Carbon and hydrogen stable isotope fractionation during aerobic bacterial degradation of aromatic hydrocarbons

13C/(12)C and D/H stable isotope fractionation during aerobic degradation was determined for Pseudomonas putida strain mt-2, Pseudomonas putida strain F1, Ralstonia pickettii strain PKO1, and Pseudomonas putida strain NCIB 9816 grown with toluene, xylenes, and naphthalene. Different types of initial reactions used by the respective bacterial strains could be linked with certain extents of stable isotope fractionation during substrate degradation.     

The metabolism of caffeine by a Pseudomonas putida strain

1) A bacterium capable of growing aerobically with caffeine (1,3,7-trimethylxanthine) as sole source of carbon and nitrogen was isolated from soil. The morphological and physiological characteristics of the bacterium were examined. The organism was identified as a strain of Pseudomonas putida and is referred to as Pseudomonas putida C1. 15 additional caffeine-degrading bacteria were isolated, and all of them were also identified as Pseudomonas putida strains. The properties of the isolates are discussed in comparison with 6 Pseudomonas putida strains of the American Type Culture Collection. 2) The degradation of caffeine by Pseudomonas putida C1 was investigated; the following 14 metabolites were identified: 3,7-dimethylxanthine (theobromine), 1,7-dimethylxanthine, 7-methylxanthine, xanthine, 3,7-dimethyluric acid, 1,7-dimethyluric acid, 7-methyluric acid, uric acid, allantoin, allantoic acid, ureidoglycolic acid, glyoxylic acid, urea, and formaldehyde. Formaldehyde has been demonstrated to be the product of oxidative N-demethylation mediated by an inducible demethylase. A pathway of caffeine degradation is proposed.     

Enhanced tolerance to naphthalene and enhanced rhizoremediation performance for Pseudomonas putida KT2440 via the NAH7 catabolic plasmid

In this work, we explore the potential use of the Pseudomonas putida KT2440 strain for bioremediation of naphthalene-polluted soils. Pseudomonas putida strain KT2440 thrives in naphthalene-saturated medium, establishing a complex response that activates genes coding for extrusion pumps and cellular damage repair enzymes, as well as genes involved in the oxidative stress response. The transfer of the NAH7 plasmid enables naphthalene degradation by P. putida KT2440 while alleviating the cellular stress brought about by this toxic compound, without affecting key functions necessary for survival and colonization of the rhizosphere. Pseudomonas putida KT2440(NAH7) efficiently expresses the Nah catabolic pathway in vitro and in situ, leading to the complete mineralization of [(14)C]naphthalene, measured as the evolution of (14)CO(2), while the rate of mineralization was at least 2-fold higher in the rhizosphere than in bulk soil.     

Amplification of toluene dioxygenase genes in a hybrid Pseudomonas strain to enhance the biodegradation of benzene, toluene, and p-xylene mixture

A hybrid metabolic pathway through which benzene, toluene, and p-xylene (BTX) mixture could be simultaneously mineralized was previously constructed in Pseudomonas putida TB101 (Lee, Roh, Kim, Biotechnol. Bioeng 43: 1146-1152, 1994). In this work, we improved the performance of the hybrid pathway by cloning the todC1C2BA genes in the broad-host-range multicopy vector RSF1010 and by introducing the resulting plasmid pTOL037 into P. putida mt-2 which harbors the archetypal TOL plasmid. As a result, a new hybrid strain, P. putida TB103, possessing the enhanced activity of toluene dioxygenase in the hybrid pathway was constructed. The degradation rates of benzene, toluene, and p-xylene by P. putida TB103 were increased by about 9.3-, 3.7-, and 1.4-fold, respectively, compared with those by previously constructed P. putida TB101. Apparently, this improved capability of P. putida TB103 for the degradation of BTX mixture resulted from the amplification of the todC1C2BA genes. Furthermore, a relatively long lag period for benzene degradation observed when P. putida TB101 was used for the degradation of BTX mixture at low dissolved oxygen (DO) tension disappeared when P. putida TB103 was employed. (c) 1995 John Wiley & Sons, Inc.     

Trichloroethylene degradation by Escherichia coli containing the cloned Pseudomonas putida F1 toluene dioxygenase genes

Toluene dioxygenase from Pseudomonas putida F1 has been implicated as an enzyme capable of degrading trichloroethylene. This has now been confirmed with Escherichia coli JM109(pDTG601) that contains the structural genes (todC1C2BA) of toluene dioxygenase under the control of the tac promoter. The extent of trichloroethylene degradation by the recombinant organism depended on the cell concentration and the concentration of trichloroethylene. A linear rate of trichloroethylene degradation was observed with the E. coli recombinant strain. In contrast, P. putida F39/D, a mutant strain of P. putida F1 that does not contain cis-toluene dihydrodiol dehydrogenase, showed a much faster initial rate of trichloroethylene degradation which decreased over time.     

Trichloroethylene degradation by Escherichia coli containing the cloned Pseudomonas putida F1 toluene dioxygenase genes.

Toluene dioxygenase from Pseudomonas putida F1 has been implicated as an enzyme capable of degrading trichloroethylene. This has now been confirmed with Escherichia coli JM109(pDTG601) that contains the structural genes (todC1C2BA) of toluene dioxygenase under the control of the tac promoter. The extent of trichloroethylene degradation by the recombinant organism depended on the cell concentration and the concentration of trichloroethylene. A linear rate of trichloroethylene degradation was observed with the E. coli recombinant strain. In contrast, P. putida F39/D, a mutant strain of P. putida F1 that does not contain cis-toluene dihydrodiol dehydrogenase, showed a much faster initial rate of trichloroethylene degradation which decreased over time.     

Expression of dibenzothiophene-degradative genes in two Pseudomonas species

The genes encoding dibenzothiophene (DBT) degradation in Pseudomonas alcaligenes strain DBT2 were cloned into plasmid pC1 by other workers. This plasmid was conjugally transferred into a spontaneous variant of Pseudomonas sp. HL7b (designated HL7bR) incapable of oxidizing DBT (Dbt- phenotype). Acquisition of plasmid pC1 simultaneously restored oxidation of DBT and naphthalene to the transconjugant, although the primary DBT metabolite produced by transconjugant HL7bR(pC1) corresponded to that produced by wild-type strain DBT2 rather than that from wild-type strain HL7b. Inducers of the naphthalene pathway (naphthalene, salicylic acid, and 2-aminobenzoate) stimulated DBT oxidation in transconjugant HL7bR(pC1) when present at 0.1 mM concentrations but had no effect on wild-type strain HL7b. Higher concentrations (5 mM) of salicylic acid and naphthalene were inhibitory to DBT oxidation in all strains. DNA-DNA hybridization was not observed between plasmid pC1 and genomic DNA from strains HL7b or HL7bR, nor between authentic naphthalene-degradative genes (plasmid NAH2) and either plasmid pC1 or strain HL7b, despite the observation that the degradative genes encoded on plasmid pC1 functionally resembled broad-specificity naphthalene-degradative genes. Transconjugant HL7bR(pC1) is a mosaic of the parental types regarding DBT metabolite production, regulation, and use of carbon sources.     

Location and sequence of the todF gene encoding 2-hydroxy-6-oxohepta-2,4-dienoate hydrolase in Pseudomonas putida F1

The gene (todF) encoding 2-hydroxy-6-oxohepta-2,4-dienoate hydrolase in Pseudomonas putida F1 was shown to be located upstream of the todC1C2BADE genes. The latter form part of the tod operon and encode the enzymes responsible for the initial reactions in toluene degradation. The nucleotide (nt) sequence of todF was determined and the deduced amino acid (aa) sequence revealed that the hydrolase contains 276 aa with a Mr of 30,753. The deduced aa sequence was 63.5% homologous to that reported for 2-hydroxymuconic semialdehyde hydrolase which is involved in phenol degradation by Pseudomonas CF600.     

Factors influencing the ability of Pseudomonas putida strains epI and II to degrade the organophosphate ethoprophos

Two strains of Pseudomonas putida (epI and epII), isolated previously from ethoprophos-treated soil, were able to degrade ethoprophos (10 mg 1(-1)) in a mineral salts medium plus nitrogen (MSMN) in less than 50 h with a concurrent population growth. Addition of glucose or succinate to MSMN did not influence the degrading ability of Ps. putida epI, but increased the lag phase before rapid degradation commenced with Ps. putida epII. The degrading ability of the two isolates was lost when the pesticide provided the sole source of phosphorus. Degradation of ethoprophos was most rapid when bacterial cultures were incubated at 25 and 37 degrees C. Pseudomonas putida epI was capable of completely degrading ethoprophos at a slow rate at 5 degrees C, compared with Ps. putida epII which could not completely degrade ethoprophos at the same time. Pseudomonas putida epI was capable of degrading ethoprophos when only 60 cells ml(-1) were used as initial inoculum. In contrast, Ps. putida epII was able to totally degrade ethoprophos when inoculum densities of 600 cells ml(-1) or higher were used. In general, longer lag phases accompanied the lower inoculum levels. Both isolates rapidly degraded ethoprophos in MSMN at pHs ranging from 5.5 to 7.6, but not at pH 5 or below.     

Degradation of lawsone by Pseudomonas putida L2

From humus obtained from Stuttgart, a bacterium was isolated with lawsone (2-hydroxy-1,4-naphthoquinone) as selective source of carbon. This bacterium is capable of utilizing lawsone as sole source of carbon and energy. Morphological and physiological characteristics of the bacterium were examined and it was identified as a strain of Pseudomonas putida. The organism is referred to as Pseudomonas putida L2. The degradation of lawsone by Pseudomonas putida L2 was investigated. Salicylic acid and catechol were isolated and identified as metabolites. In lawsone-induced cells of Pseudomonas putida L2, salicylic acid is converted to catechol by salicylate 1-monooxygenase. Catechol 1,2-dioxygenase catalyses ortho-fission of catechol which is then metabolized via the beta-ketoadipate pathway. Formation of cis,cis-muconate and beta-ketoadipate was demonstrated by enzyme assays. Salicylate 1-monooxygenase and catechol 1,2-dioxygenase are induced sequentially. The enzymes of the beta-ketoadipate pathway are also inducible. Naphthoquinone hydroxylase, however, was demonstrated in induced and non-induced cells. This constitutive enzyme enables Pseudomonas putida L2 to degrade various 1,4-naphthoquinones in experiments with resting cells.     

Bacterial Transformations of 1,2,3,4-Tetrahydrodibenzothiophene and Dibenzothiophene

The transformations of 1,2,3,4-tetrahydrodibenzothiophene (THDBT) were investigated with pure cultures of hydrocarbon-degrading bacteria. Metabolites were extracted from cultures with dichloromethane (DCM) and analyzed by gas chromatography (GC) with flame photometric, mass, and Fourier transform infrared detectors. Three 1-methylnaphthalene (1-MN)-utilizing Pseudomonas strains oxidized the sulfur atom of THDBT to give the sulfoxide and sulfone. They also degraded the benzene ring to yield 3-hydroxy-2-formyl-4,5,6,7-tetrahydrobenzothiophene. A cell suspension of a cyclohexane-degrading bacterium oxidized the alicyclic ring to give a hydroxy-substituted THDBT and a ketone, and it oxidized the aromatic ring to give a phenol, but no ring cleavage products were detected. GC analyses with an atomic emission detector, using the sulfur-selective mode, were used to quantify the transformation products from THDBT and dibenzothiophene (DBT). The cyclohexane degrader oxidized 19% of the THDBT to three metabolites. The cometabolism of THDBT and DBT by the three 1-MN-grown Pseudomonas strains resulted in a much greater depletion of the condensed thiophenes than could be accounted for in the metabolites detected by GC analysis, but there was no evidence of sulfate release from DBT. These 1-MN-grown strains transiently accumulated 3-hydroxy-2-formylbenzothiophene (HFBT) from DBT, but it was subsequently degraded. On the other hand, Pseudomonas strain BT1d, which was maintained on DBT as a sole carbon source, accumulated 52% of the sulfur from DBT as HFBT over 7 days, and, in total, 82% of the sulfur from DBT was accounted for by the GC method used. Lyophilization of cultures grown on 1-MN with DBT and methyl esterification of the residues gave improved recoveries of total sulfur over that obtained by DCM extraction and GC analysis. This suggested that the further degradation of HFBT by these cultures leads to the formation of organosulfur compounds that are too polar to be extracted with DCM. We believe that this is the first attempt to quantify the products of DBT degradation by the so-called Kodama pathway.     

Phenanthrene degradation by bacteria of the genera Pseudomonas and Burkholderia in model soil systems

Degradation of phenanthrene by strains Pseudomonas putida BS3701 (pBS1141, pBS1142), Pseudomonas putida BS3745 (pBS216), and Burkholderia sp. BS3702 (pBS1143) was studied in model soil systems. The differences in accumulation and uptake rate of phenanthrene intermediates between the strains under study have been shown, Accumulation of 1-hydroxy-2-naphthoic acid in soil in the course of phenanthrene degradation by strain BS3702 (pBS143) in a model system has been revealed. The efficiency of phenanthrene biodegradation was assessed using the mathematical model proposed previously for assessment of naphthalene degradation efficiency. The efficiency of degradation of both phenanthrene and the intermediate products of its degradation in phenanthrene-contaminated soil is expected to increase with the joint use of strains P. putida BS3701 (pBS1141, pBS1142) and Burkholderia sp. BS3702 (pBS1143).     

Catabolism of biphenyl by Pseudomonas putida BS 893 strain containing the biodegradation plasmid pBS241

The isolation and identification of biphenyl catabolism products in Pseudomonas putida BS 893 (pBS241) showed the presence of benzoic, m-hydroxybenzoic and cinnamic acids. The two latter compounds were not found in biphenyl degradation by other bacterial strains. P. putida BS 893 (pBS241) differed from other biphenyl-positive Pseudomonas strains in the enzyme activity. These differences may stem from peculiarities in the pathway of biphenyl catabolism controlled by plasmid pBS241.     

石油生物脱硫菌Pseudomonas stutzeri UP-1的筛选

以二苯并噻吩(DBT)为模型化合物,筛选到一株能有效降解DBT的菌株,根据其菌落的形态特征、生理生化特征和分子生物学鉴定方法,确定其为Pseudomonms stutzer UP1。该菌株对DBT具有较强的降解能力,降解终产物为水溶性物质。通过对降解产物的分析,初步推断DBT的降解符合Kodama机理。     

Novel organic solvent-responsive expression vectors for biocatalysis: application for development of an organic solvent-tolerant biodesulfurizing strain

Biodesulfurization is an attractive alternative to hydrodesulfurization for lowering the sulfur content of petroleum products. However, the fuel oils are toxic to microorganisms, which have seriously hindered the application of biodesulfurization. Here, a solvent-tolerant desulfurizing bacterium, Pseudomonas putida DS23, was developed using one of the organic solvent-responsive expression vectors newly constructed for biocatalysis, in which gene expression could be regulated in an organic solvent-dependent fashion. The biodesulfurizing activity of P. putida DS23 could be induced by all the organic solvents used. P. putida DS23 cells induced by n-hexane were able to degrade 56% of 0.5 mM DBT in 12 h in the biphasic reaction containing 33.3% (v/v) n-hexane, while the strain induced by isopropyl β-D-1-thiogalactopyranoside could only degrade 26% of 0.5 mM DBT. These results suggested that use of the constructed organic solvent-responsive expression vectors can facilitate the biphasic biocatalysis involving organic solvents.     

Pseudomonas kuykendallii sp. nov.: A Novel γ-Proteobacteria Isolated From a Hexazinone Degrading Bioreactor

Three strains of Gram-negative bacteria designated strains H2(T), H6, and H7 were isolated from bioreactors that degraded the herbicide hexazinone. Similar morphological characteristics, cellular fatty acid profiles, and 16S rRNA gene sequences show that the isolates are members of the same species. These characteristics also show that the isolates belong to the genus Pseudomonas with P. graminis, P. putida, and P. stutzeri as close relatives. The 16S rRNA gene of the H2(T) strain differed from that of type strains for P. graminis, P. putida, and P. stutzeri by 1.9, 2.5, and 2.7 %, respectively, indicating that the H2(T), H6, and H7 strains are related to P. graminis, P. putida, and P. stutzeri but are different enough to represent a novel species. The G+C content of the three strains averaged 61.2 ± 0.8 mol% which is similar to the values reported for P. graminis (61), P. putida (61.6), and P. stutzeri (62.2-65.5). The major cellular fatty acids present in the H2(T) strain were C(18:1) ω7c/C (18:1) ω6c (34.3 %), C(16:1) ω6c/C(16:1) ω7c (27.4 %), C(16:0) (20.6 %), C(12:0) (7.9 %), C(12:0) 3-OH (4.5 %), and C(10:0) 3-OH (3.1 %). The name Pseudomonas kuykendallii sp. nov. is proposed for these bacteria.     

Pseudomonas japonica sp. nov., a novel species that assimilates straight chain alkylphenols

A bacterial strain, WL(T), which was isolated from an activated sludge, was able to degrade alkylphenols. 16S rDNA sequence analysis indicated that strain WL(T) belonged to the genus Pseudomonas (sensu stricto) and formed a monophyletic clade with the type strain of Pseudomonas graminis and other members in the Pseudomonas putida subcluster with sequence similarity values higher than 97%. Genomic relatedness based on DNA-DNA hybridization of strain WL(T) to these strains is 2-41%. Strain WL(T) contained ubiquinone-9 as the main respiratory quinone, and the G+C content of DNA was 66 mol%. The organism contained hexadecanoic acid (16:0), hexadecenoic acid (16:1) and octadecenoic acid (18:1) as major cellular fatty acids. The hydroxy fatty acids detected were 3-hydroxydecanoic acid (3-OH 10:0), 3-hydroxydodecanoic acid (3-OH 12:0) and 2-hydroxydodecanoic acid (2-OH 12:0). These results, as well as physiological and biochemical characteristics clearly indicate that the strain WL(T) represents a new Pseudomonas species, for which the name Pseudomonas japonica is proposed. The type strain is strain WL(T) (=IAM 15071T=TISTR 1526T).     

A common pathway of sulfide oxidation by sulfate-reducing bacteria

Abstract Pseudomonas putida strain DMB capable of growing on 3,4-dimethylbenzoic acid as the only C and energy source was isolated by enrichment techniques. It does not utilize for growth or cooxidize the other dimethylbenzoate isomers tested. 3,4-Dimethylsalicylic acid, 3,4-dimethylphenol and 3,4-dimethylcatechol were isolated and identified by nuclear magnetic resonance and mass spectra in the reaction mixture of P. putida washed cells. The detection of the two first metabolites suggests that the initial step in the degradation of 3,4-dimethylbenzoic acid is the formation of 3,4-dimethylcyclohexa-3,5-diene-1, 2-diol-1-carboxylic acid which underwent an acid-catalyzed dehydration yielding 3,4-dimethylsalicylic acid and 3,4-dimethylphenol. Further degradation proceeds through 3,4-dimethylcatechol via the meta pathway.     

Cloning and nucleotide sequence analysis of xylE gene responsible for meta-cleavage of 4-chlorocatechol from Pseudomonas sp. S-47

Pseudomonas sp. S-47 expresses catechol 2,3-dioxygenase (C230) catalyzing the conversion of 4-chlorocatechol (4CC) as well as catechol to 5-chloro-2-hydroxymuconic semialdehyde and 2-hydroxymuconic semialdehyde, respectively, through meta-ring cleavage. The xylE gene encoding C230 for meta-cleavage was cloned from strain S-47 and its nucleotide sequence was analyzed. The pRES101 containing the xylE gene exhibited high C230 activity toward catechol and 4CC without altering the substrate specificity from natural strain. The xylE gene was composed of 924 bp and encoded polypeptide of molecular mass 35 kDa containing 307 amino acids. A deduced amino acid sequence of the C230 from strain S-47 exhibited over 80% identity with those of Pseudomonas putida mt-2, Pseudomonas putida G7, and Pseudomonas sp. CF600. However, it shows below 45% identity with those of Pseudomonas cepacia LB400 and Pseudomonas sp. KKS102. The C230 of strain S-47 was conserved in the amino acids (His150, His214, Glu261) for metal binding ligands and those (His199, His242, and Tyr251) for catalytic sites. Therefore, Pseudomonas sp. S-47 can be explained as acting by degrading catechol as well as 4CC by xylE-encoding C230 which was fused by N domain of nahH and C domain of dmpB from other Pseudomonas strains.