Expression of benzene dioxygenase from Pseudomonas putida ML2 in cis -1,2-cyclohexanediol-degrading pseudomonads (original) (raw)
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Enzyme and Microbial Technology, 1988
A Pseudomonas sp. able to grow on benzene as sole carbon and energy source was isolated from a mixture of soil and water samples. Experiments with whole cells and enzyme studies showed that benzene was metabolized via cis-l,2-dihydroxycyclohexa-3,5-diene (cis-benzeneglycol) to catechol. A mutant of this strain, which lacked a functional cis-benzeneglycol dehydrogenase, converted benzene almost quantitatively to cis-benzeneglycol. Addition of benzene to a carbon-limited chemostat in which this mutant was growing on succinic acid, resulted in an accumulation of cis-benzeneglycol in the medium. Under the conditions used cis-benzeneglycol was produced at a rate of about 1.4 10-6 mmol s-1 (mg protein)-]. In order to predict the cis-benzeneglycol concentration at various times, a mathematical model is described that fitted rather well for both benzene-transport-limited and ldnetically limited production conditions. However, during prolonged continuous production under carbon-limited conditions, the mutant cells were outcompeted by revertants after about three days. To circumvent this problem, the chemostat was operated under nitrogen-limited conditions, which resulted in a very stable continuous cis-benzeneglycol production process for 10 days.
Degradation of cyclohexane and cyclohexanone by Bacillus lentus strain LP32
A Gram-positive bacterium, Bacillus lentus LP32, originally isolated on the basis of its ability to utilise pyrene as sole source of carbon was found to be able to grow luxuriantly on alicyclic compounds as sole substrates. It showed poor growth on anthracene, naphthalene, 1-naphthol and phenanthrene. Growth rate on cyclohexane was 1.32 d -1 , while doubling time was 0.76 d. The corresponding values for growth on cyclohexanone were 0.77 d -1 and 1.29 d, respectively. Within 10 days, the amount of cyclohexane in culture reduced from 317.62 to 102.55 mgl -1 , then to 23.04 mgl -1 on day 18. On cyclohexanone, substrate concentration decreased from 287.56 mgl -1 to 101.66 mgl -1 in 10 days before declining to 24.21 mgl -1 on day 18. The rate of degradation when growing on cyclohexane was 23.50 mgl -1 d -1 in the first 10 days and 9.93 mgl -1 d -1 between day 10 and day 18, with 67.71% degradation in 10 days and overall percentage degradation of 92.43%. On cyclohexanone, the corresponding values were 18.59 and 9.68 mg l -1 d -1 as well as 64.65 and 91.58%, respectively. This organism is a potential candidate for bioremediation purpose.
Applied and Environmental Microbiology, 2015
Pseudomonas veronii 1YdBTEX2, a benzene and toluene degrader, and Pseudomonas veronii 1YB2, a benzene degrader, have previously been shown to be key players in a benzene-contaminated site. These strains harbor unique catabolic pathways for the degradation of benzene comprising a gene cluster encoding an isopropylbenzene dioxygenase where genes encoding downstream enzymes were interrupted by stop codons. Extradiol dioxygenases were recruited from gene clusters comprising genes encoding a 2-hydroxymuconic semialdehyde dehydrogenase necessary for benzene degradation but typically absent from isopropylbenzene dioxygenase-encoding gene clusters. The benzene dihydrodiol dehydrogenase-encoding gene was not clustered with any other aromatic degradation genes, and the encoded protein was only distantly related to dehydrogenases of aromatic degradation pathways. The involvement of the different gene clusters in the degradation pathways was suggested by real-time quantitative reverse transcrip...
Enzymes involved in the biodegradation of hexachlorocyclohexane: A mini review
The scope of this paper encompasses the following subjects: (i) aerobic and anaerobic degradation pathways of hexachlorocyclohexane (HCH); (ii) important genes and enzymes involved in the metabolic pathways of -HCH degradation; (iii) the instrumental methods for identifying and quantifying intermediate metabolites, such as gas chromatography coupled to mass spectrometry (GC-MS) and other techniques. It can be concluded that typical anaerobic and aerobic pathways of -HCH are well known for a few selected microbial strains, although less is known for anaerobic consortia where the possibility of synergism, antagonism, and mutualism can lead to more particular routes and more effective degradation of -HCH. Conversion and removals in the range 39%-100% and 47%e100% have been reported for aerobic and anaerobic cultures, respectively. Most common metabolites reported for aerobic degradation of lindane are pentachlorocyclohexene (-PCCH), 2,5-dichlorobenzoquinone (DCBQ), Chlorohydroquinone (CHQ), chlorophenol, and phenol, whereas PCCH, isomers of trichlorobenzene (TCB), chlorobenzene, and benzene are the most typical metabolites found in anaerobic pathways. Enzyme and genetic characterization of the involved molecular mechanisms are in their early infancy; more work is needed to elucidate them in the future. Advances have been made on identification of enzymes of Sphingomonas paucimobilis where the gene LinB codifies for the enzyme haloalkane dehalogenase that acts on 1,3,4,6-tetrachloro 1,4-cyclohexadiene, thus debottlenecking the pathway. Other more common enzymes such as phenol hydroxylase, catechol 1,2-dioxygenase, catechol 2,3-dioxygenase are also involved since they attack intermediate metabolites of lindane such as catechol and less substituted chlorophenols. Chromatography coupled to mass spectrometric detector, especially GC-MS, is the most used technique for resolving for -HCH metabolites, although there is an increased participation of HPLC-MS methods. Scintillation methods are very useful to assess final degradation of -HCH.
Applied and environmental microbiology, 1996
Pseudomonas sp. strain JR1, recently isolated with isopropylbenzene (IPB) as the inducer substrate for trichloroethene (TCE) oxidation (B. Dabrock, J. Riedel, J. Bertram, and G. Gottschalk, Arch. Microbiol. 158:9-13, 1992), is able to degrade IPB via the meta-cleavage pathway. The genes encoding the first three enzymes in the catabolism of isopropylbenzene were isolated from a genomic library with the broad-host-range cosmid vector pWE15. A 7.6-kb fragment from a 37.7-kb primary cosmid clone was subcloned and sequenced. It contained seven complete open reading frames, designated ipbA1A2orf3A3A4BC. ipbA codes for the three subunits of a multicomponent IPB dioxygenase, ipbB codes for 2,3-dihydro-2,3-dihydroxy-IPB dehydrogenase, and ipbC codes for 3-isopropylcatechol 2,3-dioxygenase. The deduced amino acid sequences of ipbA1A2A3A4BC exhibited the highest homologies with the corresponding proteins of biphenyl-degradative pathways in gramnegative and gram-positive bacteria. The gene products of the ipb genes were identified by an in vitro transcription-translation system on the basis of their expected molecular masses. IPB dioxygenase and 3-isopropylcatechol 2,3-dioxygenase expressed in E. coli oxidized a wide range of alkyl aromatic compounds. Incubation of E. coli cells carrying ipbA1A2A3A4 with IPB and 18 O 2 yielded reaction products containing both atoms of molecular oxygen, which is in accordance with a dioxygenation reaction. E. coli recombinants harboring and expressing the IPB dioxygenase exhibited the ability to degrade TCE. The ipbA1A2A3A4carrying E. coli strain required neither IPB nor isopropyl--D-thiogalactopyranoside for induction; the rate of TCE degradation was comparable to that by fully induced Pseudomonas strain JR1.
Applied and environmental microbiology, 1997
The degradation of toluene by Pseudomonas putida F1 and of chlorobenzenes by Burkholderia sp. strain PS12 is initiated by incorporation of dioxygen into the aromatic nucleus to form cis-dihydrodihydroxybenzenes. Toluene-grown cells of P. putida F1 and 3-chlorobenzoate-grown cells of Burkholderia sp. strain PS12 were found to monooxygenate the side chain of 2- and 3-chlorotoluene to the corresponding chlorobenzyl alcohols. Further metabolism of these products was slow, and the corresponding chlorobenzoates were usually observed as end products, whereas the 3-chlorobenzoate produced from 3-chlorotoluene in Burkholderia sp. strain PS12 was metabolized further. Escherichia coli cells containing the toluene dioxygenase genes from P. putida F1 oxidized 2- and 3-chlorotoluene to the corresponding chlorobenzyl alcohols as major products, demonstrating that this enzyme is responsible for the observed side chain monooxygenation. Two methyl- and chloro-substituted 1,2-dihydroxycyclohexadienes ...
Journal of bacteriology, 1994
In Pseudomonas paucimobilis UT26, gamma-hexachlorocyclohexane (gamma-HCH) is converted to 2,5-dichloro-2,5-cyclohexadiene-1,4-diol (2,5-DDOL), which is then metabolized to 2,5-dichlorohydroquinone. Here, we isolated from the genomic library of UT26 two genes which expressed 2,5-DDOL dehydrogenase activity when they were transformed into P. putida and Escherichia coli. Both gene products had an apparent molecular size of 28 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The first gene, named linC, located separately from the two genes (linA and linB) which we had already cloned as genes involved in the gamma-HCH degradation. The other, named linX, located about 1 kb upstream of the linA gene encoding gamma-HCH dehydrochlorinase. A gamma-HCH degradation-negative mutant, named UT72, which lacked the whole linC gene but had the intact linX gene was isolated. The linC gene given in a plasmid could complement UT72. These results strongly suggest that the linC gene but n...
Kinetics of 1,4-Dioxane Biodegradation by Monooxygenase-Expressing Bacteria
Environmental Science & Technology, 2006
1,4-Dioxane is a probable human carcinogen, and an important emerging water contaminant. In this study, the biodegradation of dioxane by 20 bacterial isolates was evaluated, and 13 were found to be capable of transforming dioxane. Dioxane served as a growth substrate for Pseudonocardia dioxanivorans CB1190 and Pseudonocardia benzenivorans B5, with yields of 0.09 g protein g dioxane -1 and 0.03 g protein g dioxane -1 , respectively. Cometabolic transformation of dioxane was observed for monooxygenaseexpressing strains that were induced with methane, propane, tetrahydrofuran, or toluene including Methylosinus trichosporium OB3b, Mycobacterium vaccae JOB5, Pseudonocardia K1, Pseudomonas mendocina KR1, Ralstonia pickettii PKO1, Burkholderia cepacia G4, and Rhodococcus RR1. Product toxicity resulted in incomplete dioxane degradation for many of the cometabolic reactions. Brief exposure to acetylene, a known monooxygenase inhibitor, prevented oxidation of dioxane in all cases, supporting the hypothesis that monooxygenase enzymes participated in the transformation of dioxane by these strains. Further, Escherichia coli TG1/pBS(Kan) containing recombinant plasmids derived from the toluene-2-and toluene-4monooxygenases of G4, KR1 and PKO1 were also capable of cometabolic dioxane transformation. Dioxane oxidation rates measured at 50 mg/L ranged from 0.01 to 0.19 mg hr -1 mg protein -1 for the metabolic processes, 0.1-0.38 mg hr -1 mg protein -1 for cometabolism by the monooxygenaseinduced strains, and 0.17-0.60 mg hr -1 mg protein -1 for the recombinant strains. Dioxane was not degraded by M. trichosporium OB3b expressing particulate methane monooxygenase, Pseudomonas putida mt-2 expressing a toluene side-chain monooxygenase, and Pseudomonas JS150 and Pseudomonas putida F1 expressing toluene-2,3dioxygenases. This is the first study to definitively show the role of monooxygenases in dioxane degradation using several independent lines of evidence and to describe the kinetics of metabolic and cometabolic dioxane degradation.
Applied Microbiology and Biotechnology, 1997
Batch cultures of Pseudomonas fluorescens (strain P2a) maintained under carbon-limiting conditions in the presence of chrysene and other aromatics, survived starvation with no detectable changes in cell number for at least 4 months. P2a also demonstrated high dioxygenase levels after growth on benzoate and catechol. To characterize this strain further, early stationary-phase cells were resuspended in fresh mineral medium containing different aromatics, to evaluate enzyme expression in the presence of high-molecular-mass (isocyclic and heterocyclic) compounds. Results demonstrated effects on catechol 1,2-dioxygenase modulation by the model compounds used, confirming a low substrate specificity for this enzyme. The increases of specific activity observed in the presence of heterocyclic compounds were higher than those observed with isocyclic compounds.
Journal of Bacteriology, 2008
Proteomics and targeted gene disruption were used to investigate the catabolism of benzene, styrene, biphenyl, and ethylbenzene in Rhodococcus jostii RHA1, a well-studied soil bacterium whose potent polychlorinated biphenyl (PCB)-transforming properties are partly due to the presence of the related Bph and Etb pathways. Of 151 identified proteins, 22 Bph/Etb proteins were among the most abundant in biphenyl-, ethylbenzene-, benzene-, and styrene-grown cells. Cells grown on biphenyl, ethylbenzene, or benzene contained both Bph and Etb enzymes and at least two sets of lower Bph pathway enzymes. By contrast, styrene-grown cells contained no Etb enzymes and only one set of lower Bph pathway enzymes. Gene disruption established that biphenyl dioxygenase (BPDO) was essential for growth of RHA1 on benzene or styrene but that ethylbenzene dioxygenase (EBDO) was not required for growth on any of the tested substrates. Moreover, whole-cell assays of the ⌬bphAa and etbAa1::cmrA etbAa2::aphII mutants demonstrated that while both dioxygenases preferentially transformed biphenyl, only BPDO transformed styrene. Deletion of pcaL of the -ketoadipate pathway disrupted growth on benzene but not other substrates. Thus, styrene and benzene are degraded via meta-and ortho-cleavage, respectively. Finally, catalases were more abundant during growth on nonpolar aromatic compounds than on aromatic acids. This suggests that the relaxed specificities of BPDO and EBDO that enable RHA1 to grow on a range of compounds come at the cost of increased uncoupling during the latter's initial transformation. The stress response may augment RHA1's ability to degrade PCBs and other pollutants that induce similar uncoupling.