Journal of Environmental Biotechnology
Vol. 4, No. 1, 41–47, 2004

Original paper (regular paper)

Characterization of a Second 2,3-Dihydro-2,3-Dihydroxybiphenyl
Dehydrogenase Gene of the Polychlorinated Biphenyl
Degrader Rhodococcus sp. Strain RHA1
MASAYUKI SAKAI, EIJI MASAI*, KATSUMI SUGIYAMA, CHIGUSA TAKAHASHI,
AKIHIRO YAMADA, KEISUKE MIYAUCHI, and MASAO FUKUDA
Department of Bioengineering, Nagaoka University of Technology, Kamitomioka, Nagaoka, Niigata 940 2188, Japan* TEL: +81–258–47–9428 FAX: +81–258–47–9450* E-mail: emasai@vos.nagaokaut.ac.jp(Received; 30 March, 2004/Accepted; 16 June, 2004)
In a previous study, we characterized the bphAC1B genes of Rhodococcus sp. RHA1, which participate in the conversionof biphenyl to the meta-cleavage product and are on the 1,100-kb linear plasmid. In this study, we analyzed the 4.4-kb SphI
fragment of RHA1, which hybridized to the 1.5-kb EcoRI fragment carrying bphC1 and bphB. The nucleotide sequence of
this fragment showed the presence of a second 2,3-dihydro-2,3-dihydroxybiphenyl dehydrogenase gene, bphB2. The bphB2
gene consists of an 810-bp open reading frame encoding a polypeptide of molecular mass 28,234 Da. The nucleotide anddeduced amino acid sequences of bphB2 showed 76% and 81% identity, respectively, with those of the RHA1 bphB gene.
The etbA4 gene and orf3 whose deduced amino acid sequences are similar to ferredoxin reductase and aldehyde dehydrogenase, respectively, were identified as near to bphB2. The etbA4-bphB2-orf3 gene cluster was approximately 5-kb upstream ofthe previously characterized etbA1-etbA2-etbC-bphD1-bphE2-bphF2 gene cluster on the 450-kb linear plasmid. Reverse
transcription-PCR analysis showed that the bphB2 and bphB genes were transcribed in biphenyl- and ethylbenzene-growing
cells. The bphB2 gene was expressed in Escherichia coli to find the substrate preference, and BphB2 showed a higher activity toward the dihydrodiol compound of toluene than that of biphenyl. The insertional inactivation of the bphB gene in
RHA1 resulted in reduced growth on biphenyl. These results suggest that both bphB and bphB2 genes participate in biphenyl degradation in this strain.
Key words: PCB degradation, Rhodococcus, 2,3-dihydro-2,3-dihydroxybiphenyl dehydrogenase, bphB

1.

Introduction

Polychlorinated biphenyls (PCBs) are compounds of considerable environmental concern because of their toxicity
and recalcitrance. Microbial degradation is one of the mosteffective procedures to remove PCBs from the environment.
Many PCB-degrading bacteria have been isolated, and they
commonly cometabolize PCBs through the biphenyl catabolic pathway. In this pathway, biphenyl is transformed to
2,3-dihydro-2,3-dihydroxybiphenyl (23DDB) by a multicomponent biphenyl dioxygenase (BphA). 23DDB is converted
to 2,3-dihydroxybiphenyl (23DHBP) by 23DDB dehydrogenase (BphB), and 23DHBP is cleaved at the 1,2 position(meta-ring cleavage) by 23DHBP dioxygenase (BphC).
The ring cleavage product, 2-hydroxy-6-oxo-6-phenylhexa2,4-dienoate (HOPD), is hydrolyzed to benzoate and2-hydroxypenta-2,4-dienoate (HPD) by HOPD hydrolase(BphD) (Fig. 1A), and the resulting HPD is further converted to tricarboxylic acid cycle intermediates by HPD
hydratase, 4-hydroxy-2-oxovalerate aldolase, and acetalde-

hyde dehydrogenase (BphE, BphF, and BphG, respectively).
Rhodococcus sp. RHA1 is a strong Gram-positive PCB
degrader, which efficiently transforms the mixture of Kanechlors 200, 300, 400, and 500, including mono- to octachlorobiphenyls, for three days. To clarify why this strainshows the strong PCB degradation ability and to obtain insight into evolutional aspects of the degradation genes foraromatic compounds, we isolated and characterized thebphAC1B13) and etbCbphD112) genes, which are on the
1,100- and 450-kb linear plasmids, respectively. These genes
have roles in the transformation of biphenyl to benzoate
and HPD. Benzoate10) and HPD19) degradation genes havealso been characterized. In addition to bphC1 and etbC, wefound previously four other extradiol dioxygenase genes in
RHA118), and two sets of the bphA1A2 homologs were
found just upstream of etbC (etbA1A2; Fig. 1B) and downstream of bphF2 (ebdA1A2A3)22,26). Asturias et al.3,4) and
Maeda et al.11) have reported on multiple bphC in Rhodococcus globerulus P6 and Rhodococcus erythropolis
TA421, respectively. Two 23DDB dehydrogenase genes

SAKAI et al.

42

Fig. 1. Proposed transformation pathway of biphenyl to benzoate and HPD in Rhodococcus sp. RHA1 (A) and genetic map of the 15-kb
DNA fragment carrying etbA4-bphB2-orf3 (B). (A) Compounds: I, biphenyl; II, 23DDB; III, 23DHBP; IV, HOPD; V, benzoate; VI,
HPD. The gene product responsible for each reaction step is indicated above an arrow and is described in the text. (B) The 3’ end of the
DNA fragment includes etbA1-etbA2-etbC-bphD1 whose nucleotide sequences were found in a previous studies12,26). Abbreviations: E,
EcoRI; H, HindIII; K, KpnI; S, SalI; Sp, SphI; X, XhoI.

(bphB) have also been found in TA4212). Therefore theexistence of multiple bph genes seems to be a marked
characteristic of rhodococcal biphenyl and PCB degraders.
In this study, the bph gene homologs in RHA1 werescreened by using Southern hybridization analysis using a
RHA1 gene probe containing bphC1 and bphB. We foundanother 23DDB dehydrogenase gene (bphB2), and its geneproduct expressed in Escherichia coli was characterized.
The participation by two bphB genes of RHA1 in biphenyl
degradation was examined.

2.

Materials and Methods

Bacterial strains and plasmids. A PCB degrader, Rhodococcus sp. RHA1, was grown in Luria-Bertani medium(LB), diluted LB, and W-minimal medium (W)13) suppliedwith 0.2% biphenyl, vapor of ethylbenzene, or 0.2% succinate. R. erythropolis IAM1399, E. coli MV1190, and E.
coli DH5 were used as host strains. A Rhodococcus-E. coli
shuttle vector pK4 9) , charomid 9–36 16) , pUC119 23) ,pTrc99A1), and pKK223-37) were used as cloning vehicles.
Preparation of substrates. To prepare 23DDB frombiphenyl, R. erythropolis IAM1399 cells containing
pKRA17 carrying bphA1A2A3A4 in pK421) were used.
IAM1399 cells containing pKRA17 were grown in LB containing 100 mg/l of kanamycin. The cells were collected andwere incubated with 0.2% biphenyl in a 200 ml W medium
for 6 h at 30°C. The supernatant of the culture was collected by centrifugation and was saturated by NaCl. The
metabolites were extracted by ethyl acetate and weredried in vacuo. This extract was separated by using silica gel
chromatography (Wakogel C-200, Wako), using a solvent
consisted of chloroform and acetone (4:1). The 23DDB
fraction was trimethylsilylated and analyzed by gas chromatography-mass spectrometry (GC-MS) (model 5971A,
Hewlett-Packard Co.). The GC-MS condition was the sameas described previously12).
Cloning and DNA sequencing. Total DNA of RHA1
was prepared as described previously13). A gene library containing the HindIII fragments of RHA1 total DNA in charomid 9–36 was constructed as described previously12).
Southern and colony hybridization were done by using the
DIG-system (Roche). The 5.7-kb EcoRI and 9.9-kb HindIII
fragments carrying the partial bphB2 gene in charomid 9–
36 were cloned into pUC119, and the resulting plasmids
were designated pUH5 and pETQ1, respectively. Subclones
and deletion clones of these fragments were produced byusing restriction enzymes and exonuclease III. Nucleotide
sequencing was done by using the dideoxy termination
method20) with an ALFred DNA sequencer (Pharmacia).
The sequences were analyzed by using the GeneWorks programs (Intelligenetics, Inc). Nucleotide and amino acid se-Two bphB genes in Rhodococcus

quences were aligned by using the CLUSTAL W program.
Phylogenetic analysis was done by using the neighborjoining method17), and graphics for phylogenetic trees were
produced by using the TreeView program14). To find the expression of bphB2, the 1.0-kb XhoI-KpnI fragment carrying bphB2 was cloned into pTrc99A to produce pTB2.
Enzyme assay. 2,3-dihydro-2,3-dihydroxytoluene (23DDT;Fluka) or 23DDB (100 µM) were used as substrates andwere incubated with the cell extract (43 µg) of E. coli
MV1190 cells containing pTB2 and 100 µM NAD+ or
NADP+ in 50 mM KH2PO4-NaOH buffer (pH 7.4). The dihydrodiol dehydrogenase activity was assayed by measuring
the increase in the absorbance at 340 nm derived from theformation of NADH (ε 340=6,000 M -1cm -1; pH 7.4) or
NADPH (ε340=5,800 M-1cm-1; pH 7.4) by using a spectrophotometer (Biospec 1600, Shimadzu).
Preparation of cell extracts. E. coli MV1190 cells containing pTB2 were grown in LB containing 100 mg/l of ampicillin and 1 mM isopropyl-β-D-thiogalactopyranoside as
described previously12), and the cells were disrupted by sonication. The supernatant after centrifugation (10,000×g for
20 min at 4°C) was used as the cell extracts.
Reverse transcription-PCR (RT-PCR) analysis. RHA1
cells were grown in LB or W medium containing each of biphenyl, ethylbenzene, and succinate, and the total RNA wasprepared by using the method described by Ausubel et al.5).
RT-PCR was done by using a BcaBest RNA PCR kit ver.1.1
according to the instructions of the manufacturer (Takara
Shuzo). The following primers were used to amplify the internal regions of bphB and bphB2:
bphB forward, 5’-AACGACTTCGGCGAGGATGTCC-3’;
bphB reverse, 5’-GGTTGCCGTCCTCGAGTTCG-3’;
bphB2 forward, 5’-TTGCGGTTATCACCGGTGGC-3’;
bphB2 reverse, 5’-TTGATCAGATCTGCGAGCGG-3’.
Disruption of bphB in RHA1. The 0.6-kb HindIIIXhoI fragment carrying the truncated bphB gene whose 5’
and 3’ termini were deleted was cloned into pUCKmD,
which contains the aphII gene in pUC1919). The resulting
plasmid pDB1 was introduced into RHA1 cells by electroporation. Transformants were selected on a diluted LB agar
plate containing 50 mg/l of kanamycin, and underwent colony PCR analysis to examine the insertion of pDB1 into
the bphB gene in RHA1 by a single crossover. The primers
used for the colony PCR were:
bphB forward 2,5’-CGCTGGTCGAGCGATTCC-3’;
aphII reverse, 5’-TCCATCTTGTTCAATCATGC-3’.
Nucleotide sequence accession number. The nucleotide sequence of this study was deposited in the DDBJ,
EMBL, and GenBank nucleotide sequence databases underaccession no. AB127606.

3.

Results and Discussion

Isolation and sequencing of the bphB2 gene. Southern hybridization analysis of the RHA1 total DNA showedthat the 1.9-kb EcoRI fragment probe carrying the RHA1

43

bphC1 and bphB genes hybridized to the 5.7-kb EcoRI
fragment in addition to the 1.9-kb fragment (data notshown). This result suggested the presence of another bphCor bphB homolog in the RHA1 genome. To isolate the bph
gene homolog, the 5.7-kb EcoRI fragment was isolated
from the charomid library containing the EcoRI fragmentsof RHA1 total DNA by using a colony hybridization technique. This fragment was cloned into pUC119, and the resulting plasmid was designated pUH5. The nucleotide sequence of the 3’ end of the fragment suggested the presenceof a bphB homolog, but this gene seemed to be truncated.
To isolate the entire gene, the adjacent 9.9-kb HindIII fragment was isolated from the charomid library containing the
HindIII digests of RHA1 total DNA. Fig. 1B shows the restriction map of the 15-kb DNA fragment containing theoverlapping 5.7-kb EcoRI and 9.9-kb HindIII fragments.
The nucleotides of the 4.4-kb SphI fragment in thecloned fragments were sequenced. Three open reading
frames (ORFs) preceded by the putative ribosome bindingsites were found. The second ORF of 810 bp encoding 270
amino acid residues showed 76% and 81% identity with the
RHA1 bphB gene at the nucleotide and amino acid level,
respectively. We therefore designated this gene as bphB2.
The deduced amino acid sequences at positions 9–37
showed a high sequence similarity with the region thought
to participate in the NAD+ binding25) in BphB and TodD3).
Fig. 2 shows the phylogenetic tree of dehydrogenases fordihydrodiol of monoaryl, biaryl, and polycyclic aromatichydrocarbons. The biphenyl dihydrodiol dehydrogenases ofGram-positive bacteria including R. globerulus P63) andRhodococcus sp. M524) together with one of R. erythropolis
TA421 BphB are almost identical (99% identity) and wererelatively similar to BphB of Gram-negative aromatic compound-degrading bacteria (approximately 60% identity).
However, RHA1 BphB and BphB2 showed moderate similarity with biphenyl dihydrodiol dehydrogenases of Grampositive and -negative bacteria (approximately 50% identity), and they are not grouped with other biphenyl
dihydrodiol dehydrogenases in the phylogenetic tree. This
fact suggests that RHA1 bphBs evolved separately fromother biphenyl dihydrodiol dehydrogenases, and that one
bphB gene might have been duplicated in the RHA1 genome. Recently, the rhodococcal dihydrodiol dehydrogenase
gene, akbB of Rhodococcus sp. DK17, that is identical to
RHA1 bphB2, has appeared in databases (AAR90124).
An ORF of 1,272 bp was found upstream of bphB2 (Fig.
1B). From the amino acid sequence similarity of this ORF
with a ferredoxin reductase component (PhnD) of phenanthrene dioxygenase of Nocardioides sp. KP715) (37% identity), this ORF was predicted to encode a ferredoxin reductase and was designated as etbA4. As indicated for PhnD,
EtbA4 is distantly related to other ferredoxin reductases.
The genes that encode iron-sulfur proteins and ferredoxin
were not found proximal to etbA4, but etbA4 might encode
a functional ferredoxin reductase, which participates in hydroxylation of aromatic compounds together with other

44

SAKAI et al.

Fig. 2. Phylogenetic tree of RHA1 BphB and BphB2 with the aryl dihydrodiol dehydrogenases. The scale corresponds to a genetic distanceof 0.1 substitution at each position (10% difference). Accession numbers for the sequences are as follows: BphB_KF707, 23DDB dehydrogenase of Pseudomonas pseudoalcaligenes KF707 (P08694); BphB_LB400, 23DDB dehydrogenase of Burkholderia fungorum
LB400 (CAA46909); BphB_B-356, 23DDB dehydrogenase of Comamonas testosteroni B-356 (AAB18304); BphB_A5, 23DDB dehydrogenase of Ralstonia oxalatica A5 (CAD61143); BphB_KKS102, 23DDB dehydrogenase of Pseudomonas sp. KKS102 (BAA04140);
CumB_IP01, 2,3-dihydro-2,3-dihydroxycumene dehydrogenase of Pseudomonas fluorescens IP01 (BAA07079); EbdB_01G3, cis-2,3dihydroxy-2,3-hydroalkylbenzene dehydrogenase of Pseudomonas putida 01G3 (CAB99200); TcbB_P51, chlorobenzene glycol dehydrogenase of Pseudomonas sp. P51 (AAC43636); TodD_F1, 23DDT dehydrogenase of P. putida F1 (P13859); BpdD_M5, 23DDB dehydrogenaseof Rhodococcus sp. M5 (AAB07754); BphB#1_TA421, 23DDB dehydrogenase of R. erythropolis TA421 (BAA25607); BphB_P6,
23DDB dehydrogenase of R. globerulus P6 (CAA53296); BphB_RHA1, 23DDB dehydrogenase of Rhodococcus sp. RHA1 (BAA06873);
IpbB_I1, isopropylbenzene dihydrodiol dehydrogenase of Rhodococcus sp. I1 (CAA06877); BphB2_RHA1, 23DDB dehydrogenase of
RHA1 (BAD03967); DoxE_C18, cis-1,2-dihydro-1,2-dihydroxynaphthalene dehydrogenase of Pseudomonas sp. C18 (D49343);
PahB_OUS82, naphthalene/phenanthrene dihydrodiol dehydrogenase of P. putida OUS82 (BAA20393); BphB#2_TA421, 23DDB
dehydrogenase of TA421 (BAA25626); ThnB_TFA, 1,2-dihydroxy-1,2,5,6,7,8-hexahydronaphthalene dehydrogenase of Sphingopyxis
macrogoltabida TFA (AAN26445); PhnB_RP007, naphthalene/phenanthrene dihydrodiol dehydrogenase of Burkholderia sp. RP007(AAD09874); DbtB_DBT1, dihydrodiol dehydrogenase of Burkholderia sp. DBT1 (AAK62355).

Fig. 3. Agarose gel electrophoresis of RT-PCR products of bphB and bphB2. Total RNAs isolated from the RHA1 cells grown on biphenyl(lanes 1, 2, 9, and 10), ethylbenzene (lanes 3, 4, 11, and 12), LB (lanes 5, 6, 13, and 14), and succinate (lanes 7, 8, 15, and 16) were used
for the templates for RT-PCR. Lanes 1 to 8 and 9 to 16 were amplifications with the bphB and bphB2 primer sets, respectively. The sizesof molecular weight markers in lane M are indicated on the left side of the gel. Odd-numbered lanes are controls without reverse transcriptase.

bphA components, including bphA1A2A3, etbA1A2, andebdA1A2A3. The functional analysis of etbA4 is currently
underway in our laboratory.
The third ORF of 1,371 bp showed 64% and 36% aminoacid sequence identities with 6-oxohexanoate dehydrogenase(ChnE) of Rhodococcus sp. Phi28) and succinic semialdehyde dehydrogenase (GabD) of E. coli6), respectively. This
result suggests that orf3 encodes a semialdehyde dehydroge-

Two bphB genes in Rhodococcus

Fig. 4. Substrate preference of BphB2. 23DDT (DDT) and
23DDB (DDB) were used as substrates. NAD+ or NADP+ wasadded to the reaction mixture, and the activity was assayed bymeasuring the increase in the amount of NADH or NADPH
formed from NAD+ or NADP+, respectively. Each value is theaverage±standard deviation (error bar) of at least three measurements.

nase, but the function of orf3 remains to be clarified.
Nucleotide sequencing of the 3’ terminus of the 9.9-kb
HindIII fragment showed that this region includes thebphD1 and partial bphE2 genes, which we characterized in45

a previous study (Fig. 1B)12). This result indicates that thebphB2 gene cluster is approximately 5-kb upstream of
etbA1 on the RHA1 450-kb linear plasmid22).
RT-PCR analysis of the bphB and bphB2 genes in
RHA1. To obtain an insight into the roles of bphB andbphB2 in the degradation of aromatic compounds, we examined each of the bphB transcripts in RHA1 cells grownon various carbon sources. Total RNAs were extracted from
RHA1 cells grown in biphenyl, ethylbenzene, succinate, and
LB and then underwent RT-PCR analysis using each bphB
internal primer. The PCR products were analyzed by using
agarose gel electrophoresis (Fig. 3). Both RT-PCR products
of bphB and bphB2 of the expected sizes (600 bp) were obtained from the total RNAs of the cells grown in biphenyl
and ethylbenzene. However, no PCR product was obtained
from the total RNAs of the cells grown in LB and succinate.
These results indicate that the transcription of bphB andbphB2 were induced during the growth in biphenyl and ethylbenzene, suggesting these genes participate in the degradation of biphenyl and ethylbenzene.
Substrate preference of BphB2. The 1.0-kb XhoIKpnI fragment carrying bphB2 was cloned into pTrc99A
to produce pTB2 (Fig. 1B), which was used for the bphB2
expression in E. coli MV1190. The gene product of bphB2
was observed by using SDS-PAGE (data not shown), andthe molecular mass was estimated as 33 kDa, which is close
to the molecular mass calculated from the deduced aminoacid sequence of bphB2 (28,234 Da). The cell extract of E.
coli containing pTB2 was used for the enzyme assay with

Fig. 5. Disruption of bphB in Rhodococcus sp. RHA1. (A) Schematic overview of the disruption of bphB by integration of pDB1. The
bphB disruption was accomplished by a single crossover between bphB on the 1,100-kb linear plasmid and the truncated bphB in pDB1.
≫ and ≪ represent 5’ and 3’ deletions, respectively. (B) PCR analysis of the bphB disruption mutant (RDB1) using the bphB forward 2
and aphII reverse primers. The bphB forward 2 primer was complementary to the 5’ region of bphB, which is not included in the truncated bphB of pDB1. The sizes of molecular weight markers in lane M are indicated on the left side of the gel.

SAKAI et al.

46

the dihydrodiol compounds of biphenyl and toluene,
23DDB and 23DDT, as substrates (Fig. 4). The BphB2 enzyme showed NAD+-dependent activities toward both substrates, but 23DDT was preferable to 23DDB as a substrate.
Comparing the substrate preferences between BphB2 and
BphB was interesting, but we failed to express bphB in E.
coli using tac or trc promoters.
Disruption of bphB in RHA1. To examine the participation of bphB in the degradation of biphenyl and ethylbenzene, the bphB gene in RHA1 was disrupted by homologous recombination using pUC19-based plasmid pDB1,
which carries aphII and the truncated bphB gene whose 5’
and 3’ termini were deleted (Fig. 5A). The single crossover
between bphB on the 1,100-kb linear plasmid and the truncated bphB in pDB1 was expected to result in an insertionof the entire pDB1 plasmid into the linear plasmid, in which
the region of pDB1 containing aphII and the vector was
sandwiched between the 3’- and 5’-truncated bphB genes.
pDB1 was introduced into RHA1 by electroporation, and akanamycin-resistant transformant, RDB1, was obtained.
The colony PCR analysis of RDB1 indicated the expecteddisruption of bphB (Fig. 5B). We tried to disrupt bphB2 byusing the same strategy, but the mutant was not obtained.
To examine the growth of RDB1 on biphenyl and ethylbenzene, RDB1 and RHA1 containing a vector, pK4 (Kmr),
were streaked on W medium containing biphenyl or ethylbenzene with kanamycin. RDB1 showed reduced growth on
biphenyl compared with RHA1. This result strongly suggests that bphB participates in biphenyl degradation. The
enzyme activity of the bphB2 gene product seemed to support the growth of RDB1 on biphenyl. Therefore, most
likely is that RHA1 needs both bphB and bphB2 for maximum growth on biphenyl. However, RDB1 grew normallyon ethylbenzene, suggesting that bphB2 has a major role in
ethylbenzene degradation. However, further research isneeded to clarify the contribution of bphB and bphB2 todegradation of biphenyl and ethylbenzene.

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