Localization and organization of phenol degradation genes ofPseudomonas putida strain H

The genetic organization of the DNA region encoding the phenol degradation pathway ofPseudomonas putida H has been investigated. This strain can utilize phenol or some of its methylated derivatives as its sole source of carbon and energy. The first step in this process is the conversion of phenol into catechol. Catechol is then further metabolized via themeta-cleavage pathway into TCA cycle intermediates. Genes encoding these enzymes are clustered on the plasmid pPGH1. A region of contiguous DNA spanning about 16 kb contains all of the genetic information necessary for inducible phenol degradation. The analysis of mutants generated by insertion of transposons and cassettes indicates that all of the catabolic genes are contained in a single operon. This codes for a multicomponent phenol hydroxylase andmeta-cleavage pathway enzymes. Catabolic genes are subject to positive control by the gene product(s) of a second locus.     

Isolation and characterization of phenanthrene-degrading <Emphasis Type="Italic">Sphingomonas paucimobilis</Emphasis> strain ZX4

Phenanthrene-degrading bacterium strain ZX4 was isolated from an oil-contaminated soil, and identified as Sphingomonas paucimobilis based on 16S rDNA sequence, cellular fatty acid composition, mol% G + C and Biolog-GN tests. Besides phenanthrene, strain ZX4 could also utilize naphthalene, fluorene and other aromatic compounds. The growth on salicylic acid and catechol showed that the strain degraded phenanthrene via salicylate pathway, while the assay of catechol 2, 3-dioxygenase revealed catechol could be metabolized through meta-cleavage pathway. Three genes, including two of meta-cleavage operon genes and one of GST encoding gene were obtained. The order of genes arrangement was similar to S-type meta-pathway operons. The phylogenetic trees based on 16S rDNA sequence and meta-pathway gene both revealed that strain ZX4 is clustered with strains from genus Sphingomonas.     

Catabolic degradation of 4-chlorobiphenyl byPseudomonas sp. DJ-12 via consecutive reaction ofmeta-cleavage and hydrolytic dechlorination

Pseudomonas sp. strain DJ-12 is a bacterial isolate capable of degrading 4-chlorobiphenyl (4CBP) as a carbon and energy source. The catabolic degradation of 4CBP by the strain DJ-12 was studied along with the genetic organization of the genes responsible for the crucial steps of the catabolic degradation. The catabolic pathway was characterized as being conducted by consecutive reactions of themeta-cleavage of 4CBP, hydrolytic dechlorination of 4-chlorobenzoate (4CBA), hydroxylation of 4-hydroxybenzoate, andmeta-cleavage of protocatechuate. ThepcbC gene responsible for themeta-cleavage of 4CBP only showed a 30 to 40% homology in its deduced amino acid sequence compared to those of the corresponding genes from other strains. The amino acid sequence of 4CBA-CoA dechlorinase showed an 86% homology with that ofPseudomonas sp. CBS3, yet only a 50% homology with that ofArthrobacter spp. However, thefcb genes for the hydrolytic dechlorination of 4CBA inPseudomonas sp. DJ-12 showed an uniquely different organization from those of CBS3 and other reported strains. Accordingly, these results indicate that strain DJ-12 can degrade 4CBP completely viameta-cleavage and hydrolytic dechlorination using enzymes that are uniquely different in their amino acid sequences from those of other bacterial strains with the same degradation activities.     

Localization and organization of phenol degradation genes ofPseudomonas putida strain H

The genetic organization of the DNA region encoding the phenol degradation pathway ofPseudomonas putida H has been investigated. This strain can utilize phenol or some of its methylated derivatives as its sole source of carbon and energy. The first step in this process is the conversion of phenol into catechol. Catechol is then further metabolized via themeta-cleavage pathway into TCA cycle intermediates. Genes encoding these enzymes are clustered on the plasmid pPGH1. A region of contiguous DNA spanning about 16 kb contains all of the genetic information necessary for inducible phenol degradation. The analysis of mutants generated by insertion of transposons and cassettes indicates that all of the catabolic genes are contained in a single operon. This codes for a multicomponent phenol hydroxylase andmeta-cleavage pathway enzymes. Catabolic genes are subject to positive control by the gene product(s) of a second locus.     

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.     

Molecular characterization of the alpha subunit of multicomponent phenol hydroxylase from 4-chlorophenol-degrading Pseudomonas sp. strain PT3

Multicomponent phenol hydroxylases (mPHs) are diiron enzymes that use molecular oxygen to hydroxylate a variety of phenolic compounds. The DNA sequence of the alpha subunit (large subunit) of mPH from 4-chlorophenol (4-CP)-degrading bacterial strain PT3 was determined. Strain PT3 was isolated from oil-contaminated soil samples adjacent to automobile workshops and oil stations after enrichment and establishment of a chlorophenol-degrading consortium. Strain PT3 was identified as a member of Pseudomonas sp. based on sequence analysis of the 16S rRNA gene fragment. The 4-CP catabolic pathway by strain PT3 was tentatively proposed to proceed via a meta-cleavage pathway after hydroxylation to the corresponding chlorocatechol. This hypothesis was supported by polymerase chain reaction (PCR) detection of the LmPH encoding sequence and UV/VIS spectrophotometric analysis of the culture filtrate showing accumulation of 5-chloro-2-hydroxymuconic semialdehyde (5-CHMS) with λ_max 380. The detection of catabolic genes involved in 4-CP degradation by PCR showed the presence of both mPH and catechol 2,3-dioxygenase (C23DO). Nucleotide sequence analysis of the alpha subunit of mPH from strain PT3 revealed specific phylogenetic grouping to known mPH. The metal coordination encoding regions from strain PT3 were found to be conserved with those from the homologous dinuclear oxo-iron bacterial monooxygenases. Two DE(D)XRH motifs was detected in LmPH of strain PT3 within an approximate 100 amino acid interval, a typical arrangement characteristic of most known PHs.     

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.     

Detection and characterisation of catechol 2,3-dioxygenase in an indigenous soil <Emphasis Type="Italic">Pseudomonad</Emphasis> by MALDI-TOF MS using a column separation

The key enzyme catalyzing the second step in the phenol degradation meta-cleavage pathway (C23O) has been purified to homogeneity from a new bacterial strain, which belongs to genus Pseudomonas. The species was growing on phenol as carbon source. The C23O was detected and identified by absorption spectroscopy. The protein was isolated using sucrose density centrifugation and anion exchange chromatography. The purified protein showed a molecular mass of 32 kDa to sodium dodecyl sulfate polyacrylamid gel electrophoresis and an isoelectric point of 5 estimated by analytical isoelectrical focusing. Matrix-assisted laser desorption ionization-time of flight mass spectrometry and peptide mapping was attempted for the identification of the isolated protein and proteins involved in the metabolic pathway.     

Comparison of the downstream pathways for degradation of nitrobenzene by Pseudomonaspseudoalcaligenes JS45 (2-aminophenol pathway) and by Comamonas sp. JS765 (catechol pathway)

Nitrobenzene is degraded by Pseudomonas pseudoalcaligenes JS45 via 2-aminophenol to 2-aminomuconic semialdehyde, which is further degraded to pyruvate and acetaldehyde. Comamonas sp. JS765 degrades nitrobenzene via catechol to 2-hydroxymuconic semialdehyde. In this study we examined and compared the late steps of degradation of nitrobenzene by these two microorganisms in order to reveal the biochemical relationships of the two pathways and to provide insight for further investigation of their evolutionary history. Experiments showed that 2-hydroxymuconate, the product of the dehydrogenation of 2-hydroxymuconic semialdehyde, was degraded to pyruvate and acetaldehyde by crude extracts of Comamonas sp. JS765, which indicated the operation of a classical catechol meta-cleavage pathway. The semialdehyde dehydrogenases from Comamonas sp. JS765 and P. pseudoalcaligenes JS45 were able to metabolize both 2-amino- and 2-hydroxymuconic semialdehyde, with strong preference for the physiological substrate. 2-Aminomuconate was not a substrate for 4-oxalocrotonate decarboxylase from either bacterial strain. The close biochemical relationships among the classical catechol meta-cleavage pathway in Comamonas sp. JS765, 2-aminophenol meta-cleavage pathways in P. pseudoalcaligenes JS45, and an alternative 2-aminophenol meta-cleavage pathway in Pseudomonas sp. AP-3 suggest a common evolutionary origin. Received: 23 November 1998 / Accepted: 3 February 1999     

Location and organization of the dimethylphenol catabolic genes of Pseudomonas CF600

Summary The gene organization of the phenol catabolic pathway of Pseudomonas CF600 has been investigated. This strain can grow on phenol and some methylated phenols by virtue of an inducible phenol hydroxylase and meta-cleavage pathway enzymes. The genes coding for these enzymes are located on pVI150, an IncP-2 degradative mega plasmid of this strain. Twenty-three kilobases of contiguous DNA were isolated from lambda libraries constructed from strains harbouring wild type and Tn5 insertion mutants of pV1150. A 19.9 kb region of this DNA has been identified which encodes all the catabolic genes of the pathway. Using transposon mutagenesis, polypeptide analysis and expression of subfragments of DNA, the genes encoding the first four enzymatic steps of the pathway have been individually mapped and found to lie adjacent to each other. The order of these genes is the same as that for isofunctional genes of TOL plasmid pWWO and plasmid NAH7.     

Degradation 1,2-dimethylbenzene by Corynebacterium strain C125

In an attempt to obtain bacteria growing on 1,2-dimethylbenzene as sole carbon and energy source two different strains were isolated. One was identified as an Arthrobacter strain, the other as a Corynebacterium strain. Corynebacterium strain C125 was further investigated. The organism was not capable to grow on 1,3- and 1,4-dimethylbenzene. cis-1,2-Dihydroxycyclohexa-3,5-diene oxidoreductase and 3,4-dimethylcatechol-2,3-dioxygenase activity was found in cell extracts. When 3,4-dimethylcatechol was added to cell extract of 1,2-dimethylbenzene-grown cells, first a compound with the spectral properties of 2-hydroxy-5-methyl-6-oxo-2,4-heptadienoate was formed and subsequently acetate was produced. It is proposed that dioxygenases are involved in the initial steps of 1,2-dimethylbenzene degradation, and ring opening proceeds via meta-cleavage.     

Degradation of 2-sec-butylphenol: 3-sec-butylcatechol,2-hydroxy-6-oxo-7-methylnona-2,4-dienoic acid,and 2-methylbutyric acid as intermediates

Pseudomonas sp. strain HBP1 Prp, a mutant of strain HBP1 that was originally isolated on 2-hydroxybiphenyl, was able to grow on 2-sec-butylphenol as the sole carbon and energy source. During growth on 2-sec-butylphenol, 2-methylbutyric acid transiently accumulated in the culture medium. Its concentration reached a maximum after 20 hours and was below detection limit at the end of the growth experiment. The first three enzymes of the degradation pathway — a NADH-dependent monooxygenase, a metapyrocatechase, and ameta-fission product hydrolase — were partially purified. The product of the the monooxygenase reaction was identified as 3-sec-butylcatechol by mass spectrometry. This compound was a substrate for the metapyrocatechase and was converted to 2-hydroxy-6-oxo-7-methylnona-2,4-dienoic acid which was identified by gas chromatography-mass spectrometry of its trimethylsilyl-derivative. The cofactor independentmeta-cleavage product hydrolase used 2-hydroxy-6-oxo-7-methylnona-2,4-dienoic acid as a substrate. All three enzymes showed highest activities for 2-hydroxybiphenyl and its metabolites, respectively, indicating that 2-sec-butylphenol is metabolized via the same pathway as 2-hydroxybiphenyl.     

Carbon and energy fluxes during haloadaptation of<Emphasis Type="Italic"> Halomonas</Emphasis> sp. EF11 growing on phenol

The haloalkaliphile Halomonas sp. EF11 can grow on phenol as sole source for carbon and energy, while maintaining an osmotic equilibrium predominantly by adjusting levels of a certain compatible solute. To determine the energy costs of haloadaptation and the fate of substrate-carbon, the strain was grown continuously in an isothermal compensation calorimeter, keeping all conditions constant except salinity. As salinity increased, slight linear reductions in exothermic heat flow and biomass formation occurred, and 1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) synthesis increased linearly. However, beyond a certain salinity threshold the stationary phenol concentration increased exponentially, while heat flow fell sharply, indicating intoxication or wash-out. The clear transition point between the phases, where ectoine formation peaked, suggests that calorimetric measurements could be used to control the conversion of growth-inhibiting substrates (like phenol) into ectoine and to optimize the process. Enthalpy balance and chemical determinations revealed acetate and formate were formed as side products when the C/N ratio in the feed was low, while 2-muconic acid semialdehyde and formate were produced when the ratio was high. These findings indicate that phenol assimilation occurs via the meta pathway. However, enzyme assays implied that assimilation occurs via the ortho and meta pathways at a low C/N ratio and exclusively via the meta pathway at a high C/N ratio.Communicated by W.D. Grant     

Isolation and Characterization of Phenol-catabolizing Bacteria from a Coking Plant

New phenol degrading bacteria with high biodegradation activity and high tolerance were isolated as Burkholderia cepacia PW3 and Pseudomonas aeruginosa AT2. Both isolates could grow aerobically on phenol as a sole carbon source even at 3 g/l. The whole-cell kinetic properties for phenol degradation by strains PW3 and AT2 showed a V_max of 0.321 and 0.253 mg/l/min/(mg protein), respectively. The metabolic pathways for phenol biodegradation in both strains were assigned to the meta-cleavage activity of catechol 2,3-dioxygenase.     

Microorganisms degrading chlorobenzene via a meta-cleavage pathway harbor highly similar chlorocatechol 2,3-dioxygenase-encoding gene clusters

Pseudomonas putida GJ31 harbors a degradative pathway for chlorobenzene via meta-cleavage of 3-chlorocatechol. Pseudomonads using this route for chlorobenzene degradation, which was previously thought to be generally unproductive, were isolated from various contaminated environments of distant locations. The new isolates, Pseudomonas fluorescens SK1 (DSM16274), Pseudomonas veronii 16-6A (DSM16273), Pseudomonas sp. strain MG61 (DSM16272), harbor a chlorocatechol 2,3-dioxygenase (CbzE). The cbzE-like genes were cloned, sequenced, and expressed from the isolates and a mixed culture. The chlorocatechol 2,3-dioxygenases shared 97% identical amino acids with CbzE from strain GJ31, forming a distinct family of catechol 2,3-dioxygenases. The chlorocatechol 2,3-dioxygenase, purified from chlorobenzene-grown cells of strain SK1, showed an identical N-terminal sequence with the amino acid sequence deduced from cloned cbzE. In all investigated chlorobenzene-degrading strains, cbzT-like genes encoding ferredoxins are located upstream of cbzE. The sequence data indicate that the ferredoxins are identical (one amino acid difference in CbzT of strain 16-6A compared to the others). In addition, the structure of the operon downstream of cbzE is identical in strains GJ31, 16-6A, and SK1 with genes cbzX (unknown function) and the known part of cbzG (2-hydroxymuconic semialdehyde dehydrogenase) and share 100% nucleotide sequence identity with the entire downstream region. The current study suggests that meta-cleavage of 3-chlorocatechol is not an atypical pathway for the degradation of chlorobenzene.This publication is dedicated to the memory of Olga V. Maltseva, who contributed greatly to our current knowledge of biochemistry of degradative pathways for chloroaromatic compounds.This publication is dedicated to Prof. Dr. Hans G. Schlegel in honor of his 80th birthday.     

Induction of ortho- and meta-cleavage pathways in Pseudomonas in biodegradation of high benzoate concentration: MS identification of catabolic enzymes

The degradation pathways of benzoate at high concentration in Pseudomonas putida P8 were directly elucidated through mass spectrometric identification of some key catabolic enzymes. Proteins from P. putida P8 grown on benzoate or succinate were separated using two-dimensional gel electrophoresis. For cells grown on benzoate, eight distinct proteins, which were absent in the reference gel patterns from succinate-grown cells, were found. All the eight proteins were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry as catabolic enzymes involved in benzoate degradation. Among them, CatB (EC5.5.1.1), PcaI (EC2.8.3.6), and PcaF (EC2.3.1.174) were the enzymes involved in the ortho-cleavage pathway; DmpC (EC1.2.1.32), DmpD (EC3.1.1.-), DmpE (EC4.2.1.80), DmpF (EC1.2.1.10), and DmpG (EC4.1.3.-) were the meta-cleavage pathway enzymes. In addition, enzyme activity assays showed that the activities of both catechol 1,2-dioxygenase (C12D; EC1.13.11.1) and catechol 2,3-dioxygenase (C23D; EC1.13.11.2) were detected in benzoate-grown P. putida cells, undoubtedly suggesting the simultaneous expression of both the ortho- and the meta-cleavage pathways in P. putida P8 during the biodegradation of benzoate at high concentration.     

Gas chromatographic determination and mechanism of formation of D-amino acids occurring in fermented and roasted cocoa beans, cocoa powder, chocolate and cocoa shell

Summary. Pseudomonas sp. strain phDV1, being a phenol degrading bacterium, has been found to utilize phenol as sole carbon source via the meta pathway. Blue native polyacrylamide gel electrophoresis (BN-PAGE) is widely used for the analysis of oligomeric state and molecular mass non-dissociated protein complexes. In this study, a number of proteomic techniques were used to investigate the oligomeric state enzymes involved in the aromatic degradation pathway. In particular, the Pseudomonas sp. strain phDV1 proteome was monitored under two different growth substrate conditions, using glucose or phenol as sole carbon source. The protein complexes map was compared by BN-PAGE after fractionation by sucrose density centrifugation of the cell extracts. Multiple differences were detected. Further, analysis and identification of the subunit composition of these complexes was carried out using MALDI-TOF MS, allowing the identification of 49 proteins. Additionally, functional information regarding protein–protein interactions was assembled, by coupling 2-D BN-PAGE with MALDI-TOF MS. Application of this functional proteomics method resulted in an higher number of the identified proteins.     

Specific ratio and survival of Pseudomonas CF600 as co-culture for phenol degradation in continuous cultivation

Studies were carried out to understand parallel survival of two strains when cultivated as co-culture on a single carbon source in continuous cultivation. Strains used were Pseudomonas sp. strain CF600 that is reported for degradation of phenol; and HKR1 a lab strain, which was isolated from a site contaminated with phenol. In continuous cultivation Pseudomonas sp. CF600 showed an accumulation of colored intermediate, 2-hydroxy muconic semialdehyde (HMS), when fed with phenol as a sole source of carbon under dissolved oxygen limiting condition (40% saturation level). Under the same cultivation condition when it was co-cultured with strain HKR1, complete degradation of phenol was observed with no accumulation of intermediate. Different dilution rates (0.03, 0.15, and 0.30) were set in the bioreactor during cultivation. It was also observed that both the strains follow a typical cell density ratio of 1:18 as strain HKR1: Pseudomonas sp. CF600 irrespective of the dilution rates used in the study to favor degradation of phenol. Pseudomonas sp. CF600 is reported to degrade phenol via a plasmid-encoded pathway (pVI150). The enzymes for this meta-cleavage pathway are clustered on 15 genes encoded by a single operon, the dmp operon. PCR using primers from the different catabolic loci of dmp operon, demonstrated that the strain HKR1 follows a different metabolic pathway for intermediate utilization.     

The meta cleavage operon of TOL degradative plasmid pWWO comprises 13 genes

Summary The meta-cleavage operon of TOL plasmid pWWO of Pseudomonas putida encodes a set of enzymes which transform benzoate/toluates to Krebs cycle intermediates via extradiol (meta-) cleavage of (methyl)catechol. The genetic organization of the operon was characterized by cloning of the meta-cleavage genes into an expression vector and identification of their products in Escherichia coli maxicells. This analysis showed that the meta-cleavage operon contains 13 genes whose order and products (in kilodaltons) are The xyIXYZ genes encode three subunits of toluate 1,2-dioxygenase. The xylL, xyIE, xyIG, xylF, xylJ, xylK, xylI and xylH genes encode 1,2-dihydroxy-3,5-cyclohexadiene-1-carboxylate dehydrogenase, catechol 2,3-dioxygenase, 2-hydroxymuconic semialdehyde dehydrogenase, 2-hydroxymuconic semialdehyde hydrolase, 2-oxopent-4-enoate hydratase, 4-hydroxy-2-oxovalerate aldolase, 4-oxalocrotonate decarboxylase and 4-oxalocrotonate tautomerase, respectively. The functions of xyIT and xylQ are not known at present. The comparison of the coding capacity and the sizes of the products of the meta-cleavage operon genes indicated that most of the DNA between xyIX and xyIH consists of coding sequences.     

Metabolism of 2-aminophenol by Pseudomonas sp. AP-3: modified meta-cleavage pathway

A novel pathway for 2-aminophenol metabolism by Pseudomonas sp. AP-3 is proposed. The proposed pathway is similar to that known for meta-cleavage of catechol except that one of the hydroxyl groups on the metabolites is replaced by an amino group. During the degradation of 2-aminophenol, 2-amino-2,4-pentadienoic acid is the last metabolite containing an amino group. We, therefore, propose a modified meta-cleavage pathway for the 2-aminophenol metabolism. Received: 27 November 1997 / Accepted: 14 May 1998