Genetics and biochemistry of phenol degradation byPseudomonas sp. CF600

Pseudomonas sp. strain CF600 is an efficient degrader of phenol and methylsubstituted phenols. These compounds are degraded by the set of enzymes encoded by the plasmid locateddmpoperon. The sequences of all the fifteen structural genes required to encode the nine enzymes of the catabolic pathway have been determined and the corresponding proteins have been purified. In this review the interplay between the genetic analysis and biochemical characterisation of the catabolic pathway is emphasised. The first step in the pathway, the conversion of phenol to catechol, is catalysed by a novel multicomponent phenol hydroxylase. Here we summarise similarities of this enzyme with other multicomponent oxygenases, particularly methane monooxygenase (EC 1.14.13.25). The other enzymes encoded by the operon are those of the well-knownmeta-cleavage pathway for catechol, and include the recently discoveredmeta-pathway enzyme aldehyde dehydrogenase (acylating) (EC 1.2.1.10). The known properties of thesemeta-pathway enzymes, and isofunctional enzymes from other aromatic degraders, are summarised. Analysis of the sequences of the pathway proteins, many of which are unique to themeta-pathway, suggests new approaches to the study of these generally little-characterised enzymes. Furthermore, biochemical studies of some of these enzymes suggest that physical associations betweenmeta-pathway enzymes play an important role. In addition to the pathway enzymes, the specific regulator of phenol catabolism, DmpR, and its relationship to the XylR regulator of toluene and xylene catabolism is discussed.     

Molecular cloning and mapping of phenol degradation genes from Bacillus stearothermophilus FDTP-3 and their expression in Escherichia coli.

Two genes of the meta pathway of phenol degradation were cloned from a phenol-utilizing strain of Bacillus stearothermophilus and were mapped by subcloning and by use of a Tn5 insertion mutation. They code for phenol hydroxylase and catechol 2,3-dioxygenase, respectively. The gene encoding catechol 2,3-dioxygenase, which is more thermostable than catechol 2,3-dioxygenase encoded by the other gene, shares rather limited homology with that from Pseudomonas putida.     

Molecular cloning and mapping of phenol degradation genes from Bacillus stearothermophilus FDTP-3 and their expression in Escherichia coli.

Two genes of the meta pathway of phenol degradation were cloned from a phenol-utilizing strain of Bacillus stearothermophilus and were mapped by subcloning and by use of a Tn5 insertion mutation. They code for phenol hydroxylase and catechol 2,3-dioxygenase, respectively. The gene encoding catechol 2,3-dioxygenase, which is more thermostable than catechol 2,3-dioxygenase encoded by the other gene, shares rather limited homology with that from Pseudomonas putida.     

Plasmid-coded degradation of salicylate using the catechol cleavage pathway of the host

Degradation rates of salicylate and phenol by Pseudomonas putida PpG1064 carrying the nahG gene on a multicopy plasmid were compared with those in NAH-carrying P. putida. Degradation rates of salicylate and phenol and the growth rate of the recombinant were higher than those in NAH-carrying P. putida in SP medium. The catechol 1,2 oxygenase activity of the recombinant in Sp medium was about twice that of the catechol 2,3 oxygenase and catechol 1,2 oxygenase activities of NAH-carrying P. putida. It was suggested that in simultaneous degradation of phenol and salicylate, the recombinant stimulated its ortho cleavage pathway and attained the higher degradation rates and growth rate.     

Phenol Degradation by Immobilized Cells of Arthrobacter citreus

An aerobic microorganism with an ability to utilize phenol as carbon and energy source was isolated from a hydrocarbon contamination site by employing selective enrichment culture technique. The isolate was identified as Arthrobacter citreus based on morphological, physiological and biochemical tests. This mesophilic organism showed optimal growth at 25°C and at pH of 7.0. The phenol utilization studies with Arthrobacter citreus showed that the complete assimilation occurred in 24 hours. The organism metabolized phenol up to 22 mM concentrations whereas higher levels were inhibitory. Thin layer chromatography, UV spectral and enzyme analysis were suggestive of catechol, as a key intermediate of phenol metabolism. The enzyme activities of phenol hydroxylase and catechol 2,3-dioxygenase in cell free extracts of Arthrobacter citreus were indicative of operation of a meta-cleavage pathway for phenol degradation. The organism had additional ability to degrade catechol, cresols and naphthol. The degradation rates of phenol by alginate and agar immobilized cells in batch fermentations showed continuous phenol metabolism for a period of eight days.     

Analysis of bacterial degradation pathways for long-chain alkylphenols involving phenol hydroxylase, alkylphenol monooxygenase and catechol dioxygenase genes

Eighteen 4-t-octylphenol-degrading bacteria were isolated and screened for the presence of degradative genes by polymerase chain reaction method using four designed primer sets. The primer sets were designed to amplify specific fragments from multicomponent phenol hydroxylase, single component monooxygenase, catechol 1,2-dioxygenase and catechol 2,3-dioxygenase genes. Seventeen of the 18 isolates exhibited the presence of a 232 bp amplicon that shared 61-92% identity to known multicomponent phenol hydroxylase gene sequences from short and/or medium-chain alkylphenol-degrading strains. Twelve of the 18 isolates were positive for a 324 bp region that exhibited 78-95% identity to the closest published catechol 1,2-dioxygenase gene sequences. The two strains, Pseudomonas putida TX2 and Pseudomonas sp. TX1, contained catechol 1,2-dioxygenase genes also have catechol 2,3-dioxygenase genes. Our result revealed that most of the isolated bacteria are able to degrade long-chain alkylphenols via multicomponent phenol hydroxylase and the ortho-cleavage pathway.     

Isolation and characterization of four novel Gram-positive bacteria associated with the rhizosphere of two endemorelict plants capable of degrading a broad range of aromatic substrates

Four new Gram-positive, phenol-degrading strains were isolated from the rhizospheres of endemorelict plants Ramonda serbica and Ramonda nathaliae known to exude high amounts of phenolics in the soil. Isolates were designated Bacillus sp. PS1, Bacillus sp. PS11, Streptomyces sp. PS12, and Streptomyces sp. PN1 based on 16S rDNA sequence and biochemical analysis. In addition to their ability to tolerate and utilize high amounts of phenol of either up to 800 or up to 1,400 mg l⁻¹ without apparent inhibition in growth, all four strains were also able to degrade a broad range of aromatic substrates including benzene, toluene, ethylbenzene, xylenes, styrene, halogenated benzenes, and naphthalene. Isolates were able to grow in pure culture and in defined mixed culture on phenol and on the mixture of BTEX (benzene, toluene, ethylbenzene, and xylenes) compounds as a sole source of carbon and energy. Pure culture of Bacillus sp. PS11 yielded 1.5-fold higher biomass amounts in comparison to mixed culture, under all conditions. Strains successfully degraded phenol in the soil model system (2 g kg⁻¹) within 6 days. Activities of phenol hydroxylase, catechol 1,2-dioxygenase, and catechol 2,3-dioxygenase were detected and analyzed from the crude cell extract of the isolates. While all four strains use ortho degradation pathway, enzyme indicative of meta degradation pathway (catechol 2,3-dioxygenase) was also detected in Bacillus sp. PS11 and Streptomyces sp. PN1. Phenol degradation activities were induced 2 h after supplementation by phenol, but not by catechol. Catechol slightly inhibited activity of catechol 2,3-dioxygenase in strains PS11 and PN1.     

Proteogenomic Elucidation of the Initial Steps in the Benzene Degradation Pathway of a Novel Halophile,Arhodomonas sp. Strain Rozel,Isolated from a Hypersaline Environment

Lately, there has been a special interest in understanding the role of halophilic and halotolerant organisms for their ability to degrade hydrocarbons. The focus of this study was to investigate the genes and enzymes involved in the initial steps of the benzene degradation pathway in halophiles. The extremely halophilic bacteria Arhodomonas sp. strain Seminole and Arhodomonas sp. strain Rozel, which degrade benzene and toluene as the sole carbon source at high salinity (0.5 to 4 M NaCl), were isolated from enrichments developed from contaminated hypersaline environments. To obtain insights into the physiology of this novel group of organisms, a draft genome sequence of the Seminole strain was obtained. A cluster of 13 genes predicted to be functional in the hydrocarbon degradation pathway was identified from the sequence. Two-dimensional (2D) gel electrophoresis and liquid chromatography-mass spectrometry were used to corroborate the role of the predicted open reading frames (ORFs). ORFs 1080 and 1082 were identified as components of a multicomponent phenol hydroxylase complex, and ORF 1086 was identified as catechol 2,3-dioxygenase (2,3-CAT). Based on this analysis, it was hypothesized that benzene is converted to phenol and then to catechol by phenol hydroxylase components. The resulting catechol undergoes ring cleavage via the meta pathway by 2,3-CAT to form 2-hydroxymuconic semialdehyde, which enters the tricarboxylic acid cycle. To substantiate these findings, the Rozel strain was grown on deuterated benzene, and gas chromatography-mass spectrometry detected deuterated phenol as the initial intermediate of benzene degradation. These studies establish the initial steps of the benzene degradation pathway in halophiles.     

Role of Catechol and the Methylcatechols as Inducers of Aromatic Metabolism in Pseudomonas putida

Pseudomonas putida NCIB 10015 metabolizes phenol and the cresols (methylphenols) by the meta pathway and metabolizes benzoate by the ortho pathway. Growth on catechol, an intermediate in the metabolism of both phenol and benzoate, induces both ortho and meta pathways; growth on 3- or 4-methylcatechols, intermediates in the metabolism of the cresols, induces only the meta pathway to a very limited degree. Addition of catechol at a growth-limiting rate induces virtually no meta pathway enzymes, but high levels of ortho pathway enzymes. The role of catechol and the methylcatechols as inducers is discussed. A method is described for assaying low levels of catechol 1,2-oxygenase in the presence of high levels of catechol 2,3-oxygenase and vice versa.     

Organization and Regulation of meta Cleavage Pathway Genes for Toluene and o-Xylene Derivative Degradation in Pseudomonas stutzeri OX1

Pseudomonas stutzeri OX1 meta pathway genes for toluene and o-xylene catabolism were analyzed, and loci encoding phenol hydroxylase, catechol 2,3-dioxygenase, 2-hydroxymuconate semialdehyde dehydrogenase, and 2-hydroxymuconate semialdehyde hydrolase were mapped. Phenol hydroxylase converted a broad range of substrates, as it was also able to transform the nongrowth substrates 2,4-dimethylphenol and 2,5-dimethylphenol into 3,5-dimethylcatechol and 3,6-dimethylcatechol, respectively, which, however, were not cleaved by catechol 2,3-dioxygenase. The identified gene cluster displayed a gene order similar to that of the Pseudomonas sp. strain CF600 dmp operon for phenol catabolism and was found to be coregulated by the tou operon activator TouR. A hypothesis about the evolution of the toluene and o-xylene catabolic pathway in P. stutzeri OX1 is discussed.     

Growth rate-dependent expression of phenol-assimilation pathways in Alcaligenes eutrophus JMP 134–the influence of formate as an auxiliary energy source on phenol conversion characteristics

 Alcaligenes eutrophus JMP 134 was continuously (carbon-source-limited) grown on phenol to determine the maximum growth rates (μ_max) as a function of the phenol assimilation pathways expressed. During growth on phenol as the sole source of carbon and energy, an almost exclusive expression of the ortho cleavage pathway (catechol 1,2-dioxygenase) was observed at initially low growth rates. This allowed a μ_max of 0.28 h^-1. The induction of the meta cleavage pathway (catechol 2,3-dioxygenase), which appeared at around 0.25 h^-1, resulted in a further increase in the growth rate to 0.40 h^-1 after the enzyme activities of this pathway had been correspondingly expressed. Hence, two maximum growth rates, one for the ortho and one for the meta cleavage pathway, exist for the growth of A. eutrophus JMP 134 on phenol. Growth on phenol was stimulated by formate, which served as an auxiliary energy source in this strain. The simultaneous utilization of phenol and formate at a molar ratio of 1:5.2 resulted in an increase of the yield coefficient from about 0.75 g dry mass/g phenol to 1.25 g/g. Furthermore, formate exerted a pronounced effect on the growth rate. At a molar ratio of phenol to formate of 1:4.2, the growth rate was increased to 0.42 h^-1, despite the exclusive induction of the ortho cleavage pathway. The meta cleavage pathway was expressed during growth on this substrate mixture at about 0.4 h^-1. However, this did not enable a significant increase of the growth rate beyond 0.4 h^-1. This is attributed to an exhaustion of the capacity for formate oxidation at this rate. The results are discussed with respect to energy production capabilities when phenol is assimilated as an energy-deficient heterotrophic substrate. Received: 13 November 1995/Received revision: 15 April 1996/Accepted: 22 April 1996     

Biochemical,Transcriptional and Translational Evidences of the Phenol-meta-Degradation Pathway by the Hyperthermophilic Sulfolobus solfataricus 98/2

Phenol is a widespread pollutant and a model molecule to study the biodegradation of monoaromatic compounds. After a first oxidation step leading to catechol in mesophilic and thermophilic microorganisms, two main routes have been identified depending on the cleavage of the aromatic ring: ortho involving a catechol 1,2 dioxygenase (C12D) and meta involving a catechol 2,3 dioxygenase (C23D). Our work aimed at elucidating the phenol-degradation pathway in the hyperthermophilic archaea Sulfolobus solfataricus 98/2. For this purpose, the strain was cultivated in a fermentor under different substrate and oxygenation conditions. Indeed, reducing dissolved-oxygen concentration allowed slowing down phenol catabolism (specific growth and phenol-consumption rates dropped 55% and 39%, respectively) and thus, evidencing intermediate accumulations in the broth. HPLC/Diode Array Detector and LC-MS analyses on culture samples at low dissolved-oxygen concentration (DOC  =  0.06 mg.L⁻¹) suggested, apart for catechol, the presence of 2-hydroxymuconic acid, 4-oxalocrotonate and 4-hydroxy-2-oxovalerate, three intermediates of the meta route. RT-PCR analysis on oxygenase-coding genes of S. solfataricus 98/2 showed that the gene coding for the C23D was expressed only on phenol. In 2D-DIGE/MALDI-TOF analysis, the C23D was found and identified only on phenol. This set of results allowed us concluding that S. solfataricus 98/2 degrade phenol through the meta route.     

Novel metabolic pathway for salicylate biodegradation via phenol in yeast <Emphasis Type="Italic">Trichosporon moniliiforme</Emphasis>

A novel metabolic pathway was found in the yeast Trichosporon moniliiforme WU-0401 for salicylate degradation via phenol as the key intermediate. When 20 mM salicylate was used as the sole carbon source for the growth of strain WU-0401, phenol was detected as a distinct metabolite in the culture broth. Analysis of the products derived from salicylate or phenol through reactions with resting cells and a cell-free extract of strain WU-0401 indicated that salicylate is initially decarboxylated to phenol and then oxidized to catechol, followed by aromatic ring cleavage to form cis-cis muconate.     

Two cDNAs from the purple sea urchin,<Emphasis Type="Italic"> Strongylocentrotus purpuratus</Emphasis>, encoding mosaic proteins with domains found in factor H,factor I,and complement components C6 and C7

The vertebrate complement system is composed of about 30 serum and cell surface proteins that make up three activation pathways, a lytic pathway, and a set of proteins that regulate complement. Regulatory proteins are required for host protection against autologous complement attack and to control the amplification feedback loop of the alternative pathway. Purple sea urchin, Strongylocentrotus purpuratus, homologues of complement C3 (SpC3) and factor B (SpBf) have been identified, suggesting the presence of an alternative complement pathway. This implies that echinoderms require a complement regulatory system for the same reasons that it is required in higher vertebrates. Two cDNAs, Sp5 and Sp5013, have been characterized from coelomocytes and the deduced structures of the encoded mosaic proteins, SpCRL (S. purpuratus complement related protein, long form) and SpCRS (short form), have domains that are also found in regulatory proteins such as factor H and factor I and the terminal pathway components C6 and C7. These domains include multiple short consensus repeats, a fucolectin domain, Ser/Thr/Pro-rich regions, a Cys-rich region, and a factor I-membrane attack complex domain. The genes are constitutively expressed in all tissues of the sea urchin and are not induced in response to immune challenge. Multiple bands of varying intensity on both genome blots and RNA blots suggest that Sp5 and Sp5013 are members of a small gene family and that they might undergo alternative splicing. Based on the domains present in SpCRL and SpCRS, they might be either examples of complement regulatory proteins or members of the terminal pathway of complement.     

Phenol and Benzoate Metabolism by Pseudomonas putida: Regulation of Tangential Pathways

Catechol occurs as an intermediate in the metabolism of both benzoate and phenol by strains of Pseudomonas putida. During growth at the expense of benzoate, catechol is cleaved ortho (1,2-oxygenase) and metabolized via the beta-ketoadipate pathway; during growth at the expense of phenol or cresols, the catechol or substituted catechols formed are metabolized by a separate pathway following meta (2,3-oxygenase) cleavage of the aromatic ring of catechol. It is possible to explain the mutually exclusive occurrence of the meta and ortho pathway enzymes in phenol- and benzoate-grown cells of P. putida on the basis of differences in the mode of regulation of these two pathways. By use of both nonmetabolizable inducers and blocked mutants, gratuitous synthesis of some of the meta pathway enzymes was obtained. All four enzymes of the meta pathway are induced by the primary substrate, cresol or phenol, or its analogue. Three enzymes of the ortho pathway that catalyze the conversion of catechol to beta-ketoadipate enol-lactone are induced by cis,cis-muconate, produced from catechol by 1,2-oxygenase-mediated cleavage. Observations on the differences in specificity of induction and function of the two pathways suggest that they are not really either tangential or redundant. The meta pathway serves as a general mechanism for catabolism of various alkyl derivatives of catechol derived from substituted phenolic compounds. The ortho pathway is more specific and serves primarily in the catabolism of precursors of catechol and catechol itself.     

Changes in structure,activity and metabolism of aerobic granules as a microbial response to high phenol loading

Four column-type sequential aerobic sludge blanket reactors were fed with phenol as the sole carbon and energy source and operated at loading rates of 1.0, 1.5, 2.0 and 2.5 kg phenol m^–3 day^–1. The results indicated that phenol loading exerted a profound influence on the structure, activity and metabolism of the aerobic granules. Compact granules with good settling ability were maintained at loadings up to 2.0 kg phenol m^–3 day^–1, and structurally weakened granules with enhanced production of extracellular polymers and proteins and significantly lower hydrophobicities were observed at the highest loading of 2.5 kg phenol m^–3 day^–1. Specific oxygen uptake rate, catechol 2,3-dioxygenase (C23O) and catechol 1,2-dioxygenase (C12O) activities peaked at a loading of 2.0 kg phenol m^–3 day^–1, and declined thereafter. Granules degraded phenol completely in all four reactors, mainly through the meta cleavage pathway as C23O activities were significantly higher than C12O activities. At the highest loading applied, the anabolism and catabolism of microorganisms were regulated such that phenol degradation proceeded exclusively via the meta pathway, apparently to produce more energy for overstimulation of protein production against phenol toxicity. This work contributes to a better understanding of the ability of aerobic granules to handle high-strength industrial wastewaters containing chemicals that are normally inhibitory to microbial growth.     

Simultaneous degradation of p-nitrophenol and phenol by a newly isolated Nocardioides sp

A p-nitrophenol (PNP)- and phenol-mineralizing bacterium (strain NSP41) was isolated from an industrial wastewater and identified as a member of the genus Nocardioides. PNP was degraded via a hydroquinone pathway, and phenol was degraded through a catechol pathway in strain NSP41. Both enzyme systems for the degradation of PNP and phenol were induced simultaneously in the presence of both compounds. Although both enzyme systems were induced at the same time, PNP and phenol were degraded by the hydroquinone and catechol pathway, respectively. However, during the simultaneous degradation in the low phenol concentration, after the exhaustion of phenol, some PNP was transformed by the catechol pathway and 4-nitrocatechol was transiently accumulated. Kinetically, the addition of phenol greatly enhanced the apparent PNP degradation rate, which may be due to the increased cell mass by the assimilation of phenol.     

Induction of phenol-metabolizing enzymes in Trichosporon cutaneum

Some aspects of the induction of enzymes participating in the metabolism of phenol and resorcinol in Trichosporon cutaneum were studied using intact cells and cell-free preparations.Activities of phenol hydroxylase (1.14.13.7), catechol 1,2-oxygenase (1.13.11.1), cis,cis-muconate cyclase (5.5.1.-), delactonizing enzyme(s) and maleolylacetate reductase were 50–400 times higher in fully induced cells than in noninduced cells.In addition to phenol and resorcinol, also catechol, cresols and fluorophenols could induce phenol hydroxylase.The induction was severely inhibited by phenol concentrations higher than 1 mM. Using optimum inducer concentrations (0.01–0.10 mM), it took more than 8 h to obtain full induction, whether in proliferating or in nonproliferating cells.Phenol hydroxylase, catechol 1,2-oxygenase and cis,cis-muconate cyclase were induced simultaneously. The synthesis of the de-lactonizing activity was delayed in relation to these three preceeding enzymes of the pathway.High glucose concentration (over 15 mM) inhibited completely the induction of phenol oxidation by nonproliferating cells. It also inhibited phenol oxidation by pre-induced cells.Among the NADPH-generating enzymes, the activity of iso-citrate dehydrogenase was elevated in cells grown on phenol and resorcinol instead of glucose.     

Identification and Characterization of Acinetobacter sp. CNU961 Able to Grow with Phenol at High Concentrations

An Acinetobacter sp., strain CNU961, with a higher tolerance to phenol was isolated, and identified through a set of taxonomic studies and a genetic complementation test. Enzymatic and mutagenic studies found that the strain dissimilate phenol by hydroxylation to catechol followed by an ortho-ring cleavage pathway to further mineralize it. The phenol hydroxylase, which is an inducible enzyme and requires NADPH for optimum activity, was not inhibited by phenol at concentrations up to 0.5 mM. The different kinetic behaviors of the enzyme activities on NADPH and on phenol reflected that the phenol hydroxylase of strain CNU961 is a multisubunit allosteric enzyme consisting of heterogeneous polypeptides.     

The evolution of pathways for aromatic hydrocarbon oxidation inPseudomonas

The organisation and nucleotide sequences coding for the catabolism of benzene, toluene (and xylenes), naphthalene and biphenylvia catechol and the extradiol (meta) cleavage pathway inPseudomonas are reviewed and the various factors which may have played a part in their evolution are considered. The data suggests that the complete pathways have evolved in a modular way probably from at least three elements. The commonmeta pathway operons, downstream from the ferredoxin-like protein adjacent to the gene for catechol 2,3-dioxygenase, are highly homologous and clearly share a common ancestry. This common module may have become fused to a gene or genes the product(s) of which could convert a stable chemical (benzoate, salicylate, toluene, benzene, phenol) to catechol, thus forming the lower pathway operons found in modern strains. The upper pathway operons might then have been acquired as a third module at a later stage thus increasing the catabolic versatility of the host strains.