Enzymes of naphthalene metabolism by <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> 26K Strain

The ability of Pseudomonas fluorescens 26K strain to utilize naphthalene at concentrations up to 600 mg/liter as the sole source of carbon and energy in mineral liquid media was shown. Using HPLC, TLC, and mass-spectrometry, the intermediates of naphthalene transformation by this strain were identified as naphthalene cis-1,2-dihydrodiol, salicylaldehyde, salicylate, catechol, 2-hydroxymuconic semialdehyde, and 1-naphthol. Catechol 2,3-dioxygenase (a homotetramer with native molecular mass 125 kDa) and NAD⁺-dependent homohexameric naphthalene cis-1,2-dihydrodiol dehydrogenase with native molecular mass 160 kDa were purified from crude extract of the strain and characterized. NAD⁺-dependent homodimeric salicylaldehyde dehydrogenase with molecular mass 110 kDa was purified and characterized for the first time. Based on the data, a pathway of naphthalene degradation by P. fluorescens 26K is suggested.     

Conversion of cis‐2‐carboxycyclohexylacetyl‐CoA in the downstream pathway of anaerobic naphthalene degradation

The cyclohexane derivative cis‐2‐(carboxymethyl)cyclohexane‐1‐carboxylic acid [(1R,2R)‐/(1S,2S)‐2‐(carboxymethyl)cyclohexane‐1‐carboxylic acid] has previously been identified as metabolite in the pathway of anaerobic degradation of naphthalene by sulfate‐reducing bacteria. We tested the corresponding CoA esters of isomers and analogues of this compound for conversion in cell free extracts of the anaerobic naphthalene degraders Desulfobacterium strain N47 and Deltaproteobacterium strain NaphS2. Conversion was only observed for the cis‐isomer, verifying that this is a true intermediate and not a dead‐end product. Mass‐spectrometric analyses confirmed that conversion is performed by an acyl‐CoA dehydrogenase and a subsequent hydratase yielding an intermediate with a tertiary hydroxyl‐group. We propose that a novel kind of ring‐opening lyase is involved in the further catabolic pathway proceeding via pimeloyl‐CoA. In contrast to degradation pathways of monocyclic aromatic compounds where ring‐cleavage is achieved via hydratases, this lyase might represent a new ring‐opening strategy for the degradation of polycyclic compounds. Conversion of the potential downstream metabolites pimeloyl‐CoA and glutaryl‐CoA was proved in cell free extracts, yielding 2,3‐dehydropimeloyl‐CoA, 3‐hydroxypimeloyl‐CoA, 3‐oxopimeloyl‐CoA, glutaconyl‐CoA, crotonyl‐CoA, 3‐hydroxybutyryl‐CoA and acetyl‐CoA as observable intermediates. This indicates a link to central metabolism via β‐oxidation, a non‐decarboxylating glutaryl‐CoA dehydrogenase and a subsequent glutaconyl‐CoA decarboxylase.     

Biodegradation of phenanthrene by<Emphasis Type="Italic"> Pseudomonas</Emphasis> sp. strain PP2: novel metabolic pathway,role of biosurfactant and cell surface hydrophobicity in hydrocarbon assimilation

Pseudomonas sp. strain PP2 isolated in our laboratory efficiently metabolizes phenanthrene at 0.3% concentration as the sole source of carbon and energy. The metabolic pathways for the degradation of phenanthrene, benzoate and p-hydroxybenzoate were elucidated by identifying metabolites, biotransformation studies, oxygen uptake by whole cells on probable metabolic intermediates, and monitoring enzyme activities in cell-free extracts. The results obtained suggest that phenanthrene degradation is initiated by double hydroxylation resulting in the formation of 3,4-dihydroxyphenanthrene. The diol was finally oxidized to 2-hydroxymuconic semialdehyde. Detection of 1-hydroxy-2-naphthoic acid, alpha-naphthol, 1,2-dihydroxy naphthalene, and salicylate in the spent medium by thin layer chromatography; the presence of 1,2-dihydroxynaphthalene dioxygenase, salicylaldehyde dehydrogenase and catechol-2,3-dioxygenase activity in the extract; O(2) uptake by cells on alpha-naphthol, 1,2-dihydroxynaphthalene, salicylaldehyde, salicylate and catechol; and no O(2) uptake on o-phthalate and 3,4-dihydroxybenzoate supports the novel route of metabolism of phenanthrene via 1-hydroxy-2-naphthoic acid --> [alpha-naphthol] --> 1,2-dihydroxy naphthalene --> salicylate --> catechol. The strain degrades benzoate via catechol and cis,cis-muconic acid, and p-hydroxybenzoate via 3,4-dihydroxybenzoate and 3-carboxy- cis,cis-muconic acid. Interestingly, the culture failed to grow on naphthalene. When grown on either hydrocarbon or dextrose, the culture showed good extracellular biosurfactant production. Growth-dependent changes in the cell surface hydrophobicity, and emulsification activity experiments suggest that: (1) production of biosurfactant was constitutive and growth-associated, (2) production was higher when cells were grown on phenanthrene as compared to dextrose and benzoate, (3) hydrocarbon-grown cells were more hydrophobic and showed higher affinity towards both aromatic and aliphatic hydrocarbons compared to dextrose-grown cells, and (4) mid-log-phase cells were significantly (2-fold) more hydrophobic than stationary phase cells. Based on these results, we hypothesize that growth-associated extracellular biosurfactant production and modulation of cell surface hydrophobicity plays an important role in hydrocarbon assimilation/uptake in Pseudomonas sp. strain PP2.     

Naphthalene degradation via salicylate and gentisate by Rhodococcus sp. strain B4.

Rhodococcus sp. strain B4, isolated from a soil sample contaminated with polycyclic aromatic hydrocarbons, grows with naphthalene as the sole source of carbon and energy. Salicylate and gentisate were identified as intermediates in the catabolism of naphthalene. In contrast to the well-studied catabolic pathway encoded by the NAH7 plasmid of Pseudomonas putida, salicylate does not induce the genes of the naphthalene-degradative pathway in Rhodococcus sp. strain B4. The key enzymes of naphthalene degradation in Rhodococcus sp. strain B4 have unusual cofactor requirements. The 1,2-dihydroxynaphthalene oxygenase activity depends on NADH and the salicylate 5-hydroxylase requires NADPH, ATP, and coenzyme A.     

Degradation of Anthracene by Mycobacterium sp. Strain LB501T Proceeds via a Novel Pathway, through o-Phthalic Acid

Mycobacterium sp. strain LB501T utilizes anthracene as a sole carbon and energy source. We analyzed cultures of the wild-type strain and of UV-generated mutants impaired in anthracene utilization for metabolites to determine the anthracene degradation pathway. Identification of metabolites by comparison with authentic standards and transient accumulation of o-phthalic acid by the wild-type strain during growth on anthracene suggest a pathway through o-phthalic acid and protocatechuic acid. As the only productive degradation pathway known so far for anthracene proceeds through 2,3-dihydroxynaphthalene and the naphthalene degradation pathway to form salicylate, this indicates the existence of a novel anthracene catabolic pathway in Mycobacterium sp. LB501T.     

Structural and Functional Variation in Genetic Systems for Catabolism of Polycyclic Aromatic Hydrocarbons in Pseudomonas putida Strains

The genetic control of naphthalene, phenanthrene, and anthracene biodegradation was studied in three Pseudomonas putida strains isolated from coal tar- and oil-contaminated soils. These strains isolated from different geographical locations contained similar catabolic plasmids controlling the first steps of naphthalene conversion to salicylate (the nah1operon), functionally inoperative salicylate hydroxylase genes, and genes of the metha-pathway of catechol degradation (the nah2 operon). Salicylate oxidation in these strains is determined by genes located in trans-position relative to the nah1 operon: in strains BS202 and BS3701, they are located on the chromosome, and in the strain BS3790, on the second plasmid.     

Salicylate degradation by a cold-adapted <Emphasis Type="Italic">Pseudomonas</Emphasis> sp.

Pseudomonas sp. strain MC1 was characterized as a cold-adapted, naphthalene-degrading bacterium that is able to grow in a broad temperature range of 5–30°C. MC1 harbors a catabolic plasmid, designated pYIC1, which is almost identical to the archetypal NAH7 plasmid from the mesophilic bacterium Pseudomonas putida G7. On pYIC1, the catabolic genes for naphthalene degradation are clustered in two operons: nahAa-Ab-Ac-Ad-B-F-C-Q-E-D encoding the conversion of naphthalene to salicylate, and nahG-T-H-I-N-L-O-M-K-J encoding the conversion of salicylate through meta-cleavage pathway to pyruvate and acetyl CoA. NahH, the bona fide extradiol dioxygenase in MC1 salicylate metabolism, is thermolabile and is a cold-adapted enzyme. The thermal profiles of the NahH enzyme activities expressed in different hosts indicate the presence of a factor(s) or mechanism(s) to protect the thermolabile NahH enzyme (100% aa identity with MC1 counterpart) in G7. Overall, the results reported in the present work suggest that the thermolabile NahH might be a product of the cold-adaptation process of MC1 and thus contribute to the survival and growth ability of MC1 on salicylate and naphthalene in cold environments.     

Isolation and characterization of naphthalene-catabolic genes and plasmids from oil-contaminated soil by using two cultivation-independent approaches

Two different cultivation-independent approaches were applied to isolate genes for naphthalene dioxygenase (NDO) from oil-contaminated soil in Japan. One approach was the construction of a broad-host-range cosmid-based metagenomic DNA library, and the other was the so-called exogenous plasmid isolation technique. Our screening of NDO genes in both approaches was based on the functional complementation of Pseudomonas putida strains which contained Tn4655K, a transposon carrying the entire set of naphthalene-catabolic (nah) genes but lacking the NDO-encoding gene. We obtained in the former approach a cosmid clone (pSLX928-6) that carried an nah upper pathway operon for conversion of naphthalene to salicylate, and this operon showed a significantly high level of similarity to the corresponding operon on an IncP-9 naphthalene-catabolic plasmid, pDTG1. In the latter approach, the microbial fraction from the soil was mated with a plasmid-free P. putida strain containing a chromosomal copy of Tn4655K, and transconjugants were obtained that received either a 200- or 80-kb plasmid containing all the nah genes for the complete degradation of naphthalene. Subsequent analysis revealed that (1) both plasmids belong to the IncP-9 incompatibility group; (2) their nah upper pathway operons are significantly similar, but not completely identical, to those of pDTG1 and pSLX928-6; and (3) these plasmids carried genes for the salicylate metabolism by the meta-cleavage pathway. A.O. and R.M. contributed equally to this work.     

Metabolic regulation and chromosomal localization of carbaryl degradation pathway in Pseudomonas sp. strains C4, C5 and C6

Pseudomonas sp. strains C4, C5 and C6 degrade carbaryl (1-naphthyl N-methylcarbamate) via 1-naphthol, 1,2-dihydroxynaphthalene, salicylate and gentisate. Carbon source-dependent metabolic studies suggest that enzymes responsible for carbaryl degradation are probably organized into ‘upper’ (carbaryl to salicylate), ‘middle’ (salicylate to gentisate) and ‘lower’ (gentisate to TCA cycle) pathway. Carbaryl and 1-naphthol were found to induce all carbaryl pathway enzymes, while salicylate and gentisate induce middle and lower pathway enzymes. The strains were found to harbor plasmid(s), and carbaryl degradation property was found to be stable. Genes encoding enzymes of the degradative pathway such as 1-naphthol 2-hydroxylase, salicylaldehyde dehydrogenase, salicylate 5-hydroxylase and gentisate 1,2-dioxygenase were amplified from chromosomal DNA of these strains. The gene-specific PCR products were sequenced from strain C6, and phylogenetic tree was constructed. Southern hybridization and PCR analysis using gel eluted DNA as template supported the presence of pathway genes onto the chromosome and not on the plasmid(s).     

Isolation and characterisation of new Planococcus sp. strain able for aromatic hydrocarbons degradation

New Planococcus sp. strain S5 able to grow on salicylate or benzoate as sole carbon source was isolated from activated sludge adapted to sodium salicylate degradation. S5 was determined to be a strictly aerobic, gram-positive, catalase positive, oxidase negative, non-motile, non-spore forming coccus. The strain harboured a plasmid, named pLS5. The S5 strain when grown on salicylate expressed both catechol 1,2-dioxygenase and catechol 2,3-dioxygenase activities and degraded this substrate by both the ortho and meta pathways while grown on benzoate expressed only catechol 1,2-dioxygenase activity. Curing of the plasmid from the strain showed that plasmid pLS5 was involved in salicylate degradation by the meta pathway.     

Functional characterization of a gene cluster involved in gentisate catabolism in Rhodococcus sp. strain NCIMB 12038

Rhodococcus sp. strain NCIMB 12038 utilizes naphthalene as a sole source of carbon and energy, and degrades naphthalene via salicylate and gentisate. To identify the genes involved in this pathway, we cloned and sequenced a 12-kb DNA fragment containing a gentisate catabolic gene cluster. Among the 13 complete open reading frames deduced from this fragment, three (narIKL) have been shown to encode the enzymes involved in the reactions of gentisate catabolism. NarI is gentisate 1,2-dioxygenase which converts gentisate to maleylpyruvate, NarL is a mycothiol-dependent maleylpyruvate isomerase which catalyzes the isomerization of maleylpyruvate to fumarylpyruvate, and NarK is a fumarylpyruvate hydrolase which hydrolyzes fumarylpyruvate to fumarate and pyruvate. The narX gene, which is divergently transcribed with narIKL, has been shown to encode a functional 3-hydroxybenzoate 6-monooxygenase. This led us to discover that this strain is also capable of utilizing 3-hydroxybenzoate as its sole source of carbon and energy. Both NarL and NarX were purified to homogeneity as His-tagged proteins, and they were determined by gel filtration to exist as a trimer and a monomer, respectively. Our study suggested that the gentisate degradation pathway was shared by both naphthalene and 3-hydroxybenzoate catabolism in this strain.     

Phylogenetic analysis of the genes for naphthalene and phenanthrene degradation in Burkholderia sp. strains

The genetic systems responsible for naphthalene and phenanthrene catabolism have been analyzed in the five strains of Burkholderia sp. isolated from soil samples (West Siberia) contaminated by heavy residual fuel and in the laboratory collection strain Burkholderia sp. BS3702 isolated from soil samples of the coke gas plant (Vidnoe, Moscow oblast). The results of this work demonstrate that naphthalene and phenanthrene degradation in the above strains is encoded by the sequences not homologous to the classical nah genes of pseudomonades. In the Burkholderia sp. BS3702 strain, the initial stages of phenanthrene degradation and the subsequent stages of salicylate degradation are controlled by the sequences of different evolutionary descent (phn and nag genes).     

Degradation of anthracene by Mycobacterium sp. strain LB501T proceeds via a novel pathway,through o-phthalic acid

Mycobacterium sp. strain LB501T utilizes anthracene as a sole carbon and energy source. We analyzed cultures of the wild-type strain and of UV-generated mutants impaired in anthracene utilization for metabolites to determine the anthracene degradation pathway. Identification of metabolites by comparison with authentic standards and transient accumulation of o-phthalic acid by the wild-type strain during growth on anthracene suggest a pathway through o-phthalic acid and protocatechuic acid. As the only productive degradation pathway known so far for anthracene proceeds through 2,3-dihydroxynaphthalene and the naphthalene degradation pathway to form salicylate, this indicates the existence of a novel anthracene catabolic pathway in Mycobacterium sp. LB501T.     

An Acidic Polysaccharide in Seeds of Pisum sativum L.

A phenanthrene-assimilating bacterium which belongs to the genus Aeromonas was isolated from soil. The cells which adapted to phenanthrene required a growth lag time on a naphthalene medium. The cells oxidized l-hydroxy-2-naphthoate (1H2NA), 2-carboxybenzaldehyde (2CBAL), o-phthalate (OPA) and protocatechuate (PCA) but did not oxidize salicylaldehyde (SAL), salicylate (SA) and catechol (CAT) which are intermediates in naphthalene catabolism. Using the cell-free extract, the same results were obtained in oxidative capacity. The intact cells metabolized 1H2NA and 2CBAL without the lag time, giving 2CBAL and PCA, respectively. The ammonium sulfate-treated extract prepared from the cells grown in phenanthrene medium, converted 1H2NA to 2CBAL and 2CBAL to OPA. It was suggested that the Aeromonas sp. degraded phenanthrene through OPA.     

Catechol ring-cleavage in Pseudomonas cepacia: the simultaneous induction of ortho and meta pathways

This work demonstrates the ring-cleavage pathways of catechol on Pseudomonas cepacia ATCC 29351, formed upon its growth on salicylate and benzoate, each as a sole carbon source. When grown on salicylate, P. cepacia induces only the catechol ortho pathway by its induction of catechol 1,2-dioxygenase. However, interestingly, benzoate-grown cells induce the ortho and meta pathways for the biodegradation of catechol, by inducing simultaneously catechol 1,2-dioxygenase and 2,3-dioxygenase, respectively, in the ratio of 7:1. The results indicate that P. cepacia ATCC 29351 possesses the genetic capacity for enzymes of both the ortho- and meta-cleavage pathways of benzoate degradation, although the phenotypic expression for the ortho pathway is higher. The simultaneous induction of catechol 1,2- and 2,3-dioxygenase is not detected in salicylate degradation. Although catechol is the metabolic intermediate for both salicylate and benzoate, catechol did not induce either pathway when used as a sole carbon source.     

Metabolism of Naphthalene, 1-Naphthol, Indene, and Indole by Rhodococcus sp. Strain NCIMB 12038

The regulation of naphthalene and 1-naphthol metabolism in a Rhodococcus sp. (NCIMB 12038) has been investigated. The microorganism utilizes separate pathways for the degradation of these compounds, and they are regulated independently. Naphthalene metabolism was inducible, but not by salicylate, and 1-naphthol metabolism, although constitutive, was also repressed during growth on salicylate. The biochemistry of naphthalene degradation in this strain was otherwise identical to that found in Pseudomonas putida, with salicylate as a central metabolite and naphthalene initially being oxidized via a naphthalene dioxygenase enzyme to cis-(1R,2S)-1,2-dihydroxy-1,2-dihydronaphthalene (naphthalene cis-diol). A dioxygenase enzyme was not expressed under growth conditions which facilitate 1-naphthol degradation. However, biotransformations with indene as a substrate suggested that a monooxygenase enzyme may be involved in the degradation of this compound. Indole was transformed to indigo by both naphthalene-grown NCIMB 12038 and by cells grown in the absence of an inducer. Therefore, the presence of a naphthalene dioxygenase enzyme activity was not necessary for this reaction. Thus, the biotransformation of indole to indigo may be facilitated by another type of enzyme (possibly a monooxygenase) in this organism.     

A Gene Cluster Encoding Steps in Conversion of Naphthalene to Gentisate in Pseudomonas sp. Strain U2

Pseudomonas sp. strain U2 was isolated from oil-contaminated soil in Venezuela by selective enrichment on naphthalene as the sole carbon source. The genes for naphthalene dioxygenase were cloned from the plasmid DNA of strain U2 on an 8.3-kb BamHI fragment. The genes for the naphthalene dioxygenase genes nagAa (for ferredoxin reductase), nagAb (for ferredoxin), and nagAc and nagAd (for the large and small subunits of dioxygenase, respectively) were located by Southern hybridizations and by nucleotide sequencing. The genes for nagB (for naphthalene cis-dihydrodiol dehydrogenase) and nagF (for salicylaldehyde dehydrogenase) were inferred from subclones by their biochemical activities. Between nagAa and nagAb were two open reading frames, homologs of which have also been identified in similar locations in two nitrotoluene-using strains (J. V. Parales, A. Kumar, R. E. Parales, and D. T. Gibson, Gene 181:57–61, 1996; W.-C. Suen, B. Haigler, and J. C. Spain, J. Bacteriol. 178:4926–4934, 1996) and a naphthalene-using strain (G. J. Zylstra, E. Kim, and A. K. Goyal, Genet. Eng. 19:257–269, 1997). Recombinant Escherichia coli strains with plasmids carrying this region were able to convert salicylate to gentisate, which was identified by a combination of gas chromatography-mass spectrometry and nuclear magnetic resonance. The first open reading frame, designated nagG, encodes a protein with characteristics of a Rieske-type iron-sulfur center homologous to the large subunits of dihydroxylating dioxygenases, and the second open reading frame, designated nagH, encodes a protein with limited homology to the small subunits of the same dioxygenases. Cloned together in E. coli, nagG, nagH, and nagAb, were able to convert salicylate (2-hydroxybenzoate) into gentisate (2,5-dihydroxybenzoate) and therefore encode a salicylate 5-hydroxylase activity. Single-gene knockouts of nagG, nagH, and nagAb demonstrated their functional roles in the formation of gentisate. It is proposed that NagG and NagH are structural subunits of salicylate 5-hydroxylase linked to an electron transport chain consisting of NagAb and NagAa, although E. coli appears to be able to partially substitute for the latter. This constitutes a novel mechanism for monohydroxylation of the aromatic ring. Salicylate hydroxylase and catechol 2,3-dioxygenase in strain U2 could not be detected either by enzyme assay or by Southern hybridization. However growth on both naphthalene and salicylate caused induction of gentisate 1,2-dioxygenase, confirming this route for salicylate catabolism in strain U2. Sequence comparisons suggest that the novel gene order nagAa-nagG-nagH-nagAb-nagAc-nagAd-nagB-nagF represents the archetype for naphthalene strains which use the gentisate pathway rather than the meta cleavage pathway of catechol.     

trans-o-Hydroxybenzylidenepyruvate Hydratase-Aldolase as a Biocatalyst

The hydratase-aldolase-catalyzed conversion of trans-o-hydroxybenzylidenepyruvate to salicylaldehyde and pyruvate is an intermediate reaction in the conversion of naphthalene to salicylate by bacteria. Here, a variety of aromatic aldehydes and some nonaromatic aldehydes together with pyruvate have been shown to be substrates for aldol condensations catalyzed by this enzyme in extracts of the recombinant strain Escherichia coli JM109(pRE701). Some of the products of these reactions were also compared as substrates in the opposite (hydration-aldol cleavage) reaction.