Analysis of duplicated gene sequences associated with tfdR and tfdS in Alcaligenes eutrophus JMP134.

Plasmid pJP4 of Alcaligenes eutrophus JMP134 encodes the degradation of 2,4-dichlorophenoxyacetic acid. A 1.2-kb BamHI-XhoI region of the restriction fragment BamHI-E has been proposed to contain the regulatory gene tfdR (A. R. Harker, R. H. Olsen, and R. J. Seidler, J. Bacteriol. 171:314-320, 1989; B. Kaphammer, J. J. Kukor, and R. H. Olsen, J. Bacteriol. 172:2280-2286, 1990). When sequenced and analyzed, the region is shown to contain two incomplete open reading frames (ORFs) positioned divergently. The complete DNA sequence for one of the two ORFs was obtained by sequencing the adjacent restriction fragment BamHI-F. The DNA sequence reveals 100% identify with the regulatory gene tfdS of pJP4. An XbaI-PstI fragment, containing the complete ORF, encodes a 32,000-Da protein which binds to the promoter regions upstream from tfdA and tfdDII. The deduced amino acid sequence of the complete ORF shows similarity with sequences of activator proteins TcbR, CatM, and CatR of the LysR family. The complete ORF represents the regulatory gene tfdR. The deduced amino acid sequence of the incomplete ORF, situated divergently from tfdR, indicates similarity to chloromuconate cycloisomerases produced by genes tfdD and tcbD of plasmids pJP4 and pP51, respectively. This ORF is identified as part of a putative isofunctional gene, tfdDII.     

Nucleotide sequence of the 2,3-dihydroxybiphenyl dioxygenase gene of Pseudomonas pseudoalcaligenes.

2,3-Dihydroxybiphenyl dioxygenase, which catalyzes ring metacleavage of 2,3-dihydroxybiphenyl, is encoded by the bphC gene of Pseudomonas pseudoalcaligenes KF707 (K. Furukawa and T. Miyazaki, J. Bacteriol. 166:392-398, 1986). We determined the nucleotide sequence of a DNA fragment of 2,040 base pairs which included the bphC gene. The fragment included one open reading frame of 912 base pairs to accommodate the enzyme. The predicted processed amino acid sequence of the enzyme subunit consisted of 302 residues, and its 12 NH2-terminal residues were in perfect agreement with those determined for the enzyme. Approximately 10 base pairs upstream from the initiation codon for 2,3-dihydroxybiphenyl dioxygenase, there was a base sequence complementary to the 3' end of the 16S rRNA from Pseudomonas aeruginosa. There was no promoterlike sequence in the region upstream of the bphC gene, but another long open reading frame was present. A putative bphD gene encoding a metacleavage compound-hydrolyzing enzyme was suggested in the region downstream of the bphC gene.     

Toluene degradation by Pseudomonas putida F1. Nucleotide sequence of the todC1C2BADE genes and their expression in Escherichia coli

The nucleotide sequence of the todC1C2BADE genes which encode the first three enzymes in the catabolism of toluene by Pseudomonas putida F1 was determined. The genes encode the three components of the toluene dioxygenase enzyme system: reductaseTOL (todA), ferredoxinTOL (todB), and the two subunits of the terminal dioxygenase (todC1C2); (+)-cis-(1S, 2R)-dihydroxy-3-methylcyclohexa-3,5-diene dehydrogenase (todD); and 3-methylcatechol 2,3-dioxygenase (todE). Knowledge of the nucleotide sequence of the tod genes was used to construct clones of Escherichia coli JM109 that overproduce toluene dioxygenase (JM109(pDT-601]; toluene dioxygenase and (+)-cis-(1S, 2R)-dihydroxy-3-methylcyclohexa-3,5-diene dehydrogenase (JM109(pDTG602]; and toluene dioxygenase, (+)-cis-(1S, 2R)-dihydroxy-3-methylcyclohexa-3,5-diene dehydrogenase, and 3-methylcatechol 2,3-dioxygenase (JM109(pDTG603]. The overexpression of the tod-C1C2BADE gene products was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The three E. coli JM109 strains harboring the plasmids pDTG601, pDTG602, and pDTG603, after induction with isopropyl-beta-D-thiogalactopyranoside, oxidized toluene to (+)-cis-(1S, 2R)-dihydroxy-3-methylcyclohexa-3,5-diene, 3-methylcatechol, and 2-hydroxy-6-oxo-2,4-heptadienoate, respectively. The tod-C1C2BAD genes show significant homology to the reported nucleotide sequence for benzene dioxygenase and cis-1,2-dihydroxycyclohexa-3,5-diene dehydrogenase from P. putida 136R-3 (Irie, S., Doi, S., Yorifuji, T., Takagi, M., and Yano, K. (1987) J. Bacteriol. 169, 5174-5179). In addition, significant homology was observed between the nucleotide sequences for the todDE genes and the sequences reported for cis-1,2-dihydroxy-6-phenylcyclohexa-3,5-diene dehydrogenase and 2,3-dihydroxybiphenyl-1,2-dioxygenase from Pseudomonas pseudoalcaligenes KF707 (Furukawa, K., Arimura, N., and Miyazaki, T. (1987) J. Bacteriol. 169, 427-429).     

Bacterial aromatic ring-cleavage enzymes are classified into two different gene families

Dioxygenases that catalyze the cleavage of the aromatic ring are classified into two groups according to their mode of ring fission. Substrates of ring-cleavage dioxygenases usually contain hydroxyl groups on adjacent aromatic carbons, and intradiol enzymes cleave the ring between these two hydroxyl groups. Extradiol enzymes in contrast cleave the ring between one hydroxylated carbon and its adjacent nonhydroxylated carbon. In this study, we determined the complete nucleotide sequence of nahC, the structural gene for 1,2-dihydroxynaphthalene dioxygenase encoded in the NAH7 plasmid of Pseudomonas putida. This enzyme is an extradiol ring-cleavage enzyme that cleaves the first ring of 1,2-dihydroxynaphthalene. The amino acid sequence of the dioxygenase deduced from the DNA sequence demonstrated that the molecular weight of the enzyme is 33,882. This result was in agreement with those of maxicell analyses that showed that the nahC product was a 36-kDa protein. Interestingly, the amino acid sequence of 1,2-dihydroxynaphthalene dioxygenase was 50% homologous with that of 2,3-dihydroxybiphenyl dioxygenase, which catalyzes extradiol cleavage of the first ring of 2,3-dihydroxybiphenyl (Furukawa, K., Arimura, N., and Miyazaki, T. (1987) J. Bacteriol. 169, 427-429). The amino acid sequence similarity of 1,2-dihydroxynaphthalene dioxygenase with catechol 2,3-dioxygenase, which is an authentic extradiol dioxygenase, was rather low (16%). However, a statistical analysis by the method of S. B. Needleman and C. D. Wunsch [1970) J. Mol. Biol. 48, 443-453) clearly showed that these two dioxygenases are evolutionarily related. Therefore, these extradiol enzymes are considered as products of the same gene superfamily. From the significant sequence similarity between intradiol enzymes, it has been shown (Neidle, E. L., Harnett, C., Bonitz, S., and Ornston, L. N. (1988) J. Bacteriol. 170, 4874-4880) that intradiol enzymes evolved from a common ancestor. Comparison of the amino acid sequence of extradiol enzymes with those of intradiol dioxygenases did not show any significant global or localized similarity.     

The atzB gene of Pseudomonas sp. strain ADP encodes the second enzyme of a novel atrazine degradation pathway.

We previously reported the isolation of a 21.5-kb genomic DNA fragment from Pseudomonas sp. strain ADP, which contains the atzA gene, encoding the first metabolic step for the degradation of the herbicide atrazine (M. de Souza, L. P. Wackett, K. L. Boundy-Mills, R. T. Mandelbaum, and M. J. Sadowsky, Appl. Environ. Microbiol. 61:3373-3378, 1995). In this study, we show that this fragment also contained the second gene of the atrazine metabolic pathway, atzB. AtzB catalyzed the transformation of hydroxyatrazine to N-isopropylammelide. The product was identified by use of high-performance liquid chromatography, mass spectrometery, and nuclear magnetic resonance spectroscopy. Tn5 mutagenesis of pMD1 was used to determine that atzB was located 8 kb downstream of atzA. Hydroxyatrazine degradation activity was localized to a 4.0-kb ClaI fragment, which was subcloned into the vector pACYC184 to produce plasmid pATZB-2. The DNA sequence of this region was determined and found to contain two large overlapping divergent open reading frames, ORF1 and ORF2. ORF1 was identified as the coding region of atzB by demonstrating that (i) only ORF1 was transcribed in Pseudomonas sp. strain ADP, (ii) a Tn5 insertion in ORF2 did not disrupt function, and (iii) codon usage was consistent with ORF1 being translated. AtzB had 25% amino acid identity with TrzA, a protein that catalyzes a hydrolytic deamination of the s-triazine substrate melamine. The atzA and atzB genes catalyze the first two steps of the metabolic pathway in a bacterium that rapidly metabolizes atrazine to carbon dioxide, ammonia, and chloride.     

Nucleotide sequence of the gene encoding the repressor for the histidine utilization genes of Klebsiella aerogenes.

The hutC gene of Klebsiella aerogenes encodes a repressor that regulates expression of the histidine utilization (hut) operons. The DNA sequence of a region known to contain hutC was determined and shown to contain two long rightward-reading open reading frames (ORFs). One of these ORFs was identified as the 3' portion of the hutG gene. The other ORF was the hutC gene. The repressor predicted from the hutC sequence contained a helix-turn-helix motif strongly similar to that seen in other DNA-binding proteins, such as lac repressor and the catabolite gene activator protein. This motif was located in the N-terminal portion of the protein, and this portion of the protein seemed to be sufficient to allow repression of the hutUH operon but insufficient to allow interaction with the inducer. The presence of a promoterlike sequence and a ribosome-binding site immediately upstream of the hutC gene explained the earlier observation that hutC can be transcribed independently of the other hut operon genes. The predicted amino acid sequence of hut repressor strongly resembled that of the corresponding protein from Pseudomonas putida (S. L. Allison and A. T. Phillips, J. Bacteriol. 172:5470-5476, 1990). An unexpected, leftward-reading ORF extending from about the middle of hutC into the preceding (hutG) gene was also detected. The deduced amino acid sequence of this leftward ORF was quite distinct from that of an unexpected ORF of similar size found immediately downstream of the P. putida hutC gene. The nonstandard codon usage of this leftward ORF and the expression of repressor activity from plasmids with deletions in this region made it unlikely that this ORF was necessary for repressor activity.     

2,4-Dinitrotoluene dioxygenase from Burkholderia sp. strain DNT: similarity to naphthalene dioxygenase.

2,4-Dinitrotoluene (DNT) dioxygenase from Burkholderia sp. strain DNT catalyzes the initial oxidation of DNT to form 4-methyl-5-nitrocatechol (MNC) and nitrite. The displacement of the aromatic nitro group by dioxygenases has only recently been described, and nothing is known about the evolutionary origin of the enzyme systems that catalyze these reactions. We have shown previously that the gene encoding DNT dioxygenase is localized on a degradative plasmid within a 6.8-kb NsiI DNA fragment (W.-C. Suen and J. C. Spain, J. Bacteriol. 175:1831-1837, 1993). We describe here the sequence analysis and the substrate range of the enzyme system encoded by this fragment. Five open reading frames were identified, four of which have a high degree of similarity (59 to 78% identity) to the components of naphthalene dioxygenase (NDO) from Pseudomonas strains. The conserved amino acid residues within NDO that are involved in cofactor binding were also identified in the gene encoding DNT dioxygenase. An Escherichia coli clone that expressed DNT dioxygenase converted DNT to MNC and also converted naphthalene to (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. In contrast, the E. coli clone that expressed NDO did not oxidize DNT. Furthermore, the enzyme systems exhibit similar broad substrate specificities and can oxidize such compounds as indole, indan, indene, phenetole, and acenaphthene. These results suggest that DNT dioxygenase and the NDO enzyme system share a common ancestor.     

Replication origin region of the chromosome of alkaliphilic Bacillus halodurans C-125.

An 18.5-kb DNA fragment containing the oriC region of the chromosome of the alkaliphilic Bacillus halodurans C-125 was obtained by PCR and sequenced. Sixteen open reading frames (ORFs) were identified in this region. A sequencing similarity search using the BSORF database found that ORF1 to 13 all had significant similarities to gene products of Bacillus subtilis. Three other ORFs (ORF14-16) of unknown function were positioned down-stream of gyrB instead of rrnO, which is found in the same region in the case of B. subtilis. The ORF organization from gidA to gyrA was the same as that of B. subtilis. The gene organization and the location of the DnaA-box region were also similar to those of the chromosomes of other bacteria, such as Escherichia coli and Pseudomonas putida. There were two DnaA-box clusters (Box-region C and R) with a consensus sequence TTATCCACA on both sides of the dnaA gene but another DnaA box cluster (Box-region L) which is found in the region between thdF and jag in B. subtilis was not found in the corresponding region in the case of alkaliphilic Bacillus halodurans C-125.     

The osa gene of pSa encodes a 21.1-kilodalton protein that suppresses Agrobacterium tumefaciens oncogenicity.

The incompatibility group W plasmid pSa suppresses Agrobacterium tumefaciens oncogenicity (J. Loper and C. Kado, J. Bacteriol. 139:591-596, 1979). The oncogenic suppressive activity was localized to a 3.1-kb region of pSa by Tn5 mutagenesis and deletion analysis. Within this fragment, a 1.1-kb subclone bearing oncogenic suppressive activity was subjected to further characterization. Nucleotide sequencing of the 1.1-kb fragment revealed a 570-bp open reading frame (ORF1) that has a coding capacity for a protein of 21.1 kDa. Sequencing of flanking regions revealed a second ORF (ORF2) located 3 bp upstream of ORF1, with a coding capacity for a protein of 22.8 kDa. Gene fusions of these ORFs to a T7 phi 10 expression system in Escherichia coli resulted in the synthesis of polypeptides of the predicted sizes. An E. coli promoter consensus sequence was not found in the expected positions in the region preceding ORF1. However, several sequences with similarity to the consensus -10 sequence of the A. tumefaciens vir gene promoters were found upstream of ORF1. Potential translational start signals are upstream of ORF1 and ORF2. These sequences showed no significant similarity at the nucleotide or amino acid levels with those in available data bases. However, the C-terminal portion of the ORF1 protein is rich in hydrophobic residues. Perhaps oncogenicity suppression is effected by an association of this protein with the Agrobacterium membrane such that T-DNA transfer is blocked.     

Phthalate catabolic gene cluster is linked to the angular dioxygenase gene in <Emphasis Type="Italic">Terrabacter</Emphasis> sp. strain DBF63

Phthalate is a metabolic intermediate of the pathway of fluorene (FN) degradation via angular dioxygenation. A gene cluster responsible for the conversion of phthalate to protocatechuate was cloned from the dibenzofuran (DF)- and FN-degrading bacterium Terrabacter sp. strain DBF63 and sequenced. The genes encoding seven catabolic enzymes, oxygenase large subunit of phthalate 3,4-dioxygenase (phtA1), oxygenase small subunit of phthalate 3,4-dioxygenase (phtA2), cis-3,4-dihydroxy-3,4-dihydrophthalate dehydrogenase (phtB), [3Fe-4S] or [4Fe-4S] type of ferredoxin (phtA3), ferredoxin reductase (phtA4), 3,4-dihydroxyphthalate decarboxylase (phtC) and putative regulatory protein (phtR), were found in the upstream region of the angular dioxygenase gene (dbfA1A2), encoded in this order. Escherichia coli carrying phtA1A2BA3A4 genes converted phthalate to 3,4-dihydroxyphthalate, and the 3,4-dihydroxyphthalate decarboxylase activity by E. coli cells carrying phtC was finally detected with the introduction of a Shine-Dalgarno sequence in the upstream region of its initiation codon. Homology analysis on the upstream region of the pht gene cluster revealed that there was an insertion sequence (IS) (ISTesp2; ORF14 and its flanking region), part of which was almost 100% identical to the orf1 and its flanking region adjacent to the extradiol dioxygenase gene ( bphC1) involved in the DF degradation of Terrabacter sp. strain DPO360 [Schmid et al. (1997) J Bacteriol 179:53-62]. This suggests that ISTesp2 plays a role in the metabolism of aromatic compounds in Terrabacter sp. strains DBF63 and DPO360.     

Nucleotide sequence and functional analysis of the meta-cleavage pathway involved in biphenyl and polychlorinated biphenyl degradation in Pseudomonas sp. strain KKS102.

Pseudomonas sp. strain KKS102 is able to degrade biphenyl and polychlorinated biphenyls via the meta-cleavage pathway. We sequenced the upstream region of the bphA1A2A3BCD (open reading frame 1 [ORF1]) A4 and found four ORFs in this region. As the deduced amino acid sequences of the first, second, and third ORFs are homologous to the meta-cleavage enzymes from Pseudomonas sp. strain CF600 (V. Shingler, J. Powlowski, and U. Marklund, J. Bacteriol. 174:711-724, 1992), these ORFs have been named bphE, bphG, and bphF, respectively. The fourth ORF (ORF4) showed homology with ORF3 from Pseudomonas pseudoalcaligenes KF707 (K. Taira, J. Hirose, S. Hayashida, and K. Furukawa, J. Biol. Chem. 267:4844-4853, 1992), whose function is unknown. The functions of meta-cleavage enzymes (BphE, BphG, and BphF) were analyzed by using crude extracts of Escherichia coli which expressed the encoding genes. The results showed that bphE, bphG, and bphF encode 2-hydroxypenta-2,4-dienoate hydratase, acetaldehyde dehydrogenase (acylating), and 4-hydroxy-2-oxovalerate aldolase, respectively. The biphenyl and polychlorinated biphenyl degradation pathway of KKS102 is encoded by 12 genes in the order bphEGF (ORF4)A1A2A3BCD (ORF1)A4. The functions of ORF1 and ORF4 are unknown. The features of this bph gene cluster are discussed.     

Site-specific recombination system encoded by toluene catabolic transposon Tn4651

The 56-kb class II toluene catabolic transposon Tn4651 from Pseudomonas putida plasmid pWW0 is unique in that (i) its efficient resolution requires, in addition to the 0.2-kb resolution (res) site, the two gene products TnpS and TnpT and (ii) the 2.4-kb tnpT-res-tnpS region is 48 kb apart from the tnpA gene (M. Tsuda, K.-I. Minegishi, and T. Iino, J. Bacteriol. 171:1386-1393, 1989). Detailed analysis of the 2.4-kb region revealed that the tnpS and tnpT genes encoding the putative 323- and 332-amino-acid proteins, respectively, were transcribed divergently with an overlapping 59-bp sequence in the 203-bp res site. The motifs (the R-H-R-Y tetrad in domains I and II with proper spacing) commonly conserved in the integrase family of site-specific recombinases were found in TnpS. In contrast, TnpT did not show any significant amino acid sequence homology to the other proteins that are directly or indirectly involved in recombination. Analysis of site-specific recombination under the Escherichia coli recA cells indicated that (i) the site-specific resolution between the two copies of the res site on a single molecule was catalyzed by TnpS, (ii) the functional res site was located within a 95-bp segment, and (iii) TnpT appeared to have the role of enhancing the site-specific resolution. It was also found that TnpS catalyzed the site-specific recombination between the res sites located at two different molecules to form a cointegrate molecule. Site-specific mutagenesis of the conserved tyrosine residue in TnpS led to the loss of both the resolution and the integration activities, indicating that such a residue took part in both types of recombination.     

Gene components responsible for discrete substrate specificity in the metabolism of biphenyl (bph operon) and toluene (tod operon).

bph operons coding for biphenyl-polychlorinated biphenyl degradation in Pseudomonas pseudoalcaligenes KF707 and Pseudomonas putida KF715 and tod operons coding for toluene-benzene metabolism in P. putida F1 are very similar in gene organization as well as size and homology of the corresponding enzymes (G. J. Zylstra and D. T. Gibson, J. Biol. Chem. 264:14940-14946, 1989; K. Taira, J. Hirose, S. Hayashida, and K. Furukawa, J. Biol. Chem. 267:4844-4853, 1992), despite their discrete substrate ranges for metabolism. The gene components responsible for substrate specificity between the bph and tod operons were investigated. The large subunit of the terminal dioxygenase (encoded by bphA1 and todC1) and the ring meta-cleavage compound hydrolase (bphD and todF) were critical for their discrete metabolic specificities, as shown by the following results. (i) Introduction of todC1C2 (coding for the large and small subunits of the terminal dioxygenase in toluene metabolism) or even only todC1 into biphenyl-utilizing P. pseudoalcaligenes KF707 and P. putida KF715 allowed them to grow on toluene-benzene by coupling with the lower benzoate meta-cleavage pathway. Introduction of the bphD gene (coding for 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase) into toluene-utilizing P. putida F1 permitted growth on biphenyl. (ii) With various bph and tod mutant strains, it was shown that enzyme components of ferredoxin (encoded by bphA3 and todB), ferredoxin reductase (bphA4 and todA), and dihydrodiol dehydrogenase (bphB and todD) were complementary with one another. (iii) Escherichia coli cells carrying a hybrid gene cluster of todClbphA2A3A4BC (constructed by replacing bphA1 with todC1) converted toluene to a ring meta-cleavage 2-hydroxy-6-oxo-hepta-2,4-dienoic acid, indicating that TodC1 formed a functional multicomponent dioxygenase associated with BphA2 (a small subunit of the terminal dioxygenase in biphenyl metabolism), BphA3, and BphA4.     

Cloning and sequencing of a dehalogenase gene encoding an enzyme with hydrolase activity involved in the degradation of gamma-hexachlorocyclohexane in Pseudomonas paucimobilis.

In Pseudomonas paucimobilis UT26, gamma-hexachlorocyclohexane (gamma-HCH) is converted by two steps of dehydrochlorination to a chemically unstable intermediate, 1,3,4,6-tetrachloro-1,4-cyclohexadiene (1,4-TCDN), which is then metabolized to 2,5-dichloro-2,5-cyclohexadiene-1,4-diol (2,5-DDOL) by two steps of hydrolytic dehalogenation via the chemically unstable intermediate 2,4,5-trichloro-2,5-cyclohexadiene-1-ol (2,4,5-DNOL). To clone a gene encoding the enzyme responsible for the conversion of the chemically unstable intermediates 1,4-TCDN and 2,4,5-DNOL, a genomic library of P. paucimobilis UT26 was constructed in Pseudomonas putida PpY101LA into which the linA gene had been introduced by Tn5. An 8-kb BglII fragment from one of the cosmid clones, which could convert gamma-HCH to 2,5-DDOL, was subcloned, and subsequent deletion analyses revealed that a ca. 1.1-kb region was responsible for the activity. Nucleotide sequence analysis revealed an open reading frame (designated the linB gene) of 885 bp within the region. The deduced amino acid sequence of LinB showed significant similarity to hydrolytic dehalogenase, DhlA (D. B. Janssen, F. Pries, J. van der Ploeg, B. Kazemier, P. Terpstra, and B. Witholt, J. Bacteriol. 171:6791-6799, 1989). The protein product of the linB gene was 32 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Not only 1-chlorobutane but also 1-chlorodecane (C10) and 2-chlorobutane, which are poor substrates for other dehalogenases, were good substrates for LinB, suggesting that LinB may be a member of haloalkane dehalogenases with broad-range specificity for substrates.     

Analysis of cumene (isopropylbenzene) degradation genes from Pseudomonas fluorescens IP01.

We obtained the DNA fragments encoding 2-hydroxy-6-oxo-7-methylocta-2,4-dienoic acid (HOMODA) hydrolase in the cumene (isopropylbenzene) degrader Pseudomonas fluorescens strain IP01 via PCR using two synthesized oligonucleotides corresponding to the conserved regions within known meta-cleavage compound hydrolases. Following colony hybridization using the amplified DNA as a probe, a 4.5-kb HindIII fragment was isolated from P. fluorescens IP01. After determining the nucleotide sequence of this fragment, three open reading frames (ORF11 [cumH], ORF12 [cumD], and ORF13) were identified. The deduced amino acid sequence of ORF12 showed homology with meta-cleavage compound hydrolases encoded by the tod, dmp, xyl, and bph operons. Although the product of ORF12 was found to exhibit HOMODA and 2-hydroxy-6-oxohepta-2,4-dienoic acid (HOHDA) hydrolase activities, it did not exhibit 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) hydrolase activity. The deduced amino acid sequence of ORF11 showed 40.4% homology with the sequence of todX in Pseudomonas putida F1 (Y. Wang, M. Ralings, D. T. Gibson, D. Labbé, H. Bergeron, R. Brousseau, and P. C. K. Lau, Mol. Gen. Genet. 246:570-579, 1995). The nucleotide sequence of ORF13 and its flanking region showed strong homology (91.0%) with IS52 from Pseudomonas savastanoi (Y. Yamada, P.-D. Lee, and T. Kosuge, Proc. Natl. Acad. Sci. USA 83:8263-8267, 1982). By characterization of cumH and cumD, the entire cum gene cluster from the cumene-degrader P. fluorescens IP01 (cumA1A2A3A4BCEGFHD) has been identified.     

Organization and sequence analysis of the 2,4-dichlorophenol hydroxylase and dichlorocatechol oxidative operons of plasmid pJP4.

Growth of Alcaligenes eutrophus JMP134 on 2,4-dichlorophenoxyacetate requires a 2,4-dichlorphenol hydroxylase encoded by gene tfdB. Catabolism of either 2,4-dichlorophenoxyacetate or 3-chlorobenzoate involves enzymes encoded by the chlorocatechol oxidative operon consisting of tfdCDEF, which converts 3-chloro- and 3,5-dichlorocatechol to maleylacetate and chloromaleylacetate, respectively. Transposon mutagenesis has localized tfdB and tfdCDEF to EcoRI fragment B of plasmid pJP4 (R. H. Don, A. J. Wieghtman, H.-J. Knackmuss, and K. N. Timmis, J. Bacteriol. 161:85-90, 1985). We present the complete nucleotide sequence of tfdB and tfdCDEF contained within a 7,954-base-pair HindIII-SstI fragment from EcoRI fragment B. Sequence and expression analysis of tfdB in Escherichia coli suggested that 2,4-dichlorophenol hydroxylase consists of a single subunit of 65 kilodaltons. The amino acid sequences of proteins encoded by tfdD and tfdE were found to be 63 and 53% identical to those of functionally similar enzymes encoded by clcB and clcD, respectively, from plasmid pAC27 of Pseudomonas putida. P. putida(pAC27) can utilize 3-chlorocatechol but not dichlorinated catechols. A region of DNA adjacent to clcD in pAC27 was found to be 47% identical in amino acid sequence to tfdF, a gene important in catabolizing dichlorocatechols. The region in pAC27 does not appear to encode a protein, suggesting that the absence of a functional trans-chlorodienelactone isomerase may prevent P. putida(pAC27) from utilizing 3,5-dichlorocatechol.