Aug 22, 1989 - polypeptides of the ligA and ligB gene products were identified by ..... 3 (pRD369), the 5' terminus of the ligA gene is deleted by 369 base pairs ...
Vol. 172, No. 5
JOURNAL OF BACTERIOLOGY, May 1990, p. 2704-2709 0021-9193/90/052704-06$02.00/0 Copyright X 1990, American Society for Microbiology
Molecular Cloning of the Protocatechuate 4,5-Dioxygenase Genes of Pseudomonas paucimobilis YOICHI NODA,' SEIJI NISHIKAWA,2 KOH-ICHI SHIOZUKA,1 HIROSHI KADOKURA,l HARUSHI NAKAJIMA,l KOJI YODA,' YOSHIHIRO KATAYAMA,2 NORIYUKI MOROHOSHI,2 TAKAFUSA HARAGUCHI,2 AND MAKARI YAMASAKI1* Department of Agricultural Chemistry, The University of Tokyo, Bunkyo-ku, Tokyo 113,1 and Faculty of Agriculture, Tokyo Noko University, Fuchu, Tokyo 183,2 Japan Received 22 August 1989/Accepted 5 February 1990
We determined the nucleotide sequence of a 1.9-kilobase fragment of Pseudomonas paucimobilis SYK6 chromosomal DNA that included genes encoding protocatechuate 4,5-dioxygenase, the enzyme responsible for the aromatic ring fission of protocatechuate. Two open reading frames of 417 and 906 base pairs were found that had no homology with previously reported sequences, including those encoding protocatechuate 3,4-dioxygenase. Since both open reading frames were indispensable for the enzyme activity, they should encode the subunits of protocatechuate 4,5-dioxygenase. We named these genes ligA and ligB. Protocatechuate 4,5-dioxygenase was efficiently expressed in Escherichia coli with the aid of the lac promoter, and the polypeptides of the ligA and ligB gene products were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino acid sequencing.
Lignins are major structural components of vascular plants and contain a large fraction of the carbon in the biosphere. They are constructed with various intermonomer linkages between phenylpropanes with guaiacyl, syringyl, p-hydroxyphenyl, and biphenyl nuclei. They are difficult to utilize as natural resources because of their extreme stability. If efficient degradation of lignins by microorganisms becomes possible, we will be able to make good use of them. Pseudomonas paucimobilis SYK6 is able to grow on dihydrodivanilic acid as a sole carbon source (16). This strain can also grow on syringate, 3-methylgailate, vanillate, and other dimeric model compounds of lignins. EcoRI fragments of P. paucimobilis SYK6 chromosomal DNA were inserted in a plasmid vector pKT230 (4) and introduced into P. putida PpY1100. A 10.5-kilobase EcoRI fragment cloned in pVA01 conferred on Pseudomonas putida the ability to grow on vanillate (17) or protocatechuate. Gas chromatography-mass spectrometry showed that vanillate is degraded via protocatechuate in the transformant. We report here the nucleotide sequence of a 1.9-kilobase XbaI-SalI subfragment that encodes protocatechuate 4,5dioxygenase. The enzyme was expressed in Escherichia coli with the aid of the lac promoter. This is the first report of cloning and sequencing of the protocatechuate 4,5-dioxygenase gene.
SalI sites of plasmid vector pUC118 or pUC119, yielding pPC118 or pPC119, respectively (20). We used a Kilosequence kit (Takara Shuzo) to construct various deletion derivatives of pPC118 and pPC119 (14). Nucleotide sequences of these deletion derivatives were determined by the method of Sanger et al. (27) with a Sequenase sequencing kit (U.S. Biochemical Corp.). Both single- and doublestranded DNAs were used for sequencing. Sequences were analyzed with a sequence analysis program (SDC GENETYX-CD; Software Development Co.) on an NEC PC9801F personal computer. Protocatechuate 4,5-dioxygenase assay. When the optical density at 550 nm (OD550) of the culture of E. coli harboring the plasmids reached 0.1, isopropyl-p-D-thiogalactoside was added to a final concentration of 1 mM. When the OD550 reached 0.8, the whole culture (10 ml) was contrifuged, and cells were suspended in 0.5 ml of ice-cold 67 mM Tris hydrochloride (pH 7.6). The cells were disrupted by sonication for 3 min, and ethanol was added to a final concentration of 10%. The samples were placed on ice until assay. One gram of protocatechuate was dissolved in 4 ml of water by adding a small amount of NaOH solution, and 5 ,ul of this solution was added to 10 ml of 100 mM Tris hydrochloride (pH 9.5). The 0.2 ml of lysate was added to 2.8 ml of 100 mM Tris hydrochloride (pH 9.5) at room temperature, and the reaction was started by mixing 1 ,u of protocatechuate solution. The activity of protocatechuate 4,5-dioxygenase was determined by measuring the A410 of the reaction mixture by the method of Ono et al. (26). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The overnight culture of E. coli harboring the plasmids was used to inoculate fresh medium. When the OD550 of the culture reached 0.1, isopropyl-p-D-thiogalactoside was added to a final concentration of 1 mM. When the OD550 reached 0.8, 1 ml of the culture was centrifuged, and cells were suspended in 50 ,ul of lysis buffer. Samples were boiled for 2 min, and 10 ,ul of each was loaded on 15% polyacrylamide gel. After electrophoresis the gel was stained with Coomassie brilliant blue. Amino acid sequence. Plasmid pRD65, one of the deletion
MATERIALS AND METHODS Bacterial strain, medium, and growth conditions. E. coli JM109, used as a host strain, was grown at 37°C in NB medium containing 1.8% nutrient broth and 0.2% yeast extract. Ampicillin was added to the selective medium to a final concentration of 100 mg per liter. Enzymes and reagents. Restriction enzymes and T4 DNA ligase were obtained from Boehringer Mannheim Biochemicals or Takara Shuzo Co. [32P]dCTP (specific activity, >400 Ci/mol) was from Amersham Corp. Subcloning and nucleotide sequencing. The 1.9-kilobase XbaI-SalI fragment was subcloned between the XbaI and *
Corresponding author. 2704
PSEUDOMONAS PROTOCATECHUATE 4,5-DIOXYGENASE GENE
VOL. 172, 1990
derivatives used for nucleotide sequencing, has a ligA gene deleted 65 nucleotides from its 5' terminus and connected to the lacZ gene in the vector pUC118 in the same reading frame. The LacZ-LigA fusion protein and the LigB protein should be produced after induction of the lac promoter. E. coli harboring pRD65 was cultured in a 5-liter flask containing 1 liter of NB medium. When the OD550 of the culture reached 0.9, isopropyl-p-D-thiogalactoside was added to a final concentration of 1 mM. After 2 h, the whole culture was centrifuged, and cells were suspended in 50 mM Tris hydrochloride (pH 7.6) and disrupted in a French pressure cell. Since the LacZ-LigA and LigB proteins were found to be in the form of inclusion bodies, the rapidly sedimenting fraction of the cell lysate was collected by centrifugation for 10 min at 8,000 x g. Then the precipitate was suspended in 50 mM Tris hydrochloride-2.5 mM EDTA. The LacZ-LigA and LigB proteins were separated from other proteins by sucrose density gradient centrifugation for 14 h at 110,000 x g at 4°C. The sucrose density gradient was a stepwise gradient of 15, 53, and 70% (wt/vol) sucrose in 50 mM Tris hydrochloride2.5 mM EDTA. Samples were fractionated from the bottom of the tube. A small amount of each fraction was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis to determine the protein composition of each fraction. The fractions enriched in LacZ-LigA and LigB were subjected to preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and proteins were transferred to a polyvinylidene difluoride membrane (Immobilon; Millipore Corp.). The areas of the two protein bands were cut out, and the amino acid sequence of each protein was determined by
1.4
1.3
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m 0.9 0 0.7
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XbaI
S aI
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3.5
6.7
7.8
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l.9kb FIG. 2. Regions of the fragment encoding protocatechuate 4,5dioxygenase. Various parts of the 10.5-kilobase (kb) EcoRI fragment were subcloned in the vectors as indicated. The arrowheads indicate the direction of transcription from the lac promoter. The enzyme activity of each fragment in E. coli is shown within parentheses to the right.
using a 477A gas-phase amino acid sequencer (Applied Biosystems Inc.). RESULTS Characterization of the enzyme encoded in the cloned fragment. Protocatechuate 3,4-dioxygenase (EC 1.13.11.3) (9, 18, 24, 25) and protocatechuate 4,5-dioxygenase (EC 1.13.1.8) (5, 26, 33) have been reported in soil bacteria. Protocatechuate is cleaved by the latter enzyme in P. paucimobilis SYK-6 (16). The lysates of transformants [either P. putida PpyllOO (pVAO1) or E. coli MC1061(pVAO1)] developed the yellow color of ot-hydroxy-,3-carboxymuconate semialdehyde (Xmax, 410 nm at pH 9.5) after the addition of protocatechuate. Host cell lysates did not have this activity. This confirmed that the ring fission enzyme coded in pVAO1 is protocatechuate 4,5-dioxygenase. In E. coli the expression of the enzyme was dependent on the lac promoter (Fig. 1). Regions of the fragment encoding protocatechuate 4,5dioxygenase. Subcloning and deletion analysis indicated the region encoding the enzyme resides near one end of the EcoRI fragment (Fig. 2). Moreover, some E. coli transformants that had subfragments on pUC18 or pUC19 expressed protocatechuate 4,5-dioxygenase when the direction of the insert was the same as the direction of the transcription from the lac promoter on the vectors (Fig. 2). The shortest fragment that expressed the enzyme was the 1.9-kilobase XbaI-SalI fragment.
pgp I fgA
40
I.
1.4 1.9 2.4
0.5
20
2705
80
100
120
140
160
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ligai
X 1500
1000
1900
180
TIME(SEC)
FIG. 1. Detection of protocatechuate 4,5-dioxygenase activity in E. coli. pPC118 and pPC119 have the same XbaI-SalI fragment in pUC118 and pUC119, respectively. The direction of the insertion of the fragment is different in the two plasmids. Cell lysates of E. coli harboring the plasmid pPC118 (0) or pPC119 (O) and the substrate were mixed at 0 s, and the A410 of the reaction mixture was determined.
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4
-
FIG. 3. Sequencing strategy for the 1.9-kilobase XbaI-SalI fragment. Horizontal arrows indicate the extent and the direction of sequencing. The locations of the ligA and ligB genes are shown.
2706
J. BACTERIOL.
NODA ET AL.
TCTAGAGCTTTGTTTCCCGTGATTTCCGAGCCGCAGGATGTGCCCATCCTGCTCGAAATCACTAAAGA3j9CCAT
77
1 ORF ATG ACC
GAG AAG AAA GAG AGA ATC GAC GTT CAC GCC TAT CTC GCC GAG TTT GAC GAC ATT CCC GGC ACC CGC GTG TTC ACC GCC CAG CGC Met Thr Glu Lys LYs Glu Arg lie AsP Val His Ala Tyr Leu Ala Glu Phe Asp Asp lie Pro Gly Thr Arg Val Phe Thr Ala Gin Arg
167
GCG CGC AAG GGC TAT AAT CTC AAC CAG TTC GCG ATG AGC CTG ATG AAG GCC GAG AAC CGC GAG CGG TTC AAG GCC GAC GAG AGC GCC TAT Ala Arg LYs GlY Tyr Asn Leu Asn Gln Phe Ala Met Ser Leu Met Lys Ala Glu Asn Arg Glu Arg Phe Lys Ala Asp Glu Ser Ala Tyr
257
CTG GAC GAG TGG AAC CTC ACG CCC GCC GCC AAG GCC GCC GTG CTG GCC CGC GAC TAC AAT GCG ATG ATC GAC GAG GGC GGG AAT GTC TAT Leu Asp Glu Trp Asn Leu Thr Pro Ala Ala LYs Ala Ala Val Leu Ala Arg Asp Tyr Asn Ala Met lie Asp Glu Gly Gly Asn Val Tyr
347
TTC CTG TCC AAG CTG TTC TCG ACC GAC GGC AAG AGC TTC CAG TTC GCC GCC GGC TCG ATG ACC GGC ATG ACT CAG GAA GAA TAT GCA CAG Phe Leu Ser Lys Leu Phe Ser Thr Asp Gly LyS Ser Phe Gln Phe Ala Ala Gly Ser Met Thr Gly Met Thr Gln Glu Glu Tyr Ala Gln
437
ATG ATG ATC GAT GGC GGC CGT TCG CCC GCG GGT GTG CGC TCG ATC AAG Met Met lie Asp GlY Gly Arg Ser Pro Ala Gly Val Arg Ser lie Lys
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525
TCC AGC CAC ATT CCC GCG CTC GGC GCG GCC ATC CAG ACC GGC ACC AGC GAC AAT GAT TAC TGG GGC CCG GTG TTC AAG GGC TAC CAG CCG Ser Ser His lie Pro Ala Leu GlY Ala Ala lie Gln Thr Gly Thr Ser Asp Asn Asp Tyr Trp Gly Pro Val Phe Lys Gly Tyr Gln Pro
615
ATC CGC GAC TGG ATC AAG CAG CCC GGC AAC ATG CCG GAC GTC GTG ATC CTG GTC TAT AAC GAC CAT GCC TCG GCC TTC GAC ATG AAC ATC lie Arg Asp Trp lie Lys Gln Pro GlY Asn Met Pro Asp Val Val lie Leu Val TYr Asn AsP His Ala Ser Ala Phe Asp Met Asn Ile
705
ATC CCG ACC TTC GCG ATC GGC TGC GCG GAA ACG TTC AAG CCC GCC GAC GAG GGA TGG GGC CCG CGC CCG GTG CCC GAC GTG AAG GGC CAT lie Pro Thr Phe Ala lie GlY CYs Ala Glu Thr Phe Lys Pro Ala Asp Glu Gly Trp GlY Pro Arg Pro Val Pro Asp Val Lys Gly His
795
CCG GAC CTT GCC TGG CAC ATC GCC CAG AGC CTG ATC CTC GAC GAG TTC GAC ATG ACC ATC ATG AAC CAG ATG GAC GTC GAT CAT GGC TGC Pro Asp Leu Ala Trp His lie Ala Gin Ser Leu lie Leu AsP Glu Phe Asp Met Thr lie Met Asn Gin Met Asp Val Asp His GlY Cys
885
ACC GTG CCG CTC TCG ATG ATC TTC GGC GAG CCC GAG GAA TGG CCG TGC AAG GTC ATC CCC TTC CCG GTC AAT GTC GTC ACT TAT CCG CCG Thr Val Pro Leu Ser Met lie Phe GlY Glu Pro Glu Glu Trp Pro Cys Lys Val lie Pro Phe Pro Val Asn Val Val Thr Tyr Pro Pro
975
CCG TCG GGC AAG CGC TGC TTC GCG CTC GGT GAC AGC ATC CGC GCC GCG GTC GAG AGC TTC CCG GAA GAC CTC AAC GTC CAT GTC TGG GGC Pro Ser Gly LYs Arg Cys Phe Ala Leu GlY Asp Ser lie Arg Ala Ala Val Glu Ser Phe Pro Glu Asp Leu Asn Val His Val Trp GlY
1065
ACC GGC GGC ATG AGC CAC CAG CTT CAG GGC CCG CGC GCC GGC CTC ATC AAC AAG GAG TTC GAC CTG AAC TTC ATC GAC AAG CTG ATC AGC Thr Gly Gly Met Ser His Gln Leu Gln Gly Pro Arg Ala Gly Leu lie Asn Lys Glu Phe Asp Leu Asn Phe lie Asp LYs Leu lie Ser
1155
GAC CCC GAG GAG CTG AGC AAG ATG CCG CAC ATC CAG TAT CTG CGC GAA AGC GGA TCG GAA GGC GTC GAG CTG GTC ATG TGG CTC ATC ATG Asp Pro Glu Glu Leu Ser LYs Met Pro His lie Gln Tyr Leu Arg Glu Ser Gly Ser Glu Gly Val Glu Leu Val Met Trp Leu lie Met
1245
CGC GGC GCG CTG CCG GAG AAG GTG CGG GAT CTC TAC ACC TTC TAT CAC ATC CCG GCC TCC AAC ACC GCG CTC GGC GCG ATG ATC CTG CAG Arg Gly Ala Leu Pro Glu Lys Val Arg Asp Leu Tyr Thr Phe TYr His Ile Pro Ala Ser Asn Thr Ala Leu Gly Ala Met lie Leu Gln
1335
CCG GAG GAG ACC GCA GGT ACA CCG CTC GAA CCG CGC AAG GTG ATG AGC GGA CAC AGC CTG GCC CAG GCC TGA TCGTCCACTAGGGCGGATCGACAT Pro Glu Glu Thr Ala Gly Thr Pro Leu Glu Pro Arg Lys Val Met Ser GlY His Ser Leu Ala Gln Ala **
1431
TCAGATGATGGCGTGGCGAAAATGGTGGTTTTTCGGGACCCGGAGCGCAGCGTACTCAAGGTACGTGAGCACCGGAAGCCCGGGAAAGCGCCATTTGCAGACCGCCAGCGCTGAATGTCG
1551
ATCCGCCCTAGCCCTCTCCCCTCTCGAGGGGAGAGGGTTGGGAGAGGGGTCGCCACGCTGCGGCGTGTCAGCCCGCTCCTCTCCTCCCCGCCCCCCTTCCCGGCCCTCTCCCTCGCAGGT
1671
GAGAGGGCGCTCATGTGAAAGGACTGCCCCATGAGAATAGCTCTCGCAGGCGCCGGCGCATTCGGCGAAAAACATCTCGACGGTCTCAAGAATATCGACGGCGTCGAGATCGTCTCGATC
1791
y Tyr ***
ATCAGCCGCAAGGCCGAGCAGGCCGCGGAAGTCGCCGCGAAATATGGCGCGAAGCACAGCGGCACCGATCTTTCCGAAGCGCTTGCCCGTGACGATGTCGAC
1893
FIG. 4. Nucleotide sequence of the XbaI-SalI fragment. The sense DNA strand is shown. ORF1 and ORF2 correspond to the ligA and ligB genes, respectively. The Shine-Dalgamo (SD) sequences are indicated by thick lines under the sequence.
Sequencing of the 1.9-kilobase fragment including protocatechuate 4,5-dioxygenase genes. To determine the nucleotide sequence, the 1.9-kilobase XbaI-SalI fragment cloned in plasmid pUC118 or pUC119 was used. Deletion derivatives of these plasmids were constructed by using exonuclease III (14), and sequences were determined by the dideoxy method (27) (Fig. 3). Both strands were sequenced at least twice. The sequence of the 1.9-kilobase fragment is presented in Fig. 4. A computer analysis of the sequence revealed two open reading frames. The direction of the open reading frames coincided with that of the lacZ transcription when the protocatechuate 4,5-dioxygenase is expressed. The first
open reading frame (ORF1) had the initiation codon (ATG) at position 78 and the termination codon (TGA) at position 495, encoding 139 amino acids. The second open reading frame (ORF2) immediately followed ORF1 and has the initiation codon (ATG) at position 499 and the termination codon (TGA) at position 1405, encoding 302 amino acids. These genes had a large amount of G+C (61% for ORF1 and 64% for ORF2), and the occurrence of G+C in the third base of each codon was very high (85% for ORF1 and 89% for ORF2). This is characteristic of Pseudomonas chromosomal genes (21, 31). There are sequences similar to ribosomal binding sites (the Shine-Dalgarno sequence) upstream of the
PSEUDOMONAS PROTOCATECHUATE 4,5-DIOXYGENASE GENE
VOL. 172, 1990
ACTIVITY
I
906bp lig B
417bp
lig A 2
2707
Lig A ACC ATG ATT ACG AAT TCG AGC TCG ACC CGC GTG TTC ACC GCC CAG
CGC GCG CGC AAG GGC
T M I T N S S S T R V F T A T M I T N S S S T R V F T AO A T
R A R K G R A ? K G
1 LQcZ LigA
LigB
,
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E>
4
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M A R V T T G I A R V T T ? I T
FIG. 5. Deletion analysis of the ligA and ligB genes: 1 (pBD149), the 3' terminus of the ligB gene is deleted by 149 base pairs; 2 (pRD65), the 5' terminus of the ligA gene is deleted by 65 base pairs; 3 (pRD369), the 5' terminus of the ligA gene is deleted by 369 base pairs; 4 (pAF287), the ligA gene is inactivated by cleaving and filling in at the EcoT14I site (A).
putative initiation codon in both genes (GAGG for ORFi and GGAGG for ORF2). These sequences are complementary to the 3'-terminal sequence of E. coli 16S rRNA (28). Since Pseudomonas aeruginosa 16S rRNA has a similar 3'-terminal sequence (6, 29), it is probable that P. paucimobilis also has a similar 3'-terminal sequence in the 16S rRNA. Deletion analysis of ligA and ligB genes. To determine whether the two open reading frames predicted are actually related to the activity of protocatechuate 4,5-dioxygenase, we assayed the enzyme of E. coli carrying deletion plasmids that had been used in the sequencing of the 1.9-kilobase fragment. The bases deleted are accurately known in these plasmids. We also constructed a frameshift mutant of ligA by cleaving and filling in at the EcoT141 site at position 287. The frameshift mutation replaced 36 codons of the LigA protein with 86 codons of another reading frame (Fig. 5). Only the transformant that had both intact genes showed the activity. This means that both genes are indispensable for the enzyme activity. pcrer,
A
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FIG. 6. Detection of the LigA and LigB proteins. Total cellular proteins of strains grown in the presence (lanes 2, 4, 6, 8, and 10) or absence (lanes 1, 3, 5, 7, and 9) of isopropyl-o-D-thiogalactoside (IPTG) were electrophoresed on sodium dodecyl sulfate-15% polyacrylamide gels containing 0.4% (A) or 0.6% (B) bisacrylamide. (A) Lanes: 1 and 2, JM109(pPC118); 3 and 4, JM109(pPC119); 5 and 6, JM109(pRD369); 7 and 8, JM109(pRD65); 9 and 10, JM109 (host). (B) Strain is JM109(pPC118). The arrowhead indicates the LigA protein.
T S S T ? ?
FIG. 7. Amino acid sequence of the LigA and LigB proteins. The amino acid sequence deduced from the nucleotide sequence is shown above the line, and those determined by an amino acid sequencer are shown below the line. The first eight residues of the LigA protein are derived from the lacZ portion of the vector.
Detection of the ligA and ligB gene products. To detect the ligA and ligB gene products in E. coli, cellular proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Fig. 6A). Since strain JM109(pRD65), which carries the lacZ-ligA protein fusion gene (see Materials and Methods), began to lyse when grown in the presence of isopropyl-p-D-thiogalactoside, cells were harvested when the ODsso reached 0.3. Thus the amount of protein is low in lane 8 of Fig. 6A. A 34-kilodalton (kDa) protein was found in lanes 2, 6, and 8. The molecular mass of the protein was close to that of the ligB gene product, 33,300 Da, which was deduced from the DNA sequence. This protein band was present only when the cells were grown in the presence of isopropyl-P-D-thiogalactoside. This indicated that the 34kDa protein is the ligB gene product. A 15-kDa protein was found in lane 8. Because this plasmid, pRD65, has a defective ligA gene with 65 nucleotides deleted from the 5' terminus, this protein is not an intact LigA protein. The 5' terminus of the lacZ gene in the vector pUC118 was joined to the deleted ligA gene in the same reading frame. This should result in the production of a LacZ-LigA fusion protein. The molecular mass of 15 kDa agrees well with this prediction. To detect the LigA protein, we used a polyacrylamide gel with a higher concentration of bisacrylamide to improve the separation (Fig. 6B). A distinct band corresponding to the LigA protein was found when the cell was cultured in the presence of isopropyl-p-D-thiogalactoside. Amino acid sequences of the LigA and LigB proteins. Fractionation of the cellular proteins revealed that when E. coli carrying plasmid pRD65 was induced by isopropyl3-D-thiogalactoside, the 34- and 15-kDa proteins were overproduced as inclusion bodies in the cell (data not shown). Taking advantage of this fact, we purified these proteins by centrifugation in a sucrose density gradient. Then the two proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins were transferred to a polyvinylidene difluoride membrane, the area of each protein band was cut out, and the amino acid sequence was determined by using a gas-phase amino acid sequencer. As for the LacZ-LigA protein, although 20 residues were determined from its N terminus, the first 8 residues belonged to the LacZ protein; so we virtually determined 12 residues of the LigA protein from its 23rd amino acid. As for the LigB protein, 19 residues were determined from its N terminus (Fig. 7). Some residues could not be determined precisely. For serine, the peak was too small. Because we used Tris-glycine buffer in blotting, the peaks of glycine were always too large to be estimated. The sequences were fairly
2708
NODA ET AL.
identical with those predicted by the DNA sequence of the ligA and ligB genes. This indicates that the isopropylP-D-thiogalactoside-inducible proteins are the products of the ligA and ligB genes and that these genes are actually expressed in E. coli.
DISCUSSION Protocatechuate 3,4-dioxygenase, which catalyzes the intradiol cleavage of protocatechuate, was purified from P. aeruginosa and shown to be an oligomer with a molecular mass of 587 kDa containing 12 copies each of ot (200 anmino acids, 22.3 kDa) and P (238 amino acids, 26.6 kDa) subunits (15, 18, 25). The three-dimensional structure of this enzyme has been solved by X-ray crystallography, and its unusual mixed P barrels and the location of the active site are known. Protocatechuate 4,5-dioxygenase, which catalyzes the extradiol cleavage of protocatechuate, has been partially purified from P. testosteroni (5) or a Pseudomonas sp. (26), and kinetic studies were done (33). The molecular mass is reported to be 140 kDa (5) or 150 kDa (26). It has also been reported that this enzyme contains iron and is composed of two subunits (a, Mr 17,000; 13, Mr -33',800) in a 1:1 ratio (1-3). In this study the nucleotide sequences of two tandemly placed genes concerning protocatechuate 4,5-dioxygenase were determined. Since both genes were essential for the enzyme activity and the m'olecular masses of the subunits deduced from the nucleotide sequence were consistent with those reported by Arciero et al. '(1), the ligA and ligB genes should encode the subunits of protocatechuate 4,5-dioxygenase. The amino acid sequences of five extradiol enzymes have been reported so far: catechol 2,3-dioxygenase (XylE) (22), catechol 2,3-dioxygenase (NahN) (11, 13), 1,2-dihydroxynaphthalene dioxygenase (NahC) (12), and two 2,3-dihydroxybiphenyl dioxygenases (BphC) (8, 30). Harayama and Rekik reported similarities among the sequences and some highly conserved regions, and consequently they proposed that these five extradiol enzymes share a common origin (12). We compared the amino acid sequences of LigA and LigB with those of the five extradiol enzymes to see whether protocatechuate 4,5-dioxygenase shares a common ancestor with the others. We could not find any sequence similarities between either LigA or LigB and the others. As for the subunit composition, protocatechuate 4,5-dioxygenase is composed of heterogeneous a and ,B subunits, whereas the five extradiol enzymes are composed of homogeneous subunits. So we concluded that protocatechuate 4,5-dioxygenase has a different''origin from that of the five extradiol enzymes. We also examined sequences of four intradiol enzymes, catechol 1,2-dioxygenase (CatA) (23), chlorocatechol dioxygenase (ClcA) (7), catechol 1,2-dioxygenase (TfdC) (10), and protocatechuate 3,4-dioxygenase (5, 19), but we could not find any homology with those of LigA or LigB. We also searched in the protein databases (NBRFPDB, release 22.0; SWISS-PROT, release' 11.0) for homologous sequences (32) with LigA or LigB, but we failed to find any. The termination codon of ORF1 (ligA) and the initiation codon of ORF2 (ligB) are separated by only one nucleotide. Since both genes were expressed by placing the lac promoter in front of the ligA gene, ligA and ligB genes should be comprised in an operon. Since the 1.9-kilobase fragment did not express itself in either P. putida or E. coli without the aid of an exogenous promoter, the intrinsic promoter should be in the 5' region upstream of the ligA gene. There might be
J. BACTERIOL.
other genes concerned with lignin degradation between the intrinsic promoter of the operon and the ligA gene. On the other hand, there were no other long open reading frames in the 3' region downstream of the ligB gene, even though we could not find a characteristic sequence for the transcription terminator. The ligA and ligB genes were expressed in E. coli by using the lac promoter. We found that when the ligA gene was deleted by 65 nuileotides from the N terminus, the LacZLigA and LigB Oroteins were overproduced and formed inclusion bodies in the cell (Fig. 6, lane 8), in contrast to the relatively low expression of the intact genes (Fig. 6, lane 2). Some possible explanations are as follows: (i) a transcription terminator exists upstream of ligA; (ii) deletion in the 5' region of ligA increases the stability of the hybrid plasmid; (iii) deletion in the 5' region of ligA increases the copy number of the hybrid plasmid. Further experiments will be needed to elucidate the mechanism of this regulation. ACKNOWLEDGMENTS We thank Takashi Kamakura for his advice in performing the protein sequencing and Jurate Tsurufuji for critical reading of the
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