salicylates and naphthalene

In pseudomonads, naphthalene is catabolized in a series of reactions to salicylic acid, which is further degraded via the catechol meta-cleavage, ortho-cleavage, or gentisic acid pathway to Krebs cycle intermediates. The naphthalene catabolic genes have been located on self-transmissible plasmids, in most cases, and implicated to have chromosomal locations in other cases. The best-studied naphthalene catabolic plasmid is NAH7. It carries two operons, one of which enables the host to utilize naphthalene and the other to utilize salicylate as a carbon and energy source. The product of another NAH7 gene, nahR, is required to turn on both operons in the presence of the inducer, salicylate. Several different naphthalene and salicylate catabolic plasmids have been shown to share sequence homology with NAH7. These plasmids can undergo structural alterations involving insertions and deletions during conjugations and changes in nutritional conditions. Available evidence suggests that salicylate catabolic plasmids can form from the naphthalene catabolic plasmids by structural alterations of the plasmid DNA. The gene organization and regulation, as well as the genetic instability of the naphthalene catabolic plasmids, are reminiscent of the TOL plasmids and suggest that the naphthalene catabolic plasmids and other catabolic plasmids may have evolved in a short period of time by acquiring and modifying preevolved gene clusters from host chromosomes or other plasmids.

Topics: Naphthalenes; Plasmids; Pseudomonas; Salicylates; Salicylic Acid

Topics: Alkanes; Biodegradation, Environmental; Naphthalenes; Polycyclic Aromatic Hydrocarbons; Salicylates

Naphthalene, as a component of crude oil, is a common environmental pollutant. Biochemical and genetic aspects of naphthalene catabolism have been examined in most detail in the bacteria of Pseudomonas genus. In pseudomonads, the key intermediate in naphthalene degradation is salicylate. In this study, we investigated the ability of Rhodococcus opacus strain 3D to utilize naphthalene as a sole carbon and energy source. The characteristic feature of this strain is the inability to grow in the mineral medium supplemented with salicylate (typical intermediate of naphthalene degradation in Gram-negative bacteria). The absence of salicylate hydroxylase activity and salicylate accumulation in the course of R. opacus 3D cultivation in the mineral medium supplemented with naphthalene indicated existence of an alternative pathway of naphthalene oxidation. At the same time, R. opacus 3D was able to use monoaromatic compounds (salts of gentisic, ortho-phthalic, and 2-hydroxycinnamic acids and coumarin) as growth substrates. Based on the analysis of enzymatic activities, identification of the reaction intermediates, genetic determinants, and growth substrates, we concluded that R. opacus 3D carries out naphthalene degradation through an alternative pathway via formation of ortho-phthalic acid, which is untypical for pseudomonads. Using mass spectrometry, we showed for the first time that salicylic acid associate formed in trace amounts in the process of naphthalene degradation is not further metabolized and accumulated in the growth medium in a form of a dimer.

Topics: Carbon; Cinnamates; Dimerization; Mass Spectrometry; Metabolic Networks and Pathways; Mixed Function Oxygenases; Naphthalenes; Phthalic Acids; Pseudomonas; Rhodococcus; Salicylates; Sewage; Wastewater; Water Pollutants, Chemical; Water Pollution; Water Purification

The pollution of aquatic environments by drugs is a problem for which scarce research has been conducted in regards of their removal. Amycolatopsis sp. Poz 14 presents the ability to biotransformation naphthalene at high efficiency, therefore, in this work this bacterium was proposed as an assimilator of naproxen and carbamazepine. Growth curves at different concentrations of naproxen and carbamazepine showed that Amycolatopsis sp. Poz 14 is able to utilize these drugs at a concentration of 50 mg L

Topics: Actinomycetales; Biodegradation, Environmental; Biotransformation; Carbamazepine; Carbon; Catechol 1,2-Dioxygenase; Catechols; Dioxygenases; Environmental Pollution; Gentisates; Hydroxybenzoate Ethers; Kinetics; Metabolic Networks and Pathways; Mixed Function Oxygenases; Naphthalenes; Naproxen; Salicylates

The salophen copper(II) complex was successfully used for the efficient synthesis of new 1,2,3-triazoles based on the naphthalene-1,4-dione scaffold. The reaction of 2-chloro-3-(prop-2-yn-1-yloxy)naphthalene-1,4-dione or 2,3-bis(prop-2-yn-1-yloxy)naphthalene-1,4-dione with aromatic azides in the presence of a low copper catalyst (loading 1 mol-%) afforded 2-chloro-3-[(1-phenyl-1H-1,2,3-triazol-4-yl)methoxy]naphthalene-1,4-dione or 2,3-bis[(1-phenyl-1H-1,2,3-triazol-4-yl)methoxy]naphthalene-1,4-dione, respectively. The advantages of these reactions are short reaction times, high-to-excellent reaction yields, operational simplicity, and mild experimental conditions. The new 1,2,3-triazoles obtained were screened for their in vitro antibacterial activities and were subjected to molecular docking studies.

Topics: Anti-Bacterial Agents; Click Chemistry; Coordination Complexes; Copper; Hydrogen Bonding; Hydrophobic and Hydrophilic Interactions; Microbial Sensitivity Tests; Molecular Docking Simulation; Naphthalenes; Salicylates; Thermodynamics; Triazoles

Recombinant microbial biosensors are known to be simple, cheap, and very efficient monitoring tools for detecting various environmental pollutants in the field. However, although various recombinant microbial biosensors have been developed for aqueous-phase samples, very few are applicable to the gas phase. Here, we report a recombinant Escherichia coli biosensor that can be used to monitor polycyclic aromatic hydrocarbons (PAHs) in both gas and aqueous phases by color development. Among the PAHs, naphthalene and salicylate are often used as model compounds, since they are less toxic than other options and they are widely used in various applications. Here, recombinant E. coli cells carrying nahR (encoding the NahR regulatory protein for naphthalene degradation)::lac Z fusion genes were constructed and suspended (for aqueous measurements) or co-immobilized (for gaseous measurements) with chlorophenol red-ß-D-galactopyranoside (CPRG). Biosensing was then performed by ß-galactosidase, which hydrolyzed CPRG as a substrate, developing detectable red color with the naked eye. The system showed selective responses to salicylate and naphthalene. Importantly, its response to naphthalene was much more sensitive (about 10(5)-fold) in the gas phase compared to the aqueous phase. Thus, this system could potentially be used for the instrument-free, color-change-based monitoring of gaseous pollutants.

Topics: Air Pollutants; Biosensing Techniques; Colorimetry; Escherichia coli; Naphthalenes; Polycyclic Aromatic Hydrocarbons; Recombinant Fusion Proteins; Salicylates; Water Pollutants, Chemical

The Pseudomonas putida АК5 that was isolated from the slime pit of a Nizhnekamsk oil chemical factory can metabolize naphthalene via salicylate and gentisate. Catabolic genes are localized on non-conjugative IncP-7 plasmid pAK5 of about 115 kb in size. The "classical"nah-1 operon and the novel sgp-operon (salicylate-gentisate pathway) are both involved in naphthalene degradation by P. putida АК5, that was first described for Pseudomonas. The sgp-operon includes six open reading frames (ORFs) (sgpAIKGHB). The four ORFs code for the entire salicylate 5-hydroxylase - oxidoreductase component (sgpA), large and small subunits of the oxigenase component (sgpG and sgpH) and 2Fe-2S ferredoxin (sgpB). Genes for gentisate 1, 2-dioxygenase (sgpI) and fumarylpyruvate hydrolase (sgpK) are located in salicylate 5-hydroxylase genes clustering between sgpA and sgpG. The putative positive regulator for the sgp-operon (sgpR) was found upstream of the sgpA gene and oriented in the opposite direction from sgpA. The putative maleylacetoacetate isomerase gene is located apart, directly downstream from the sgp-operon. The sgp-operon organization and phylogenetic analysis of deduced amino acid sequences indicate that this operon has a mosaic structure according to the modular theory of the evolution of modern catabolic pathways.

Topics: Amino Acid Sequence; Bacterial Proteins; cis-trans-Isomerases; Dioxygenases; DNA, Bacterial; Gentisates; Hydrolases; Mixed Function Oxygenases; Molecular Sequence Data; Naphthalenes; Open Reading Frames; Operon; Oxidoreductases; Phylogeny; Pseudomonas putida; Salicylates

The classical salicylaldehyde dehydrogenases found in naphthalene-degrading bacteria are denoted as NahF. In addition to NahF, NahV, and its corresponding gene nahV, were found here in multiple naphthalene-degrading bacteria isolated from industrial wastewater polluted with polycyclic aromatic hydrocarbons (PAHs). In this study, we described for the first time the biological function and regulation model of NahV for the mineralization of naphthalene by P. putida ND6 via the construction of nahF-, nahV- and regulatory gene nahR-deficient strains. The two mutants of salicylaldehyde dehydrogenase genes and wild-type Pseudomonas ND6 were compared with respect to growth rate, naphthalene degradation efficiency, protein expression level, and salicylaldehyde dehydrogenase activity. The data showed that the presence of NahV conferred a physiological advantage on P. putida ND6 for the catabolism of naphthalene in the presence of NahF. NahV could facilitate naphthalene degradation by increasing total salicylaldehyde dehydrogenase activity when both dehydrogenases are present and it could replace the function of NahF when nahF gene is deleted or mutated, thus ensuring mutants could survive in naphthalene-containing environments. To investigate regulation model of NahV, we detected the expression levels and salicylaldehyde dehydrogenase activity in the wild-type and the nahR mutant strains following cultivation in the presence of glucose±salicylate. The data demonstrated that just like the classical salicylaldehyde dehydrogenases, NahF, NahV was induced by salicylate in the presence of NahR.

Topics: Aldehyde Oxidoreductases; Culture Media; Gene Deletion; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Glucose; Industrial Microbiology; Naphthalenes; Pseudomonas putida; Salicylates; Water Microbiology

NahR, a transcriptional regulator for naphthalene degradation in response to salicylate, is a central element in the microbial biosensor for detection of naphthalene and salicylate. To maximize the sensitivity of the biosensor, we have chosen a rational design of highly-sensitive microbial biosensors by introducing site directed mutagenesis to nahR gene. Eight single mutants (N169A, N169C, N169K, N169S, R248H, R248M, R248Q, and R248Y) were made at residues 169 and 248 known as the central inducer-recognition and the C-terminal multimerization domain. The effects of these mutations were examined by monitoring expression of a firefly luciferase (luc) reporter gene under the control of NahR. We found that all mutants at residues 248 and N169C show increased sensitivity (maximum ∼50-fold) compared to wild type, respectively. R248M shows response even at toxic concentration, 5mM. The results show the feasibility and potential versatility of mutational approach for the development of the highly-sensitive microbial biosensors.

Topics: Bacterial Proteins; Biosensing Techniques; Cloning, Molecular; Luciferases, Firefly; Mutagenesis, Site-Directed; Naphthalenes; Recombinant Proteins; Salicylates; Sensitivity and Specificity; Transcription Factors

The physiological role of NahW, the second salicylate hydroxylase of Pseudomonas stutzeri AN10, has been analysed by gene mutation and further complementation. When grown on naphthalene as a unique carbon and energy source, the nahW mutant showed a strong decrease in salicylate hydroxylase activity when compared with the wild-type strain, exhibited lower specific growth rates and accumulated salicylate in culture supernatants. Similarly, lower specific growth rates and salicylate accumulation were observed for the nahW mutant when growth on naphthalene supplemented with succinate or pyruvate. When P. stutzeri AN10 was grown in Luria-Bertani medium in the presence of salicylate, or was cultivated on minimal medium supplemented with salicylate as a unique carbon and energy source, an increase in the lag phase and a decrease in the specific growth rate were observed on increasing the salicylate concentrations, suggesting a plausible toxic effect. This toxic effect of salicylate was much more evident for the nahW mutant than for the wild-type strain. Complementation of the nahW mutant restored all growth parameters. These results indicate that NahW may have two functions in P. stutzeri AN10: (1) to improve its capacity to degrade naphthalene and (2) effectively convert the salicylate produced during naphthalene degradation to tricarboxylic acid cycle intermediates, preventing its toxic effect.

Topics: Gene Knockout Techniques; Genetic Complementation Test; Mixed Function Oxygenases; Naphthalenes; Pseudomonas stutzeri; Pyruvic Acid; Salicylates; Succinic Acid

Although many nitroaromatic compounds have been in nature for only a few decades, bacteria have already evolved the ability to metabolize them. Both horizontal transfer of genes and mutagenesis induced under stressful conditions might facilitate evolution of new catabolic pathways. Nitrotoluene degradation pathways are supposedly derived from an ancestral naphthalene degradation pathway. The 2-nitrotoluene degradation genes in Acidovorax sp. strain JS42 are controlled by the transcriptional activator NtdR, which differs from NagR, the activator of the naphthalene degradation operon in Ralstonia sp. strain U2, by only five amino acids. Both regulators respond to salicylate, an intermediate of naphthalene degradation, but NtdR also recognizes a wide range of nitroaromatic compounds. In this issue of Molecular Microbiology, Ju et al. present results of site-directed mutagenesis of NtdR and NagR and show how the nitrotoluene-responsive regulator NtdR can be generated from a NagR-like ancestor by only a few mutations. The reconstructed hypothetical pathway for the evolution of NtdR from NagR demonstrates stepwise broadening of the effector range of the evolving protein without loss of the original activity. These results provide strong evidence for the idea that promiscuity of proteins is an important step in the evolution of new functions.

Topics: Bacterial Proteins; Biotransformation; Comamonadaceae; Evolution, Molecular; Metabolic Networks and Pathways; Mutagenesis, Site-Directed; Naphthalenes; Ralstonia; Salicylates; Toluene

The NagR protein is a response regulatory protein found in the bacterium Ralstonia sp. U2 that is involved in sensing for salicylic acid and the subsequent induction of the operon just upstream of its gene. The genes encoded for in this operon are involved in the degradation of salicylic acid. Escherichia coli strain RFM443 carrying a fusion of the Photorhabdus luminescens luxCDABE operon with the nagR gene and upstream region of the nagAa gene was constructed and characterized with respect to its optimum temperature, its response time and kinetics, and its ability to detect numerous benzoic acid derivatives. Although capable of detecting 0.5 mM salicylic acid at any temperature between 28 and 40 degrees C, this E. coli strain, labeled DNT5, showed its greatest relative activity at 30 degrees C, i.e., the temperature at which the largest induction was seen. Furthermore, experiments done with numerous benzoic acid derivatives found the NagR protein to be responsive to only a few of the compounds tested, including salicylic acid and 3-methyl salicylic acid, and acetyl salicylic acid was the strongest inducer. The lower limits of detection for these compounds with E. coli strain DNT5 were also established, with the native inducer, salicylic acid, giving the most sensitive response and detectable down to a concentration of about 2 microM. A second lux fusion plasmid was also constructed and transformed into an NahR background, Pseudomonas putida KCTC1768. Within this strain, NAGK-1768, the supplemental activity of the NahR protein on the nagAa promoter, was shown to extend both the range of chemicals detected and the sensitivity.

Topics: Benzene; Benzoic Acid; Biosensing Techniques; Biotechnology; Cloning, Molecular; Escherichia coli; Naphthalenes; Operon; Photorhabdus; Plasmids; Pseudomonas putida; Ralstonia; Recombinant Fusion Proteins; Salicylates; Salicylic Acid; Sensitivity and Specificity; Temperature; Time Factors

Bacterial chemotaxis may have a significant impact on the structure and function of bacterial communities. Quantification of chemotactic motion is necessary to identify chemoeffectors and to determine the bacterial transport parameters used in predictive models of chemotaxis. When the chemotactic bacteria consume the chemoeffector, the chemoeffector gradient to which the bacteria respond may be significantly perturbed by the consumption. Therefore, consumption of the chemoeffector can confound chemotaxis measurements if it is not accounted for. Current methods of quantifying chemotaxis use bacterial concentrations that are too high to preclude chemoeffector consumption or involve ill-defined conditions that make quantifying chemotaxis difficult. We developed a method of quantifying bacterial chemotaxis at low cell concentrations ( approximately 10(5) CFU/ml), so metabolism of the chemoeffector is minimized. The method facilitates quantification of bacterial-transport parameters by providing well-defined boundary conditions and can be used with volatile and semivolatile chemoeffectors.

Topics: Bacteriological Techniques; Chemotaxis; Colony Count, Microbial; Models, Biological; Naphthalenes; Pseudomonas putida; Salicylates

In Ralstonia sp. strain U2, the nag catabolic genes, which encode the enzymes for the pathway that catabolizes naphthalene via the alternative ring cleavage gentisate pathway, are transcribed as an operon under the same promoter. nagR, which encodes a LysR-type transcriptional regulator, is divergently transcribed compared to the nag catabolic genes. A 4-bp frameshift deletion in nagR demonstrated that NagR is required for expression of the nag operon. The transcriptional start of the nag operon was mapped, and a putative -10, -35 sigma(70)-type promoter binding site was identified. Further upstream, a site proximal to the promoter was identified as a site that has bases which have been found to be conserved in the activator-binding motif of other naphthalene pathways. Transcriptional fusion studies demonstrated that NagR regulates the expression of the nag operon positively in the presence of salicylate and to a lesser extent in the presence of 2-nitrobenzoate. Mutation of the LysR-type activator-binding motif in the nag promoter-proximal region resulted in a loss of inducibility of a lacZ reporter gene transcriptionally fused to nagAa, the first gene of the operon. However, other mutations in the region increased the effectiveness of salicylate as an inducer.

Topics: Bacterial Proteins; Base Sequence; Betaproteobacteria; Gene Deletion; Gene Expression Regulation, Bacterial; Molecular Sequence Data; Naphthalenes; Operon; Promoter Regions, Genetic; Salicylates; Transcription Factors; Transcription, Genetic

We report an integrated CMOS microluminometer optimized for the detection of low-level bioluminescence as part of the bioluminescent bioreporter integrated circuit (BBIC). This microluminometer improves on previous devices through careful management of the sub-femtoampere currents, both signal and leakage, that flow in the front-end processing circuitry. In particular, the photodiode is operated with a reverse bias of only a few mV, requiring special attention to the reset circuitry of the current-to-frequency converter (CFC) that forms the front-end circuit. We report a sub-femtoampere leakage current and a minimum detectable signal (MDS) of 0.15 fA (1510 s integration time) using a room temperature 1.47 mm2 CMOS photodiode. This microluminometer can detect luminescence from as few as 5000 fully induced Pseudomonas fluorescens 5RL bacterial cells.

Topics: Biosensing Techniques; Electronics; Environmental Monitoring; Genes, Reporter; Luminescent Measurements; Nanotechnology; Naphthalenes; Pseudomonas fluorescens; Salicylates; Signal Processing, Computer-Assisted

The genes from the oxygenase cluster nagAaGHAbAcAd of naphthalene-degrading Ralstonia sp. strain U2 were cloned and overexpressed. Salicylate 5-hydroxylase (S5H) activity, converting salicylate to gentisate, was present in vitro only in the single extract of cells with overexpressed nagAaGHAb or in a mixture of three cell extracts containing, respectively, NagGH (the oxygenase components), NagAa (ferredoxin reductase), and NagAb (ferredoxin). Each of the three extracts required for S5H activity was rate limiting in the presence of excess of the others but, when in excess, did not affect the rate of catalysis. S5H catalyzed the 5-hydroxylation of the aromatic rings of 3- and 4-substituted salicylates. However, the methyl group of 5-methylsalicylate was hydroxylated to produce the 5-hydroxymethyl derivative and the 6-position on the ring of 5-chlorosalicylate was hydroxylated, producing 5-chloro-2,6-dihydroxybenzoate. In an assay for the nag naphthalene dioxygenase (NDO) based on the indole-linked oxidation of NADH, three extracts were essential for activity (NagAcAd, NagAa, and NagAb). NDO and S5H were assayed in the presence of all possible combinations of the nag proteins and the corresponding nah NDO proteins from the "classical" naphthalene degrader P. putida NCIMB9816. All three oxygenase components functioned with mixed combinations of the electron transport proteins from either strain. The S5H from strain U2 is a unique monooxygenase which shares sequence similarity with dioxygenases such as NDO but is also sufficiently similar in structure to interact with the same electron transport chain and probably does so in vivo during naphthalene catabolism in strain U2.

Topics: Dioxygenases; Electron Transport; Ferredoxin-NADP Reductase; Ferredoxins; Gram-Negative Aerobic Rods and Cocci; Mixed Function Oxygenases; Multienzyme Complexes; Naphthalenes; Oxygenases; Recombinant Proteins; Salicylates

Miscible-displacement experiments were conducted to examine the impact of microbial lag and bacterial cell growth on the transport of salicylate, a model hydrocarbon compound. The impacts of these processes were examined separately, as well as jointly, to determine their relative effects on biodegradation dynamics. For each experiment, a column was packed with porous medium that was first inoculated with bacteria that contained the NAH plasmid encoding genes for the degradation of naphthalene and salicylate, and then subjected to a step input of salicylate solution. The transport behavior of salicylate was non-steady for all cases examined, and was clearly influenced by a delay (lag) in the onset of biodegradation. This microbial lag, which was consistent with the results of batch experiments, is attributed to the induction and synthesis of the enzymes required for biodegradation of salicylate. The effect of microbial lag on salicylate transport was eliminated by exposing the column to two successive pulses of salicylate, thereby allowing the cells to acclimate to the carbon source during the first pulse. Elimination of microbial lag effects allowed the impact of bacterial growth on salicylate transport to be quantified, which was accomplished by determining a cell mass balance. Conversely, the impact of microbial lag was further investigated by performing a similar double-pulse experiment under no-growth conditions. Significant cell elution was observed and quantified for all conditions/systems. The results of these experiments allowed us to differentiate the effects associated with microbial lag and growth, two coupled processes whose impacts on the biodegradation and transport of contaminants can be difficult to distinguish.

Topics: Bacteria; Biodegradation, Environmental; Kinetics; Models, Theoretical; Naphthalenes; Plasmids; Population Dynamics; Porosity; Salicylates; Soil Microbiology; Soil Pollutants; Time Factors; Water Pollutants, Chemical

Pseudomonas stutzeri strain AN10 is a naphthalene-degrading strain whose dissimilatory genes are chromosomally encoded. We sequenced the entire naphthalene-degradation lower pathway of P. stutzeri AN10, this being, together with the upper-pathway reported previously (Bosch R. et al., 1999a. Gene 236, 149-157) the first complete DNA sequence for an entire naphthalene-catabolic pathway. Eleven open reading frames were identified. The nahGTHINLOMKJ genes encode enzymes for the metabolism of salicylate to pyruvate and acetyl-CoA, and nahR encodes the NahR regulatory protein. Our findings suggest that catabolic modules were recruited through transposition events and recombination among tnpA-like genes, and subsequent rearrangements and deletions of non-essential DNA fragments allowed the formation of the actual catabolic pathway. Our results also suggest that the genes encoding the xylene/toluene-degradation enzymes of P. putida mt-2 (pWW0) have coexisted with the nah genes of the P. stutzeri AN10 ancestral genome. This could allow the selection, via recombination events among homologous genes, for a combination of genes enabling the metabolism of a given aromatic compound in the ancestral host strain. Such events accelerate the evolution of modern catabolic pathways and provide new genetic material to the environment, ultimately resulting in improved, natural, bioremediation potential.

Topics: Amino Acid Sequence; Bacterial Proteins; Base Sequence; Chromosome Mapping; Chromosomes, Bacterial; Cloning, Molecular; DNA, Bacterial; Evolution, Molecular; Gene Expression Regulation, Bacterial; Molecular Sequence Data; Naphthalenes; Operon; Promoter Regions, Genetic; Pseudomonas; Salicylates; Sequence Analysis, DNA; Transcription Factors

Two genes, nahG and nahW, encoding two independent salicylate 1-hydroxylases have been identified in the naphthalene-degrading strain Pseudomonas stutzeri AN10. While nahG resides in the same transcriptional unit as the meta-cleavage pathway genes, forming the naphthalene degradation lower pathway, nahW is situated outside but in close proximity to this transcriptional unit. The nahG and nahW genes of P. stutzeri AN10 are induced and expressed upon incubation with salicylate, and the enzymes that are encoded, NahG and NahW, are involved in naphthalene and salicylate metabolism. Both genes, nahG and nahW, have been cloned in Escherichia coli JM109. The overexpression of these genes yields peptides with apparent molecular masses of 46 kDa (NahG) and 43 kDa (NahW), respectively. Both enzymes exhibit broad substrate specificities and metabolize salicylate, methylsalicylates, and chlorosalicylates. However, the relative rates by which the substituted analogs are transformed differ considerably.

Topics: Amino Acid Sequence; Biodegradation, Environmental; Enzyme Induction; Genes, Bacterial; Isoenzymes; Mixed Function Oxygenases; Molecular Sequence Data; Naphthalenes; Pseudomonas; Salicylates; Sequence Homology, Amino Acid; Substrate Specificity

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 gent

Topics: Base Sequence; Biodegradation, Environmental; Cloning, Molecular; Dioxygenases; Enzyme Induction; Escherichia coli; Ferredoxin-NADP Reductase; Ferredoxins; Genes, Bacterial; Gentisates; Mixed Function Oxygenases; Models, Biological; Molecular Sequence Data; Multienzyme Complexes; Multigene Family; Naphthalenes; Oxygenases; Pseudomonas; Salicylates; Salicylic Acid; Sequence Analysis, DNA

The sequence of a 2,437-bp DNA segment from the naphthalene upper catabolic pathway operon of plasmid NAH7 was determined. This segment contains three large open reading frames designated nahQ', nahE, and nahD. The first of these is the 3' end of an open reading frame that has no known function, the second (993 bp) encodes trans-o-hydroxybenzylidenepyruvate hydratase-aldolase (deduced molecular weight, 36,640), and the third (609 bp) encodes 2-hydroxychromene-2-carboxylate isomerase (deduced molecular weight, 23,031). This DNA has a high degree of sequence homology (greater than 91% for the first 2161 bp) with a DNA segment from the dox (dibenzothiophene oxidation) operon of Pseudomonas sp. strain C18, which encodes a pathway analogous to that encoded by NAH7. However, 84 bp downstream from nahD, the last gene in the nah operon, this homology ends. This 84-bp sequence at the downstream end of nah and dox homology has 76% homology to a sequence that occurs just upstream of the nah promoter in NAH7. These directly repeated 84-bp sequences thus encompass the upper-pathway nah operon and constitute the ends of a highly conserved region.

Topics: Amino Acid Sequence; Base Sequence; Hydro-Lyases; Intramolecular Oxidoreductases; Isomerases; Molecular Sequence Data; Naphthalenes; Plasmids; Pseudomonas putida; Salicylates; Salicylic Acid; Sequence Homology, Amino Acid; Sequence Homology, Nucleic Acid

An optical whole-cell biosensor based on a genetically engineered bioluminescent catabolic reporter bacterium was developed for continuous on-line monitoring of naphthalene and salicylate bioavailability and microbial catabolic activity potential in waste streams. The bioluminescent reporter bacterium, Pseudomonas fluorescens HK44, carries a transcriptional nahG-luxCDABE fusion for naphthalene and salicylate catabolism. Exposure to either compound resulted in inducible bioluminescence. The reporter culture was immobilized onto the surface of an optical light guide by using strontium alginate. This biosensor probe was then inserted into a measurement cell which simultaneously received the waste stream solution and a maintenance medium. Exposure under defined conditions to both naphthalene and salicylate resulted in a rapid increase in bioluminescence. The magnitude of the response and the response time were concentration dependent. Good reproducibility of the response was observed during repetitive perturbations with either naphthalene or salicylate. Exposure to other compounds, such as glucose and complex nutrient medium or toluene, resulted in either minor bioluminescence increases after significantly longer response times compared with naphthalene or no response, respectively. The environmental utility of the biosensor was tested by using real pollutant mixtures. A specific bioluminescence response was obtained after exposure to either an aqueous solution saturated with JP-4 jet fuel or an aqueous leachate from a manufactured-gas plant soil, since naphthalene was present in both pollutant mixtures.

Topics: Base Sequence; Biosensing Techniques; DNA, Bacterial; Environmental Monitoring; Molecular Sequence Data; Naphthalenes; Pseudomonas fluorescens; Salicylates; Salicylic Acid; Water Pollutants

The reactions involved in the bacterial metabolism of naphthalene to salicylate have been reinvestigated by using recombinant bacteria carrying genes cloned from plasmid NAH7. When intact cells of Pseudomonas aeruginosa PAO1 carrying DNA fragments encoding the first three enzymes of the pathway were incubated with naphthalene, they formed products of the dioxygenase-catalyzed ring cleavage of 1,2-dihydroxynaphthalene. These products were separated by chromatography on Sephadex G-25 and were identified by 1H and 13C nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry as 2-hydroxychromene-2-carboxylate (HCCA) and trans-o-hydroxybenzylidenepyruvate (tHBPA). HCCA was detected as the first reaction product in these incubation mixtures by its characteristic UV spectrum, which slowly changed to a spectrum indicative of an equilibrium mixture of HCCA and tHBPA. Isomerization of either purified product occurred slowly and spontaneously to give an equilibrium mixture of essentially the same composition. tHBPA is also formed from HCCA by the action of an isomerase enzyme encoded by plasmid NAH7. The gene encoding this enzyme, nahD, was cloned on a 1.95-kb KpnI-BglII fragment. Extracts of Escherichia coli JM109 carrying this fragment catalyzed the rapid equilibration of HCCA and tHBPA. Metabolism of tHBPA to salicylaldehyde by hydration and aldol cleavage is catalyzed by a single enzyme encoded by a 1-kb MluI-StuI restriction fragment. A mechanism for the hydratase-aldolase-catalyzed reaction is proposed. The salicylaldehyde dehydrogenase gene, nahF, was cloned on a 2.75-kb BamHI fragment which also carries the naphthalene dihydrodiol dehydrogenase gene, nahB. On the basis of the identification of the enzymes encoded by various clones, the gene order for the nah operon was shown to be p, A, B, F, C, E, D.

Topics: Aldehyde Oxidoreductases; Aldehydes; Biodegradation, Environmental; Cloning, Molecular; DNA, Recombinant; Escherichia coli; Gene Deletion; Hydro-Lyases; Intramolecular Oxidoreductases; Isomerases; Models, Biological; Naphthalenes; Naphthols; Oxidoreductases; Plasmids; Pseudomonas aeruginosa; Restriction Mapping; Salicylates; Salicylic Acid; Spectrophotometry, Ultraviolet

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.

Topics: Biodegradation, Environmental; Gentisates; Hydroxybenzoates; Mixed Function Oxygenases; NADP; Naphthalenes; Oxygen Consumption; Rhodococcus; Salicylates; Salicylic Acid; Soil Microbiology; Species Specificity

The addition of specific nontoxic inducers of catabolic operons to contaminated sites is an approach that may enhance the efficiency of in situ biodegradation. We determined the genetic response of six pseudomonads to salicylate (also known as 2-hydroxybenzoate) added directly to 50 g of nonsterile soil samples. The strains, isolated from a polyaromatic hydrocarbon-contaminated soil, metabolized naphthalene as the sole source of available carbon, and their DNA sequences show significant homology to the nahAB genes of the degradative plasmid NAH7. Duplicate nonsterile soil cultures were incubated for up to 30 days. Experimental soil cultures were seeded with naphthalene-degrading strains (10(8) CFU g-1) originally isolated from the soil and amended with salicylate (16 or 160 micrograms g-1). Soil samples were analyzed periodically for the population density of heterotrophic bacteria and naphthalene degraders and for the abundance of the naphthalene-degradative genotype in the bacterial community. At 160 micrograms g-1, salicylate sustained the density of naphthalene degraders at the introduced density for 30 days in addition to producing a two- to sixfold increase in the occurrence in the bacterial community of DNA sequences homologous to the nah operon. No change in recoverable bacterial population densities was observed when soil samples were amended with 16 micrograms of salicylate g-1, but this concentration of salicylate induced a significant increase in the level of nah-related genes in the population.

Topics: Biodegradation, Environmental; Colony Count, Microbial; DNA, Bacterial; Genes, Bacterial; Genotype; Naphthalenes; Pseudomonas; Salicylates; Salicylic Acid; Soil Microbiology; Transcription, Genetic

Two bacterial strains were isolated from a bacterial community formed of nine strains, selected from a marine sediment on a seawater medium with naphthalene as sole carbon source. The two strains studied in the present work were the only strains of this community able to grow in pure culture on naphthalene; therefore, they were called "primary" strains. The seven other strains were maintained in the community by using metabolic intermediates of the two primary strains; they were called "auxiliary" strains. Regulation of naphthalene metabolism was studied for the two primary strains. They oxidized naphthalene into catechol, which was degraded only by the meta pathway. For Pseudomonas Lav. 4, naphthalene oxygenase and salicylate hydroxylase were inducible; catechol 2,3-dioxygenase was constitutive. For Moraxella Lav. 7, naphthalene oxygenase was constitutive; salicylate hydroxylase and catechol 2,3-oxygenase were inducible. The Moraxella strain carries two cryptic plasmids, about 63- and 85-kb in molecular size. In the bacterial community culture medium, Moraxella Lav. 7 prevented accumulation of 2-hydroxymuconate semialdehyde formed by Pseudomonas Lav. 4. The auxiliary strains take up formic, acetic, pyruvic, propionic, and succinic acids released by the two primary strains.

Topics: Biodegradation, Environmental; Catechols; Culture Media; Moraxella; Naphthalenes; Oxygenases; Plasmids; Pseudomonas; Salicylates; Seawater; Succinates; Water Microbiology; Water Pollution, Chemical

The regulated meta pathway operon for the catabolism of salicylate on the naphthalene plasmid pWW60-22 was cloned into the broad-host-range vector pKT230 on a 17.5 kbp BamHI fragment. The recombinant plasmid conferred the ability to grow on salicylate when mobilized into plasmid-free Pseudomonas putida PaW130. A detailed restriction map of the insert was derived and the locations of some of the genes were determined by subcloning and assaying for their gene products in Escherichia coli and P. putida hosts. The existence of a regulatory gene was demonstrated by the induction of enzyme activities in the presence of salicylate. DNA-DNA hybridization indicated a high degree of structural homology between the pWW60-22 operon and the analogous meta pathway operon on TOL plasmid pWW53-4. The data are consistent with the structural genes being arranged in an identical linear array and suggest an evolutionary link between the two catabolic systems.

Topics: Biological Evolution; Cloning, Molecular; DNA; Genes, Regulator; Genes, Viral; Naphthalenes; Nucleic Acid Hybridization; Operon; Plasmids; Pseudomonas; Recombination, Genetic; Restriction Mapping; Salicylates; Salicylic Acid

The in vivo transcription start sites of the nah and sal operons of the NAH7 plasmid were determined by S1 nuclease mapping and the nucleotide sequence surrounding these transcription start sites was determined. Since expression of both of these operons is coordinately controlled by the product of the transcriptional activator gene nahR, the sequences were compared to locate potential sites involved in common regulation. In the 100-base-pair region preceding transcription start sites of both operons, three regions of extensive homology were found and may be involved in nahR-mediated transcriptional control: between -80 and -60 with 81% homology; between -40 and -28 with 75% homology; between -1 and +15 with 70% homology. Comparison of the promoter sequences of nah and sal with the analogous sequences of the xylABC and xylDEFG operons of the TOL plasmid showed little homology between the 5' regions of these two sets of positively regulated hydrocarbon degradation operons. In addition, the transcription start site of the nahR regulatory gene was located and its promoter sequence was determined. The nahR promoter overlapped at the -35 position of the sal promoter; however, the nahR gene is transcribed in the opposite direction. Sequences similar to the consensus sequences of Escherichia coli promoters (at -35 and -10) were found in nah, sal, and nahR at the appropriate positions.

Topics: Chromosome Mapping; Cloning, Molecular; DNA, Bacterial; Endonucleases; Gene Expression Regulation; Genes, Bacterial; Genes, Regulator; Naphthalenes; Operon; Promoter Regions, Genetic; Pseudomonas; Salicylates; Salicylic Acid; Sequence Homology, Nucleic Acid; Single-Strand Specific DNA and RNA Endonucleases; Transcription, Genetic

The product of the nahR gene, a salicylate-dependent activator of transcription of the nah and sal hydrocarbon degradation operons of the NAH7 plasmid, was identified and characterized after synthesis in Escherichia coli maxicells. The nahR gene product had a subunit molecular weight of 36,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas gel filtration analysis of the nondenatured nahR protein indicated a molecular weight in excess of 250,000. However, DNase I treatment of this high-molecular-weight complex shifted the apparent molecular weight of the nahR protein to 40,000. Various upstream portions of the sal operon promoter were transcriptionally fused to the E. coli galactokinase gene. Fusion plasmids containing the sal promoter sequence from --83 to 27 (relative to the transcription start site) showed salicylate-inducible expression of galactokinase in the presence of the cloned nahR gene, while expression of galactokinase from a fusion plasmid containing the sal promoter sequence from --45 to 27 was not induced by the nahR gene and salicylate. Results suggest that the nahR gene product is a 36-kilodalton polypeptide which exerts its salicylate-dependent activation of transcription of the sal operon by interacting with the promoter sequence in the region of --83 to --45 base pairs before the transcription start site.

Topics: Bacterial Proteins; Base Sequence; Chromatography, Gel; Chromosome Deletion; Cloning, Molecular; DNA, Bacterial; Genes, Bacterial; Genes, Regulator; Naphthalenes; Operon; Promoter Regions, Genetic; Salicylates; Salicylic Acid; Transcription, Genetic

The catabolism of naphthalene and salicylate is specified by two operons on an 80 Kb metabolic plasmid, NAH7. These operons, nah and sal, are carried on the contiguous 30 Kb EcoRI-A, C fragments, and are under positive control of a regulator region, nahR. Five Nah Sal Tn5 insertion mutants form two complementation groups: A = nahR203, nahR204; and B = nahR201, nahR202, nahR205. The physical and genetic maps assign the nahR location to the 15.7-17.2 Kb region of the EcoRI-A fragment, with suggestion of more than one control gene.

Topics: Cloning, Molecular; Deoxyribonuclease HindIII; DNA Restriction Enzymes; DNA, Recombinant; Escherichia coli; Genes, Regulator; Naphthalenes; Nucleic Acid Hybridization; Operon; Plasmids; Pseudomonas; Salicylates; Salicylic Acid

Pseudomonas putida PMD-1 dissimilates naphthalene (Nah), salicylate (Sal), and benzoate (Ben) via catechol which is metabolized through the meta (or alpha-keto acid) pathway. The ability to utilize salicylate but not naphthalene was transferred from P. putida PMD-1 to several Pseudomonas species. Agarose gel electrophoresis of deoxyribonucleic acid (DNA) from PMD-1 and Sal+ exconjugants indicated that a plasmid (pMWD-1) of 110 megadaltons is correlated with the Sal+ phenotype; restriction enzyme analysis of DNA from Sal+ exconjugants indicated that plasmid pMWD-1 was transmitted intact. Enzyme analysis of Sal+ exconjugants demonstrated that the enzymes required to oxidize naphthalene to salicylate are absent, but salicylate hydroxylase and enzymes of the meta pathway are present. Thus, naphthalene conversion to salicylate requires chromosomal genes, whereas salicylate degradation is plasmid encoded. Comparison of restriction digests of plasmid pMWD-1 indicated that it differs considerably from the naphthalene and salicylate degradative plasmids previously described in P. putida.

Topics: Catechol 1,2-Dioxygenase; Catechol 2,3-Dioxygenase; Chromosomes, Bacterial; Conjugation, Genetic; Dioxygenases; Genes, Bacterial; Naphthalenes; Oxygenases; Plasmids; Pseudomonas; Salicylates; Salicylic Acid

Topics: Naphthalenes; Oxidation-Reduction; Pseudomonas aeruginosa; Salicylates; Salicylic Acid

Topics: Albuminuria; Benzenesulfonates; Humans; Indicators and Reagents; Naphthalenes; Peptides; Salicylates