Cloning of bacterial genes specifying degradation of 4-chlorobiphenyl from Pseudomonas putida OU83.

Genes capable of 4-chlorobiphenyl (4-CBP) degradation were cloned from 4-CBP-degrading Pseudomonas putida OU83 by using a genomic library which was constructed in the broad-host-range cosmid vector pCP13. P. putida AC812 containing chimeric cosmid-expressing enzymes involved in the 4-CBP degradation pathway were identified by detecting 3-phenylcatechol dioxygenase activity (3-PDA). Chimeric cosmid clones pOH83, pOH84, pOH85, pOH87, and pOH88 positive for 3-PDA grew in synthetic basal medium containing 4-CBP (5 mM) as a carbon source. Restriction digestion analysis of recombinant cosmids showed DNA inserts ranging from 6 to 30 kilobase pairs. Southern hybridization data revealed that the cloned DNA inserts originated from strain OU83. Gas chromatography-mass spectrometry analysis of the metabolites of P. putida AC812(pOH88) incubated with 4-CBP and 4'-chloro-3-phenylcatechol showed the formation of 4-chlorobenzoic acid and benzoic acid. These results demonstrate that the cloned DNA fragments contain genes encoding for chlorobiphenyl dioxygenase (cbpA), dihydrodiol dehydrogenase (cbpB), 4'-chloro-3-phenylcatechol dioxygenase (cbpC), a meta-cleavage compound (a chloro derivative of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate) hydrolase (cbpD), and a new dechlorinating activity (dcpE). The location of the cbpC gene specifying 3-PDA was determined by subcloning an EcoRI DNA fragment (9.8 kilobase pairs) of pOH88 in plasmid vector pUC19. The cloned gene encoding 3-PDA was expressed in Escherichia coli HB101 and had substrate specificity only for 3-phenylcatechol and 4'-chloro-3-phenylcatechol.     

Cloning of genes specifying carbohydrate catabolism in Pseudomonas aeruginosa and Pseudomonas putida.

A 6.0-kilobase EcoRI fragment of the Pseudomonas aeruginosa PAO chromosome containing a cluster of genes specifying carbohydrate catabolism was cloned into the multicopy plasmid pRO1769. The vector contains a unique EcoRI site for cloning within a streptomycin resistance determinant and a selectable gene encoding gentamicin resistance. Mutants of P. aeruginosa PAO transformed with the chimeric plasmid pRO1816 regained the ability to grow on glucose, and the following deficiencies in enzyme or transport activities corresponding to the specific mutations were complemented: glcT1, glucose transport and periplasmic glucose-binding protein; glcK1, glucokinase; and edd-1, 6-phosphogluconate dehydratase. Two other carbohydrate catabolic markers that are cotransducible with glcT1 and edd-1 were not complemented by plasmid pRO1816: zwf-1, glucose-6-phosphate dehydrogenase; and eda-9001, 2-keto-3-deoxy-6-phosphogluconate aldolase. However, all five of these normally inducible activities were expressed at markedly elevated basal levels when transformed cells of prototrophic strain PAO1 were grown without carbohydrate inducer. Vector plasmid pRO1769 had no effect on the expression of these activities in transformed mutant or wild-type cells. Thus, the chromosomal insert in pRO1816 contains the edd and glcK structural genes, at least one gene (glcT) that is essential for expression of the glucose active transport system, and other loci that regulate the expression of the five clustered carbohydrate catabolic genes. The insert in pRO1816 also complemented the edd-1 mutation in a glucose-negative Pseudomonas putida mutant but not the eda-1 defect in another mutant. Moreover, pRO1816 caused the expression of high specific activities of glucokinase, an enzyme that is naturally lacking in these strains of Pseudomonas putida.     

Rapid purification of an active recombinant His-tagged 2,3-dihydroxybiphenyl 1,2-dioxygenase from Pseudomonas putida OU83

2,3-Dihydroxybiphenyl 1,2-dioxygenase (2,3-DBPD) is an extradiol-type dioxygenase that catalyzes the aromatic ring fission of 2,3-dihydroxybiphenyl, the third step in the biphenyl degradation pathway. The nucleotide sequence of the Pseudomonas putida OU83 gene bphC, which encodes 2,3-DBPD, was cloned into a plasmid pQE31. The His-tagged 2,3-DBPD produced by a recombinant Escherichia coli strain, SG13009(pREP4)(pAKC1), and purified with a Ni-nitrilotriacetic acid resin affinity column using the His-bind Qiagen system. The His-tagged 2,3-DBPD construction, carrying a single 6×His tail on the N-terminal of the polypeptide, was active. SDS-PAGE analysis of the purified active 2,3-DBPD gave a single band of 34 kDa; this is in agreement with the size of the bphC coding region. The K_m for 2,3-dihydroxybiphenyl was 14.5±2 μM. The enzyme activity was enhanced by ferrous ion but inhibited by ferric ion. The enzyme activity was inhibited by thiol-blocking reagents and heavy metals HgCl₂, CuSO₄, NiSO₄, and CdCl₂. The yield was much higher and the time required to purify recombinant 2,3-DBPD from clone pAKC1 was faster than by the conventional chromatography procedures.     

Supra-operonic clustering of genes specifying glucose dissimilation in Pseudomonas putida.

Summary The linkage arrangements of genes governing glucolysis in Pseudomonas putida have been determined by transductional analysis. Five genes (gdh, kgtA, kgtB, edd and eda), comprising at least three operons, are cotransducible with each other, but not with ggu (glucose and gluconate uptake) nor with genes of a known supra-operonic cluster of genes specifying enzymes of other dissimilatory pathways, nor with a biochemically uncharacterized his marker. It thus appears that P. putida may have more than one chromosomal region in which genes with dissimilatory function are clustered in a supra-operonic fashion.     

Purification of 2,3-dihydroxybiphenyl 1,2-dioxygenase from Pseudomonas putida OU83 and characterization of the gene (bphC).

The 2,3-dihydroxybiphenyl 1,2-dioxygenase (2,3-DBPD) of Pseudomonas putida OU83 was constitutively expressed and purified to apparent homogeneity. The apparent molecular mass of the native enzyme was 256 kDa, and the subunit molecular mass was 32 kDa. The data suggested that 2,3-DBPD was an octamer of identical subunits. The nucleotide sequence of a DNA fragment containing the bphC region was determined. The deduced protein sequence for 2,3-DBPD consisted of 292 amino acid residues, with a calculated molecular mass of 31.9 kDa, which was in agreement with data for the purified 2,3-DBPD. Nucleotide and amino acid sequence analyses of the bphC gene and its product, respectively, revealed that there was a high degree of homology between the OU83 bphC gene and the bphC genes of Pseudomonas cepacia LB400 and Pseudomonas pseudoalcaligenes KF707.     

Cloning of genes degrading 3-chlorobenzoate from Pseudomonas putida strain 87]

The ability of Pseudomonas putida strain 87 to catabolize 3-chlorobenzoate was shown to be mediated by genes of pBS109 plasmid. The plasmid may be transferred by conjugation into P. aeruginosa PAO2175. It seems possible that the pBS109 plasmid codes for pyrocatechase II specific for halogenated catechol, but not catechol. The genes specifying utilization of 3-chlorobenzoate from pBS109 plasmid were cloned in the 5.5 kb BgIII fragment by using broad-host cloning system. The resulting pBS110 plasmid was transferred into P. putida, which results in utilization of 3-chlorobenzoate by transconjugants.     

Cloning and expression of pca genes from Pseudomonas putida in Escherichia coli

Beta-Ketoadipate elicits expression of five structural pca genes encoding enzymes that catalyse consecutive reactions in the utilization of protocatechuate by Pseudomonas putida. Three derivatives of P. putida PRS2000 were obtained, each carrying a single copy of Tn5 DNA inserted into a separate region of the genome and preventing expression of different sets of pca genes. Selection of Tn5 in or near the pca genes in these derivatives was used to clone four structural pca genes and to enable their expression as inserts in pUC19 carried in Escherichia coli. Three of the genes were clustered as components of an apparent operon in the order pcaBDC. This observation indicates that rearrangement of the closely linked genes accompanied divergence of their evolutionary homologues, which are known to appear in the order pcaDBC in the Acinetobacter calcoaceticus pcaEFDBCA gene cluster. Additional evidence for genetic reorganization during evolutionary divergence emerged from the demonstration that the P. putida pcaE gene lies more than 15 kilobase pairs (kbp) away from the pcaBDC operon. An additional P. putida gene, pcaR, was shown to be required for expression of the pca structural genes in response to beta-ketoadipate. The regulatory pcaR gene is located about 15 kbp upstream from the pcaBDC operon.     

Cloning of Pseudomonas sp. strain CBS3 genes specifying dehalogenation of 4-chlorobenzoate.

The degradation of 4-chlorobenzoate (4-CBA) by Pseudomonas sp. strain CBS3 is thought to proceed first by the dehalogenation of 4-CBA to 4-hydroxybenzoate (4-HBA), which is then metabolized following the protocatechuate branch of the beta-ketoadipate pathway. The cloning of the 4-CBA dehalogenation system was carried out by constructing a gene bank of Pseudomonas sp. strain CBS3 in Pseudomonas putida. Hybrid plasmid pPSA843 contains a 9.5-kilobase-pair fragment derived from the chromosome of Pseudomonas sp. strain CBS3. This plasmid confers on P. putida the ability to dehalogenate 4-CBA and grow on 4-CBA as the only source of carbon. However, pPSA843 did not complement mutants of P. putida unable to grow on 4-HBA (POB-), showing that the genes involved in the metabolism of 4-HBA were not cloned. Subcloning of Pseudomonas sp. strain CBS3 genes revealed that most of the insert is required for the dehalogenation of 4-CBA, suggesting that more than one gene product is involved in this dehalogenation.     

Cloning of genes for naphthalene metabolism in Pseudomonas putida.

Plasmid pIG7 DNA cloned in Pseudomonas putida with the broad-host-range vectors pRK290 and pKT240 expresses the genes encoding nephthalene oxidation in the presence of the intermediate substrate, salicylate, or the gratuitous inducer, anthranilate. Two operons, nahAF and nahGK, cloned from the EcoRI fragment A (25 kilobases) are under wild-type regulation by the nahR locus. Deletion plasmids provide a restriction map of both operons. Double transformants containing structural and regulatory cistron nahR in trans are used to demonstrate positive control of expression.     

Cloning and expression of the plasmid-encoded benzene dioxygenase genes from Pseudomonas putida ML2

Three different bld mutants from S. griseus ATCC 10137 were isolated by nitrosoguanidine mutagenesis. They simultaneously lost the capability of antibiotic production and the formation of pigments. The three bld mutants were differently affected by different carbon sources. Two of these mutants showed a high efficiency of transformation with several plasmid vectors, in contrast to the low efficiency of transformation showed by the wild type. We showed that S. griseus ATCC 10137 and the three bld mutants possess an enzymatic activity that protects their DNAs against the digestion by SacI. Antibiotic and pigment production, and low transformability with plasmid DNA were together restored in spontaneous spo+ revertants.     

Cloning, physical mapping and expression of chromosomal genes specifying degradation of the herbicide 2,4,5-T by Pseudomonas cepacia AC1100

A genomic library of total DNA of Pseudomonas cepacia AC1100 was constructed on a broad-host-range cosmid vector pCP13 in Escherichia coli AC80. A 25-kb segment was isolated from the library which complemented a Tn5-generated, 2,4,5-trichlorophenoxyacetic acid-negative (2,4,5-T-) mutant, P. cepacia PT88. This mutation was partially characterized and appeared to be lacking functional enzyme required for metabolism of an intermediate of the 2,4,5-T pathway, recently identified as 5-chloro-1,2,4-trihydroxybenzene [Chapman et al., Abstr. Soc. Environ. Toxicol. Chem. USA 8 (1987) 127]. A simple colorimetric assay was developed to detect the presence of this active enzyme in intact cells and was used to determine the expression of complementing genes. Subcloning experiments showed that a 4-kb BamHI-PstI fragment and a 290-bp PstI-EcoRI fragment, separated by 1.3-kb, were required for complementation. Both fragments are identified to be chromosomal in origin. Hybridization studies using the subcloned fragments revealed that in addition to a Tn5 insertion, mutant PT88 contained an extensive chromosomal deletion accounting for its 2,4,5-T- phenotype. The cloned fragments did not show homology to plasmid DNAs carrying degradative genes for toluene, naphthalene and 3-chlorobenzoate.     

Complete sequence and genetic organization of pDTG1, the 83 kilobase naphthalene degradation plasmid from Pseudomonas putida strain NCIB 9816-4

The complete 83,042 bp sequence of the circular naphthalene degradation plasmid pDTG1 from Pseudomonas putida strain NCIB 9816-4 was determined in order to examine the process by which the nah and sal operons may have been compiled and distributed in nature. Eighty-nine open reading frames were predicted using computer analyses, comprising 80.0% of the pDTG1 DNA sequence. The most distinctive feature of the plasmid is the upper and lower naphthalene degradation operons, which occupy 9.5 kb and 13.4 kb regions, respectively, bordered by numerous defective mobile genetic element fragments. Identified on this plasmid were homologues of genes required for large plasmid replication, maintenance, and conjugation, as well as transposases, resolvases, and integrases, suggesting an evolution that involved the lateral transfer of DNA between bacterial species. Also found were genes that contain a high degree of sequence similarity to other known degradation genes, as well as genes involved in chemotaxis. Although the incompatibility group designation of pDTG1 remains unresolved, striking sequence organization and homology exists between the plasmid backbones of pDTG1 and the IncP-9 toluene-degradation plasmid pWW0, which suggests a divergent evolution from a progenitor plasmid prior to degradative gene incorporation.     

Characterization of genes involved in the initial reactions of 4-chloronitrobenzene degradation in Pseudomonas putida ZWL73

The genes encoding enzymes involved in the initial reactions during degradation of 4-chloronitrobenzene (4CNB) were characterized from the 4CNB utilizer Pseudomonas putida ZWL73, in which a partial reductive pathway was adopted. A DNA fragment containing genes coding for chloronitrobenzene nitroreductase (CnbA) and hydroxylaminobenzene mutase (CnbB) were PCR-amplified and subsequently sequenced. These two genes were actively expressed in Escherichia coli, and recombinant E. coli cells catalyzed the conversion of 4CNB to 2-amino-5-chlorophenol, which is the ring-cleavage substrate in the degradation of 4CNB. Phylogenetic analyses on sequences of chloronitrobenzene nitroreductase and hydroxylaminobenzene mutase revealed that these two enzymes are closely related to the functionally identified nitrobenzene nitroreductase and hydroxylaminobenzene mutase from Pseudomonas strains JS45 and HS12. The nitroreductase from strain ZWL73 showed a higher specific activity toward 4CNB than nitrobenzene (approximately at a ratio of 1.6:1 for the recombinant or 2:1 for the wild type), which is in contrast to the case where the nitroreductase from nitrobenzene utilizers Pseudomonas pseudoalcaligenes JS45 with an apparently lower specific activity against 4CNB than nitrobenzene (0.16:1) [Kadiyala et al. Appl Environ Microbiol 69:6520–6526, 2003]. This suggests that the nitroreductase from 4-chloronitrobenzene utilizer P. putida ZWL73 may have evolved to prefer chloronitrobenzene to nitrobenzene as its substrate.Y.X. and J.-F.W. equally contributed to this work.     

Control of meta-cleavage degradation of 4-hydroxyphenylacetate in Pseudomonas putida.

Synthesis of enzymes of the 4-hydroxyphenylacetate meta-cleavage pathway was studied in Pseudomonas putida wild-type strain P23X1 (NCIB 9865) and mutant strains which had either structural or regulatory gene mutations. Induction studies with mutant strains each defective in an enzyme of the pathway showed that 4-hydroxyphenylacetate induced the hydroxylase and that 3,4-dihydroxyphenylacetate induced the 2,3-oxygenase, aldehyde dehydrogenase, isomerase, decarboxylase, and hydratase. This showed that the hydroxylase structural gene does not exist in an operon that contains any other structural gene of this meta pathway. Studies of mutant strains that synthesized constitutively the 2,3-oxygenase and subsequent enzymes suggested that the regulation of synthesis of these enzymes was coincident, and, in such strains, the hydroxylase was inducible only. Observations made with a putative polarity mutant that lacked 2,3-oxygenase activity suggested that the structural genes encoding this enzyme and subsequent enzymes of the pathway exist in the same operon. Studies of a regulatory mutant strain that was defective in the induction of the 2,3-oxygenase and subsequent enzymes suggest that the 2,3-oxygenase operon is under positive control.     

Molecular cloning of salicylate hydroxylase genes from Pseudomonas cepacia and Pseudomonas putida

The sal gene encoding Pseudomonas cepacia salicylate hydroxylase was cloned and the sal encoding Pseudomonas putida salicylate hydroxylase was subcloned into plasmid vector pRO2317 to generate recombinant plasmids pTK3 and pTK1, respectively. Both cloned genes were expressed in the host Pseudomonas aeruginosa PAO1. The parental strain can utilize catechol, a product of the salicylate hydroxylase-catalyzed reaction, but not salicylate as the sole carbon source for growth due to a natural deficiency of salicylate hydroxylase. The pTK1- or pTK3-transformed P. aeruginosa PAO1, however, can be grown on salicylate as the sole carbon source and exhibited activities for the cloned salicylate hydroxylase in crude cell lysates. In wild-type P. cepacia as well as in pTK1- or pTK3-transformed P. aeruginosa PAO1, the presence of glucose in addition to salicylate in media resulted in lower efficiencies of sal expression P. cepacia apparently can degrade salicylate via the meta cleavage pathway which, unlike the plasmid-encoded pathway in P. putida, appears to be encoded on chromosome. As revealed by DNA cross hybridizations, the P. cepacia hsd and ht genes showed significant homology with the corresponding plasmid-borne genes of P. putida but the P. cepacia sal was not homologous to the P. putida sal. Furthermore, polyclonal antibodies developed against purified P. cepacia salicylate hydroxylase inactivated the cloned P. cepacia salicylate hydroxylase but not the cloned P. putida salicylate hydroxylase in P. aeruginosa PAO1. It appears that P. cepacia and P. putida salicylate hydroxylases, being structurally distinct, were probably derived through convergent evolution.