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.     

Cloning and characterization of a FAD-monooxygenase gene ( cadA) involved in degradation of chloranilic acid (2,5-dichloro-3,6-dihydroxybenzo-1,4-quinone) in Pseudomonas putida TQ07

A bacterium culture was isolated on the basis of its ability to degrade chloranilic acid, and was later identified as Pseudomonas putida (TQ07). Several transposon insertion mutants unable to degrade chloranilic acid were selected. The characterization of the site of insertion of one of these mutants led to the identification of the cadA gene encoding an enzyme with significant homology with FAD-monooxygenases involved in the degradation of aromatic and chloroaromatic compounds. The finding that, after replacing the mutant allele with the wild-type one, the strain recovered the wild-type pattern of "halo" formation (a zone of clearing color on agar plates around TQ07 colonies that degrade chloranilic acid) and degradation of chloranilic acid, unequivocally assigned cadA a function in the metabolism of this compound. We also found that most of the transposon insertion mutants unable to degrade chloranilic acid are clustered in a 10-kb region of the P. putida genome that is encoded in a megaplasmid or in an unstable chromosomal region. Electronic Publication     

Cloning and sequencing of the gene for a Pseudomonas paucimobilis enzyme that cleaves beta-aryl ether.

We isolated Pseudomonas paucimobilis SYK-6, which was able to degrade various dimeric lignin compounds (Y. Katayama, S. Nishikawa, M. Nakamura, K. Yano, M. Yamasaki, N. Morohoshi, and T. Haraguchi, Mokuzai Gakkaishi 33:77-79, 1987). This metabolic process is a distinct characteristic of this bacterium, which is equipped with an enzymatic modification system for various dimeric lignin compounds involved in the tricarboxylic acid cycle. Cleavage of the beta-aryl ether linkage is essential in this process, because this linkage is the most abundant (approximately 50%) in lignin. Here, we report the isolation and characterization of the beta-etherase gene, which contains an open reading frame of 843 bp and which we call ligE. This gene was expressed in Escherichia coli, and the enzyme had the same kinetic properties as the P. paucimobilis SYK-6 enzyme.     

Identification and characterization of genes involved in the downstream degradation pathway of gamma-hexachlorocyclohexane in Sphingomonas paucimobilis UT26

Sphingomonas paucimobilis UT26 utilizes gamma-hexachlorocyclohexane (gamma-HCH) as a sole source of carbon and energy. In our previous study, we cloned and characterized genes that are involved in the conversion of gamma-HCH to maleylacetate (MA) via chlorohydroquinone (CHQ) in UT26. In this study, we identified and characterized an MA reductase gene, designated linF, that is essential for the utilization of gamma-HCH in UT26. A gene named linEb, whose deduced product showed significant identity to LinE (53%), was located close to linF. LinE is a novel type of ring cleavage dioxygenase that catalyzes the conversion of CHQ to MA. LinEb expressed in Escherichia coli transformed CHQ and 2,6-dichlorohydroquinone to MA and 2-chloromaleylacetate, respectively. Our previous and present results indicate that UT26 (i) has two gene clusters for degradation of chlorinated aromatic compounds via hydroquinone-type intermediates and (ii) uses at least parts of both clusters for gamma-HCH utilization.     

Molecular cloning of a Pseudomonas paucimobilis gene encoding a 17-kilodalton polypeptide that eliminates HCl molecules from gamma-hexachlorocyclohexane.

Pseudomonas paucimobilis UT26 is capable of growing on gamma-hexachlorocyclohexane (gamma-HCH). A genomic library of P. paucimobilis UT26 was constructed in Pseudomonas putida by using the broad-host-range cosmid vector pKS13. After 2,300 clones were screened by gas chromatography, 3 clones showing gamma-HCH degradation were detected. A 5-kb fragment from one of the cosmid clones was subcloned into pUC118, and subsequent deletion and gas chromatography-mass spectrometry analyses revealed that a fragment of ca. 500 bp was responsible for the conversion of gamma-HCH to 1,2,4-trichlorobenzene via gamma-pentachlorocyclohexene. Nucleotide sequence analysis revealed an open reading frame (linA) of 465 bp within the fragment. The nucleotide sequence of the linA gene and the deduced amino acid sequence showed no similarity to any known sequences. The product of the linA gene was 16.5 kDa according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Cloning and characterization of plasmid-encoded genes for the degradation of 1,2-dichloro-, 1,4-dichloro-, and 1,2,4-trichlorobenzene of Pseudomonas sp. strain P51.

Pseudomonas sp. strain P51 is able to use 1,2-dichlorobenzene, 1,4-dichlorobenzene, and 1,2,4-trichlorobenzene as sole carbon and energy sources. Two gene clusters involved in the degradation of these compounds were identified on a catabolic plasmid, pP51, with a size of 110 kb by using hybridization. They were further characterized by cloning in Escherichia coli, Pseudomonas putida KT2442, and Alcaligenes eutrophus JMP222. Expression studies in these organisms showed that the upper-pathway genes (tcbA and tcbB) code for the conversion of 1,2-dichlorobenzene and 1,2,4-trichlorobenzene to 3,4-dichlorocatechol and 3,4,6-trichlorocatechol, respectively, by means of a dioxygenase system and a dehydrogenase. The lower-pathway genes have the order tcbC-tcbD-tcbE and encode a catechol 1,2-dioxygenase II, a cycloisomerase II, and a hydrolase II, respectively. The combined action of these enzymes degrades 3,4-dichlorocatechol and 3,4,6-trichlorocatechol to a chloromaleylacetic acid. The release of one chlorine atom from 3,4-dichlorocatechol takes place during lactonization of 2,3-dichloromuconic acid.     

Cloning and sequencing of the genes involved in glyphosate utilization by Pseudomonas pseudomallei.

Thirty-four strains of Pseudomonas pseudomallei isolated from soil were selected for their ability to degrade the phosphonate herbicide glyphosate. All strains tested were able to grow on glyphosate as the only phosphorus source without the addition of aromatic amino acids. One of these strains, P. pseudomallei 22, showed 50% glyphosate degradation in 40 h in glyphosate medium. From a genomic library of this strain constructed in pUC19, we have isolated a plasmid carrying a 3.0-kb DNA fragment which confers to E. coli the ability to use glyphosate as a phosphorus source. This 3.0-kb DNA fragment from P. pseudomallei contained two open reading frames (glpA and glpB) which are involved in glyphosate tolerance and in the modification of glyphosate to a substrate of the Escherichia coli carbon-phosphorus lyase. glpA exhibited significant homology with the E. coli hygromycin phosphotransferase gene. It was also found that the hygromycin phosphotransferase genes from both P. pseudomallei and E. coli confer tolerance to glyphosate.     

Cloning, sequencing, and characterization of a gene cluster involved in EDTA degradation from the bacterium BNC1

EDTA is a chelating agent, widely used in many industries. Because of its ability to mobilize heavy metals and radionuclides, it can be an environmental pollutant. The EDTA monooxygenases that initiate EDTA degradation have been purified and characterized in bacterial strains BNC1 and DSM 9103. However, the genes encoding the enzymes have not been reported. The EDTA monooxygenase gene was cloned by probing a genomic library of strain BNC1 with a probe generated from the N-terminal amino acid sequence of the monooxygenase. Sequencing of the cloned DNA fragment revealed a gene cluster containing eight genes. Two of the genes, emoA and emoB, were expressed in Escherichia coli, and the gene products, EmoA and EmoB, were purified and characterized. Both experimental data and sequence analysis showed that EmoA is a reduced flavin mononucleotide-utilizing monooxygenase and that EmoB is an NADH:flavin mononucleotide oxidoreductase. The two-enzyme system oxidized EDTA to ethylenediaminediacetate (EDDA) and nitrilotriacetate (NTA) to iminodiacetate (IDA) with the production of glyoxylate. The emoA and emoB genes were cotranscribed when BNC1 cells were grown on EDTA. Other genes in the cluster encoded a hypothetical transport system, a putative regulatory protein, and IDA oxidase that oxidizes IDA and EDDA. We concluded that this gene cluster is responsible for the initial steps of EDTA and NTA degradation.     

Cloning and sequencing of the Pseudomonas aeruginosa PAK pilin gene

A 1.2-kilobase (kb) HindIII restriction fragment containing the pilin gene from Pseudomonas aeruginosa PAK has been cloned and sequenced. The pilin protein is 144 amino acids in length with a positively charged leader sequence of 6 amino acids. There is probably only one copy of the gene per chromosome.     

Cloning of a gene cluster encoding biphenyl and chlorobiphenyl degradation in Pseudomonas pseudoalcaligenes.

A gene cluster encoding biphenyl- and chlorobiphenyl-degrading enzymes was cloned from a soil pseudomonad into Pseudomonas aeruginosa PAO1161. Chromosomal DNA from polychlorinated biphenyl-degrading Pseudomonas pseudoalcaligenes KF707 was digested with restriction endonuclease XhoI and cloned into the unique XhoI site of broad-host-range plasmid pKF330. Of 8,000 transformants tested, only 1, containing the chimeric plasmid pMFB1, rendered the host cell able to convert biphenyls and chlorobiphenyls to ring meta cleavage compounds via dihydrodiols and dihydroxy compounds. The chimeric plasmid contained a 7.9-kilobase XhoI insert. Subcloning experiments revealed that the genes bphA (encoding biphenyl dioxygenase), bphB (encoding dihydrodiol dehydrogenase), and bphC (encoding 2,3-dihydroxybiphenyl dioxygenase) were coded for by the 7.9-kilobase fragment. The gene order was bphA-bphB-bphC. The hydrolase activity, which converted the intermediate meta cleavage compounds to the final product, chlorobenzoic acids, and was encoded by a putative bphD gene, was missing from the cloned 7.9-kilobase fragment.     

Cloning and expression in Pseudomonas aeruginosa of a gene involved in the production of alginate.

Pseudomonas aeruginosa strains isolated from patients with cystic fibrosis commonly produce a capsule-like exopolysaccharide called alginate. The alginate-producing (Alg+) phenotype results in a mucoid colony morphology and is an unstable trait. A mutant of P. aeruginosa FRD (a cystic fibrosis isolate) was obtained which was temperature sensitive for alginate production ( Algts ). At elevated growth temperatures (41 degrees C), no alginate was detected in culture supernatants of the Algts mutant, but yields of alginate increased as the temperature of incubation was reduced. The mutation responsible for the Algts phenotype, alg-50(Ts), has been mapped to a region of the FRD chromosome closely linked to trp-2. The alg-50(Ts) marker did not map near the met-l-linked chromosomal mutations responsible for the instability of the Alg+ phenotype. A broad host range cosmid cloning system based upon derivatives of plasmid RK2 was used to construct a P. aeruginosa clone bank. After transfer of the clone bank to the Algts mutant, hybrid plasmids were obtained which complemented the Algts defect. Deletion mapping of the original 20.3 kilobases of P. aeruginosa DNA cloned showed that a 4.7-kilobase fragment would complement the alg-50(Ts) mutation.     

Cloning and sequencing of the serine dehydrogenase gene from Agrobacterium tumefaciens

The structural gene for NADP+-dependent serine dehydrogenase [EC 1.1.1.-] from Agrobacterium tumefaciens ICR 1600 was cloned into Escherichia coli cells and its complete DNA sequence was analyzed. The gene encodes a polypeptide containing 249 amino acid residues. The enzyme had high sequence similarity to short-chain alcohol dehydrogenases from bacteria and unknown proteins of Haemophilus influenzae, Escherichia coli, and Saccharomyces cerevisiae.     

Cloning and characterization of a Pseudomonas aeruginosa gene involved in the negative regulation of phosphate taxis.

Pseudomonas aeruginosa PAO1 exhibited a positive chemotactic response to P(i). The chemotactic response was induced by P(i) limitation. An alkaline phosphatase (AP) constitutive mutant showed a chemotactic response to P(i), regardless of whether the cells were starved for P(i). Sequence analysis and complementation studies showed that the P. aeruginosa phoU gene was involved both in the regulation of AP expression and in the induction of P(i) taxis. However, unlike AP expression, P(i) taxis was not regulated by the phoB gene product.     

Cloning, sequencing and analysis of a gene encoding Escherichia coli proline dehydrogenase

Abstract Using a genomic subtraction technique, we cloned a DNA sequence that is present in wild-type Escherichia coli strain CSH4 but is missing in a presumptive proline dehydrogenase deletion mutant RM2. Experimental evidence indicated that the cloned fragment codes for proline dehydrogenase (EC 1.5.99.8) since RM2 cells transformed with a plasmid containing this sequence was able to survive on minimal medium supplemented with proline as the sole nitrogen and carbon sources. The cloned DNA fragment has an open reading frame of 3942 bp and encodes a protein of 1313 amino acids with a calculated M _r of 143 808. The deduced amino acid sequence of the E. colli proline dehydrogenase has an 84.9% homology to the previously reported Salmonella typhimurium putA gene but it is 111 amino acids longer at the C-terminal than the latter.     

Cloning and sequencing of the gene encoding the 72-kilodalton dehydrogenase subunit of alcohol dehydrogenase from Acetobacter aceti.

A genomic library of Acetobacter aceti DNA was constructed by using a broad-host-range cosmid vector. Complementation of a spontaneous alcohol dehydrogenase-deficient mutant resulted in the isolation of a plasmid designated pAA701. Subcloning and deletion analysis of pAA701 limited the region that complemented the deficiency in alcohol dehydrogenase activity of the mutant. The nucleotide sequence of this region was determined and showed that this region contained the full structural gene for the 72-kilodalton dehydrogenase subunit of the alcohol dehydrogenase enzyme complex. The predicted amino acid sequence of the gene showed homology with sequences of methanol dehydrogenase structural genes of Paracoccus denitrificans and Methylobacterium organophilum.