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

Abstract

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 EcoRl-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.The biodegradation of polycyclic aromatic hydrocarbons (PAHs) has been intensively studied. Many bacterial strains have been shown to degrade naphthalene, fluorene, or phenanthrene, often using these PAHs as sole carbon sources (3, 5). One naphthalene degradation system, NAH7, which was isolated from Pseudomonas putida PpG7, has been well characterized both genetically and biochemically (25). Another system, isolated from P. putida NCIB 9816, has been examined by several investigators (4, 22a).Other systems capable of transforming naphthalene, fluorene, or phenanthrene have also been described. A 9.8-kb DNA fragment was cloned and sequenced from a soil isolate that degrades naphthalene and dibenzothiophene, Pseudomonas sp. strain C18 (8). confusion has arisen in the literature because several variants derived from NCIB 9816 have been used by different investigators (25). These strains vary in their regulation of the naphthalene degradation pathway and in plasmid numbers and plasmid sizes. Yen and Serdar (25) resolved their pedigree and assigned to each of these variants a reference number. Genetic analysis was conducted with NCIB 9816-2 by Connors and Barnsley (6), with NCIB 9816-3 (P. putida PGB1) by Cane and Williams (4), and with NCIB 9816-4 by Serdar (22a). The genes encoding the dioxygenase enzyme complex were cloned and sequenced by Kurkelar and coworkers (15) from an unspecified NCIB 9816 strain and by Simon and coworkers (23) from NCIB 9816-4.Here, we report the preliminary characterization of multiple PAH degradation by a single gene cluster cloned from the...

Recent Advances in Petroleum Microbiology

“…Overall, the broad PAH-degrading capabilities in many strains may be attributed to relaxed initial enzyme specificity for PAHs (low and high molecular weight and methyl substituted), the presence of multiple oxygenases, and the presence of multiple metabolic pathways or multiple genes for isofunctional pathways (83,112,160,249,220,330,396,399,418,437,519,520,532,641,677). Finally, the presence of both alkane and aromatic compound-degrading genes within single strains appears to be common (120,301,576,578,641,662).…”

Formation of indigo and related compounds from indolecarboxylic acids by aromatic acid-degrading bacteria: chromogenic reactions for cloning genes encoding dioxygenases that act on aromatic acids

“…A useful chromogenic property of several of these enzyme systems is the ability to oxygenate the heterocyclic ring of indole, facilitating indigo formation (17). This property has been valuable in the cloning of aromatic hydrocarbon dioxygenase genes (10,11,16,29,30,45) and in the development of processes for the industrial production of indigo (32).Dioxygenase reactions leading to 1,2-diphenol formation and ring cleavage are also employed by bacteria in the catabolism of a number of aromatic carboxylic acids. This is the case for compounds such as benzoic acid (35, 36), phthalic acid (2, 15, 38), phenylpropionic (hydrocinnamic) acid (4, 42), m-and p-toluic acids (1, 43), and p-cumic acid (8).…”

“…A useful chromogenic property of several of these enzyme systems is the ability to oxygenate the heterocyclic ring of indole, facilitating indigo formation (17). This property has been valuable in the cloning of aromatic hydrocarbon dioxygenase genes (10,11,16,29,30,45) and in the development of processes for the industrial production of indigo (32).…”

“…Since the discovery that E. coli carrying cloned naphthalene dioxygenase genes produces the water-insoluble blue dye indigo from indole (17), that transformation has been the basis for the identification of recombinant bacteria carrying not only naphthalene dioxygenase genes (11,29,30,45) but also genes encoding other aromatic hydrocarbon dioxygenases, including toluene dioxygenase (40) and isopropylbenzene dioxygenase (10,16), as well as 2,4-dinitrotoluene dioxygenase (41). The transformation of indole to indigo also has potential for use in the identification of recombinant bacteria carrying xylene monooxygenase genes (26,31).…”

Biochemical and genetic characterization of 2-carboxybenzaldehyde dehydrogenase, an enzyme involved in phenanthrene degradation by Nocardioides sp. strain KP7

“…The enzymatic steps for the decomposition of the first and second rings of phenanthrene in the salicylate route resemble those of the first ring of naphthalene. It has been demonstrated that a common set of enzymes was responsible for the decomposition of the first and second rings of phenanthrene, as well as for the first ring of naphthalene (26,34,36). The PAH-degrading pathway via salicylate found in Burkholderia cepacia F297 seems to exhibit quite broad substrate specificity, as this pathway degrades a wide variety of polycyclic aromatic compounds, including fluorene, (methyl-)naphthalene, phenanthrene, anthracene, and dibenzothiophene (12).…”