Vol. 174, No. 22

JOURNAL OF BACrERIOLOGY, Nov. 1992, p. 7253-7261

0021-9193/92/227253-09$02.00/0 Copyright ) 1992, American Society for Microbiology

Identification of a New Gene, tmoF, in the Pseudomonas mendocina KR1 Gene Cluster Encoding Toluene-4-Monooxygenase KWANG-MU YEN* AND MICHAEL R. KARL Amgen Inc., Amgen Center, Thousand Oaks, California 91320-1789 Received 8 May 1992/Accepted 11 September 1992

Five genes, tmoABCDE, encoding toluene4-monooxygenase (T4MO) were previously mapped to a 3.6-kb region of a 10.2-kb SacI DNA fragment isolated from Pseudomonas mendocina KR1 (K.-M. Yen, M. R. Karl, L. M. Blatt, M. J. Simon, R. B. Winter, P. R. Fausset, H. S. Lu, A. A. Harcourt, and K. K. Chen, J. Bacteriol. 173:5315-5327, 1991). In this report, we describe the identification and characterization of a DNA region in the SacI frgment whose expression enhances the T4MO activity determined by the tmoABCDE gene cluster. This region was mapped immediately downstream of the putative transcription termination sequence previously located at the end of the tmoABCDE gene cluster (Yen et al., J. Bacteriol., 1991) and was found to stimulate T4MO activity two- to threefold when expressed in Escherichia coi or Pseudomonas putid. Determination of the nucleotide sequence of this region revealed an open reading frame (ORF) of 978 bp. Expression of the ORF resulted in the synthesis of an -37-kDa polypeptide whose N-terminal amino acid sequence completely matched that of the product predicted from the ORF. The ORF thus defines a gene, which has now been designated hnoF. The TmoF protein shares amino acid sequence homology with the reductases of several mono- and dioxygenase systems. In addition, the reductase component of the naphthalene dioxygenase system, encoded by the nahA. gene of plasmid NAH7 from P. puda G7, could largely replace the TmoF protein in stimulating T4MO activity, and TmoF could partially replace the NahA., protein in forming active naphthalene dioxygenase. The overall properties of tmoF suggest that it is a member of the T4MO gene cluster and encodes the NADH:ferredoxin oxidoreductase of the T4MO system. A monooxygenase system in Pseudomonas mendocina KR1 allowing the hydroxylation of toluene and formation of p-cresol was recently reported (23). This toluene-4-monoxygenase (T4MO) system has been resolved into three functional components (23). The properties of these three components suggested an electron flow in this enzyme system from NADH to a flavin-containing NADH:ferredoxin oxidoreductase, to a ferredoxin protein, and finally to a colorless iron-containing oxygenase (23). A similar functional arrangement of three components has been reported for the wellcharacterized naphthalene dioxygenase (NDO) system in Pseudomonas putida NCIB 9816 (5, 8, 9) and toluene dioxygenase system in P. putida Fl (19-21). Structurally, four polypeptides are required to assemble each of these two enzyme systems, since in each system the oxygenase component consists of two subunits (17, 29). Interestingly, five polypeptides are required for the assembly of the T4MO system in Escherichia coli (28). The complexity of the T4MO enzyme system, however, is not unique. Two other monooxygenase systems are known to consist of five polypeptides, namely, the methane monooxygenase (MMO) system in Methylococcus capsulatus (Bath), which catalyzes the oxidation of methane to methanol (4), and the phenol hydroxylase (DMP) system in Pseudomonas sp. strain CF600, which catalyzes the hydroxylation of phenol and some of its methylated derivatives to catechol and related compounds (13, 14). One of the T4MO proteins has been tentatively identified as a ferredoxin on the basis of its spectrophotometric properties (23) and its amino acid sequence homology with the ferredoxin proteins from the NDO, toluene dioxy*

genase, and benzene dioxygenase systems (28). Several other T4MO polypeptides have been shown to share amino acid sequence homology with some DMP components (28). Two of the T4MO polypeptides might be subunits of the oxygenase component in the T4MO system (28). The structure of the T4MO NADH:ferredoxin oxidoreductase was completely unknown (23, 28). Five T4MO genes, tmoABCDE, were previously mapped to a 3.6-kb region of a 10.2-kb Sacl DNA fragment (Fig. 1) isolated from P. mendocina KR1 (28). Downstream from this gene cluster, a DNA sequence reminiscent of a rho-independent transcription terminator was located (28). In this report, we describe the identification and characterization of a new gene, tmoF, whose expression enhances T4MO activity. We have mapped this gene immediately downstream of the putative transcription termination sequence at the end of the tmoABCDE cluster and determined its nucleotide sequence. The properties of tmoF suggest that it encodes the NADH: ferredoxin oxidoreductase component of the T4MO system.

MATERIAIS AND METHODS Construction of plasmids carrying the T4MO gene(s). Cloning vectors pCFM1146 and pKMY319 and plasmids pMY421, pKMY277, pKMY341, and pKMY336 (Fig. 1) have been described previously (26, 28). Construction of plasmid pKMY340 (Fig. 1) involved the use of plasmids pKMY277 (Fig. 1) and pMY421. Deletion of a 2.3-kb BamHI fragment of pKMY277 extending from coordinates 7.9 to 10.2 (Fig. 1) generated a plasmid designated pKMY280 (Fig. 1). Replacement of the 2-kb region of pMY421 extending from coordinates 3.1 to 5.1 (Fig. 1) in an Asp718-BamHI fragment with the 4.8-kb Asp718-BamHI fragment of

Corresponding author. 7253

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FIG. 1. Restriction map of P. mendocina KR1 DNA fragment carrying the T4MO genes, tmoABCDEF, and plasmid constructs containing various parts of this fragment. The sizes and transcriptional directions of the T4MO genes are marked with arrows. The lines underneath the restriction map denote regions of the P. mendocina KR1 DNA fragment contained in the plasmids indicated. Plasmids pMY601 and pMY603 also contain the nahA. gene at the locations indicated. The size of nahA. is shown, and its transcriptional direction is indicated by an arrow. a

pKMY280 extending from coordinates 3.1 to 7.9 (Fig. 1) produced pKMY340. Plasmid pKMY321 (Fig. 1) was constructed by cloning the entire insert of pMY421 as an XbaI-XhoI fragment into pKMY319. Plasmid pMY486 (Fig. 1) was generated by cloning the entire insert of pKMY341 (Fig. 1) as an XbaI-SacI fragment into pKMY319. Construction of plasmid pMY6l7 involved a number of intermediate plasmids. Deletion of a HindIII fragment from the 5' end of the insert in pKMY341 generated plasmid pMY460 (Fig. 1). Cloning of the SspI fragment of pKMY336 extending from coordinates 4.8 to 5.2 (Fig. 1) into the HpaI site of the E. coli cloning vector pCFM4722 (1) produced plasmid pMY497 (Fig. 1). In pMY497, the nucleotide sequence shown in Fig. 2 was generated at the junction between the 5' end of the tmoF sequence derived from pKMY336 and the pCFM4722 sequence. This resulted in the

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replacement of the coding sequence for the ribosome binding site (RBS) of tmoF mRNA and the three N-terminal amino acids, MFN, of the TmoF polypeptide with a DNA sequence encoding a new ribosome binding site and the amino acid sequence MV (Fig. 2). Replacement of the 5' end of the HindIII-XhoI region of the insert of pMY460 as a ClaI-XhoI fragment with the -0.3-kb ClaI-XhoI fragment of pMY497 containing the modified 5' end of tmoF generated a modified tmoF gene, tmoF*, in a plasmid designated pMY498 (Fig. 1). Elimination of the EcoRI site downstream of tmoF* in pMY498 by EcoRI digestion, filling in the ends, and bluntend ligation generated plasmid pMY613. Insertion of an EcoRI linker at the XbaI site upstream of tmoF* in pMY6l3 produced plasmid pMY614. Insertion of anXbal linker at the ClaI site upstream of the created EcoRI site in pMY614 generated plasmid pMY6l5. Insertion of an EcoRI linker at the PpuMI site of pKMY336 (Fig. 1) between tmoE and tmoF generated plasmid pMY499. Insertion of the pMY499 region extending from coordinates 1.2 to 4.7 (Fig. 1) as an XbaI-EcoRI fragment into the XbaI and EcoRI sites of pMY615 produced plasmid pMY6l6 (Fig. 1). In pMY6l6, a new tmoABCDEF* gene cluster was assembled in which the putative transcription terminator between tmoE and tmoF had been deleted and the 5' end of tmoF had been modified. Cloning of the tmoABCDEF* cluster as an XbaI-SacI fragment of pMY6l6 into the XbaI and SacI sites of pKMY319 generated plasmid pMY617. Construction of plasmids carrying the NDO gene(s). The nahAbAd4d genes and nahAa gene were derived from recombinant plasmids pACGG and pACG8 obtained from C. Serdar

tmoF, A NEW MEMBER OF P. MENDOCINA KR1 T4MO GENES

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and D. Murdock at Amgen. pACGG is an E. coli pACl plasmid (15) carrying the NDO gene cluster of plasmid NAH7 (17) (Fig. 3), and pACG8 is a derivative of pACGG with part of the nahAb gene and the nahACAd genes located downstream of the XhoI site at coordinate 2.1 deleted (Fig. 3). Construction of plasmid pMY603 (Fig. 1), which contains the nahA. gene, involved construction of plasmids pMY496 and pMY601. Deletion of the nucleotide sequence between coordinates 0.05 and 0.6 of pACG8 (Fig. 3) by digestion with SacII, blunting the end, attachment of an EcoRI linker, redigestion with EcoRI, and ligation generated plasmid pMY496 (Fig. 3). Replacement of the EcoRI-XhoI fragment of pMY499 extending from coordinates 4.7 to 5.1 (Fig. 1) with the EcoRI-XhoI fragment of pMY496 extending from coordinates 0.6 to 2.1 (Fig. 3) produced plasmid pMY601 (Fig. 1). Cloning of the region of pMY601 carrying the tmoABCDE-nahAa gene cluster as an XbaI-XhoI fragment into pKMY319 generated pMY603. Construction of plasmid pMY619 (Fig. 3), a derivative of pKMY319 (26) carrying the tmoF*-nahA,A($d gene cluster which can be induced from the PG promoter in pKMY319, involved construction of a number of intermediate plasmids. Cloning of the BamHI-XhoI fragment of pACGl extending from coordinates 4.0 to 4.7 (Fig. 3) into the BamHI and Sall sites of the E. coli cloning vector pUC18 (25) produced plasmid pMY606 (Fig. 3). Cloning of the EcoRI-BamHI fragment of pACG1 extending from coordinates 0.05 to 4.0 (Fig. 3) into the EcoRI and BamHI sites of pMY606 produced plasmid pMY607 (Fig. 3). In pMY607, the entire NDO gene cluster was reassembled to contain appropriate 5'- and 3'-flanking restriction sites for further manipulation. Insertion of a SacI linker at the HindIII site downstream of the NDO gene cluster in pMY607 generated plasmid pMY608 and insertion of an XbaI linker at the EcoRI site upstream of the NDO gene cluster in pMY608 generated plasmid pMY609 (Fig. 3). Replacement of the XbaI-Eco47III fragment of pMY609 extending from coordinates 0.05 to 1.8 (Fig. 3) with the XbaI-XmaI fragment of pMY498 containing the modified tmoF gene, tmoF* (for details of the construction of pMY498, see above and Fig. 1), generated plasmid pMY611 (Fig. 3). Cloning of the XbaI-SacI fragment of pMY611 carrying the tmoF*-nahAbAcAd gene cluster into the XbaI and Sacl sites of pKMY319 completed construction of plasmid pMY619. Construction of plasmid pMY618 (Fig. 3), a derivative of pKMY319 carrying the nahAbACAd genes which can be induced from the PG promoter in pKMY319, involved construction of plasmid pMY610 (Fig. 3). Insertion of an XbaI linker at the Eco47III site at the 3' end of nahAa in pMY608 generated pMY610. Cloning of the region of pMY610 extending from coordinates 1.8 to 4.7 (Fig. 3) as an XbaI-SacI fragment into the XbaI and Sacl sites of pKMY319 generated pMY618. Plasmid pMY612 (Fig. 3) was used in the construction of plasmid pMY620 (Fig. 3), which is a derivative of pKMY319 and carries the nahAAbAdAd gene cluster which can be induced from the PG promoter of pKMY319. Replacement of the XbaI-XhoI fragment of pMY609 (Fig. 3) extending from coordinates 0.05 to 2.1 with the region of pMY496 extending from coordinates 0.6 to 2.1 (Fig. 3) as an XbaI-X7hoI fragment produced pMY612. Cloning of the entire insert of pMY612 extending from coordinates 0.6 to 4.7 (Fig. 3) as an XbaI-SacI fragment into the XbaI and SacI sites of pKMY319 generated plasmid pMY620. Nucleotide sequence accession number. The nucleotide

TABLE 1. Effects of tmoF, tmoF*, and nah/A8 genes on the tmoABCDE-determined T4MO activity Induced T4MO

Hoste

Plasmid'

Pertinent genotype

E. coli FM5

pCFM1146 tmoABCDE pMY421 pKMY336 tmoABCDEF pKMY340 tmoABCDEF

E. coli HB101

pKMY319 pKMY321 tmoABCDE pMY486 tmoABCDEF

pMY603

pMY617

sp (nol min-1 mg of act

protein-L)0.1 ± 0.02 (2) 10 ± 0.9 (3) 19 ± 4.2 (3) 20 ± 4.5 (3)

0.2 5.8 15 tmoABCDE-nZhAa 10 18 tmoABCDEF*

± 0.1 (6) ± ± ± ±

1.3 (5) 1.2 (6) 3.3 (5) 3.5 (5)

P. putida KT2440 pKMY319

0.2 ± 0.1 (4) 4.7 ± 1.1 (5) pKMY321 tmoABCDE 16 ± 4.9 (4) pMY486 tmoABCDEF pMY6O3 tmoABCDE-nahAa 13 ± 5.3 (5)

"All of the bacterial strains and growth and induction conditions (including temperature induction conditions of E. coli FM5 derivatives and sodium salicylate induction conditions of other strains) were described previously (28).

b Construction of plasmids was described in Materials and Methods. The T4MO assay was performed as described previously (28). Specific activities presented are means ± standard deviations, and the numbers of independent determinations are shown in parentheses.

sequence of the tmoF gene has been submitted to GenBank and assigned accession number M95045. RESULTS Identification of the T4MO-enhancing activity. Previously we demonstrated that a plasmid (pMY421) carrying an insert which extends from the HindIII site at coordinate 1.2 to the XhoI site at coordinate 5.1 in the Sacl fragment shown in Fig. 1 contains all of the five genes, tmoABCDE, required for the synthesis of the T4MO system in E. coli (28). However, we subsequently found that extension of the insert to the XmaI site at coordinate 5.9 (Fig. 1) included a DNA sequence which upon expression, consistently enhanced the tmoABCDE-determined T4MO activity. Further extension of the insert beyond the XmaI site did not produce higher T4MO activity. These results were obtained from cells carrying plasmids pKMY336 and pKMY340. These two plasmids, like pMY421, are derivatives of the E. coli plasmid vector pCFM1146 and contain the tmoABCDE gene cluster which can be induced by heat from a phage lambda PL promoter (28). Unlike pMY421, pKMY336 carries an insert extending from the HindIII site at coordinate 1.2 to the XmaI site at coordinate 5.9 (Fig. 1) and pKMY340 carries an insert extending from the same HindIII site to the BamHI site at coordinate 7.9 (Fig. 1). Under induction conditions, plasmids pKMY336 and pKMY340 both produced twice as much T4MO activity as did plasmid pMY421 in E. coli FM5 (28) (Table 1). This T4MO-enhancing activity was not simply an oddity attributable to E. coli FM5 and could be observed in other bacterial strains as well. When the inserts in pMY421 and in pKMY336 were cloned into the broad-host-range expression vector pKMY319 (26) and the resulting plasmids pKMY321 and pMY486 (Fig. 1), respectively, were induced with sodium salicylate in both E. coli HB101 (28) and P. putida KT2440 (28), the T4MO-enhancing activity from pMY486 was observed in both hosts. Compared with the

tmoF, A NEW MEMBER OF P. MENDOCINA KR1 T4MO GENES

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00 00 000

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3550 GGT CTTCCCCTTCGGCATTGCTGAAGGGGCTTTTTAGAGACGTTATCTATGTTCAATATTCAATCGGATGATCTCCTGCACCATTTTGAGGCGGATAGTA (TmoF)M F N I Q S D D L L H H F E A D S N

3650 ATGACACTCTACTTAGTGCTGCT CTACGTGCT GAATTGGTAT T TCCATATGAGTGTAACT CAGGAGGGT GCGGCGCAT GTAAGAT CGAGCTGCT TGAGGG D

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4250 TGAATACGCTGA\TTGCCCAAGGGTAT GAAAAACCGAT TACGGT GTT TTACGGT TCACGGCTAGAAGCT GAACTGGAAGCGGCCGAAACCCT GT T TGGGT G N

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FIG. 4. Nucleotide sequence of the tmoF gene and the inferred amino acid sequence of its product. The sequence was determined by a method previously described (28). The sequenced region extends from coordinates 4.6 to 5.9 shown in Fig. 1 and overlaps the sequence reported previously (28). The putative transcription termination sequence determined previously (28) is underlined. The bases that are complementary to the 3' end of E. coli 16S rRNA and appear to determine the ribosome binding site (16) are marked with open circles. The sequence presented here is numbered in relation to the tmoABCDE sequence previously reported (28).

level of T4MO activity produced from plasmid pKMY321, an approximately threefold stimulation of the T4MO activity was observed in E. coli HB101 and P. putida KT2440 carrying plasmid pMY486 (Table 1). Since the tmoE gene ends immediately upstream of the putative transcription terminator (28), the results in Table 1 indicated that the DNA region between the putative transcription terminator and the XmaI site at coordinate 5.9 (Fig. 1) determined a T4MOenhancing activity. Identification of a new gene, tmoF, encoding T4MO-enhancing activity and determination of its nucleotide sequence. To determine whether the T4MO-enhancing activity detected from pKMY336 was specified by a new gene, a plasmid, pMY440, was constructed by deleting a 3.4-kb HindIII fragment extending from coordinates 1.2 to 4.6 (Fig. 1) from the 5' end of the insert in pKMY336 (Fig. 1) and the remaining P. mendocina KR1 DNA fragment in pMY440 extending from coordinates 4.6 to 5.9 (Fig. 1) was sequenced in its entirety in both orientations. A complete open reading frame consisting of 978 bp and running with the same transcriptional direction as that of the tmoABCDE cluster was identified immediately downstream of the putative transcription terminator at the end of the tmoABCDE cluster (Fig. 4). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of crude extracts of induced E. coli cells carrying pMY440 revealed the synthesis of an -37-kDa polypeptide from the insert in pMY440 (Fig. 5). The polypeptide band in the gel was electroblotted onto a polyvinyl difluoride membrane, stained, excised, and sequenced in a protein sequencer as described previously (28). The determined N-terminal amino acid sequence, MFNIQSDDLLH HFE, completely matched the N-terminal amino acid sequence of the protein predicted from the open reading frame (Fig. 4). The open reading frame thus defines a gene. This gene was designated tmoF. The nucleotide sequence of

tmoF predicted a polypeptide product with a molecular weight of 35,983 which approximately matched the estimated molecular weight of the polypeptide produced from pMY440. The G+C content of the tmoF sequence is 47.8%

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7258

J. BACTERIOL.

YEN AND KARL 0000

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DfpP

80

FNIQSDDLLHHFEA DSNDTLLSAALRAELVFP YECNSGGCGACKIELLEGEVSNLWPD APG M -yNv--e-----iE- e--qTiL-AALR-gv-lP f-C--G-Ca-CKvqvvEGEV-- -e A-M M-------------- -- lQ-eD-v-rF ----- eTL--AA-R-qi-iP -dC--GeCG-Cr ---- Gn --- - D---e ----------- ----S-------------qTiL--ALh-gi-FP -dC--GsCG-CK--Li-G-V-e L--- a M -e---e --- --- d-eD-v-rF ---n--eTv--AA-R-gi-lP -dC--GaCGACK ---- G--SM -l-IQ-nn-L--F-p g-N LL-- LR-n-V---Y-C-SG-CG-Cr--v-dG-V-d --- G M --a -------C--GGCa-CK----EGd -Sde-vi-AALR-ni-l--------v--Dg---rFEM 81

TmoF DmpP BenC

00

KTADETQSVFIAGGSGIAPMVSMVNTLIAQGYEKPITVFYGSRLEAEL EAAETLFGWKENLKLINVSSSV VGNSEK --V--- - dd-Er--q--d-iFIAGGSGL ----SMi--LL--G--r-ITLF-G-R--AEL---E--E-L-ar--Nf--v r---- -- lmlAGG-GIAPfLSMLq-L---G-E-Pv-L-fG---d-dL---E--d-L -- ----f------- -qeSq---n-T- V-vAGG-GLAPi-cvlq-m ---- Er-v-LFfG-R-q-dL--.-d--E-L ----9---- - i---- -e-S--dr---- -- lllAGG-GLAPf--Mle-i--QG-E-PL-LiYG---d-dL---d--E-f-ar--Nf- --c--r------- L-vgGG-GLAPvLSiV--- L--G---PI-LyfG-R-q-dL---E----L-a---qL-v--V---- --N-----d-L-sL ---m--- -V---V- -- -gdK- ----- FvAGG-GLAPvVSMV--m ---------- iyfG---EpEL III

400

321 TmoF

DmpP BenC

XylA

XyLZ NahAa MmoC

LGAEFYLCGPPQMINSVQKLLMIENKVPFEA IHFDRFF -G---YLCGPP-MId-----LM E-rl FE--I-meRFy------------------e-d-YLCGP--Mve-V--wL--q---P -- f-FekF-----E-YLCGPP-Mvd--e--Lv------ E- v--DRFy-------- YLCGPP-Mve-V--- i--q ---P-n f-yekF ---w---- --- P-Mve-L--v ------ P-- I--D-Fy ---iP-E- v-FekF-----s----pdiYLCGPP-MId----Lv---G---d-l-E-LE

KYPT GYVHEIIPEYME GL ---GfVHd-----fd--f ---GYV-g-I EY-----w- ---G-V-E-f-EY-----YP- -GYV-q-I --- GG-V-dvl---i ---r-

IV

FIG. 6. Homology of TmoF protein with other reductases. The amino acid sequence of TmoF is compared with those of the reductase components of the DMP system (DmpP) from Pseudomonas sp. strain CF600 (13, 14); the MMO system (MmoC) from M. capsulatus (Bath) (18), the xylene monooxygenase system (XylA) from P. putida PaWl (22), the NDO system (NahAa) from P. putida G7 (17), the benzoate dioxygenase system (BenC) from A. calcoaceticus BD413 (12), and the toluate dioxygenase system (XylZ) from P. putida PaWl (10). Amino acids identical to those in TmoF are shown in capital letters and amino acids similar (as defined previously) (28) to those in TmoF are shown in lowercase letters. In reference to TmoF, dissimilar amino acids are represented by dashes. All proteins have an N-terminal region resembling chloroplast-type ferredoxins and a C-terminal region resembling oxidoreductases of various origins (see text). The amino acids completely conserved in these reductases, as in chloroplast-type ferredoxins (11), are marked with asterisks. The highly but not completely conserved positions that exhibit some amino acid variations either in the reductases or in chloroplast-type ferredoxins (11) are marked with open circles. Highly conserved regions in the C-terminal portion of the reductases are underlined.

and is similar to the G+C content of the tmoABCDE cluster

(49.1%). Function of the tmoF gene. To investigate the role of tmoF in the T4MO system, the deduced amino acid sequence of the TmoF polypeptide was compared with those of other gene products. It was found that TmoF shares homology with the oxidoreductase components of several mono- and dioxygenase systems, including the reductase components of the DMP, MMO, and xylene monooxygenase systems encoded by the dmpP gene of plasmid pVI150 from Pseudomonas sp. strain CF600 (13, 14), the mmoC gene of M. capsulatus (Bath) (22), and the xyA gene of TOL plasmid pWWO from P. putida PaWl (18) and the reductase components of naphthalene, benzoate, and toluate dioxygenase systems encoded by the nahA. gene of plasmid NAH7 from P. putida G7 (17), the benC gene of Acinetobacter calcoaceticus BD413 (12), and the xylZ gene of TOL plasmid pWWO

(10). An alignment of the amino acid sequences of these reductases with that of TmoF showing identical and similar amino acids is presented in Fig. 6. These reductase proteins all have molecular weights similar to that of TmoF and share a significant number of identical and similar amino acids with TmoF (Table 2). The DmpP, BenC, XylA, XylZ, and MmoC proteins were each shown to have an N-terminal region resembling chloroplast-type ferredoxins and a C-terminal region resembling oxidoreductases of bacterial, yeast, plant, animal, and human origins (12, 14, 18). As shown in Fig. 6, these properties are also shared by the TmoF and NahAa proteins. In the N-terminal portion of each of the proteins shown in Fig. 6, many amino acids either completely or highly conserved in chloroplast-type ferredoxins (11) are also completely or mostly conserved in these proteins. The amino acids completely conserved, as in chloroplast-type ferredoxins, in-

tmoF, A NEW MEMBER OF P. MENDOCINA KR1 T4MO GENES

VOL. 174,- 1992

TABLE 2. Homology between TmoF protein and the oxidoreductase components of some mono- and dioxygenase enzyme systems

Reductase'

Mol wtb

DmpP BenC

38,500 38,800 38,600 38,200 35,500 36,200

MmoC

XylA NahAa XyIZ

% of amino acid residues in TmoF homologous to those in other reductasesc Identical amino Similar amino acids acids

32.5 28.8 27.0 26.7 25.8 25.2

22.1 19.3 21.2 16.6 18.4 21.8

and dioxygenase systems in which these reductases play an electron-transferring role are described in the text. b The molecular weights of the reductase proteins are determined from nucleotide sequences (10, 12, 13, 17, 18, 22). c The percentages were calculated on the basis of amino acid alignment presented in Fig. 6. Similar amino acids are as defined previously (28). a

The

mono-

clude two glycine residues and the four cysteine residues at positions 53, 58, 61, and 96 (Fig. 6), which may be involved in the coordination of the two iron atoms in the [2Fe-2S] cluster. In the C-terminal portion of these reductase proteins, regions of significant homology can be identified (Fig. 6). Region I contains sequences previously proposed to be involved in the binding of flavin adenine dinucleotide and region III contains sequences proposed to be involved in the binding of NAD (12). The proteins BenC, XylA, XylZ, and MmoC, each of which was presumably formed during evolution by the fusion of a ferredoxin to a reductase, are all capable of transferring electrons from NADH directly to the oxygenase component in their respective enzyme system. Unlike these reductases, NahA, functions with a separate ferredoxin protein in carrying out this same task (8, 9). In this respect, TmoF is similar to NahAa, since a ferredoxin is also present in the T4MO system (28). The structural and functional homologies between TmoF and the well-characterized NahA, prompted us to test the functional interchangeability between tmoF and nahAa in stimulating the tmoABCDE-determined T4MO activity and in directing the formation of active NDO. For the evaluation of nahA function in stimulating the tmoABCDE-determined T4MO activity, a tmoABCDE-nahAa gene cluster was constructed and cloned into the expression vector pKMY319 (26). The resulting plasmid pMY603 (Fig. 1) was found to give an induced level of T4MO activity in E. coli HB101 or P. putida 2440 only slightly lower than that given by plasmid pMY1486, which carries the wild-type gene cluster tmoABCDEF (Fig. 1; Table 1). This result demonstrated that the NADH:ferredoxin oxidoreductase of the NDO system encoded by plasmid NAH7 can largely replace the TmoF protein in stimulating T4MO activity in both E. coli and P. a

putida.

The nahAb and nahA4Ad genes of the NDO system encode ferredoxin and the two subunits of the terminal oxygenase component, respectively (17). In the construction of a tmoFnahAbAAd gene cluster for the evaluation of the functional similarity of tmoF to nahAa in the formation of NDO, it was desirable to remove the putative transcription termination sequence upstream of tmoF to increase the sensitivity of the test. A restriction site for the SspI enzyme (AATATT) is located immediately downstream of the tmoF sequence encoding the first three amino acids of the TmoF protein

7259

TABLE 3. Functional comparison of tmoF* with nahA. in NDO synthesis Plasmid'

Pertinent genotype genotype

pKMY319 Uninduced Induced

NDO sp act (nmol minm mg

~~~of protein1-'b

0.9 ± 0.4 (3) 0.9 ± 0.2 (3)

pMY618 Uninduced Induced

nahAbA44d

pMY619 Uninduced Induced

tmoF*-nahMbAAd

pMY620 Uninduced Induced

nahAAbA4.Ad

1.2 ± 0.2 (2) 2.9 ± 0.7 (3)

1.0 ± 0.3 (3) 7.5 ± 0.8 (3) 1.0 ± 0.2 (3) 70 ± 15 (3)

a All plasmids were carried in E. coli HB101, and construction of plasmids was described in Materials and Methods. b Conditions of NDO assay were identical to those of T4MO assay (28) except that 60 nmol of [14CJnaphthalene (8 mCi/mmol; Sigma) was used in each assay. Specific activities presented are means ± standard deviations, and the numbers of independent determinations are shown in parentheses.

(Fig. 4). The putative transcription termination sequence and the region encoding the ribosome binding site of tmoF mRNA and the three N-terminal amino acids, MFN, of the TmoF protein were removed at the SspI site and were replaced with a DNA sequence encoding a new ribosome binding site and the amino acids MV (Materials and Methods) (Fig. 2). These sequence changes did not lead to a significantly higher TmoF activity and nevertheless resulted in a modified tmoF gene, tmoF*, that remained active in stimulating the tmoABCDE-determined T4MO activity. As shown in Table 1, plasmid pMY617, which carries the gene cluster tmoABCDEF* that can be induced from the PG promoter (Materials and Methods), expressed T4MO activity in E. coli at a level slightly higher than that expressed by plasmid pMY486, which carries the wild-type gene cluster tmoABCDEF (Fig. 1). To determine whether tmoF could replace nahAa in directing NDO synthesis, the assembled tmoF*-nahAb,ACAd cluster, the wild-type nahAaAbACAd cluster, and a derivative of the latter cluster lacking nahiAa were individually cloned into the expression vector pKMY319, and the induced NDO activities from the resulting plasmids pMY619, pMY620, and pMY618, respectively, were compared. As shown in Table 3, induction of NDO from all three plasmids could be detected in E. coli B101. The low-level induction from plasmid pMY618 (Table 3), which does not carry the nahAa gene, reflected the presence of a nonspecific reductase activity in the E. coli host and has also been observed by other researchers (6, 17). The induced NDO activity from plasmid pMY619, although representing only 1/10th that obtained from plasmid pMY620, was more than twofold the level obtained from plasmid pMY618 (Table 3). This result thus demonstrated that the tmoF product can partially replace the NahA. protein in the formation of NDO. In conjunction with the observations that TmoF shares amino acid sequence homology with reductases of several oxygenase systems and that NahAa can largely replace TmoF in stimulating T4MO activity, this result suggested that TmoF serves as an NADH:ferredoxin oxidoreductase in the T4MO

7260

YEN AND KARL

system. The nonspecific reductase activity in the E. coli host could account for the tmoABCDE-specified T4MO activity observed in the absence of tmoF (Table 1) (28). The presence of a similar nonspecific reductase activity in P. putida could also make TmoF dispensable in the formation of T4MO in this host (Table 1).

DISCUSSION In this study, we have identified and characterized a new P. mendocina KR1 gene, tmoF, whose expression enhanced the T4MO activity determined by the tmoABCDE cluster (28). This gene was not identified initially in our study of the T4MO gene cluster because of the nonessential role it played in T4MO synthesis in E. coli (28). In fact, tmoF is not essential in tmoABCDE-directed T4MO synthesis in any of the bacterial hosts (including three E. coli, three P. putida, and two P. mendocina strains) we have examined to date (Table 1) (27, 28). Several lines of evidence presented in Results nevertheless suggest that tmoF is a member of the T4MO gene cluster and encodes the NADH:ferredoxin oxidoreductase of the T4MO system: (i) a two- to threefold stimulation of the TmoABCDE-determined T4MO activity by TmoF (Table 1), (ii) homology of TmoF with the oxidoreductases of other mono- and dioxygenase systems (Fig. 6), (iii) partial interchangeability in function between TmoF and NahAa in stimulating the activity of T4MO (Table 1) and of NDO (Table 3), and (iv) close linkage of tmoF with the tmoABCDE cluster (Fig. 4). The TmoABCDE-determined T4MO activity in the absence of TmoF is mostly likely due to the presence of a nonspecific reductase activity in the host (see Results). However, the determination of whether TmoF is absolutely essential for T4MO activity must await further in vitro analysis using purified T4MO functional components. Expression of tmoF from plasmids pKMY336, pMY486, and pMY440 indicated that tmoF is in the same transcription unit as the tmoABCDE cluster and that the putative transcription terminator downstream of the tmoABCDE cluster allows transcriptional read-through. In the construction of plasmid pMY617, not only was the putative transcription terminator upstream of tmoF deleted, but also the nucleotide sequence encoding the ribosome binding site of TmoF mRNA and the first three amino acids of TmoF protein was changed (see Materials and Methods). The effect on the expression of tmoF or the activity of T4MO in the absence of the transcription terminator still cannot be effectively evaluated in cells carrying pMY617. The function of the putative transcription terminator remains unclear. The presence of tmoF indicates that the T4MO gene cluster consists of six genes. We have presented suggestive evidence for the roles of TmoA, TmoC, TmoE, and TmoF in T4MO synthesis (Tables 1 and 3) (28). The functions of TmoB (molecular weight, 9,600) and TmoD (molecular weight, 12,000) remain completely unknown. However, clues for a function of TmoB or TmoD might be provided by the MMO system of M. capsulatus. The MMO system consists of three components and catalyzes the oxidation of methane to methanol (2). Among its many other activities is the catalytic conversion of toluene into cresol (3), a reaction also catalyzed by the T4MO system (23). The substratebinding oxygenase component of the MMO system is a colorless iron-containing protein whose absorption spectrum shows a maximum at around 280 nm and a shoulder at around 406 nm (24). These properties are similar to those of T4MO oxygenase component, which is also a colorless

J. BACT1ERIOL.

iron-containing protein with maximum absorption at 280 nm and a shoulder at around 390 nm (23). The oxygenase component of the MMO system consists of three subunits with molecular weights of 54,000, 42,000, and 17,000 (24). The putative oxygenase component of the T4MO system was shown to consist of at least two subunits with molecular weights of 50,000 and 32,000 (23) or 58,000 and 38,500 as predicted from gene sequences (28). The possibility exists that the T4MO oxygenase contains an additional subunit encoded by the tmoB or tmoD gene. In addition to the oxygenase component, the MMO system contains a reductase component and a regulator component. The regulator is essential for monooxygenase activity and capable of converting the enzyme from an oxidase to an oxygenase (7). It is a single-subunit protein, with a molecular weight of -17,000 (7). It will be interesting to determine whether TmoB or TmoD can regulate T4MO activity. ACKNOWLEDGMENTS We are grateful to Arlette Harcourt and Sidney Suggs for determination of the nucleotide sequence of the tmoF gene and Patricia Fausset and Hsieng S. Lu for determination of the N-terminal amino acid sequence of the TmoF protein. We thank Cuneyt Serdar and Douglas Murdock for providing plasmids pACG1 and pACG8 and Joan Bennett for typing the manuscript. REFERENCES 1. Burnette, W. N., V. L. Mar, W. Cieplak, C. F. Morris, K. T. Kaljot, K. S. Marchitto, R. K. Sachdev, C. Locht, and J. M. Keith. 1988. Direct expression of Bordetella pertussis toxin subunits to high levels in Escherichia coli. Bio/Technology 6:699-706. 2. Colby, J., and H. Dalton. 1978. Resolution of the methane monooxygenase of Methylococcus capsulatus (Bath) into three components. Purification and properties of component C, a flavoprotein. Biochem. J. 171:461-468. 3. Colby, J., D. I. Stirling, and H. Dalton. 1977. The soluble methane monooxygenase of Methylococcus capsulatus (Bath). Its ability to oxygenate n-alkanes, ethers, and alicyclic, aromatic and heterocyclic compounds. Biochem. J. 165:395-402. 4. Dalton, H. 1980. Oxidation of hydrocarbons by methane monooxygenases from a variety of microbes. Adv. Appl. Microbiol. 26:71-87. 5. Ensley, B. D., and D. T. Gibson. 1983. Naphthalene dioxygenase: purification and properties of a terminal oxygenase component. J. Bacteriol. 155:505-511. 6. Ensley, B. D., T. D. Osslund, M. Joyce, and M. J. Simon. 1988. Expression and complementation of naphthalene dioxygenase activity in Escherichia coli, p. 437-455. In S. R. Hagedorn, R. S. Hanson, and D. A. Kunz (ed.), Microbial metabolism and the carbon cycle. Harwood Academic Publisher, New York. 7. Green, J., and H. Dalton. 1985. Protein B of soluble methane monooxygenase from Methylococcus capsulatus (Bath). J. Biol. Chem. 260:15795-15801. 8. Haigler, B. E., and D. T. Gibson. 1990. Purification and properties of NADH-ferredoxinNAp reductase, a component of naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816. J. Bacteriol. 172:457-466. 9. Haigler, B. E., and D. T. Gibson. 1990. Purification and properties of ferredoxinNAp, a component of naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816. J. Bacteriol. 172:465-468. 10. Harayama, S., M. Rekik, A. Bairoch, E. L. Neidle, and L. N. Ornston. 1991. Potential DNA slippage structures acquired during evolutionary divergence of Acinetobacter calcoaceticus chromosomal benABC and Pseudomonas putida TOL pWWO plasmid xyLYYZ, genes encoding benzoate dioxygenases. J. Bacteriol. 173:7540-7548. 11. Matsubara, H., and T. Hase. 1983. Phylogenetic consideration

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tmoF, A NEW MEMBER OF P. MENDOCINA KR1 T4MO GENES

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