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.     

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.     

The roles of intermediates in biodegradation of benzene,toluene, and p-xylene by Pseudomonas putida F1

Several types of biodegradation experiments with benzene, toluene, or p-xylene show accumulation of intermediates by Pseudomonas putida F1. Under aerobic conditions, the major intermediates identified for benzene, toluene, and p-xylene are catechol, 3-methylcatechol, and 3,6-dimethylcatechol, respectively. Oxidations of catechol and 3-methylcatechol are linked to biomass synthesis. When oxygen is limited in the system, phenol (from benzene) and m-cresol and o-cresol (from toluene) accumulate.     

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.     

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.     

Oil biodegradation by Bacillus strains isolated from the rock of an oil reservoir located in a deep-water production basin in Brazil

Sixteen spore forming Gram-positive bacteria were isolated from the rock of an oil reservoir located in a deep-water production basin in Brazil. These strains were identified as belonging to the genus Bacillus using classical biochemical techniques and API 50CH kits, and their identity was confirmed by sequencing of part of the 16S rRNA gene. All strains were tested for oil degradation ability in microplates using Arabian Light and Marlin oils and only seven strains showed positive results in both kinds of oils. They were also able to grow in the presence of carbazole, n-hexadecane and polyalphaolefin (PAO), but not in toluene, as the only carbon sources. The production of key enzymes involved with aromatic hydrocarbons biodegradation process by Bacillus strains (catechol 1,2-dioxygenase and catechol 2,3-dioxygenase) was verified spectrophotometrically by detection of cis,cis-muconic acid and 2-hydroxymuconic semialdehyde, and results indicated that the ortho ring cleavage pathway is preferential. Furthermore, polymerase chain reaction (PCR) products were obtained when the DNA of seven Bacillus strains were screened for the presence of catabolic genes encoding alkane monooxygenase, catechol 1,2-dioxygenase, and/or catechol 2,3-dioxygenase. This is the first study on Bacillus strains isolated from an oil reservoir in Brazil.     

Germanium accumulation by bacteria

Germanium accumulation was investigated in 23 bacterial strains. Bacillus strains accumulated the most Ge. Increasing the pH of the incubation medium from 7 to 8.5, as well as substituting catechol for glucose resulted in increased Ge accumulation. The apparent K _s and V _max of Ge accumulation in Bacillus cereus NRC 3045 were found to be 4.0 g/l and 2.2 mg/g dry wt/h, respectively. When cells from three different Bacillus strains were incubated in the presence of 2,4-dinitrophenol or toluene, Ge accumulation was completely inhibited. At 6° C, two out of three Bacillus strains showed a large decrease in Ge accumulation. In addition, non-viable Bacillus cells killed by UV irradiation did not accumulate Ge. These results strongly suggest that Ge accumulation by some Bacillus strains may be an energy-dependent process.     

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.     

Biotransformation of anisole and phenetole by aerobic hydrocarbonoxidizing bacteria

Wild type, mutant, and recombinant bacterial strains capable of oxidizing aromatic hydrocarbons were screened for their ability to oxidize anisole (methoxybenzene) and phenetole (ethoxybenzene). Toluene-induced cells ofPseudomonas putida F39/D transformed anisole to a compound tentatively identified ascis-1,2-dihydroxy-3-methoxyclohexa-3,5-diene (anisole-2,3-dihydrodiol), 2-methoxyphenol, catechol, and trace amounts of phenol while phenetole was converted primarily tocis-1,2-dihydroxy-3-ethoxycyclohexa-3,5-diene (phenetole-2,3-dihydrodiol) and 2-ethoxyphenol. Induced cells ofPseudomonas sp. NCIB 9816/11 andBeijerinckia sp. B8/36 transformed anisole to phenol, and phenetole to phenol and ethenyloxybenzene. Toluene-induced cells ofP. putida BG1 converted anisole to phenol but did not oxidize phenetole. In contrast, toluene-induced cells ofP. mendocina KR1, which oxidize toluene via monooxygenation at thepara position, transformed anisole to 4-methoxyphenol, and phenetole to 2-, 3- and 4-ethoxyphenol. The involvement of toluene and naphthalene dioxygenases in the reactions catalyzed by strains F39/D and NCIB 9816/11, respectively, was confirmed with recombinantE. coli strains expressing the cloned dioxygenase genes. The results show that the oxygenases from differentPseudomonas strains oxidize anisole and phenetole to different hydroxylated products.     

Short communication: Benzene metabolism via the intradiol cleavage in a Rhodococcus sp.

Benzene was metabolized by Rhodococcus sp. 33 through the intradiol cleavage (ortho-) pathway producing cis-benzene glycol, catechol and cis, cis-muconic acid as the intermediates. This is the first elucidation of the pathway by which benzene is degraded by a gram-positive organism. The enzyme assays have also suggested that Rhodococcus 33 does not have a fully functional tricarboxylic acid cycle but may have an operational glyoxylate bypass.     

The induction and repression of benzene and catechol oxidizing capacity of Pseudomonas putida ML2 studied in perturbed chemostat culture

The oxidation of catechol, an intermediate in benzene catabolism, was studied using transient variations in dissolved oxygen tension (DOT) when a succinate limited steady state culture of Pseudomonas putida ML2 was perturbed with a pulse of another substrate. A model was developed and tested for the effect of fluctuations in oxidizing enzyme activity on DOT. It was found that the rate of induction of catechol oxidizing enzymes was independent of dilution rate up to a relative growth rate /_max of 0.75. Only at higher dilution rates was catabolite repression observed.Abbreviations DOT dissolved oxygen tension - K _L a gas transfer coefficient - specific growth rate - _max maximum specific growth rate - K_s substrate saturation constant     

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.     

Dioxygenase activity and relative behaviour of Pseudomonas strains from soil in the presence of different aromatic compounds

Ten different Pseudomonas strains isolated from contaminated soils were tested for expression of active dioxygenases. Of these, two different clusters, related to strain origin were observed. The first included two P. fluorescens strains and two P. aeruginosa strains isolated from soils polluted with polyaromatic hydrocarbons and the second two P. cepacia strains and four P. chlororaphis strains from soils with polyphenols. All the isolates showed catechol 1,2-dioxygenase basal activity, while other dioxygenases (catechol 2,3-dioxygenase, protocatechuate 2,3-, 3,4- and 4,5-dioxygenases) were detected only after growth in the presence of suitable inducers (benzoate, catechol, salicylate, phenol). Significant induction of catechol 1,2-dioxygenase, the major activity of the tested strains, was also observed when combining starvation with the presence of high molecular weight aromatic hydrocarbons with recalcitrant structures (fluoranthene, chrysene, benzanthracene, pyrene).     

A two-step model for the kinetics of BTX degradation and intermediate formation by Pseudomonas putida F1

A two-step model is developed for the aerobic biodegradation of benzene,toluene, and p-xylene (BTX) by Pseudomonas putida F1. The model contains three unique features. First, an initial dioxygenation step transforms BTX into their catechol intermediates, but does not support biomassgrowth. Second, the benzene or toluene intermediates are mineralized, which supports biomass synthesis. Third, BTX exhibit competitive inhibition on each other's transformation, while toluene and benzenenoncompetitively inhibit the mineralization of their catechol intermediate. A suite of batch and chemostat experiments is used to systematically measure the kinetic parameters for the two-step transformations and the substrate interactions.     

Deepening TOL and TOU catabolic pathways of Pseudomonas sp. OX1: Cloning,sequencing and characterization of the lower pathways

Pseudomonas sp. OX1 is able to metabolize toluene and o-xylene through the TOU catabolic pathway, whereas its mutant M1 strain was found to be able to use m- and p-xylene as carbon and energy source, using the TOL catabolic pathway. Here we report the complete nucleotide sequence of the phe lower operon of the TOU catabolic pathway, and the sequence of the last four genes of the xyl-like lower operon of the TOL catabolic pathway. DNA sequence analysis shows the gene order within the operons to be pheCDEFGHI (phe operon) and xyl-likeQKIH (xyl-like operon), identical to the order found for the isofunctional genes of meta operons in the toluene/xylene pathway of TOL plasmid pWW0 from Pseudomonas putida mt-2 and the phenol/methylphenol pathway of pVIl50 from Pseudomonas sp. CF600. The nucleotide and the deduced amino acid sequences are homologous to the equivalent gene and enzyme sequences from other Pseudomonas meta pathways. Recombinant 2-hydroxymuconic semialdehyde dehydrogenase (HMSD) and 2-hydroxymuconic semialdehyde hydrolase (HMSH), coded by pheCD genes, respectively, and ADA and HOA enzymes from both phe and xyl operons were expressed in E. coli and steady-state kinetic analysis was carried out. The analysis of the kinetic parameters of HMSD and HMSH showed that the enzymes from Pseudomonas sp. OX1 are more specialized to channel metabolites into the two branches of the lower pathway than homologous enzymes from other pseudomonads. The kinetics parameters of recombinant ADA from phe and xyl-like operon were found to be similar to those of homologous enzymes from other Pseudomonas strains. In addition, the enzyme from xyl-like operon showed a substrate affinity three times higher than the enzyme from phe operon.     

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.     

Grouping of phenol hydroxylase and catechol 2,3-dioxygenase genes among phenol- and p-cresol-degrading Pseudomonas species and biotypes

Phenol- and p-cresol-degrading pseudomonads isolated from phenol-polluted water were analysed by the sequences of a large subunit of multicomponent phenol hydroxylase (LmPH) and catechol 2,3-dioxygenase (C23O), as well as according to the structure of the plasmid-borne pheBA operon encoding catechol 1,2-dioxygenase and single component phenol hydoxylase. Comparison of the carA gene sequences (encodes the small subunit of carbamoylphosphate synthase) between the strains showed species- and biotype-specific phylogenetic grouping. LmPHs and C23Os clustered similarly in P. fluorescens biotype B, whereas in P. mendocina strains strong genetic heterogeneity became evident. P. fluorescens strains from biotypes C and F were shown to possess the pheBA operon, which was also detected in the majority of P. putida biotype B strains which use the ortho pathway for phenol degradation. Six strains forming a separate LmPH cluster were described as the first pseudomonads possessing the Mop type LmPHs. Two strains of this cluster possessed the genes for both single and multicomponent PHs, and two had genetic rearrangements in the pheBA operon leading to the deletion of the pheA gene. Our data suggest that few central routes for the degradation of phenolic compounds may emerge in bacteria as a result of the combination of genetically diverse catabolic genes.     

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.     

Biodegradation of BTEX at high salinity by an enrichment culture from hypersaline sediments of Rozel Point at Great Salt Lake

Aims: The primary goal of this research was to assess the biodegradation of benzene, toluene, ethylbenzene and xylenes in sediment from Great Salt Lake, near Rozel Point, UT. Methods and Results: An enrichment culture that degraded benzene or toluene as the sole carbon source at high salinity was developed from a sediment sample obtained from Rozel Point. The enrichment degraded benzene or toluene within 1, 2 and 5 weeks in the presence of 14%, 23% and 29% NaCl respectively. PCR studies using degenerate primers revealed that degradation occurred primarily via catechol and the meta‐cleavage pathway. Molecular analysis showed that the Gammaproteobacteria were the dominant members of the enrichment and that shifts in community composition occurred during benzene metabolism. Conclusions: This study demonstrated that micro‐organisms at Rozel Point have the ability to degrade hydrocarbons over a broad range of salinities (1–5 mol l^?1 NaCl) and that the members of the Gammaproteobacteria class play an important role in the degradation process. Significance and Impact of the Study: These results are significant as little is known about the fate of petroleum seeps at Rozel Point. Also, the identity of microbes and the key enzymes involved in the degradation steps are important for understanding natural attenuation potential of hydrocarbons.