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

Abstract

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.

FIG. 1

Physical map and genetic organization of phenanthrene-degrading genes. Gray bars indicate the DNA inserts in the library clones. Open bars show inserts in the two clones used in this study. Arrows on the enlarged physical map show the locations and orientations of the phenanthrene-degrading genes: phdA, the α subunit of the oxygenase component; B, phdB, the β subunit of the oxygenase component; C, phdC, the ferredoxin component; phdD, the ferredoxin reductase component; phdE, dihydrodiol dehydrogenase; phdF, extradiol dioxygenase; phdG, hydratase-aldolase; phdH, aldehyde dehydrogenase. The box with oblique indicates the 302-bp PCR-amplified fragment that was used for probing the library clones. The typical expression plasmids are shown at the bottom. Circles indicate promoters. Transcription of DNA segments containing phdABCD genes cloned in pSA315 and pST46 is under the control of the lac and T7 promoters, respectively. The transcription in pHA171 of the phdABCD genes preceded by the SD sequence that is effective in E. coli (filled boxes) is under the control of the T7 promoter. The bar with three arrows indicates the previously reported sequence (AB000735 in DDBJ/GenBank/EMBL databases), arrows marked with I, J, and K showing the locations and orientations of the phenanthrene-degrading genes phdI (1-hydroxy-2-naphthoate dioxygenase), phdJ (trans-2′-carboxybenzalpyruvate hydratase-aldolase), and phdK (2-carboxybenzaldehyde dehydrogenase). The striped bar shows the 2.5-kb HindIII-BamHI DNA fragment that was used as probe in the hybridization following PFGE (Fig. 2). Restriction sites: B, BamHI; Bg, BglII; H, HindIII, M, MluI; Nc, NcoI; RI, EcoRI; V, EcoRV; X, XbaI. The asterisk indicates that the other NcoI restriction sites are not shown.

FIG. 2

Localization of phenanthrene-degrading genes on the chromosome DNA. Total DNA of Nocardioides sp. strain KP7 was subjected to PFGE (lanes 1 to 8) and hybridization with the 2.5-kb HindIII-BamHI DNA fragment shown in Fig. 1 (lanes 9 to 12). Lanes 1 and 7, S. cerevisiae YNN295 chromosomal DNA as molecular mass markers; lanes 2 and 8, ladder of phage lambda DNA concatemers as molecular mass markers; lanes 3 and 9, undigested total DNA of KP7; lanes 4 and 10, DraI-digested total DNA of KP7; lanes 5 and 11, XbaI-digested total DNA of KP7; lanes 6 and 12, HindIII-digested total DNA of KP7.

FIG. 3

Alignment of well-conserved sequence regions in the N-terminal and central part of the α subunits of ring-hydroxylating dioxygenases. The three-dimensional structure of the NdoBC oxygenase component from Pseudomonas sp. strain NCIB 9816-4 has recently been determined (28). Highlighted characters represent the residues involved in binding to the Rieske-type [2Fe-2S] cluster and to the mononuclear iron atom. Shadowed characters indicate the Asp residues hydrogen bonded to active-site ligand His on the same molecule and to [2Fe-2S] Rieske center ligand His on the neighboring α subunit. The Rieske-type [2Fe-2S] cluster-binding sequence, CXHX₁₇CX₂H, in PhdA was located at Cys91 to His114. The residues involved in coordinating the catalytic iron were also conserved as His219 and His224 (and Asp376). Asterisks indicate residues conserved in all proteins. Definition of abbreviations are given in the legend to Fig. 5.

FIG. 4

Requirements of the phdABCD genes for phenanthrene transformation activity. The removal of phenanthrene (A) and formation of the dihydrodiol (B) were monitored by HPLC at 254 nm in a suspension of BL21(DE3) cells carrying expression plasmids pHA171 (phdABCD) (□), pHA141eV (phdABC) (■), pHA151 (phdABD) (●), pHA111eV (phdAB) (◊), pHB141 (phdBCD) (⧫), pHA162 (phdACD) (▵), and pT7-7 (BglIIX) (▴). Prolonged incubation (24 h) of the cells carrying pHA171 resulted in the complete removal of phenanthrene. Therefore, the rate of dihydrodiol formation was calculated by taking the amount of dihydrodiol at 24 h as 100%. Values are averages from two independent experiments.

FIG. 5

Phylogenetic tree obtained from the alignment of PhdA with related proteins. The protein sequences of the 28 α subunits of ring-hydroxylating dioxygenases including PhdA are classified. The multiple-alignment analysis was performed with the PHYLIP software package, and the phylogenetic unrooted tree was drawn by using TreeView. The numbers on some branches refer to the percentage confidence estimated by a bootstrap analysis with 1,000 replications. The scale bar indicates the percentage divergence. The sequence abbreviations, enzyme substrates, species, and DDBJ/GenBank/EMBL references are as follows: NdoB.NCIB9816, naphthalene, P. putida NCIB 9816, M23914; PahA3.PaK1, naphthalene, P. aeruginosa PaK1, D84146; NtdAc.JS42, 2-nitrotoluene, Pseudomonas sp. strain JS42, U49504; PhnAc.RP007, phenanthrene, Burkholderia sp. strain RP007, AF061751; BnzA.BE-81, benzene, P. putida BE-81, M17904; TodC1.F1, toluene, P. putida F1, J04996; TcbAa.P51, chlorobenzene, Pseudomonas sp. strain P51, U15298; IbpA1.BD2, isopropylbenzene, Rhodococcus erythropolis BD2, U24277; BphA1.P6, biphenyl, R. globerulus P6, X80041; IbpA1.JR1, isopropylbenzene, Pseudomonas sp. strain JR1, U53507; BphA1.KKS102, biphenyl, Pseudomonas sp. strain KKS102, D17319; BphA.LB400, biphenyl, Burkholderia cepacia LB400, M86348; XylC1.RB1, substrate unknown, Cycloclasticus oligotrophus RB1, U51165; HcaA1.K12, phenylpropionate, E. coli K-12, AE000340; DxnA1.RW1, dibenzo-p-dioxin, Sphingomonas sp. strain RW1, X72850; CarAa.CB3, carbazole, Sphingomonas sp. strain CB3, AF060489; XylX.mt2, toluate, P. putida mt2, M64747; BenA.ADP1, benzoate, Acinetobacter calcoaceticus ADP1, AF009224; CbdA.2CBS, 2-halobenzoate, B. cepacia 2CBS, X79076; AntA.ADP1, anthranilate, A. calcoaceticus ADP1, AF071556; TftA.AC1100, 2,4,5-trichlorophenoxyacetic acid, B. cepacia AC1100, U11420; CmtAb.F1, p-cumate, P. putida F1, U24215; TdnA1.UCC22, aniline, P. putida UCC22, D85415; AtdA3.YAA, aniline, Acinetobacter sp. strain YAA, AB008831; NidA.I24, indene, Rhodococcus sp. strain I24, AF121905; NarAa.NCIMB12038, naphthalene, Rhodococcus sp. strain NCIMB 12038, AF082663; DitA1.BKME-9, diterpenoid, P. abietaniphila BKME-9, AF119621; PhdA.KP7, phenanthrene, Nocardioides sp. strain KP7, AB017794.