A novel phenanthrene dioxygenase from Nocardioides sp. Strain KP7: expression in Escherichia coli

Nocardioides sp. strain KP7 grows on phenanthrene but not on naphthalene. This organism degrades phenanthrene via 1-hydroxy-2-naphthoate, o-phthalate, and protocatechuate. The genes responsible for the degradation of phenanthrene to o-phthalate (phd) were found by Southern hybridization to reside on the chromosome. A 10.6-kb DNA fragment containing eight phd genes was cloned and sequenced. The phdA, phdB, phdC, and phdD genes, which encode the alpha and beta subunits of the oxygenase component, a ferredoxin, and a ferredoxin reductase, respectively, of phenanthrene dioxygenase were identified. The gene cluster, phdAB, was located 8. 3 kb downstream of the previously characterized phdK gene, which encodes 2-carboxybenzaldehyde dehydrogenase. The phdCD gene cluster was located 2.9 kb downstream of the phdB gene. PhdA and PhdB exhibited moderate (less than 60%) sequence identity to the alpha and beta subunits of other ring-hydroxylating dioxygenases. The PhdC sequence showed features of a [3Fe-4S] or [4Fe-4S] type of ferredoxin, not of the [2Fe-2S] type of ferredoxin that has been found in most of the reported ring-hydroxylating dioxygenases. PhdD also showed moderate (less than 40%) sequence identity to known reductases. The phdABCD genes were expressed poorly in Escherichia coli, even when placed under the control of strong promoters. The introduction of a Shine-Dalgarno sequence upstream of each initiation codon of the phdABCD genes improved their expression in E. coli. E. coli cells carrying phdBCD or phdACD exhibited no phenanthrene-degrading activity, and those carrying phdABD or phdABC exhibited phenanthrene-degrading activity which was significantly less than that in cells carrying the phdABCD genes. It was thus concluded that all of the phdABCD genes are necessary for the efficient expression of phenanthrene-degrading activity. The genetic organization of the phd genes, the phylogenetically diverged positions of these genes, and an unusual type of ferredoxin component suggest phenanthrene dioxygenase in Nocardioides sp. strain KP7 to be a new class of aromatic ring-hydroxylating dioxygenases.     

Occurrence of Two Resin Acid-Degrading Bacteria and a Gene Encoding Resin Acid Biodegradation in Pulp and Paper Mill Effluent Biotreatment Systems Assayed by PCR

Abstract We examined the distribution of two dehydroabietic acid-degrading bacteria, Pseudomonas abietaniphila BKME-9 and Zoogloea resiniphila DhA-35, in biotreatment systems for pulp and paper mill effluents (PPMEs) using PCR assays. These two bacteria were first isolated from two PPME biotreatment systems and can degrade both dehydroabietic acid (DhA) and other abietane resin acids. We also examined the distribution of a catabolic gene, ditA1, encoding the α subunit of an aromatic ring-hydroxylating dioxygenase involved in DhA degradation by BKME-9. PCR primers specific for the 16S rDNA of BKME-9 and of DhA-35 and specific for ditA1 were used. Among 3 laboratory- and 17 full-scale PPME biotreatment systems, 10 contained phylotype BKME-9, 3 contained phylotype DhA-35, and 11 contained ditA1, indicating the wider distribution of phylotype BKME-9 than of phylotype DhA-35. Both phylotype BKME-9 and ditA1 were detected in the biotreatment system from which BKME-9 was originally isolated in 1994, suggesting the persistance of BKME-9 in that biotreatment system. The detection limit of the PCR assay was one cell per PCR reaction, which corresponds to one BKME-9 cell per 6 × 10⁷ total sludge bacteria. A competitive PCR assay indicated that ditA1 ranged from 51 to 250 copies/mg of dry biomass. BKME-9 appears to contribute to the biodegration of resin acids in some PPME biotreatment systems. Using degenerate PCR primers and touchdown PCR, we obtained from our DhA-degrading strain collection six DNA sequences putatively homologous to that of ditA1. Cluster analysis of these DNA sequences suggests that ditA1 encodes a representative of a novel class of dioxygenase enzymes. Received: 12 February 1999; Accepted: 4 May 1999     

Isolation and characterization of genes encoding polycyclic aromatic hydrocarbon dioxygenase from acenaphthene and acenaphthylene degrading Sphingomonas sp. strain A4

Sphingomonas sp. strain A4 is capable of utilizing acenaphthene and acenaphthylene as sole carbon and energy sources, but it is unable to grow on other polycyclic aromatic hydrocarbons (PAHs). The genes encoding terminal oxygenase components of ring-hydroxylating dioxygenase (arhA1 and arhA2) were isolated from this strain by means of the ability to oxidize indole to indigo of the Escherichia coli clone containing electron transport proteins from phenanthrene-degrading Sphingobium sp. strain P2. The translated products of arhA1 and arhA2 exhibited moderate sequence identity (less than 56%) to large and small subunits of dioxygenase of other ring-hydroxylating dioxygenases. Biotransformation with recombinant E. coli clone revealed the broad substrate specificity of this oxygenase toward several PAHs including acenaphthene, acenaphthylene, naphthalene, phenanthrene, anthracene and fluoranthene. Southern hybridization analysis revealed the presence of a putative arhA1 homologue on a locus different from that of the arhA1 gene. Insertion inactivation of the arhA1 gene in strain A4 suggested that the gene but not the putative homologue one was involved in the degradation of acenaphthene and acenaphthylene in this strain.     

An alternative inverse PCR (IPCR) method to amplify DNA sequences flanking Tn5 transposon insertions

We have developed an alternative method to amplify DNA sequences flanking Tn5 transposon insertions. This method relies on the identical sequences of inverted terminal repeats, located at the 5' and 3' ends of Tn5, to determine the location and orientation of a transposon insertion within a restriction endonuclease fragment. From this information, PCR primers can be designed to selectively amplify by inverse PCR the DNA flanking one side of the transposon. This method avoids the problem of amplifying or cloning long sequences flanking Tn5. To demonstrate the applicability of this method, we generated Tn5 transposon mutants of Pseudomonas abietaniphila BKME-9 which no longer grew on dehydroabietic acid (DhA). The flanking sequence of one of the mutant (strain BKME-941) which accumulated 7-oxoDhA, was amplified.     

Identification and functional analysis of two aromatic-ring-hydroxylating dioxygenases from a sphingomonas strain that degrades various polycyclic aromatic hydrocarbons

In this study, the enzymes involved in polycyclic aromatic hydrocarbon (PAH) degradation in the chrysene-degrading organism Sphingomonas sp. strain CHY-1 were investigated. [14C]chrysene mineralization experiments showed that PAH-grown bacteria produced high levels of chrysene-catabolic activity. One PAH-induced protein displayed similarity with a ring-hydroxylating dioxygenase beta subunit, and a second PAH-induced protein displayed similarity with an extradiol dioxygenase. The genes encoding these proteins were cloned, and sequence analysis revealed two distinct loci containing clustered catabolic genes with strong similarities to corresponding genes found in Novosphingobium aromaticivorans F199. In the first locus, two genes potentially encoding a terminal dioxygenase component, designated PhnI, were followed by a gene coding for an aryl alcohol dehydrogenase (phnB). The second locus contained five genes encoding an extradiol dioxygenase (phnC), a ferredoxin (phnA3), another oxygenase component (PhnII), and an isomerase (phnD). PhnI was found to be capable of converting several PAHs, including chrysene, to the corresponding dihydrodiols. The activity of PhnI was greatly enhanced upon coexpression of genes encoding a ferredoxin (phnA3) and a reductase (phnA4). Disruption of the phnA1a gene encoding the PhnI alpha subunit resulted in a mutant strain that had lost the ability to grow on PAHs. The recombinant PhnII enzyme overproduced in Escherichia coli functioned as a salicylate 1-hydroxylase. PhnII also used methylsalicylates and anthranilate as substrates. Our results indicated that a single enzyme (PhnI) was responsible for the initial attack of a range of PAHs, including chrysene, in strain CHY-1. Furthermore, the conversion of salicylate to catechol was catalyzed by a three-component oxygenase unrelated to known salicylate hydroxylases.     

Molecular characterization and substrate preference of a polycyclic aromatic hydrocarbon dioxygenase from Cycloclasticus sp. strain A5

Cycloclasticus sp. strain A5 is able to grow with petroleum polycyclic aromatic hydrocarbons (PAHs), including unsubstituted and substituted naphthalenes, dibenzothiophenes, phenanthrenes, and fluorenes. A set of genes responsible for the degradation of petroleum PAHs was isolated by using the ability of the organism to oxidize indole to indigo. This 10.5-kb DNA fragment was sequenced and found to contain 10 open reading frames (ORFs). Seven ORFs showed homology to previously characterized genes for PAH degradation and were designated phn genes, although the sequence and order of these phn genes were significantly different from the sequence and order of the known PAH-degrading genes. The phnA1, phnA2, phnA3, and phnA4 genes, which encode the alpha and beta subunits of an iron-sulfur protein, a ferredoxin, and a ferredoxin reductase, respectively, were identified as the genes coding for PAH dioxygenase. The phnA4A3 gene cluster was located 3.7 kb downstream of the phnA2 gene. PhnA1 and PhnA2 exhibited moderate (less than 62%) sequence identity to the alpha and beta subunits of other aromatic ring-hydroxylating dioxygenases, but motifs such as the Fe(II)-binding site and the [2Fe-2S] cluster ligands were conserved. Escherichia coli cells possessing the phnA1A2A3A4 genes were able to convert phenanthrene, naphthalene, and methylnaphthalene in addition to the tricyclic heterocycles dibenzofuran and dibenzothiophene to their hydroxylated forms. Significantly, the E. coli cells also transformed biphenyl and diphenylmethane, which are ordinarily the substrates of biphenyl dioxygenases.     

Patchwork assembly of nag-like nitroarene dioxygenase genes and the 3-chlorocatechol degradation cluster for evolution of the 2-chloronitrobenzene catabolism pathway in Pseudomonas stutzeri ZWLR2-1

Pseudomonas stutzeri ZWLR2-1 utilizes 2-chloronitrobenzene (2CNB) as a sole source of carbon, nitrogen, and energy. To identify genes involved in this pathway, a 16.2-kb DNA fragment containing putative 2CNB dioxygenase genes was cloned and sequenced. Of the products from the 19 open reading frames that resulted from this fragment, CnbAc and CnbAd exhibited striking identities to the respective α and β subunits of the Nag-like ring-hydroxylating dioxygenases involved in the metabolism of nitrotoluene, nitrobenzene, and naphthalene. The encoding genes were also flanked by two copies of insertion sequence IS6100. CnbAa and CnbAb are similar to the ferredoxin reductase and ferredoxin for anthranilate 1,2-dioxygenase from Burkholderia cepacia DBO1. Escherichia coli cells expressing cnbAaAbAcAd converted 2CNB to 3-chlorocatechol with concomitant nitrite release. Cell extracts of E. coli/pCNBC exhibited chlorocatechol 1,2-dioxygenase activity. The cnbCDEF gene cluster, homologous to a 3-chlorocatechol degradation cluster in Sphingomonas sp. strain TFD44, probably contains all of the genes necessary for the conversion of 3-chlorocatechol to 3-oxoadipate. The patchwork-like structure of this catabolic cluster suggests that the cnb cluster for 2CNB degradation evolved by recruiting two catabolic clusters encoding a nitroarene dioxygenase and a chlorocatechol degradation pathway. This provides another example to help elucidate the bacterial evolution of catabolic pathways in response to xenobiotic chemicals.     

Isolation and characterization of the genes encoding a novel oxygenase component of angular dioxygenase from the gram-positive dibenzofuran-degrader Terrabacter sp. strain DBF63

A gram-positive bacterium Terrabacter sp. strain DBF63 is able to degrade dibenzofuran (DF) via initial dioxygenation by a novel angular dioxygenase. The dbfA1 and dbfA2 genes, which encode the large and small subunits of the dibenzofuran 4,4a-dioxygenase (DFDO), respectively, were isolated by a polymerase chain reaction-based method. DbfA1 and DbfA2 showed moderate homology to the large and small subunits of other ring-hydroxylating dioxygenases (less than 40%), respectively, and some motifs such as the Fe(II) binding site and the [2Fe-2S] cluster ligands were conserved in DbfA1. DFDO activity was confirmed in Escherichia coli cells containing the cloned dbfA1 and dbfA2 genes with the complementation of nonspecific ferredoxin and ferredoxin reductase component of E. coli. Under this condition, these cells exhibited angular dioxygenation of DF and dibenzo-p-dioxin, and monooxygenation of fluorene, but not angular dioxygenation of carbazole, xanthene, and phenoxathiin. Phylogenetic analysis revealed that DbfA1 formed a branch with recently reported large subunits of polycyclic aromatic hydrocarbon (PAH) dioxygenase from gram-positive bacteria but did not cluster with that of other angular dioxygenases, i.e., DxnA1 from Sphingomonas sp. strain RW1 [Armengaud, J., Happe, B., and Timmis, K. N. J. Bacteriol. 180, 3954-3966, 1998] and CarAa from Pseudomonas sp. strain CA10 [Sato, S., Nam, J.-W., Kasuga, K., Nojiri, H., Yamane, H., and Omori, T. J. Bacteriol. 179, 4850-4858, 1997].     

X-ray crystal structure of benzoate 1,2-dioxygenase reductase from Acinetobacter sp. strain ADP1

One of the major processes for aerobic biodegradation of aromatic compounds is initiated by Rieske dioxygenases. Benzoate dioxygenase contains a reductase component, BenC, that is responsible for the two-electron transfer from NADH via FAD and an iron-sulfur cluster to the terminal oxygenase component. Here, we present the structure of BenC from Acinetobacter sp. strain ADP1 at 1.5 A resolution. BenC contains three domains, each binding a redox cofactor: iron-sulfur, FAD and NADH, respectively. The [2Fe-2S] domain is similar to that of plant ferredoxins, and the FAD and NADH domains are similar to members of the ferredoxin:NADPH reductase superfamily. In phthalate dioxygenase reductase, the only other Rieske dioxygenase reductase for which a crystal structure is available, the ferredoxin-like and flavin binding domains are sequentially reversed compared to BenC. The BenC structure shows significant differences in the location of the ferredoxin domain relative to the other domains, compared to phthalate dioxygenase reductase and other known systems containing these three domains. In BenC, the ferredoxin domain interacts with both the flavin and NAD(P)H domains. The iron-sulfur center and the flavin are about 9 A apart, which allows a fast electron transfer. The BenC structure is the first determined for a reductase from the class IB Rieske dioxygenases, whose reductases transfer electrons directly to their oxygenase components. Based on sequence similarities, a very similar structure was modeled for the class III naphthalene dioxygenase reductase, which transfers electrons to an intermediary ferredoxin, rather than the oxygenase component.     

Identification and Functional Analysis of Two Aromatic-Ring-Hydroxylating Dioxygenases from a Sphingomonas Strain That Degrades Various Polycyclic Aromatic Hydrocarbons

In this study, the enzymes involved in polycyclic aromatic hydrocarbon (PAH) degradation in the chrysene-degrading organism Sphingomonas sp. strain CHY-1 were investigated. [¹⁴C]chrysene mineralization experiments showed that PAH-grown bacteria produced high levels of chrysene-catabolic activity. One PAH-induced protein displayed similarity with a ring-hydroxylating dioxygenase beta subunit, and a second PAH-induced protein displayed similarity with an extradiol dioxygenase. The genes encoding these proteins were cloned, and sequence analysis revealed two distinct loci containing clustered catabolic genes with strong similarities to corresponding genes found in Novosphingobium aromaticivorans F199. In the first locus, two genes potentially encoding a terminal dioxygenase component, designated PhnI, were followed by a gene coding for an aryl alcohol dehydrogenase (phnB). The second locus contained five genes encoding an extradiol dioxygenase (phnC), a ferredoxin (phnA3), another oxygenase component (PhnII), and an isomerase (phnD). PhnI was found to be capable of converting several PAHs, including chrysene, to the corresponding dihydrodiols. The activity of PhnI was greatly enhanced upon coexpression of genes encoding a ferredoxin (phnA3) and a reductase (phnA4). Disruption of the phnA1_a gene encoding the PhnI alpha subunit resulted in a mutant strain that had lost the ability to grow on PAHs. The recombinant PhnII enzyme overproduced in Escherichia coli functioned as a salicylate 1-hydroxylase. PhnII also used methylsalicylates and anthranilate as substrates. Our results indicated that a single enzyme (PhnI) was responsible for the initial attack of a range of PAHs, including chrysene, in strain CHY-1. Furthermore, the conversion of salicylate to catechol was catalyzed by a three-component oxygenase unrelated to known salicylate hydroxylases.     

Characterization of DitA3, the [Fe3S4] ferredoxin of an aromatic ring-hydroxylating dioxygenase from a diterpenoid-degrading microorganism

DitA3, a small soluble ferredoxin, is a component of a ring-hydroxylating dioxygenase involved in the microbial degradation of the diterpenoid, dehydroabietic acid. The anaerobic purification of a heterologously expressed his-tagged DitA3 yielded 20 mg of apparently homogeneous recombinant protein, rcDitA3, per liter of cell culture. Each mole of purified rcDitA3 contained 2.9 equivalents of iron and 4.2 equivalents of sulfur, indicating the presence of a single [Fe(3)S(4)] cluster. This conclusion was corroborated by UV-Visible absorption (epsilon(412)=13.4 mM(-1) cm(-1)) and EPR (g(x,y)=2.00 and g(z)=2.02) spectroscopies. The reduction potential of rcDitA3, determined using a highly oriented parallel graphite (HOPG) electrode, was -177.0+/-0.5 mV vs. the standard hydrogen electrode (SHE) (20 mM MOPS, 80 mM KCl, pH 7.0, 22 degrees C). This potential is similar to those of small, soluble Rieske-type ferredoxin components of aromatic-ring dihydroxylating dioxygenases. In contrast to these Rieske-type ferredoxins, DitA3 appears to exist as a dimer in solution. The dimeric ferredoxin may be more stable or may increase the catalytic efficiency of the dioxygenase by delivering the two reducing equivalents required for turnover of the oxygenase.     

Insights into the genetic diversity of initial dioxygenases from PAH-degrading bacteria

Alpha subunit genes of initial polyaromatic hydrocarbon (PAH) dioxygenases were used as targets for the PCR detection of PAH-degrading strains of the genera Pseudomonas, Comamonas and Rhodococcus which were obtained from activated sludge or soil samples. Sequence analysis of PCR products from several Pseudomonas strains showed that alpha subunits (nahAc allele) of this genus are highly conserved. PCR primers for the specific detection of alpha subunit genes of initial PAH dioxygenases from Pseudomonas strains were not suitable for detecting the corresponding genes from the genera Comamonas and Rhodococcus. Southern analysis using a heterologous gene probe derived from the P. putida OUS82 PAH dioxygenase alpha subunit identified segments of the PAH-degradation gene cluster from C. testosteroni strain H. Parts of this gene cluster containing three subunits of the initial PAH dioxygenase were isolated. These three subunits [ferredoxin (pahAb), alpha (pahAc) and beta (pahAd) subunit] were amplified by PCR as one fragment and expressed in Escherichia coli DH5alpha, resulting in an active initial dioxygenase with the ability to transform indole and phenanthrene. The DNA sequence alignment of alpha subunits from C. testosteroni H and various PAH-degrading bacteria permitted the design of new primers and oligonucleotide probes which are useful for the detection of the initial PAH dioxygenases from strains of Pseudomonas, Comamonas and Rhodococcus.     

Phthalate catabolic gene cluster is linked to the angular dioxygenase gene in <Emphasis Type="Italic">Terrabacter</Emphasis> sp. strain DBF63

Phthalate is a metabolic intermediate of the pathway of fluorene (FN) degradation via angular dioxygenation. A gene cluster responsible for the conversion of phthalate to protocatechuate was cloned from the dibenzofuran (DF)- and FN-degrading bacterium Terrabacter sp. strain DBF63 and sequenced. The genes encoding seven catabolic enzymes, oxygenase large subunit of phthalate 3,4-dioxygenase (phtA1), oxygenase small subunit of phthalate 3,4-dioxygenase (phtA2), cis-3,4-dihydroxy-3,4-dihydrophthalate dehydrogenase (phtB), [3Fe-4S] or [4Fe-4S] type of ferredoxin (phtA3), ferredoxin reductase (phtA4), 3,4-dihydroxyphthalate decarboxylase (phtC) and putative regulatory protein (phtR), were found in the upstream region of the angular dioxygenase gene (dbfA1A2), encoded in this order. Escherichia coli carrying phtA1A2BA3A4 genes converted phthalate to 3,4-dihydroxyphthalate, and the 3,4-dihydroxyphthalate decarboxylase activity by E. coli cells carrying phtC was finally detected with the introduction of a Shine-Dalgarno sequence in the upstream region of its initiation codon. Homology analysis on the upstream region of the pht gene cluster revealed that there was an insertion sequence (IS) (ISTesp2; ORF14 and its flanking region), part of which was almost 100% identical to the orf1 and its flanking region adjacent to the extradiol dioxygenase gene ( bphC1) involved in the DF degradation of Terrabacter sp. strain DPO360 [Schmid et al. (1997) J Bacteriol 179:53-62]. This suggests that ISTesp2 plays a role in the metabolism of aromatic compounds in Terrabacter sp. strains DBF63 and DPO360.