Toluene Degradation by Pseudomonas putida F1 - The Journal of ...

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Gerben J. Zylstra and David T. Gibson$. From the Department of Microbiology and Biocatalysis Research Group, the University of Iowa, Iowa City, Iowa 52242.

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 264, No. 25, Issue of September 5. pp. 14940-14946,1989 Printed in U.S. A.

Toluene Degradationby Pseudomonas putida F1 NUCLEOTIDESEQUENCEOF COLI*


(Received for publication, March 10, 1989)

Gerben J. Zylstra andDavid T. Gibson$ From the Department of Microbiology and Biocatalysis Research Group, the University of Iowa, Iowa City, Iowa 52242

The nucleotide sequenceof the todCl C2BADE genes Pseudomonas putidu F1 (PpF1) utilizes toluene as a sole which encodethe first three enzymes in thecatabolism source of carbon and energy for growth. The catabolic pathof toluene by Pseudomonasputida F1 was determined. way, termedthe dihydrodiol pathway,is shown in Fig. 1. The genes encode the threecomponents of the toluene Tolueneisfirst oxidized to(+)-cis-(lS,2R)-dihydroxy-3dioxygenase enzyme system: reductaseToL (todA), methylcyclohexa-3,5-diene(cis-toluene dihydrodiol)’ through ferredoxinToL (todB), and the two subunits of the ter- the addition of both atoms of molecular oxygen to the arominal dioxygenase (todClC2); (+)-cis-(lS, 2R)-dihy- matic nucleus (1-3). This initial reaction is carried out by a droxy-3-methylcyclohexa-3,5-dienedehydrogenase multicomponent enzyme system, designated toluene dioxy(todD); and 3-methylcatechol 2,3-dioxygenase (todE). Knowledge of the nucleotide sequence of the tod genes genase (4),which functions to transfer electrons from NADH was used to constructclones of Escherichia coli JM109 to the terminal dioxygenase. Electrons are initially accepted from NADH by a flavoprotein, reductaseToL (5), which transthat overproduce toluene dioxygenase (JMlOS(pDTfers G601)); toluene dioxygenase and (+)-cis-(lS, ZR)-dih- them to a small iron-sulfur protein, ferredoxinToL (6). ydroxy-3-methylcyclohexa-3,5-diene dehydrogenase FerredoxinToLreduces theterminal dioxygenase, anironbeen designated ISPToL (7,8). Reduced (JM109(pDTG602)); and toluene dioxygenase, (+)-cis- sulfur protein that has (lS, 2R)-dihydroxy-3-methylcyclohexa-3,5-diene de- ISPTOLcatalyzes the addition of both atoms of molecular oxygen to toluene to formcis-toluene dihydrodiol. The latter hydrogenase, and 3-methylcatechol ZY3-dioxygenase (JMlOg(pDTG603)). The overexpression of the tod- compound is dehydrogenated in a NAD+-dependent reaction ClC2BADE gene products was detected by sodium mediated by cis-toluene dihydrodiol dehydrogenaseto form 3dodecyl sulfate-polyacrylamide gel electrophoresis. methylcatechol (9). Extradiol (metu) cleavage by 3-methylcatechol 2,3-dioxygenase leads to the formation of 2-hydroxyThe three E. coli JM109 strains harboring plasmids the pDTG601, pDTG602, and pDTG603, after induction 6-0~0-2,4-heptadienoate. Each of these enzymes has previwith isopropyl-P-D-thiogalactopyranoside,oxidized ously been purified and partially characterized(5-7, 9).’ toluene to (+)-cis-(lS, 2R)-dihydroxy-3-methylcyclo- The chemistry and biochemistry of the dihydrodiol pathway hexa-3,5-dieneY 3-methylcatechol, and 2-hydroxy-6shown in Fig. 1has been studied extensively. Our recent work oxo-2,4-heptadienoate,respectively.Thetodhas focused on the organization and regulation of the genes ClC2BAD genes show significanthomology to the re- involved in toluene catabolism. Mutants defective in each of ported nucleotide sequence for benzene dioxygenase the structuralgenes have been isolated (10,ll) and the genes and cis-1,2-dihydroxycyclohexa-3,5-diene dehydro- (designated tod) are induced coordinately (11).Transposon genase from P. putidu 136R-3 (Irie, S., Doi, S., Yori- mutagenesisexperiments revealed that the todClC2BADE fuji, T., Takagi, M., and Yano, K. (1987)J. Bacteriol. genes are arranged in an operon (12). The initialgenes of the 169, 5174-5179). In addition, significant homology tod operon were cloned intothebroad-host-range cosmid was observed betweenthe nucleotide sequencesfor the he plasmid, designated todDE genes and the sequences reported for ~ i s - 1 ~ 2 -cloning vector P L A F R ~ . ~ T resulting dihydroxy-6-phenylcyclohexa-3,5-dienedehydrogen- pDTG301, contains two EcoRI fragments clonedfrom the ase and 2,3-dihydroxybipheny1-ly2-dioxygenase from P p F l chromosome. Each EcoRI fragmentwas subcloned into the broad-host-rangevector pKT230 and oneof the resulting Pseudomonas pseudoalcaligenes KF707 (Furukawa, to encode the K., Arimura, N., and Miyazaki,T. (1987)J. Bacteriol. plasmids,designated pDTG351,wasshown todClC2BADE genes (12). Further studies involving the con169,427-429). struction of deletion derivatives of pDTG351 and analysisof their ability to complement PpFl mutants in specific tod genes have shown the location of each of the tod genes on pDTG351 (12). *This work was supported by Public Health Service Grant We now report the entire nucleotide sequence of the todGM29909 from the National Institute of General Medical Sciences. ClC2BADE genes as well as flanking sequences. Knowledge

A preliminary reportof this work was presented at the 88th American Society for Microbiology Meeting, Miami, FL, May 8-13, 1988. The costs of publication of this article were defrayed in part by the payment of page charges. This articlemusttherefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) 504996. $ T o whom correspondence and reprint requestsshould be addressed.

The abbreviations used are: cis-toluene dihydrodiol, (+)-cis-(lS, 2R)-dihydroxy-3-methylcyclohexa-3,5-diene; ISPTOL, iron-sulfur protein component of toluene dioxygenase; MSB, mineral salts basal medium; CTG, calcium chloride-thymidine-glycerol; IPTG, isopropyl-P-D-thiogalactopyranoside; TLC, thinlayer chromatography; GC/ MS, gas chromatography/mass spectroscopy; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. F.-M. Menn and D. T. Gibson, unpublished data. W. R. McCombie and D. T. Gibson, manuscript in preparation.


Degradation Toluene

by P. putida


pDTG552 (Fig. 2B). In subsequentexperiments pDTG552 was first cleaved with BamHI and the 5”protruding ends filled in with a-phosphorothioatedeoxynucleotides using Klenow fragment of DNA polymerase. The DNAwas then cleaved with XhoI followed by partial digestion with exonuclease 111. Exonuclease 111does not attackphosphorothioatelinked nucleotides and, since XhoI produces 5’ overhangs which are attacked by exonuclease 111, deletion occurs to the left on the restrictionmap shown in Fig.2B. The DNA fragments were then treated with S1 nuclease and Klenow fragment of DNA polymerase prior to ligation overnight at room temperature. After transformation into E. coli JM109, NAD’ NADH+H’ 0 2 colonies were screened to determine the extentof the plasmid cis.Toluene 3-Methylcalechol deletion. Plasmidscontaining overlapping deletions were dihydrodiol 2,3.dioxygenase dehydrogenase saved and subjected to DNA sequencing as described below. (rodD) (rod€) DNA Sequencing-The nucleotide sequence of the 6.7FIG. 1. Metabolic pathway for the oxidation of toluene to 2- kilobase pair EcoRI to XhoI fragment of DNA encoding the hydroxy-6-0~0-2,4-heptadienoate by P.putida F l . Gene des- todCl C2BADE genes was determined as described under “Exignations for individual proteins are shown in parentheses. perimental Procedures.” Plasmids to be sequenced were chosen from both the forward and reverse deletion series that of the sequence has enabled us to construct clones which had been constructed as described above. Each plasmid was overexpress these enzymes in Escherichia coli and accumulate sequenced a distance of 300-400 bases starting from the edge of the deleted region and extending into the region encoding specific intermediates in the dihydrodiol pathway. the genes of interest (Fig. 2C). Thus, both the forward and reverse strands were sequenced in their entirety to yield the EXPERIMENTALPROCEDURES4 nucleotide sequence of the 6716-base pair EcoRI to XhoI fragment shown in Fig. 3. Analysis of the sequence shows that RESULTS there are six open reading frames in the locations predicted Construction of Subclones for DNA Sequencing-The 6.7previously by plasmid deletion constructionand mutantcomkilobase pair EcoRI to XhoI fragment of pDTC351 (Fig. 2 A ) plementation studies for the todClCZBADE genes (12, Fig. contains the todClC2BADE genes responsible for the oxida- 2 A ) . The predicted amino acid sequences for the proteins are tion of toluene to 2-hydroxy-6-0~0-2,4-heptadienoate (12, Fig. also shown in Fig. 3. The calculated molecular weights for 1).The strategy used to sequence this DNA fragment is shown each protein based on the nucleotide sequence agrees quite in Fig. 2. A series of overlapping deletions from both the left well with those determined for the purified proteins (Table and right ends of the fragment were constructed and used to I). The N-terminal amino acid sequence of each of these determine the nucleotide sequence of both the forward and proteins purified from P. putida F1 has been determined reverse strands of DNA. Overlapping deletions for sequencing (Table I)5 and these agree with those determined by extrapoin the forward direction were constructed by first subcloning lation from the nucleotide sequence. the BamHI to XhoI fragment of pDTG351, containing the Construction of Clones That Overexpress the tod Gene ProdtodClC2BADE genes, into the sequencing vector pGEM3Z ucts-In order to study the individual enzymes of the tod which had been restricted withthe enzymes BamHI andSalI. operon in more detail three clones overexpressing the first The resulting plasmid, designated pDTG551 (Fig. 2B), was three enzymes of the toluene metabolic pathway were consubsequently cleaved with the enzymes KpnI and BanHI structed. The regions of the nucleotide sequence that were followed by a seriesof partial digestions with exonuclease 111. cloned into pKK223-3 to make the appropriate constructs are Cleavage with KpnI produces 3”protruding DNA ends which summarized in Table 11. The series of forward and reverse are notattacked by exonuclease 111 while cleavage with deletions obtained with exonuclease I11 as described above BamHI produces 5”protruding DNA ends which are attacked were utilized in the construction of these clones. A plasmid, by exonuclease 111. Therefore, deletion only occurs to the designated pDTG561, was shown to be deleted up to base right on the restriction map shown in Fig.2B. The DNA number 611 of the sequenced region. The 4.0-kilobase pair fragments obtainedwere successivelytreated with S1 nuclease EcoRI to PvuII fragment of pDTG561 was subcloned into and Klenow fragment of DNA polymerase prior to overnight pKK223-3 which had been cut with EcoRI and SmaI. The ligation with T4 ligase at room temperature. The ligated DNA resulting plasmid was designated pDTG601 (Table 11) and was transformed into E. coli JM109 by the calcium chloride- was designed to place the genes for the toluene dioxygenase thymidine-glycerol procedure with selection on ampicillin- complex (reductaseToL, ferredoxinToL,and ISPTOL) under concontaining L-agar. Colonies were screened for their plasmid trol of the tac promoter. A second plasmid was constructed content and the extentof nucleotide deletion. Plasmids con- utilizing a reverse deletion, designated pDTG581, that was taining overlapping deletions were saved and subjected to shown to be deleted up to base number 5322 of the sequenced DNA sequencing as described below. region. The 2.5-kilobase pair EcoRI to NotI fragment of A series of overlapping deletions for sequencing the reverse pDTG561 and the1.9-kilobase pair NotI to HindIIIfragment strand of DNA was also constructed. The EcoRI fragment of of pDTG581 were subcloned together into pKK223-3 that had pDTG351 containing the todClCZBADE genes was first sub- been cleaved with EcoRI and HindIII. This plasmid was cloned into the EcoRI site of pGEM3Z to yield the plasmid designated pDTG602 (Table 11) and placed the genes for both Portions of this paper (including “Experimental Procedures” and toluene dioxygenase and cis-toluene dihydrodiol dehydrogenFig. 2) are presented in miniprint at the endof this paper. Miniprint ase under control of the tac promoter. A third plasmid was is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that G. J. Zylstra, F:M. is available from Waverly Press. lished data.

Menn, W.-K. Yeh, and D. T. Gibson, unpub-


Toluene Degradationby P. putida

FIG. 3. Nucleotide sequenceof the P.putida F1 todCIC2BADE genes and the corresponding amino acid sequences.

TABLEI Molecular weights and N-terminalsequence of the todClC2BADE gene products from P. putida F1 Gene

todCl 2083-2643 todCP todB 2978-4207 todA 4207-5031 todD todE

Region of nucleotide sequence”

620-1969 2655-2975


Subunit molecular weight Determined termined from purified proteinb

N-terminal sequence defrom purified protein


Determined from nucleotide sequence

ISPTOLlarge subunit ISPTOLsmall subunit FerredoxinToL ReductaseToL cis-Toluene dihydrodiol dehydrogenase 3-Methylcatechol2,3dioxygenase

50,900 22,000 11,900 42,900 28,700

52,500 20,800 15,300 46,000 27,000





a The N-terminal amino acid sequences determined for the purified proteins were used to locate the correct open reading frames in the nucleotide sequence. Molecular weights of the protein subunits have been reported previously (5-7, 9).2

constructed utilizing another reverse deletion, designated pDTG582, that was shown to be deleted up to base number 6172 of the sequenced region. The 2.5-kilobase pair EcoRI to NotI fragment of pDTG561 and the3.0-kilobase pair NotI to

HindIII fragment of pDTG582 were subcloned together into pKK223-3 that had been cut with EcoRI and HindIII. This plasmid was designated pDTG603 (Table 11) and placed the genes for toluene dioxygenase, cis-toluene dihydrodiol dehy-

by P. putida

Degradation Toluene


TABLE I1 Properties of E. coli JM109 strainscarrying the recombinant plasmids pDTG601, pDTG602, and pDTG603 Plasmid Genes

Region of nucleotide sequence“

pDTG601 pDTG602

611-4675 611-5322




Metabolite formed from tolueneb


todCl C2BA todCl C2BAD todClC2BADE Toluene

Toluene dioxygenase Toluene dioxygenase cis-Toluene dihydrodiol dehydrogenase dioxygenase &-Toluene dihydrodiol dehydrogenase 3-Methylcatechol2,3dioxygenase

cis-Toluene dihydrodiol 3-Methylcatechol 2-Hydroxy-6-oxo-2,4heptadienoate

See Fig. 3.

’Metabolites were identified by comparison with authentic compounds as described in

drogenase, and 3-methylcatechol2,3-dioxygenaseunder control of the tatpromoter. Analysis of Clones That Overexpress the todClC2BADE Genes-The three clones constructed above were analyzed for their ability to overproduce the encoded enzymes under control of the tac promoter. After induction as described under “Experimental Procedures,” 1.0mlof the culture was removed, centrifuged at 13,000 x g, and the cells resuspended in 0.1 ml of SDS-PAGE loading buffer. After boiling for 5 min, each sample (20 pl) was analyzed by SDS-PAGE. Fig. 4 shows the protein profiles of the extractsobtained from JM109 cells containing either pKK223-3, pDTG601, pDTG602, or pDTG603. It can clearly be seenfrom this figure that pDTG601, pDTG602, and pDTG603 all overproduce proteins having the same molecular weight as the four components of toluene dioxygenase: reductaseToL,ferredoxinToL, and ISPToL (large and small subunits). It can also be seen that in addition bothpDTG602 and pDTG603 overproduce a protein with the same molecular weight as cis-toluene dihydrodiol dehydrogenase. JMlOg(pDTG603) should overproduce 3-methylcatechol2,3-dioxygenase in addition to toluene dioxygenase and cis-toluene dihydrodiol dehydrogenase. How-







- ”?-- ..24,000 - 4



.Ire ”


ever, overproduction of 3-methylcatechol 2,3-dioxygenase is obscured by a native JM109 protein having the same molecular weight. The overexpression of these proteinsis consistent with the theoretical expectations based on the plasmid constructs asdescribed above. Enzymatic Activity of Cloned tod Genes in E. coli JMlO9The physical overexpression of the todCl C2BADE geneproducts in E. coli JM109 shown in Fig. 4 and described above does not indicate that thesix proteins have enzymatic activity. Preliminarystudies with isopropyl-P-D-thiogalactopyranoside-induced cells of JM109(pDTG601) showed that theywere capable of oxidizing toluene to cis-toluene dihydrodiol. Formation of the dihydrodiol was linear over a 24-h time period after which the rate of product formation began to decline. The dihydrodiol was isolated and shown to have identical spectral, thin layer chromatographic, and gas chromatographic/mass spectroscopic properties to those given by cistoluene dihydrodiol formed from toluene by P. putida F39/D (1). Analogous experimentswith JM109(pDTG602) and JM109(pDTG603) showed that these strains oxidized toluene to 3-methylcatechol and 2-hydroxy-6-0~0-2,4-heptadienoate, respectively. The former compound produced by JM109(pDTG602) gave an identical R F value (0.23) on thin layer chromatography, absorption (Xmax, 275 nm), and mass (m/e 124) spectra to those given by authentic 3-methylcatechol. latter The compound produced by JM109(pDTG603) gave identical absorption spectra in acid ,,X,(, 315 nm) and base ,,,X(, 388 nm) to those previously reported for 2-hydroxy-6-0~0-2,4-heptadienoate (24). These results are summarized in Table 11. DISCUSSION




-CatecholDioxygenase Dehydrogenase

-ISP (Small)




1 -Ferredoxin

FIG. 4. SDS-PAGE of extracts from clones that overexpress the tod gene products. Extracts were prepared as described in the text from E. coli JM109 strains containing either plasmid pKK223-3 (lane C), pDTG601 (lane I ) , pDTG602 (lane 2 ) , or pDTG603 (lane 3).

Sequence Analysis-The base composition of the todClC2BADE nucleotide sequence shown in Fig. 3 is similar to that found for other P. putida genes that have been sequenced (25, 26). The G+C content of the entire sequenced region is 60%. This high G+C value is due in part to thecodon usage preference indicated by the composition of the genes that have been sequenced (Table 111). There is a preponderance (72%)of codons that have a guanine or a cytosine in the third variable position. The termination codons TGA and TAG are used (five times and one time, respectively) in preference to the termination codon TAA which was not used. Sequences homologous to known Pseudomonas promoters (27) and translation terminators typical of those found in enteric bacteria (28,29) were not detected. The endof a putativeopen reading frame was detected upstream from the genes sequenced (1520 bases in Fig. 3). The beginning of another putative reading frame was detected downstream from the genes sequenced

Toluene Degradationby P. putida


TABLE111 Codon usage for the todClC2BADEgenes from P. putida F1 Codon













TTT-Phe TTC-Phe10 TTA-Leu TTG-Leu CTT-Leu CTC-Leu CTA-Leu CTG-Leu ATT-Ile ATC-Ile ATA-Ile ATG-Met GTT-Val GTC-Val GTA-Val GTG-Val TCT-Ser TCC-Ser TCA-Ser TCG-Ser CCT-Pro CCC-Pro CCA-Pro CCG-Pro ACT-Thr ACC-Thr ACA-Thr ACG-Thr GCT-Ala GCC-Ala GCA-Ala GCG-Ala

3 14 0 1

2 0 2

0 5 0 4

2 7 0 8

1 10 1 6

6 13 2 5

14 59 3 26

4 13 0 0

1 6 0 1

0 3 0 0

2 2 0 0

4 4 1 0 0

2 0 0 0

13 38 0 1

4 7 0 19

2 6 0 5

0 1 0 312

8 7 2 21

4 7 2

3 4 9

21 32 5 69

7 6 1 11

1 3 0 4

2 1 0 3

3 1 6 9

3 2 2 3

1 9 3 6

17 22 12 36

5 12 1 15

4 9 0 6

1 2 1 3

6 13 1 9

4 6 2 3

5 5 0 7

25 47 5 43

6 12 3 14

3 6 0 4

0 2 1 6

2 3 2 2

3 2 5 7

3 1 2 6

17 26 13 39

2 16 3 7

0 4 1 4

0 5 1 5

2 6 17 7 8 0 9 1 3

3 10 0 10

13 59 13 48

2 4 2 3

0 3 3 2

0 0 0 1

2 4 1 5

0 2 0 4

1 2 5 6

5 15 11 21

5 6 3 9

0 4 0 1

3 1 2 1

1 8 2 5

1 2 1 4

0 4 2

10 25 9 22

1 15 5 7

0 4 0

1 2 1



2 4 2 2

2 10 0 4

8 42 10 34

4 11 6 13

2 14 7 6

17 71 28 64


6 19 2 14


2 0 8 4 4


2 1

1 0 0 3

4 4 19 9 24



(base 6047 to the endof the sequenced region). Both of these reading frames showed a codon usage preference similar to that of the todClC2BADE genes. Although the identities of these two genes are not known, the end of the open reading frameupstreamfrom todCl is probably part of todF, the hydrostructural gene for 2-hydroxy-6-0~0-2,4-heptadienoate lase. This is based on transpositional analyses which have indicatedthatthe gene orderinthe tod operonis todFClC2BADE (12). The sixcomplete open reading frames in the region sequenced were identified as the todClC2BADE genes by comparison of the aminoacid sequence deduced fromthe nucleotide sequence to the N-terminal amino acid sequence of the individual proteins isolated from P. putida F1. The N-terminal amino acid sequences of the proteins are shown in Table I. It is of interest to note that methionine was not the Nterminalamino acid in ferredoxinToL (todB), reductaseToL (todA), and3-methylcatechol dioxygenase (todE). In contrast a N-terminal methionine is present in both ISPTOL subunits (todClC2) and cis-toluene dihydrodiol dehydrogenase(todD). The latter protein utilizes GTG rather than ATG as the codon for the N-terminal methionine. There is a ribosome binding site (30, Shine-Dalgarno site) preceding each of the genes. The calculated molecular weightsof each of the gene products based on the nucleotide sequence are very similar to those calculated previously for the isolated proteins (Table I). Overexpression of tod Genes in E. coli-Three clones were constructed that overexpress the tod gene products in E. coli JM109 using the expressionvector pKK223-3(Table 11). PlasmidpDTG601 overexpresses the three components of

9 21 15 19

8 6

4 5 3

9 9 13

8 10 9 6

7 11 6 10

50 63 50 63

2 5 1 14

0 0 0 3

0 5 1 2

1 3 1 5

0 2 1 2

0 2 1 6

3 17 5 32

5 14 1 8

3 1 6

0 1 0 2

6 14 5 6

5 5 0 1

7 4 4 1

26 48 11 24

1 12 0 2

1 5 1 0

0 0 0 0

6 5 1 2


1 6 2 1

9 36 6 6


37 87 16 29

4 17 4 8







4 2 1 2


5 0 3


2 2513 5 6


2 1 8 25 1 5

5 5

toluene dioxygenase: ISPTOL,ferredoxinToL,and reductaseToL. Plasmid pDTG602overexpresses toluene dioxygenase and cistoluene dihydrodiol dehydrogenase. Plasmid pDTG603 overexpresses toluene dioxygenase, cis-toluene dihydrodiol dehydrogenase, and 3-methylcatechol 2,3-dioxygenase. Not only were these proteins physicallyoverexpressed as shown by SDS-PAGE studies(Fig. 4), theywere also active in termsof their ability to metabolize intermediates in the toluene catabolic pathway (Table 11). This was shown by the ability of each E. coli recombinant strain to metabolize toluene to the expected downstream metabolite (cis-toluene dihydrodiol, 3methylcatechol, or 2-hydroxy-6-0~0-2,4-heptadienoate). Functional overexpression is important when one considers that the enzymes were originally cloned from P. putida F1 andare beingoverexpressed in E. coli. Therecombinant strains are currentlybeing utilized to determine the substrate specificities of toluene dioxygenase, cis-toluene dihydrodiol dehydrogenase, and 3-methylcatechol 2,3-dioxygenase. The strains will also prove valuableas sources of pure proteinsfor future studies on the mechanism of action of enzymes involved in toluene degradation. Sequence Homologies-The nucleotidesequences of the P p F l todClC2BAD genes are very similar to the sequences reported for the genes encoding benzene dioxygenaseand cisbenzenedihydrodioldehydrogenase from P. putidastrain 136R-3 (31). The latter strain was derived from the parent organism P. putidaBE-81 by mutagenesis twice with N methyl-N’-nitro-N-nitrosoguanidine. The mutagenesis procedure was used toinactivatethecatechol 1,2- and 2,3dioxygenases present in strain BE-81 with the objective of

Toluene Degradation by P. putida


Although there is significant homology between the amino acid sequences of the 3-methylcatechol2,3-&oxygenase from MKLKGEAVLITGGASGLGRALVDRFVAE AKVAVLDKSAERLAELETOLGONVLGIVGDVRSLEDqKQAASRCVA PpFl and the2,3-dihydroxybiphenyl 1,2-dioxygenase from P. KF707, these enzymes show significant difA f c K L ~ C ~ V G ~ V ~ L ~ Q ~ I r p D D L I S E ~ E ~ M F E V N V K G Y I L A A K A A L P A L Y Q ~ K G S A I F T V S N A G F Y Ppseudoalcaligenes GG RFGKIDTLIPNAGIWDYSTALVDLPEESLOAAFOEVFHINVKGVIHAVKA LPALVASRGNVIFTISNAGFYPNG ferences in their substratespecificity. The catecholoxygenase from P. pseudoalcaligenes KF707 has been purified to homoGGVLYTAG~HAVI~IKp~H~WG~RI~IA~ILG~L~LK~OLQOKSlSTFPLDDMLKSVLPTGRAAT geneity and shown to be specific for 2,3-&hydroxybiphenyl. GGPLYlAAKqAlVGLVRELAFELAPYVRVNGVGPGGMN SDMRGPSSLGMGSKAISTVPLADMLKSVLPIGRMPE No significant activity wasobserved with catecholor 3-methAEEYAGAYVFFATRGDTV~LT~SVLNFOGGMGVRGL~EASL~AO~DKHFG (38). In contrast, purified 3-methylcatechol 2,3ylcatechol VEEYTGAYVFFATRGDAAPASGALVNYDGGLGVRGFFSGAGGNOLLEqLNlHP dioxygenase from P p F l oxidizes 3-methylcatechol, catechol, and 2,3-dihydro~ybiphenyl.~ It will be interesting to compare E. the amino acid sequences and substrate specificities of the MSIQRLGYLGFEVADVRSWRTFATTRLGMMEASASETEATFRIDSRAWRLSVSR~PA~YL~F~DSEQ~~E biphenyl dioxygenase from P. pseudoalcaligenes KF707 with HSIRSLGYMGFAWSDVRAWRSFLTQKLGLMEAGTTONGDLFRIDSRA~RlRV~~GEVOOLAFAGYEVADAAGLA~ the toluene dioxygenase from PpFl to see if the observations with the catechol dioxygenases are also true for the initial enzymes in bothmetabolic pathways.







Acknowledgments-We thank W.-K. Yeh and F.-M.Menn for determining the N-terminal sequences of ferredoxinToL and 3-methylcatechol2,3-dioxygenase, respectively.


REFERENCES 1. Gibson, D. T., Hensley, M., Yoshioka, H., and Mabry, T. J. (1970) Biochemistry 9, 1626-1630 2. Kobal, V. M., Gibson, D. T., Davis, R. E., and Garza, A. (1973) J. Am. Chem. SOC. 95,4420-4421 3. Ziffer, H., Jerina, D. M., Gibson, D. T., and Kobal, V. M. (1973) J . Am. Chem. SOC.95,4048-4049 4. Yeh, W.-K.,Gibson, D. T., andLiu, T.-N. (1977) Biochem. Biophys. Res. Commun. 78,401-410 5. Subramanian, V., Liu, T.-N., Yeh, W.-K., Narro,M., and Gibson, D. T. (1981)J. Biol. Chem. 256,2723-2730 isolating a strain that would accumulate catechol from ben6. Subramanian,V., Liu,T.-N., Yeh, W.-K., Serdar,C. M., Wackett, zene (32). Thenucleotide sequence of strain 136R-3 is almost L. P., and Gibson, D. T. (1985) J. Bid. Chem. 260,2355-2363 identical to the sequence reported here for bases 1-5292 in 7. Subramanian, V., Liu,T.-N.,Yeh,W.-K.,andGibson, D. T. PpFI. The differences are mainly due to bases present in the (1979) Biochem. Biophys. Res. Comnun. 91, 1131-1139 8. Gibson, D. T., Yeh, W.-K.,Liu, T.-N., and Subramanian, V. P p F l sequence that are absent in the sequence deduced for (1982) in Oxygenases and OxygenMetabolism (Nozaki, M., strain 136R-3. This could be due to the treatment of the Yamamoto, S., Ishimura, Y., Coon, M. J., Ernster, L.,and parent strain BE-81with N-methyl-N’-nitro-N-nitrosoguanEstabrook, R. W., eds) pp. 51-62, Academic Press, Inc., New idine prior to sequence determination. The identity of the York genes in the sequence from strain 136R-3was determined by 9. Rogers, J. E., and Gibson, D. T. (1977) J. Bacteriol. 130, 11171124 identifying the open reading frames and comparing the cal10. Finette, B. A., Subramanian, V., and Gibson, D. T. (1984) J. culated molecular weightswith those reported in the literature Bacteriol. 160, 1003-1009 for purified proteins. The problems inherent in this approach 11. Finette, B. A., and Gibson, D. T. (1988)Biocatalysis 2, 29-37 can be seen in the reported molecular weight of 17,220 for cis- 12. Zylstra, G . J., McCombie, W. R., Gibson, D. T., and Finette, B. benzene dihydrodiol dehydrogenase based on the position of A. (1988) Appl. Enuiron. Microbiol. 54, 1498-1503 the ATG codon for methionine (31). The N-terminal amino 13. Yanisch-Perron, C., Viera, J., and Messing, J. (1985)Gene (Amst.) 33,103-119 acid sequence for cis-toluene dihydrodiol dehydrogenase from P p F l begins translation with a GTG codon for methionine, 14. Gibson, D. T., Koch, J. R., and Kallio, R. E. (1968) Biochemistry 7,2653-2662 and this gives a subunit molecular weight of 28,700 which is 15. Stanier, R. Y., Palleroni, N. J., and Doudoroff, M. (1966) J . Gen. close to the value of 27,200 calculated for the purified protein Microbiol. 43, 159-271 (Table I). If the cis-benzene dihydrodiol dehydrogenase from 16. Maniatis, T. E., Fritsch, E. F.,and Sambrook, J. (1982) Molecular P. putida 136R-3 is assumedto start with a GTG codon then C1oning:A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor,NY the dehydrogenase subunit would be 112 amino acids longer 17. Birnboim, H. C., and Doly, J. (1979) Nucleic Acids Res. 7, 1513than that reported for the protein starting with ATG and 1523 would have a molecular weight of 28,700. 18. Ish-Horowitz,D., and Burke, J. F. (1981) Nucleic Acids Res. 9, There is also significant homology (61%) between the nu2989-2998 cleotide sequences for cis-toluene dihydrodiol dehydrogenase 19. Henikoff, S. (1984) Gene (Amst.) 28, 351-359 and3-methylcatechol 2,3-dioxygenase from PpFl and the 20. Sanger, F., Mickler, S., and Coulson, A. R. (1977) Proc.Natl. Acad. Sci. U. S. A . 74, 5463-5467 analogousenzymesinvolvedin biphenylcatabolism by P. 21. Tabor, S., and Richardson, C. C. (1987) Proc. Natl. Acad. Sci. pseudoalcaligenes KF707 (33). Fig. 5 shows that the amino U. S. A. 84,4767-4771 acidsequences can easily be aligned with each other. The 22. Garoff, H., and Ansorge,W. (1981)Anal. Biochem. 115,450-457 amino acid sequences forthe dihydrodiol dehydrogenases and 23. Laemmli, U. K. (1970) Nature 227, 680-685 catechol oxygenases show 58 and 54%homology, respectively. 24. Bayly, R. C., Dagley, S., and Gibson, D. T. (1966) Biochem. J . 101,293-301 In contrast, the amino acid sequences of the rneta-cleavage catechol dioxygenases encoded bythe TOL (34,35) and NAH25. Crawford, I. P., and Eberly, L. (1986)Mol. Biol. Euol. 3,436-448 (36, 37) plasmids show only 23 and 20% homology, respec- 26. Hadero, A., and Crawford, I. P. (1986) Mol. Biol. Euol. 3, 191204 tively,whenmaximallyalignedwith the 3-methylcatechol 2,3-dioxygenase from PpF1. F.”. Menn, G. J. Zylstra, and D. T. Gibson, unpublished data. FIG. 5. Comparison of amino acid sequences from P . pseudoalcaligenes KF707 and P. putida F1. A, comparison of P. putidaF1cis-toluenedihydrodioldehydrogenase (top) to P. pseudoalcaligenesKF707cis-biphenyldihydrodioldehydrogenase (bottom). Thebeginning of the KF707dehydrogenaseis inferred by homology to that reported here for P. putida F1. B, comparison of P. putida F1 3-methylcatechol2,3-dioxygenase(top) to P. pseudoalcaligenes KF707 2,3-dihydroxybiphenyl 1,2-dioxygenase (bottom). Identical amino acids are indicatedby an 1.

Toluene Degradation by P. putida


27. Deretic, V., Gill, J. F., and Chakrabarty, A. M. (1987) Biotechnology 5 , 469-477 28. Adhya, S., and Gottesman, M. (1978) Annu. Reu. Biochern. 4 7 , 967-996 29. Platt, T. (1986) Annu. Reu. Biochern. 5 5 , 339-372 30. Shine, J., and Dalgarno, L. (1975) Nature 2 5 4 , 34-38 31. hie, S., Doi, S., Yorifuji, T., Takagi, M., and Yano, K. (1987) J. Bacteriol. 1 6 9 , 5174-5179 32. Shirai, K. (1986) Agric. Biol. Chern. SO, 2875-2880 33.Furukawa, K., Arimura, N., and Miyazaki, T. (1987) J . Bacterial. 169,427-429

34. Nakai, C., Kagamiyama, H., Nozaki, M., Nakazawa, T., Inouye, S., Ebina, Y., and Nakazawa, A. (1983) J. Biol. Chern. 258, 2923-2928 M., Gaffney, D. F., Speck, D., Kaufmann, M., 35. M. Zukowski, Findeli, A., Wisecup, A., and Lecocq, J. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 1101-1105 36. Harayama, S.,Rekik, M., Wasserfallen, A., and Bairoch, A. (1987) Mol. Gen. Genet. 210,241-247 37. Ghosal, D., You, I., and Gunsalus, I. C. (1987) Gene (Amst.) 5 5 , 19-28 38.Furukawa, K., and Arimura, N. (1987) J. Bacterid. 169, 924927

SUPPLEMENTARY VATERIAL TO TOLUENE OEGRADATION BY PSEUWIIONAS PU7IDA F I : NUCLEOTIDE SEQUENCE OF THE todClCZ8UIE GENES AND THEIR EXPRESSION I N E. COLI Gerben J . I y l l t r a and David 1. Gibson EXPERIMENTAL PROCEDURES B a c t e r i 8 1s t r a i nosl.a s m i d s . and media. Escherichia c o l i s t r a i n JMlO9 (recAl cndAl gyrA996 t h r hrdRI7 Supf44 r e l A l A(1ac-prsA.9) [F' trrD36pmA8IrcIqZ6#15l, 1 3 ) was u t i l i z e d as fw a11 recombinant plarmidr. Pseudoionas putTda F1 ( 1 4 ) was t h e source otfh e 10.7 the host k i l o b a s ep a i r EcoRI fragmentcloned i n plasmid pOTG351 (12).Thisplasmidcontainsthe genes f o rt o l u e n ed i o x y g e n a r e( t o d C l C z 8 A ) ,c i s - t o l u e n ed i h y d r o d i o ld e h y d r o g e n a s e( t o d D ) , and 3a e t h y l c a t e c h a 2l . 3 - d i o x y g e m r e( t o d f ) .P l a m l d pGEM3l (Promega, Madisan, VI) was used t o C o n s t r u c tt h es u b c l o n e sn e c e s s a r yf o r DNA sequencing & I d e s c r i b e d i n t h eR e s u l t ss e c t i o n . was used t o Plasmid pKK223-3 ( P h a m a c i aP. l r c a t a w a y . NJ) containing t h et a cp r o m o t e r COnstwCtstrainsthatoverproducedthe todCJC28A. todD. and t o d f gene p r o d u c t sr e s p e c t i v e l y . ( 1 5 ) . This medium was Mlneral Salts basal medium (MSB) was prepared as d e s c r i b epdr e v i o u s l y supplemented w i t h 20 n*l glucose and 1 mn t h i a m i nfeot hrger o w tohf E . c o l i JM109. Toluene, was added i n t h ev a p o r phase. L b r o t h( t r y p t o n e , when needed f o m r e t a b o l i cs t u d i e s . l O g / l i t e r ; y e a s t extract, 5 q / l i t w ; and NaCl. S q / l i t e r l rupplementedwith0.1% qlucore served as complete medium S o l l d medium contained 2X-agar. k m p l c i l l i n , when n e e d e d t as e l e cfto r t h ep r e s e n c eo pf l a s m i d s . w a s added a t a c o n c e n t r a t i o no f 100 pg/ml. E . c o l i JMlOg was c u l t u r e d a t 37'C except where i n d i c a t e d a t h e r u l r e . QilA Techniauer.Plasmids w e ~ et r a n r f o m e di n t o E. coli JM109 by thecalciumchlorideDNA was routinely prepapedby an a l k a l i n e - s o d i u m t h y m i d i n e - g l y c e r o l (CTG) t e c h n l q u e( 1 6 ) . d o d e c y sl u l f a t ep r o c e d u r e (17. 18).Plasmids needed f o Pd e l e t i o nc o n l t r U C t i o n or f O l DNA sequencing were f u r t h e rp u r i f i e db yc e n t r i f u g a t i o n In a cesium chloride-ethidium bromide rd4 T r i s . 1 mM EDTA [pH 8.01) I t -20%. w a s s t o r e d i n TE b u f f e r ( I O d e n s i t y g r a d l e n t . DNA cleavage b y w s t r i c t i o n enzymes and l i g a t i o n w i t h 14 l i g a s e were performed 11 recamendedby t h es u p p l i e r( B e t h e r d a Research L a b o r a t o r i e s , I n c . . G n t h e r r b u r g . MD O r U. 5. B1Ochemicils. Cleveland, OH). R e s t r i c t i o nf r a g m e n t s were analyzed by 1.0% agarore gel e l e c t r o p h o r e s i si n H T T ~ P .0.089 M b o r i c x l d . 0.002 M Na2EDTA). P l a s m i dd e l e t i o n s were TBE b u f f e r( 0 . 1 c o n s t r u c t e d usxng exanucleareI11 and 8 1 nuclease (Promega,Madison, V I ) a s d e s c r i b e db y Henlkaff 119). Plasmids were sequenced by t h ed i d e o txe c h n l q u e (20) urlng modified 17 8 . Biechernicalr. Cleveland. OH) and .?.K2P dATP a s d e r c r l b e d Tabor byand polymerare Rlchardron ( 2 1 ) . The r e s u l t i n go l l g o n u c l e o t i d e r were subjected to e l e c t r o p h o r e s i s on 7.0 M " m a . 62 p o l y x r y l a m d eg e l s( 1 9 : la c r y l a m i d et ob i r a c r y l a m i d er a t i o )i n TBE b u f f e r . Wedge g e l s ( 0 . 2 mm t o 0.5 m t h i c k ) were run a t I500 v o l t s at a constant temperatureof 7OoC t o e l i m i n a t e secondary structure a s described by G a r o f f and Ansorge ( 2 2 ) . A f t e re l e c t r o p h o r e s i s t h eg e l s ,p r e v i o u s l y bound t ot h eg l a s sp l a t e rw i t h ~-(~eth~cryl~xy)propyltrilnethax~rIlane, were f i x e d i n 10% a c e t i c a c i d f o r 15 m i n m d washed i n d i s t i l l e d water f o r 15 min. Gels were d r l e d at 8o°C and exposed t o X - r a y film f o r 6 to 2 4 h.


J n d u c t i o n / E x o r e r r r o n S t u d r e r . E. c o l i JM109 s t r a i n sc o n t a i n i n gP p F I ONA c l o n e d downstvem from thetacoromoter ~n oKK223-3 flee R e r u l t r.l were .9rom to s t a t i o n a r y Phase on HSB g l u c o s es u p p l e m e n t e dw i t ht h l m i n e and m p i d l l ? n . C e l l s were s u b c u l t u r e di n t of r e s h medium (2% inoculum) and growthmanrtared bymeasuringthe t u r b i d i t y o f t h e culture at 600 nn. Yhen t h et u r b i d i t yr e a c h e d a v a l u eo f 0.7, i r o p r o p y l - 8 - 0 - t h l e g l l l c t o p y r l n o r l d e (IPTG) w a s added to a f l n a lc o n c e n t r a t , o no f1 M 4 and r n c u b a t l o n w a s c o n t i n u e df o rt h r e eh o u r s . C e l l s were h a r v e s t e d b y c e n t r i f u g a t i o n at 10,000 g (4'C) f o r 10 n i n . . washed w r t h In egual volume O f 50 mM Na/KP04 b u f f e r . pH 7.25, and rerurpended i n 0.5 volume o f t h e same b u f f e r supplemented w i t h 20 mM glucose Toluene was i n t w d u c eitdnhvea p w phase and t hcee l l s were i n c u b a t e dw i t hi l e l a t l o n at 30OC. Accumulat?on o f m t a b o l i t e s i n t h e c u l t u r e medium was p e r l o d ! c a lnl yo n l t a r ebtday k r n q a 1 m l sample, r e m o v i nt h gcee l l s by centrifugation at 13,000 9, and recording tahbes o r p t isopne ctcthorlefeasaurp e r n a t asnotl u t i o n . Absorbance exonuclease s p e c t ro cauf l t u rseu p e r n a t a n I ot l u t l o n s were o b t a i n ewdi t h an h i n c o DM-2 rpectrophotoineter.

A fctuelrt hu24 er e h was e x t r a c t e d t w c equal e with volumes Of acetate. ethyl The organic e x t r a c t s were d r i e d over m h y d r e u rr o d l u ns u l f a t e and evaporated to drynessunder vacuum at 30°C. Each r e s i d u e was r e d i l w l v e idn acetone f oa rn a l y P i s by t h il na y e r Chromatography (TLC) w i t hc h 1 o r o f o r n : r c e t o n e ( 4 : l ) I I t h eI O l Y e n t M . etabolites were a110 separated and i d e n t i f i e bd y 911 Chromatography fallowed by mass r p e c t r o r c o p y (GC/MS) u s i n g 1 F i n n i g a n MA14000 gar chromatograph-marsspectrometer. Sodium d o d e c y ls u l f a t ep o l y a c r y l a m i d eg e le l e c t r o p h o r e r l r (808-PAGE) slabgelsof whale c e l le x t r a c t s were prepared by the method o f Laenrnli ( 2 3 ) . Gels (1.0 m t h i c k ) were run at 16 mA throughthe stacking g e l (4%) and a t 24 4.m throughtheseparatinggel (12%) u n t i lt h e t r a c k l n g dyereachedthebottom O f t h eg e P l roteins were v i s u a l i z e d by r t a l n i n gw i t h 0.1% CaomarrieBlue R-250 i n 40% methanol 10% acetic a c > d s o l u t i o n S . t a n d a r dp r o t e i n s (Sigma Chemcal t o . , St. L o u i s , no) used f omr o l e c u l a r weight d e t e r m i n a t r o n r were o - l a c t a l b u m i n (14,000). soybean t r y p s ilnn h l b , t o(r2 0 , 1 0 0 ) , trypsinogen (24.000), c a r b o macn h y d r a s e (29,000). glyceraldehyde-)-phosphate dehydrogenase (36.000). egg albumin (45,0001,and bovine albumin (66.000).





f i g u r e 2: Sequencing Strategy A. R e s t r i c t i o n map o f pOTG351 showing t h e l o c a t i o n s o f t h e todCICZBME genes t 1 2 ) . B R e s t r i c t i o n maps tohpf el a s m i d s used it nhceo n s t r u c t i o n Of plasmid deletions. The ~ 1 0 i n~d i1c a t e t h e d i r e c t i o n ofexanucleare 111 d i g e s t i o n .


Strategy used i n sequencing the tod genes. Each arrow represents I d i f f e r e n t I l l - g e n e r a t celdo tnhea t was sequenced. Abbreviationr: 8 , BanHI: Bg. 89111; E . fcoRI; H, H l n d l l l : X , K p n I ; N. X o t l i P. PvuIl; S. 2.71; X, Xhol.

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