Cloning,nucleotide sequence and characterization of genes encoding naphthalene dioxygenase of Pseudomonas putida strain NCIB9816

We have cloned the naphthalene dioxygenase(ND)-coding genes from Pseudomonas putida strain NCIB9816 based on their ability to convert indole to indigo. The region coding for this enzyme activity was sequenced and three successive open reading frames were found. The corresponding gene products were identified using the T7 polymerase/promoter system. All of them are necessary for the ND activity. A comparison of the ND-coding genes with the ones coding for benzene dioxygenase revealed significant homology which was more pronounced at the nucleotide level than at the amino acid level.     

Toluene and ethylbenzene oxidation by purified naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4.

Purified naphthalene dioxygenase (NDO) from Pseudomonas sp. strain NCIB 9816-4 oxidized toluene to benzyl alcohol and benzaldehyde by reactions involving benzylic monooxygenation and dioxygen-dependent alcohol oxidation, respectively. Xylene and nitrotoluene isomers were also oxidized to substituted benzyl alcohol and benzaldehyde derivatives. NDO oxidized ethylbenzene sequentially through (S)-1-phenethyl alcohol (77% enantiomeric excess) and acetophenone to 2-hydroxyacetophenone. In addition, NDO also oxidized ethylbenzene through styrene to (R)-1-phenyl-1,2-ethanediol (74% enantiomeric excess) by reactions involving desaturation and dihydroxylation, respectively. Isotope experiments with 18O2, H2 18O, and D2O suggest that 1-phenethyl alcohol is oxidized to acetophenone by a minor reaction involving desaturation followed by tautomerization. The major reaction in the conversion of 1-phenethyl alcohol and benzyl alcohol to acetophenone and benzaldehyde, respectively, probably involves monohydroxylation to form a gem-diol intermediate which stereospecifically loses the incoming hydroxyl group to leave the carbonyl product. These results are compared with similar reactions catalyzed by cytochrome P-450.     

Isolation and preliminary characterization of the subunits of the terminal component of naphthalene dioxygenase from Pseudomonas putida NCIB 9816-4.

The terminal oxygenase component (ISPNAP) of naphthalene dioxygenase from Pseudomonas putida NCIB 9816-4 was purified to homogeneity. The protein contained approximately 4 g-atoms each of iron and acid-labile sulfide per mol of ISPNAP, and enzyme activity was stimulated significantly by addition of exogenous iron. The large (alpha) and small (beta) subunits of ISPNAP were isolated by two different procedures. The NH2-terminal amino acid sequences of the alpha and beta subunits were identical to the deduced amino acid sequences reported for the ndoB and ndoC genes from P. putida NCIB 9816 and almost identical to the NH2-terminal amino acid sequences determined for the large and small subunits of ISPNAP from P. putida G7. Gel filtration in the presence of 6 M urea gave an alpha subunit with an absorption maximum at 325 nm and broad absorption between 420 and 450 nm. The alpha subunit contained approximately 2 g-atoms each of iron and acid-labile sulfide per mol of the subunit. The beta subunit did not contain iron or acid-labile sulfide. These results, taken in conjunction with the deduced amino acid sequences of the large subunits from several iron-sulfur oxygenases, indicate that each alpha subunit of ISPNAP contains a Rieske [2Fe-2S] center.     

Plasmids Responsible for Horizontal Transfer of Naphthalene Catabolism Genes between Bacteria at a Coal Tar-Contaminated Site Are Homologous to pDTG1 from Pseudomonas putida NCIB 9816-4

The presence of a highly conserved nahAc allele among phylogenetically diverse bacteria carrying naphthalene-catabolic plasmids provided evidence for in situ horizontal gene transfer at a coal tar-contaminated site (J. B. Herrick, K. G. Stuart-Keil, W. C. Ghiorse, and E. L. Madsen, Appl. Environ. Microbiol. 63:2330–2337, 1997). The objective of the present study was to identify and characterize the different-sized naphthalene-catabolic plasmids in order to determine the probable mechanism of horizontal transfer of the nahAc gene in situ. Filter matings between naphthalene-degrading bacterial isolates and their cured progeny revealed that the naphthalene-catabolic plasmids were self-transmissible. Limited interstrain transfer was also found. Analysis of the restriction fragment length polymorphism (RFLP) patterns indicated that catabolic plasmids from 12 site-derived isolates were closely related to each other and to the naphthalene-catabolic plasmid (pDTG1) of Pseudomonas putida NCIB 9816-4, which was isolated decades ago in Bangor, Wales. The similarity among all site-derived naphthalene-catabolic plasmids and pDTG1 was confirmed by using the entire pDTG1 plasmid as a probe in Southern hybridizations. Two distinct but similar naphthalene-catabolic plasmids were retrieved directly from the microbial community indigenous to the contaminated site in a filter mating by using a cured, rifampin-resistant site-derived isolate as the recipient. RFLP patterns and Southern hybridization showed that both of these newly retrieved plasmids, like the isolate-derived plasmids, were closely related to pDTG1. These data indicate that a pDTG1-like plasmid is the mobile genetic element responsible for transferring naphthalene-catabolic genes among bacteria in situ. The pervasiveness and persistence of this naphthalene-catabolic plasmid suggest that it may have played a role in the adaptation of this microbial community to the coal tar contamination at our study site.     

Conjugal transfer and mobilization capacity of the completely sequenced naphthalene plasmid pNAH20 from multiplasmid strain Pseudomonas fluorescens PC20

The complete 83 042-bp nucleotide sequence of the IncP-9 naphthalene degradation plasmid pNAH20 from Pseudomonas fluorescens PC20 exhibits striking similarity in size and sequence to another naphthalene (NAH) plasmid pDTG1. However, the positions of insertion sequence (IS) elements significantly alter both catabolic and backbone functions provided by the two plasmids. In pDTG1, insertion of a pCAR1 IS Pre1 -like element disrupts expression of the lower naphthalene operon and this strain utilizes the chromosomal pathway for complete naphthalene degradation. In pNAH20, this operon is intact and functional. The transfer frequency of pNAH20 is 100 times higher than that of pDTG1 probably due to insertion of the pCAR1 IS Pre2 -like element into the mpfR gene coding for a putative repressor of the mpf operon responsible for mating pilus formation. We also demonstrate in situ plasmid transfer – we isolated a rhizosphere transconjugant strain of pNAH20, P. fluorescens NS8. The plasmid pNS8, a derivative of pNAH20, lacks the ability to self-transfer as a result of an additional insertion event of IS Pre2 -like element that disrupts the gene coding for VirB2-like major pilus protein MpfA. The characteristics of the strain PC20 and the conjugal transfer/mobilization capacity of pNAH20 (or its backbone) make this strain/plasmid a potentially successful tool for bioremediation applications.     

Stereospecific dihydroxylation of the styrene vinyl group by purified naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4.

Naphthalene dioxygenase (NDO) from Pseudomonas sp. strain NCIB 9816-4 adds both atoms of the dioxygen molecule to styrene to form (R)-l-phenyl-1,2-ethanediol. Product formation is tightly coupled to dioxygen consumption and NADH oxidation. NDO oxidizes styrene-d8 at almost the same initial rate as styrene. The results indicate that dioxygen activation by NDO is different from that by cytochrome P-450 and other monooxygenases, which oxidize styrene to styrene 1,2-oxide.     

Regio- and stereospecific oxidation of fluorene, dibenzofuran, and dibenzothiophene by naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4.

The regio- and stereospecific oxidation of fluorene, dibenzofuran, and dibenzothiophene was examined with mutant and recombinant strains expressing naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4. The initial oxidation products were isolated and identified by gas chromatography-mass spectrometry and nuclear magnetic resonance spectrometry. Salicylate-induced cells of Pseudomonas sp. strain 9816/11 and isopropyl-beta-D-thiogalactopyranoside-induced cells of Escherichia coli JM109(DE3)(pDTG141) oxidized fluorene to (+)-(3S,4R)-cis-3,4-dihydroxy-3,4-dihydrofluorene (80 to 90% relative yield; > 95% enantiomeric excess [ee]) and 9-fluorenol (< 10% yield). The same cells oxidized dibenzofuran to (1R,2S)-cis-1,2-dihydroxy-1, 2-dihydrodibenzofuran (60 to 70% yield; > 95% ee) and (3S,4R)-cis-3, 4-dihydroxy-3,4-dihydrodibenzofuran (30 to 40% yield; > 95% ee). Induced cells of both strains, as well as the purified dioxygenase, also oxidized dibenzothiophene to (+)-(1R,2S)-cis-1,2-dihydroxy-1, 2-dihydrodibenzothiophene (84 to 87% yield; > 95% ee) and dibenzothiophene sulfoxide (< 15% yield). The major reaction catalyzed by naphthalene dioxygenase with each substrate was stereospecific dihydroxylation in which the cis-dihydrodiols were of identical regiochemistry and of R configuration at the benzylic center adjacent to the bridgehead carbon atom. The regiospecific oxidation of dibenzofuran differed from that of the other substrates in that a significant amount of the minor cis-3,4-dihydrodiol regioisomer was formed. The results indicate that although the absolute stereochemistry of the cis-diene diols was the same, the nature of the bridging atom or heteroatom influenced the regiospecificity of the reactions catalyzed by naphthalene dioxygenase.     

Stereospecific oxidation of (R)- and (S)-1-indanol by naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4.

A recombinant Escherichia coli strain which expresses naphthalene dioxygenase (NDO) from Pseudomonas sp. strain NCIB 9816-4 oxidized (S)-1-indanol to trans-(1S,3S)-indan-1,3-diol (95.5%) and (R)-3-hydroxy-1-indanone (4.5%). The same cells oxidized (R)-1-indanol to cis-1,3-indandiol (71%), (R)-3-hydroxy-1-indanone (18.2%), and cis-1,2,3-indantriol (10.8%). Purified NDO oxidized (S)-1-indenol to both syn- and anti-2,3-dihydroxy-1-indanol.     

Prime plasmids derived from the IncP-10 plasmid R91-5 in Pseudomonas putida

Abstract Plasmid primes carrying various fragments of Pseudomonas putida chromosome have been derived from pMO22, a derivative of R91-5 loaded with Tn 501 . These prime plasmids transfer efficiently to P. aeruginosa where they effectively complement various auxotrophic markers. Proof of prime plasmid formation has been provided by the high-frequency transfer of plasmid and chromosomal markers, the unselected cotransfer of either plasmid or chromosomal markers into P. aeruginosa and by transformation of both plasmid and chromosomal markers using prime plasmid DNA. Such prime plasmids have been used to map the location of new markers on the P. putida chromosome.     

Metabolism of naphthalene, fluorene, and phenanthrene: preliminary characterization of a cloned gene cluster from Pseudomonas putida NCIB 9816.

A modified cloning procedure was used to obtain large DNA insertions (20 to 30 kb) from Pseudomonas putida NCIB 9816 that expressed polycyclic aromatic hydrocarbon (PAH) transformation activity in Escherichia coli HB101. Four subclones (16 [in both orientations], 12, and 8.5 kb in size) were constructed from the initial clones. Naphthalene, fluorene, and phenanthrene transformations were investigated in these eight NCIB 9816 clones by a simple agar plate assay method, which was developed to detect and identify potential PAH metabolites. Results indicated that the necessary genes encoding the initial ring fission of the three PAHs in E. coli cells are located in an 8.5-kb EcoRI-XhoI portion, but the lower-pathway genes are not present in a 38-kb neighborhood region. These NCIB 9816 clones could transform naphthalene and phenanthrene to salicylic acid and 1-hydroxy-2-naphthoic acid, respectively. With the same clones, fluorene was degraded to 9-hydroxyfluorene, 9-fluorenone, and two unidentified compounds. Genetic similarity between the NAH7 upper-pathway genes and the cloned NCIB 9816 genes was confirmed by Southern blot DNA-DNA hybridization. In spite of this genetic similarity, the abilities of the two clusters to transform multiple PAHs were different. Under our experimental conditions, only the metabolites from naphthalene transformation by the NAH7 clone (pE317) were detected, whereas the NCIB 9816 clones produced metabolites from all three PAHs.     

Purification and properties of ferredoxinNAP, a component of naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816.

One of the three components of the naphthalene dioxygenase occurring in induced cells of Pseudomonas sp. strain NCIB 9816 has been purified to homogeneity. The protein contained 2 g-atoms each of iron and acid-labile sulfur and had an apparent molecular weight of 13,600. The evidence indicates that it is a ferredoxin-type protein that functions as an intermediate electron transfer protein in naphthalene dioxygenase activity.     

Purification and properties of NADH-ferredoxinNAP reductase, a component of naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816.

Cells of Pseudomonas sp. strain NCIB 9816, after growth with naphthalene or salicylate, contain a multicomponent enzyme system that oxidizes naphthalene to cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. We purified one of these components to homogeneity and found it to be an iron-sulfur flavoprotein that loses the flavin cofactor during purification. Dialysis against flavin adenine dinucleotide (FAD) showed that the enzyme bound 1 mol of FAD per mol of enzyme protein. The enzyme consisted of a single polypeptide with an apparent molecular weight of 36,300. The purified protein contained 1.8 g-atoms of iron and 2.0 g-atoms of acid-labile sulfur and showed absorption maxima at 278, 340, 420, and 460 nm, with a broad shoulder at 540 nm. The purified enzyme catalyzed the reduction of cytochrome c, dichlorophenolindophenol, Nitro Blue Tetrazolium, and ferricyanide. These activities were enhanced in the presence of added FAD. The ability of the enzyme to catalyze the reduction of the ferredoxin involved in naphthalene reduction and other electron acceptors indicates that it functions as an NAD(P)H-oxidoreductase in the naphthalene dioxygenase system. The results suggest that naphthalene dioxygenase requires two proteins with three redox groups to transfer electrons from NADH to the terminal oxygenase.     

Amino acid substitutions in naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4 result in regio- and stereo-specific hydroxylation of flavanone and isoflavanone

Wild-type naphthalene dioxygenase (NDO) from Pseudomonas sp. strain NCIB 9816-4 transforms relatively planar flavone and isoflavone to cis-dihydrodiols. However, this enzyme cannot catalyze the transformation of flavanone and isoflavanone in which a phenyl group bonds to the stereogenic C2 or C3 of the C-ring. Protein modeling suggested that Phe224 in the substrate binding site of NDO may play a key role in substrate specificity toward flavanone and isoflavanone. Site-directed mutants of NDO with substitution of Phe224 with Tyr biotransformed only the (S)-stereoisomers of flavanone and isoflavanone, producing an 8-OH group on the A-ring. In contrast, the Phe224Cys and Phe224Gln substitutions, which used (2S)-flavanone as a substrate, and Phe224Lys, which transformed (2S)-flavanone and (3S)-isoflavanone, each showed lower activity than the Phe224Tyr substitution. The remainder of the tested mutants had no activity with flavanone and isoflavanone. Protein docking studies of flavanone and isoflavanone to the modeled mutant enzyme structures revealed that an expanded substrate binding site, due to mutation at 224, as well as appropriate hydrophobic interaction with the residue at 224, are critical for successful binding of the substrates. Results of this study also suggested that in addition to the previously known Phe352, the Phe224 site of NDO appears to be important site for expanding the substrate range of NDO and bringing regiospecific and stereospecific hydroxylation reactions to C8 of the flavanone and isoflavanone A-rings.     

Isopropylbenzene catabolic pathway in Pseudomonas putida RE204: nucleotide sequence analysis of the ipb operon and neighboring DNA from pRE4

Pseudomonas putida RE204 employs a set of plasmid-specified enzymes in the catabolism of isopropylbenzene (cumene) and related alkylbenzenes. A 21,768 bp segment of the plasmid pRE4, whose sequence is discussed here, includes the ipb (isopropylbenzene catabolic) operon as well as associated genetic elements. The ipb operon, ipbAaAbAcAdBCEGFHD, encodes enzymes catalyzing the conversion of isopropylbenzene to isobutyrate, pyruvate, and acetyl-coenzyme A as well as an outer membrane protein (IpbH) of uncertain function. These gene products are 75 to 91% identical to those encoded by other isopropylbenzene catabolic operons and are somewhat less similar to analogous proteins of related pathways for the catabolism of mono-substituted benzenes. Upstream of ipbAa, ipbR encodes a positive regulatory protein which has about 56% identity to XylS regulatory proteins of TOL (xylene/toluate) catabolic plasmids. This similarity and that of the DNA sequence in the proposed ipb operator-promoter region (ipbOP) to the same region of the xyl meta operon (xylOmPm) suggest that, although the IpbR and XylS regulatory proteins recognize very different inducers, their interactions with DNA to activate gene expression are similar. Upstream of ipbR is an 1196 bp insertion sequence, IS1543, related to IS52 and IS1406. Separating ipbR from ipbAa are 3 additional tightly clustered IS elements. These are IS1544, related to IS1543, IS52, and other members of the IS5 family; IS1545, related to IS1240; and IS1546, related to IS1491. Encompassing the ipb catabolic genes and the other genetic elements and separated from each other by 18,492 bp, are two identical, directly repeated 1007 bp DNA segments. Homologous recombination between these segments appears to be responsible for the occasional deletion of the intervening DNA from pRE4.     

Desaturation, dioxygenation, and monooxygenation reactions catalyzed by naphthalene dioxygenase from Pseudomonas sp. strain 9816-4.

The stereospecific oxidation of indan and indene was examined with mutant and recombinant strains expressing naphthalene dioxygenase of Pseudomonas sp. strain 9816-4. Pseudomonas sp. strain 9816/11 and Escherichia coli JM109(DE3)[pDTG141] oxidized indan to (+)-(1S)-indanol, (+)-cis-(1R,2S)-indandiol, (+)-(1S)-indenol, and 1-indanone. The same strains oxidized indene to (+)-cis-(1R,2S)-indandiol and (+)-(1S)-indenol. Purified naphthalene dioxygenase oxidized indan to the same four products formed by strains 9816/11 and JM109(DE3)[pDTG141]. In addition, indene was identified as an intermediate in indan oxidation. The major products formed from indene by purified naphthalene dioxygenase were (+)-(1S)-indenol and (+)-(1R,2S)-indandiol. The results show that naphthalene dioxygenase catalyzes the enantiospecific monooxygenation of indan to (+)-(1S)-indanol and the desaturation of indan to indene, which then serves as a substrate for the formation of (+)-(1R,2S)-indandiol and (+)-(1S)-indenol. The relationship of the desaturase, monooxygenase, and dioxygenase activities of naphthalene dioxygenase is discussed with reference to reactions catalyzed by toluene dioxygenase, plant desaturases, cytochrome P-450, methane monooxygenase, and other bacterial monooxygenases.     

The effect of transposons on the expression of the naphthalene biodegradation genes in Pseudomonas putida BS202(NPL-1) and derivative strains

NPL-1 and its derivative plasmid pBS106, which control the degradation of naphthalene and salicylate, were found to contain class II transposons of the Tn3 family. These transposons are involved in intraplasmid rearrangements, such as deletions and inversions, and can influence the expression of the catabolic and regulatory genes borne by biodegradation plasmids. The formation of a strong NahR-independent constitutive promoter by the inversion of a DNA fragment may be responsible for changing the character of naphthalene dioxygenase synthesis from inducible (in the case of plasmid NPL-1) to constitutive (in the case of plasmid NPL-41). The stability of plasmids NPL-1 and NPL-41 in Pseudomonas putida strains grown on different substrates depends on the expression of the nah and tnp genes.Translated from Mikrobiologiya, Vol. 74, No. 1, 2005, pp. 79–86.Original Russian Text Copyright © 2005 by Sokolov, Kosheleva, Filonov, Boronin.     

Involvement of the plasmid pPGH1 in the phenol degradation of Pseudomonas putida strain H

Pseudomonas putida strain H (wildtype) was shown to harbour two plasmids with a molecular mass of about 50 kb and 200–220 kb, respectively. Evidence is presented that the larger one, pPGH1, is involved in the phenol degradation via the meta-cleavage pathway.