Ethylbenzene degradation by Pseudomonas fluorescens strain CA-4

Abstract Pseudomonas fluorescens strain CA-4 is a bioreactor isolate capable of ethylbenzene degradation. Transposon mutagenesis and enzyme assays have been performed which allow us to propose the ethylbenzene degradative pathway in operation in this strain. Ethylbenzene is initially converted to 2-phenylethanol. This is degraded to phenylacetaldehyde and then to phenylacetic acid. The major inducer of the pathway is ethylbenzene itself. The pathway is regulated by the presence of non-aromatic carbon sources. Oxidation of ethylbenzene is repressed by glutamate, but not by citrate or glucose. A clone from a chromosomal library has been found to complement a mutant deficient in the ability to convert ethylbenzene to 2-phenylethanol.     

Cloning and characterization of Pseudomonas sp. strain DNT genes for 2,4-dinitrotoluene degradation.

The degradation of 2,4-dinitrotoluene (DNT) by Pseudomonas sp. strain DNT is initiated by a dioxygenase attack to yield 4-methyl-5-nitrocatechol (MNC) and nitrite. Subsequent oxidation of MNC by a monooxygenase results in the removal of the second molecule of nitrite, and further enzymatic reactions lead to ring fission. Initial studies on the molecular basis of DNT degradation in this strain revealed the presence of three plasmids. Mitomycin-derived mutants deficient in either DNT dioxygenase only or DNT dioxygenase and MNC monooxygenase were isolated. Plasmid profiles of mutant strains suggested that the mutations resulted from deletions in the largest plasmid. Total plasmid DNA partially digested by EcoRI was cloned into a broad-host-range cosmid vector, pCP13. Recombinant clones containing genes encoding DNT dioxygenase, MNC monooxygenase, and 2,4,5-trihydroxytoluene oxygenase were characterized by identification of reaction products and the ability to complement mutants. Subcloning analysis suggests that the DNT dioxygenase is a multicomponent enzyme system and that the genes for the DNT pathway are organized in at least three different operons.     

Identification of a novel dioxygenase involved in metabolism of o-xylene, toluene, and ethylbenzene by Rhodococcus sp. strain DK17

Rhodococcus sp. strain DK17 is able to grow on o-xylene, benzene, toluene, and ethylbenzene. DK17 harbors at least two megaplasmids, and the genes encoding the initial steps in alkylbenzene metabolism are present on the 330-kb pDK2. The genes encoding alkylbenzene degradation were cloned in a cosmid clone and sequenced completely to reveal 35 open reading frames (ORFs). Among the ORFs, we identified two nearly exact copies (one base difference) of genes encoding large and small subunits of an iron sulfur protein terminal oxygenase that are 6 kb apart from each other. Immediately downstream of one copy of the dioxygenase genes (akbA1a and akbA2a) is a gene encoding a dioxygenase ferredoxin component (akbA3), and downstream of the other copy (akbA1b and akbA2b) are genes putatively encoding a meta-cleavage pathway. RT-PCR experiments show that the two copies of the dioxygenase genes are operonic with the downstream putative catabolic genes and that both operons are induced by o-xylene. When expressed in Escherichia coli, AkbA1a-AkbA2a-AkbA3 transformed o-xylene into 2,3- and 3,4-dimethylphenol. These were apparently derived from an unstable o-xylene cis-3,4-dihydrodiol, which readily dehydrates. This indicates a single point of attack of the dioxygenase on the aromatic ring. In contrast, attack of AkbA1a-AkbA2a-AkbA3 on ethylbenzene resulted in the formation of two different cis-dihydrodiols resulting from an oxidation at the 2,3 and the 3,4 positions on the aromatic ring, respectively.     

Assessment of toluene/biphenyl dioxygenase gene diversity in benzene-polluted soils: links between benzene biodegradation and genes similar to those encoding isopropylbenzene dioxygenases

The PCR-single-strand conformation polymorphism (SSCP) technique was used to assess the diversity and distribution of Rieske nonheme iron oxygenases of the toluene/biphenyl subfamily in soil DNA and bacterial isolates recovered from sites contaminated with benzene, toluene, ethylbenzene, and xylenes (BTEX). The central cores of genes encoding the catalytic alpha subunits were targeted, since they are responsible for the substrate specificities of these enzymes. SSCP functional genotype fingerprinting revealed a substantial diversity of oxygenase genes in three differently BTEX-contaminated soil samples, and sequence analysis indicated that in both the soil DNA and the bacterial isolates, genes for oxygenases related to the isopropylbenzene (cumene) dioxygenase branch of the toluene/biphenyl oxygenase subfamily were predominant among the detectable genotypes. The peptide sequences of the two most abundant alpha subunit sequence types differed by only five amino acids (residues 258, 286, 288, 289, and 321 according to numbering in cumene dioxygenase alpha subunit CumA1 of Pseudomonas fluorescens IP01). However, a strong correlation between sequence type and substrate utilization pattern was observed in isolates harboring these genes. Two of these residues were located at positions contributing, according to the resolved crystal structure of cumene dioxygenase from Pseudomonas fluorescens IP01, to the inner surface of the substrate-binding pocket. Isolates containing an alpha subunit with isoleucine and leucine at positions 288 and 321, respectively, were capable of degrading benzene and toluene, whereas isolates containing two methionine substitutions were found to be incapable of degrading toluene, indicating that the more bulky methionine residues significantly narrowed the available space within the substrate-binding pocket.     

The diversity of lipases from psychrotrophic strains of Pseudomonas : a novel lipase from a highly lipolytic strain of Pseudomonas fluorescens

Strains of Pseudomonas fluorescens and Ps. fragi are the predominant psychrotrophs found in raw milk and may cause spoilage due to the secretion of hydrolytic enzymes such as lipase and protease. The diversity of lipases has been examined in Pseudomonas isolates from raw milk which represent different taxonomic groups (phenons). Significant diversity was found using both DNA hybridization and immunoblotting techniques, which has implications for the development of a diagnostic test. The lipase-encoding gene ( lipA ) was cloned from one strain, C9, of Ps. fluorescens biovar V. In contrast to previously reported lipase sequences from Ps. fluorescens , the gene encodes a lipase of M_r 33 kDa. Alignment of all known Pseudomonas and Burkholderia lipase amino acid sequences indicates the existence of two major groups, one of M_r approximately 30 kDa comprising sequences from Ps. fragi , Ps. aeruginosa , Ps. fluorescens C9 and Burkholderia , and one of approximately 50 kDa comprising Ps. fluorescens lipases. The lipase from C9 does not contain a signal peptide and is presumed to be secreted via a signal peptide-independent pathway. The lipA gene of strain C9 was disrupted by insertional mutagenesis. The mutant retained its lipolytic phenotype, strongly suggesting the presence of a second lipase in this strain.     

Engineering Pseudomonas fluorescens for biodegradation of 2,4-dinitrotoluene

Using the genes encoding the 2,4-dinitrotoluene degradation pathway enzymes, the nonpathogenic psychrotolerant rhizobacterium Pseudomonas fluorescens ATCC 17400 was genetically modified for degradation of this priority pollutant. First, a recombinant strain designated MP was constructed by conjugative transfer from Burkholderia sp. strain DNT of the pJS1 megaplasmid, which contains the dnt genes for 2,4-dinitrotoluene degradation. This strain was able to grow on 2,4-dinitrotoluene as the sole source of carbon, nitrogen, and energy at levels equivalent to those of Burkholderia sp. strain DNT. Nevertheless, loss of the 2,4-dinitrotoluene degradative phenotype was observed for strains carrying pJS1. The introduction of dnt genes into the P.fluorescens ATCC 17400 chromosome, using a suicide chromosomal integration Tn5-based delivery plasmid system, generated a degrading strain that was stable for a long time, which was designated RE. This strain was able to use 2,4-dinitrotoluene as a sole nitrogen source and to completely degrade this compound as a cosubstrate. Furthermore, P. fluorescens RE, but not Burkholderia sp. strain DNT, was capable of degrading 2,4-dinitrotoluene at temperatures as low as 10 degrees C. Finally, the presence of P. fluorescens RE in soils containing levels of 2,4-dinitrotoluene lethal to plants significantly decreased the toxic effects of this nitro compound on Arabidopsis thaliana growth. Using synthetic medium culture, P. fluorescens RE was found to be nontoxic for A.thaliana and Nicotiana tabacum, whereas under these conditions Burkholderia sp. strain DNT inhibited A.thaliana seed germination and was lethal to plants. These features reinforce the advantageous environmental robustness of P. fluorescens RE compared with Burkholderia sp. strain DNT.     

Plasmid-coded degradation of ethylbenzene and 1-phenylethanol in Pseudomonas fluorescens

Abstract Pseudomonas fluorescens EB carries genes for the catabolism of ethylbenzene and 1-phenylethanol on a plasmid. The size of the plasmid as measured by analysis of agarose electrophoresis gels after restriction endonuclease hydrolysis, was 253–267 kb. By treatment with Mitomycin C, mutants of EB strain were obtained bearing a plasmid which had undergone an extensive deletion of about 80 kb. These mutants have lost the ability to grow on ethylbenzene and 1-phenylethanol as well as to synthesize meta-cleavage enzymes.     

A novel aromatic-ring-hydroxylating dioxygenase from the diterpenoid-degrading bacterium Pseudomonas abietaniphila BKME-9

Pseudomonas abietaniphila BKME-9 is able to degrade dehydroabietic acid (DhA) via ring hydroxylation by a novel dioxygenase. The ditA1, ditA2, and ditA3 genes, which encode the alpha and beta subunits of the oxygenase and the ferredoxin of the diterpenoid dioxygenase, respectively, were isolated and sequenced. The ferredoxin gene is 9. 2 kb upstream of the oxygenase genes and 872 bp upstream of a putative meta ring cleavage dioxygenase gene, ditC. A Tn5 insertion in the alpha subunit gene, ditA1, resulted in the accumulation by the mutant strain BKME-941 of the pathway intermediate, 7-oxoDhA. Disruption of the ferredoxin gene, ditA3, in wild-type BKME-9 by mutant-allele exchange resulted in a strain (BKME-91) with a phenotype identical to that of the mutant strain BKME-941. Sequence analysis of the putative ferredoxin indicated that it is likely to be a [4Fe-4S]- or [3Fe-4S]-type ferredoxin and not a [2Fe-2S]-type ferredoxin, as found in all previously described ring-hydroxylating dioxygenases. Expression in Escherichia coli of ditA1A2A3, encoding the diterpenoid dioxygenase without its putative reductase component, resulted in a functional enzyme. The diterpenoid dioxygenase attacks 7-oxoDhA, and not DhA, at C-11 and C-12, producing 7-oxo-11, 12-dihydroxy-8,13-abietadien acid, which was identified by 1H nuclear magnetic resonance, UV-visible light, and high-resolution mass spectrometry. The organization of the genes encoding the various components of the diterpenoid dioxygenase, the phylogenetic distinctiveness of both the alpha subunit and the ferredoxin component, and the unusual Fe-S cluster of the ferredoxin all suggest that this enzyme belongs to a new class of aromatic ring-hydroxylating dioxygenases.     

Identification of nah-1 genes of the Pseudomonas putida naphthalene-degrading NPL-41 plasmid operon

Pseudomonas putida BS202 degrades naphthalene via a plasmid-encoded catabolic pathway. The nucleotide sequence of the nahC gene encoding one of this pathway enzymes, 1,2-dihydroxynaphthalene dioxygenase, has been determined. Analysis of nucleotide sequence of its flanking regions identified partially the nahF and putative nahQ genes. Comparison of these three genes with corresponding ones in the NAH7 plasmid and DOX operon showed a high degree of homology.     

XYL, a nonconjugative xylene-degradative plasmid in Pseudomonas Pxy.

Pseudomanas Pxy metabolizes p- or m-xylene through intermediate formation of the corresponding methylbenzyl alcohol and toluic acid via the meta pathway. The strain Pseudomonas Pxy spontaneously loses its ability to grow with xylene or toluate, and the rate of loss of this ability is greatly enhanced by treatment of the cells with mitomycin C. The assay of enzymes involved in xylene degradation in xylene-negative Pxy cells indicates the loss of the entire enzyme complement of the pathway. The genes specifying all the xylene-degradative enzymes, including those of the meta pathway, appear to be borne on a nonconjugative plasmid and can be transferred to xylene-negative Pxy or P. putida strain PpG1 cells only in the presence of a transfer plasmid termed factor K. When transferred to strain PpG1, the xylene-degradative plasmid, termed XYL, coexists stably with factor K, but transduction of XYL is not accompanied by a cotransfer of factor K. XYL appears to be compatible wit- all the other known degradative plasmids in P. putida. The xylene pathway is inducible in wild-type Pxy as well as in Pxy and PpG1 exconjugants, suggesting the cotransfer of regulatory genes along with the plasmid. The enzymes converting xylene to toluate are induced by xylene, methylbenzyl alcohol, or the aldehyde derivatives but not significantly by toluate, whereas catechol dioxygenase and other enzymes are induced by toluates and presumable by xylene as well.     

Characterization of Pseudomonas putida mutants unable to catabolize benzoate: cloning and characterization of Pseudomonas genes involved in benzoate catabolism and isolation of a chromosomal DNA fragment able to substitute for xylS in activation of the TOL lower-pathway promoter.

Mutants of Pseudomonas putida mt-2 that are unable to convert benzoate to catechol were isolated and grouped into two classes: those that did not initiate attack on benzoate and those that accumulated 3,5-cyclohexadiene-1,2-diol-1-carboxylic acid (benzoate diol). The latter mutants, represents by strain PP0201, were shown to lack benzoate diol dehydrogenase (benD) activity. Mutants from the former class were presumed either to carry lesions in one or more subunit structural genes of benzoate dioxygenase (benABC) or the regulatory gene (benR) or to contain multiple mutations. Previous work in this laboratory suggested that benR can substitute for the TOL plasmid-encoded xylS regulatory gene, which promotes gene expression from the OP2 region of the lower or meta pathway operon. Accordingly, structural and regulatory gene mutations were distinguished by the ability of benzoate-grown mutant strains to induce expression from OP2 without xylS by using the TOL plasmid xylE gene (encoding catechol 2,3-dioxygenase) as a reporter. A cloned 12-kb BamHI chromosomal DNA fragment from the P. aeruginosa PAO1 chromosome complemented all of the mutations, as shown by restoration of growth on benzoate minimal medium. Subcloning and deletion analyses allowed identification of DNA fragments carrying benD, benABC, and the region possessing xylS substitution activity, benR. Expression of these genes was examined in a strain devoid of benzoate-utilizing ability, Pseudomonas fluorescens PFO15. The disappearance of benzoate and the production of catechol were determined by chromatographic analysis of supernatants from cultures grown with casamino acids. When P. fluorescens PFO15 was transformed with plasmids containing only benABCD, no loss of benzoate was observed. When either benR or xylS was cloned into plasmids compatible with those plasmids containing only the benABCD regions, benzoate was removed from the medium and catechol was produced. Regulation of expression of the chromosomal structural genes by benR and xylS was quantified by benzoate diol dehydrogenase enzyme assays. The results obtained when xylS was substituted for benR strongly suggest an isofunctional regulatory mechanism between the TOL plasmid lower-pathway genes (via the OP2 promoter) and chromosomal benABC. Southern hybridizations demonstrated that DNA encoding the benzoate dioxygenase structural genes showed homology to DNA encoding toluate dioxygenase from the TOL plasmid pWW0, but benR did not show homology to xylS. Evolutionary relationships between the regulatory systems of chromosomal and plasmid-encoded genes for the catabolism of benzoate and related compounds are suggested.     

Cloning and functional analysis of aniline dioxygenase gene cluster, from Frateuria species ANA-18, that metabolizes aniline via an ortho-cleavage pathway of catechol

Genes encoding an aniline dioxygenase of Frateuria sp. ANA-18, which metabolizes aniline via the ortho-cleavage pathway of catechol, were cloned and named tdn genes. The tdn genes were located on the chromosomal DNA of this bacterium and weren't clustered with catechol-degrading gene clusters. These results show that the ANA-18 aniline-degrading gene cluster is constructionally different from Pseudomonas tdn and Acinetobacter atd gene clusters, which degrade aniline via the meta-cleavage pathway of catechol and organize catechol-metabolic genes in the gene clusters. When cloned tdnQTA1A2B genes were expressed in Eschherichia coli, aniline dioxygenase activity was observed. Southern blot analysis revealed that homologues of the tdnA1A2B genes didn't exist in strain ANA-18. Disruption of the tdnA1A2 genes gave the parent strain ANA-18 a defect in aniline metabolism. On the basis of these results, we concluded that only the cloned tdn genes function as genes encoding aniline dioxygenase in strain ANA-18 although this bacterium had two catechol-degrading gene clusters.     

Cloning and expression in Escherichia coli of the toluene dioxygenase gene from Pseudomonas putida NCIB11767

The genes encoding toluene dioxygenase, toluene cis-glycol dehydrogenase and catechol 2.3-oxygenase from Pseudomonas putida NCIB 11767 were cloned and expressed in Escherichia coli HB101 on a 20 kb fragment. The recombinant strain produced indigo and a variety of other coloured products. Although the enzymes were expressed in the absence of inducers, further induction was observed in the presence of toluene or benzene, implying the presence of regulatory elements on the 20 kb insert.     

Biodegradation of mixtures of substituted benzenes by Pseudomonas sp. strain JS150.

Pseudomonas sp. strain JS150 was isolated as a nonencapsulated variant of Pseudomonas sp. strain JS1 that contains the genes for the degradative pathways of a wide range of substituted aromatic compounds. Pseudomonas sp. strain JS150 grew on phenol, ethylbenzene, toluene, benzene, naphthalene, benzoate, p-hydroxybenzoate, salicylate, chlorobenzene, and several 1,4-dihalogenated benzenes. We designed experiments to determine the conditions required for induction of the individual pathways and to determine whether multiple substrates could be biodegraded simultaneously. Oxygen consumption studies with whole cells and enzyme assays with cell extracts showed that the enzymes of the meta, ortho, and modified ortho cleavage pathways can be induced in strain JS150. Strain JS150 contains a nonspecific toluene dioxygenase with a substrate range similar to that found in strains of Pseudomonas putida. The presence of the dioxygenase along with multiple pathways for metabolism of substituted catechols allows facile extension of the growth range by spontaneous mutation and degradation of mixtures of substituted benzenes and phenols. Chlorobenzene-grown cells of strain JS150 degraded mixtures of chlorobenzene, benzene, toluene, naphthalene, trichloroethylene, and 1,2- and 1,4-dichlorobenzenes in continuous culture. Under similar conditions, phenol-grown cells degraded a mixture of phenol, 2-chloro-, 3-chloro, and 2,5-dichlorophenol and 2-methyl- and 3-methylphenol. These results indicate that induction of appropriate biodegradative pathways in strain JS150 permits the biodegradation of complex mixtures of aromatic compounds.     

Biodegradation of mixtures of substituted benzenes by Pseudomonas sp. strain JS150.

Pseudomonas sp. strain JS150 was isolated as a nonencapsulated variant of Pseudomonas sp. strain JS1 that contains the genes for the degradative pathways of a wide range of substituted aromatic compounds. Pseudomonas sp. strain JS150 grew on phenol, ethylbenzene, toluene, benzene, naphthalene, benzoate, p-hydroxybenzoate, salicylate, chlorobenzene, and several 1,4-dihalogenated benzenes. We designed experiments to determine the conditions required for induction of the individual pathways and to determine whether multiple substrates could be biodegraded simultaneously. Oxygen consumption studies with whole cells and enzyme assays with cell extracts showed that the enzymes of the meta, ortho, and modified ortho cleavage pathways can be induced in strain JS150. Strain JS150 contains a nonspecific toluene dioxygenase with a substrate range similar to that found in strains of Pseudomonas putida. The presence of the dioxygenase along with multiple pathways for metabolism of substituted catechols allows facile extension of the growth range by spontaneous mutation and degradation of mixtures of substituted benzenes and phenols. Chlorobenzene-grown cells of strain JS150 degraded mixtures of chlorobenzene, benzene, toluene, naphthalene, trichloroethylene, and 1,2- and 1,4-dichlorobenzenes in continuous culture. Under similar conditions, phenol-grown cells degraded a mixture of phenol, 2-chloro-, 3-chloro, and 2,5-dichlorophenol and 2-methyl- and 3-methylphenol. These results indicate that induction of appropriate biodegradative pathways in strain JS150 permits the biodegradation of complex mixtures of aromatic compounds.     

Elucidation of the 4-hydroxyacetophenone catabolic pathway in Pseudomonas fluorescens ACB

The catabolism of 4-hydroxyacetophenone in Pseudomonas fluorescens ACB is known to proceed through the intermediate formation of hydroquinone. Here, we provide evidence that hydroquinone is further degraded through 4-hydroxymuconic semialdehyde and maleylacetate to beta-ketoadipate. The P. fluorescens ACB genes involved in 4-hydroxyacetophenone utilization were cloned and characterized. Sequence analysis of a 15-kb DNA fragment showed the presence of 14 open reading frames containing a gene cluster (hapCDEFGHIBA) of which at least four encoded enzymes are involved in 4-hydroxyacetophenone degradation: 4-hydroxyacetophenone monooxygenase (hapA), 4-hydroxyphenyl acetate hydrolase (hapB), 4-hydroxymuconic semialdehyde dehydrogenase (hapE), and maleylacetate reductase (hapF). In between hapF and hapB, three genes encoding a putative intradiol dioxygenase (hapG), a protein of the Yci1 family (hapH), and a [2Fe-2S] ferredoxin (hapI) were found. Downstream of the hap genes, five open reading frames are situated encoding three putative regulatory proteins (orf10, orf12, and orf13) and two proteins possibly involved in a membrane efflux pump (orf11 and orf14). Upstream of hapE, two genes (hapC and hapD) were present that showed weak similarity with several iron(II)-dependent extradiol dioxygenases. Based on these findings and additional biochemical evidence, it is proposed that the hapC and hapD gene products are involved in the ring cleavage of hydroquinone.     

Generation by a widely applicable approach of a hybrid dioxygenase showing improved oxidation of polychlorobiphenyls

Recently, a sequence-based approach has been developed for the fast isolation and characterization of class II aryl-hydroxylating dioxygenase activities (S. Kahl and B. Hofer, Microbiology 149:1475-1481, 2003). It comprises the PCR amplification of segments of alpha subunit genes of unknown sequence that encode the catalytic center and their fusion with sequences of the bphA gene cluster of Burkholderia xenovorans LB400. One of the resulting chimeric enzymes, harboring the core segment of a dioxygenase from Pseudomonas sp. strain B4-Magdeburg, has now been characterized with respect to the oxidation of chlorobiphenyls (CBs). Its substrate and product specificities differed favorably from those of the parental dioxygenase of strain LB400. The hybrid possessed a higher regiospecificity and yielded less unproductive dioxygenations at meta and para carbons. It attacked ortho-, meta-, and para-chlorinated rings with comparable efficiencies. It gave significantly higher yields in ortho,meta-dioxygenation of recalcitrant congeners containing a doubly ortho-chlorinated ring. While the parental enzyme yielded mainly unproductive meta, para dioxygenation of 2,5,4'-CB, the hybrid predominantly converted this congener into an ortho,meta-dioxygenated product. The subsequent enzymes of the LB400 catabolic pathway were able to transform most of the metabolites formed by the novel dioxygenase, indicating that the substrate ranges of these biocatalysts are not adapted to that of their initial pathway enzyme. Some of the catabolites, however, were identified as problematic for further degradation. Our results demonstrate that the outlined approach can successfully be applied to obtain novel dioxygenase specificities that favorably complement or supplement known ones.