Isolation of Terrabacter sp. Strain DDE-1, Which Metabolizes 1,1-Dichloro-2,2-Bis(4-Chlorophenyl)Ethylene when Induced with Biphenyl

Terrabacter sp. strain DDE-1, able to metabolize 1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene (DDE) in pure culture when induced with biphenyl, was enriched from a 1-1-1-trichloro-2,2-bis(4-chlorophenyl)ethane residue-contaminated agricultural soil. Gas chromatography-mass spectrometry analysis of culture extracts revealed a number of DDE catabolites, including 2-(4′-chlorophenyl)-3,3-dichloropropenoic acid, 2-(4′-chlorophenyl)-2-hydroxy acetic acid, 2-(4′-chlorophenyl) acetic acid, and 4-chlorobenzoic acid.     

Construction of a Novel Polychlorinated Biphenyl-Degrading Bacterium: Utilization of 3,4'-Dichlorobiphenyl by Pseudomonas acidovorans M3GY

Pseudomonas acidovorans M3GY is a recombinant bacterium with the novel capacity to utilize a biphenyl congener chlorinated on both rings, 3,4'-dichlorobiphenyl (3,4'-DCBP), as a sole carbon and energy source. Strain M3GY was constructed with a continuous amalgamated culture apparatus (L. Kr?ckel and D. D. Focht, Appl. Environ. Microbiol. 53:2470-2475, 1987) with P. acidovorans CC1(19), a chloroacetate and biphenyl degrader, and Pseudomonas sp. strain CB15(1), a biphenyl and 3-chlorobenzoate degrader. Genetic and phenotypic data showed the recipient parental strain to be P. acidovorans CC1 and the donor parental strain to be Pseudomonas sp. strain CB15. In growth experiments with 3,4'-DCBP as a sole source of carbon, cultures of strain M3GY increased in absorbance from 0.07 to 0.39 in 29 days while reaching a protein concentration of 58 mug ml and 67% substrate dehalogenation. 4-Chlorobenzoate was identified from culture supernatants of strain M3GY by gas chromatography-infrared spectrometry-mass spectrometry; this would be consistent with the oxidation of the m-chlorinated ring through the standard biphenyl pathway. 4-Chlorobenzoate was converted to 4-chlorocatechol, which was metabolized through the meta-fission pathway. The construction of P. acidovorans M3GY, with the novel capability to utilize 3,4'-DCBP, thus involves the complete use of meta-fission pathways for sequential rupture of the biphenyl and chlorobenzoate rings.     

Construction of a Novel Polychlorinated Biphenyl-Degrading Bacterium: Utilization of 3,4′-Dichlorobiphenyl by Pseudomonas acidovorans M3GY

Pseudomonas acidovorans M3GY is a recombinant bacterium with the novel capacity to utilize a biphenyl congener chlorinated on both rings, 3,4′-dichlorobiphenyl (3,4′-DCBP), as a sole carbon and energy source. Strain M3GY was constructed with a continuous amalgamated culture apparatus (L. Kröckel and D. D. Focht, Appl. Environ. Microbiol. 53:2470-2475, 1987) with P. acidovorans CC1(19), a chloroacetate and biphenyl degrader, and Pseudomonas sp. strain CB15(1), a biphenyl and 3-chlorobenzoate degrader. Genetic and phenotypic data showed the recipient parental strain to be P. acidovorans CC1 and the donor parental strain to be Pseudomonas sp. strain CB15. In growth experiments with 3,4′-DCBP as a sole source of carbon, cultures of strain M3GY increased in absorbance from 0.07 to 0.39 in 29 days while reaching a protein concentration of 58 μg ml^-1 and 67% substrate dehalogenation. 4-Chlorobenzoate was identified from culture supernatants of strain M3GY by gas chromatography-infrared spectrometry-mass spectrometry; this would be consistent with the oxidation of the m-chlorinated ring through the standard biphenyl pathway. 4-Chlorobenzoate was converted to 4-chlorocatechol, which was metabolized through the meta-fission pathway. The construction of P. acidovorans M3GY, with the novel capability to utilize 3,4′-DCBP, thus involves the complete use of meta-fission pathways for sequential rupture of the biphenyl and chlorobenzoate rings.     

Products Formed from Analogues of 1,1,1-Trichloro-2,2-Bis(p-Chlorophenyl) Ethane (DDT) Metabolites by Pseudomonas putida

Cultures of Pseudomonas putida growing in solutions with diphenylmethane as sole carbon source formed 1,1,1′,1′-tetraphenyldimethyl ether. The product was identified by gas chromatography, mass spectrometry, and infrared and nuclear magnetic resonance spectrometry. The formation of benzophenone, benzhydrol, and phenylglycolic acid was established by gas chromatography and mass spectrometry. Similar techniques also revealed that phenylacetic acid was a major metabolite. Resting cell suspensions converted benzhydrol to phenyl-glycolic acid and products tentatively identified as hydroxybenzhydrols and a hydroxybenzophenone. Cell suspensions of the bacterium also converted the tetraphenyldimethyl ether to benzhydrol and benzophenone. Possible pathways for the degradation of these analogues of 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) metabolites are discussed.     

Metabolism of 1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene by Alcaligenes denitrificans

Metabolism of 1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene (DDE), a persistent metabolite of 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT), by an Alcaligenes denitrificans was optimal under `non-shaking' conditions, was accelerated by adding 1 g glucose l^–1, and inhibited by 1 g sodium acetate l^–1 or 1 g sodium succinate l^–1. Addition of biphenyl, in the vapor form, to the reaction mixture did not enhance DDE metabolism. During the reaction, accumulation of conventional metabolites, 1-chloro-2,2-bis(4-chlorophenyl)ethylene (DDMU) and 4-chlorobenzoate, was not observed.     

Production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) byPseudomonas acidovorans

Summary Terpolyesters of 3-hydroxybutyrate (3HB), 4-hydroxybutyrate (4HB) and 3-hydroxyvarelate (3HV) were produced byPseudomonas acidovorans in nitrogen-free culture solutions of 1,4-butanediol and pentanol. When 1,4-butanediol was used as the sole carbon source, a polyester with an unusually high 4HB fraction of 99 mol% was produced.     

Metabolism of Lignin Model Compounds of the Arylglycerol-beta-Aryl Ether Type by Pseudomonas acidovorans D(3)

A natural bacterial isolate that we have classified as Pseudomonas acidovorans grows on the lignin model compounds 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)propane-1,3-diol (compound 1) and 1-(4-hydroxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)propane-1,3-diol (compound 1'), as well as on the corresponding 1-oxo compounds (2 and 2') as sole sources of carbon and energy. Metabolic intermediates present in cultures growing on compound 1 included compound 2, 2-methoxyphenol (guaiacol [compound 3]), beta-hydroxypro-pioveratrone (compound 4), acetoveratrone (compound 5), and veratric acid (compound 6). Also identified were compounds 1', 2', beta-hydroxypropiovanillone (compound 4'), and acetovanillone (compound 5'), indicating that 4-O demethylation also occurs. The phenolic intermediates were the same as those found in cultures growing on compound 1'. Compounds 2 and 2' were in part also reduced to compounds 1 and 1', respectively. Compound 3 was shown to be derived from the 2-methoxyphenoxy moiety. A suggested degradation scheme is as follows: compound 1-->2-->(3 + 4)-->5-->6 (and similarly for 1'). In this scheme, the key reaction is cleavage of the ether linkage between C-2 (C(beta)) of the phenylpropane moiety and the 2-methoxyphenoxy moiety in compounds 2 and 2' (i.e., beta-aryl ether cleavage). On the basis of compounds identified, viz., 3 and 4 (4'), cleavage appears formally to be reductive. Because this is unlikely, the initial cleavage products probably were not detected. The implications of these results for the enzyme(s) responsible are discussed.     

Metabolism of Lignin Model Compounds of the Arylglycerol-beta-Aryl Ether Type by Pseudomonas acidovorans D(3)

[This corrects the article on p. 2608 in vol. 53.].     

Adaptation of Pseudomonas aeruginosa to 2,2'-methylenebis (4-chlorophenol)

A culture of Pseudomonas aeruginosa, isolated from a cooling water system, was grown in the presence of sub-inhibitory concentrations of 2,2'-methylenebis(4-chlorophenol) (MBC). It adapted to increasing concentrations from an initial minimum inhibitory concentration of 36 μg ml^-1 to the highest, 80 μg ml^-1. Resistant cultures exhibited a higher survival rate when exposed to 320 μg ml^-1 than did the original strain. Lipopolysaccharide and outer membrane protein profiles were determined by SDS PAGE. No changes were detected in lipopolysaccharide profiles. The quantity of OprP, the phosphate uptake protein in the outer membrane, decreased to a low level correlating with decreased phosphate (P_i) uptake during growth. It is proposed that OprP is the place of entry for MBC and that the cell can adapt by decreasing the level of OprP in the outer membrane.     

Studies on the subunit structure of 4-deoxy-5-oxoglucarate hydro-lyase (decarboxylating) from Pseudomonas acidovorans.

1. Homogeneous preparations of D-4-deoxy-5-oxoglutarate hydro lyase (decarboxylating)(EC4.2.1.41) were analysed in the ultracentrifuge by the high-speed sedimentation-equilibrium method of Yphantis (1964). The molecular weight in 0.1 M-potassium phosphate buffer, pH 7.2, in 6M-guanidine hydrochloride and in 0.1 M-beta-mercaptoethanol in 6M-guanidine hydrochloride was 113,000, 56,000 and 30,400 respectively. Polyacrylamidegel electrophoresis in the presence of sodium dodecyl sulphate indicated a minimum molecular weight of 30,500. 2. Measurement of the thiol content of the enzyme, before and after reduction with NaBH4 or dithiothreitol under denaturing conditions, indicated the presence of eight thiol residues and two interchain disulphide bridges/enzyme molecule. 3. Amino acid analysis showed that the intact enzyme contains a total of approximately 100 arginine and lysine residues, but digestion of the enzyme with trypsin yielded about 49 peptides staining with ninhydrin in a peptide "map". 4. With the knowledge that the enzyme contains only two substrate-binding sites, it is suggested that the enzyme probably consists of four polypeptide chains arranged in an alpha2beta2 confirmation.     

Transformation of 1,1-dichloro-2,2-(4-chlorophenyl)ethane (DDD) by Ralstonia eutropha strain A5

Evidence is presented demonstrating the ability of Ralstonia eutropha A5 to degrade 1,1-dichloro-2,2-bis(4-chlorophenyl)ethane (DDD) aerobically. Strain A5 was able to effect significant transformation of [(14)C]DDD: the hexane extractable radioactivity decreased to approximately 50% of the controls while more than 25% of the total radioactivity became associated with the acidified culture supernatant. There was also an increase in the amount of radioactivity associated with the cell pellet when compared to the biotic control. A meta-fission pathway for the degradation of DDD is proposed based on the recovery of seven chlorinated metabolites identified by gas chromatography-mass spectrometry analysis.     

Cometabolism of Methyl tertiary Butyl Ether and Gaseous n-Alkanes by Pseudomonas mendocina KR-1 Grown on C5 to C8 n-Alkanes

Pseudomonas mendocina KR-1 grew well on toluene, n-alkanes (C₅ to C₈), and 1° alcohols (C₂ to C₈) but not on other aromatics, gaseous n-alkanes (C₁ to C₄), isoalkanes (C₄ to C₆), 2° alcohols (C₃ to C₈), methyl tertiary butyl ether (MTBE), or tertiary butyl alcohol (TBA). Cells grown under carbon-limited conditions on n-alkanes in the presence of MTBE (42 μmol) oxidized up to 94% of the added MTBE to TBA. Less than 3% of the added MTBE was oxidized to TBA when cells were grown on either 1° alcohols, toluene, or dextrose in the presence of MTBE. Concentrated n-pentane-grown cells oxidized MTBE to TBA without a lag phase and without generating tertiary butyl formate (TBF) as an intermediate. Neither TBF nor TBA was consumed by n-pentane-grown cells, while formaldehyde, the expected C₁ product of MTBE dealkylation, was rapidly consumed. Similar K_s values for MTBE were observed for cells grown on C₅ to C₈ n-alkanes (12.95 ± 2.04 mM), suggesting that the same enzyme oxidizes MTBE in cells grown on each n-alkane. All growth-supporting n-alkanes (C₅ to C₈) inhibited MTBE oxidation by resting n-pentane-grown cells. Propane (K_i = 53 μM) and n-butane (K_i = 16 μM) also inhibited MTBE oxidation, and both gases were also consumed by cells during growth on n-pentane. Cultures grown on C₅ to C₈ n-alkanes also exhibited up to twofold-higher levels of growth in the presence of propane or n-butane, whereas no growth stimulation was observed with methane, ethane, MTBE, TBA, or formaldehyde. The results are discussed in terms of their impacts on our understanding of MTBE biodegradation and cometabolism.     

Chiral overcrowded alkenes; asymmetric synthesis of (3S,3'S)-(M, M)-(E)-(+)-1,1',2,2',3,3',4,4'-octahydro-3,3',7,7'-tetramethyl-4, 4'-biphenanthrylidenes

An asymmetric synthesis route towards (3S,3'S)-(M,M)-(E)-(+)-1,1',2, 2',3,3',4,4'-octahydro-3,3',7,7'-tetramethyl-4,4'-biphenanthrylidene was developed using the Evans procedure as a key step. The absolute configurations of the title compound and of its parent ketone were determined by CD spectroscopy and could be correlated with the stereochemical results of the asymmetric alkylation. Furthermore, a comparison was made with the known (3R,3'R)-(P,P)-(E)-(-)-1,1',2,2', 3,3',4,4'-octahydro-3,3',7,7'-dimethyl-4,4'-biphenanthrylidene. Finally, the X-ray crystallographic analysis of (3S,3'S)-(M, M)-(E)-(+)-1,1',2,2',3,3',4,4'-octahydro-3,3',7,7'-tetramethyl-4, 4'-biphenanthrylidene is presented.     

Dissimilation of the lignin model compound veratrylglycerol-beta-(o-methoxyphenyl) ether by Pseudomonas acidovorans: initial transformations.

The initial transformadomonas acidovorans are demethylation of the 4-methoxyl group of the 3,4-dimethoxyphenyl portion of the molecule, and a NAD+-linked dehydrogenation of the benzyl alcohol group. Based on these findings and those described before, an overall degradation scheme is postulated.     

Recalcitrance of 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene to degradation by pure cultures of 1,1-diphenylethylene-degrading aerobic bacteria

1,1-Dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE) is the peri-chlorinated derivative of 1,1-diphenylethylene (DPE). Biodegradation of DDE and DPE by bacteria has so far not been shown. Pure cultures of aerobic bacteria involved in biodegradation of styrene and polychlorinated biphenyls (PCB) were therefore screened for their ability to degrade or cometabolize DPE and DDE. Styrene-metabolizing bacteria (Rhodococcus strains S5 and VLB150) grew with DPE as their sole source of carbon and energy. Polychlorinated-biphenyl-degrading bacteria (Pseudomonas fluorescens and Rhodococcus globerulus) were unable to degrade DPE even in the presence of an easily utilizable cosubstrate, biphenyl. This is the first report of the utilization of DPE as sole carbon and energy source by bacteria. All the tested bacteria failed to degrade DDE when it was provided as the sole carbon source or in the presence of the respective degradable cosubstrates. DPE transformation could also be detected in cell-free extracts of Rhodococcus S5 and VLB150, but DDE was not transformed, indicating that cell wall and membrane diffusion barriers were not limiting biodegradation. The results of the present study show that, at least for some bacteria, the chlorination of DDE is the main reason for its resistance to biodegradation by styrene and DPE-degrading bacteria. Received: 28 May 1997 / Received revision: 28 October 1997 / Accepted: 31 October 1997