed to a first-order reaction kinetic equation. The half-lives of PAEs degradation decreased significantly with increasing carbon number of the alcohol moiety. Half-lives decreased from 2.29 days for DMP to 28.4 days for DOP when the carbon number of alkyl chain increased from one for DMP to eight for DOP. The degradation rate of PAEs and the corresponding half-lives could correlate with the alkyl chain length and their octanol–water partition coefficients (K_ow) quite well for the four PAEs tested in this study.     

Aerobic and anaerobic catabolism of phthalic acid by a nitrate-respiring bacterium

A Bacillus sp., isolated by anaerobic enrichment on a o-phthalic acid-nitrate medium, grew either aerobically or anaerobically on phthalic acid. Cells grown anaerobically on phthalate immediately oxidized phthalate and benzoate with nitrate, whereas aerobic oxidation only occurred after a lag period and was inhibited by chloramphenicol. 2-Fluoro-and 3-fluorobenzoate were formed from 3-fluorophthalate by cells grown anaerobically on phthalate. Aerobically grown cells immediately oxidized phthalate, benzoate, 3-hydroxybenzoate and gentisate with oxygen. The aerobic and anaerobic route of catabolism of phthalate may thus share an initial decarboxylation to benzoate. This is the first report of the anaerobic dissimilation of phthalic acid by a pure bacterial culture.     

Oxidative metabolism of a fatty acid amide hydrolase-regulated lipid, arachidonoyltaurine

A novel class of lipids, N-acyltaurines, was recently discovered in fatty acid amide hydrolase knockout mice. In some peripheral tissues, such as liver and kidney, N-acyltaurines with long, polyunsaturated acyl chains are most prevalent. Polyunsaturated fatty acids are converted to a variety of signaling molecules by cyclooxygenases (COXs) and lipoxygenases (LOXs). The ability of COXs and LOXs to oxygenate arachidonoyltaurine was evaluated to gain insight into the potential metabolic fate of N-acyltaurines. Although arachidonoyltaurine was a poor substrate for COXs, mammalian 12 S- and 15 S-LOXs oxygenated arachidonoyltaurine with similar or better efficiency than arachidonic acid. Products of arachidonoyltaurine oxygenation were characterized by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The positional specificity of single oxygenation was retained for 15 S-LOXs. However, platelet-type 12 S-LOX produced 12- and 15-hydroxyeicosatetraenoyltaurines (HETE-Ts). Furthermore, LOXs generated dihydroxyeicosatetraenoyltaurines (diHETE-Ts). Metabolism of arachidonoyltaurine by murine resident peritoneal macrophages (RPMs) was also profiled. Arachidonoyltaurine was rapidly taken up and converted primarily to 12-HETE-T. Over prolonged incubations, RPMs also generated small amounts of diHETE-T. Oxidative metabolism of polyunsaturated N-acyltaurines may represent a pathway for the generation or termination of novel signaling molecules.