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Mycolicibacterium gadium IBE100 and Mycobacterium paragordonae IBE200 are aerobic, chemoorganoheterotrophic bacteria isolated from activated sludge from a wastewater treatment plant. They use 2-methylpropene (isobutene, 2-MP) as the sole source of carbon and energy. Here, we postulate a degradation pathway of 2-methylpropene derived from whole genome sequencing, differential expression analysis and peptide-mass fingerprinting. Key genes identified are coding for a 4-component soluble diiron monooxygenase with epoxidase activity, an epoxide hydrolase, and a 2-hydroxyisobutyryl-CoA mutase. In both strains, involved genes are arranged in clusters of 61.0 and 58.5 kbp, respectively, which also contain the genes coding for parts of the aerobic pathway of adenosylcobalamin synthesis. This vitamin is essential for the carbon rearrangement reaction catalysed by the mutase. These findings provide data for the identification of potential 2-methylpropene degraders., (© 2023 The Authors. Environmental Microbiology published by Applied Microbiology International and John Wiley & Sons Ltd.)

Omega hydroxycarboxylic acids (ω-HAs) possess two functional groups, a hydroxyl group and a carboxyl group, and are essential precursors for the production of biodegradable polyester polymers. In this work, an Acidovorax mutant was investigated as a whole-cell biocatalyst for the conversion of cycloalkanes to their respective ω-hydroxycarboxylic acids. This Acidovorax sp. strain CHX100 originated from a wastewater treatment plant and uses cyclohexane as the sole source of carbon and energy with excellent growth rates (0.199 h -1 ). The metabolic efficiency of Acidovorax CHX100 is based on a highly efficient enzyme cascade used for the mineralization of cyclohexane. A deletion of 6-hydroxyhexanoate dehydrogenase in the native cycloalkane pathway resulted in the Acidovorax sp. strain CHX100 Δ6HX mutant, which accumulated short ω-hydroxycarboxylic acids (C5 to C10) from cycloalkanes. This mutant transformed cyclopentane and cyclohexane (5 mM) to 5-hydroxypentanoic acid and 6-hydroxyhexanoic acid, respectively, with a molar conversion above 98% in 6 h. An elementary environmental and economical assessment based on E-factor and biocatalyst yield suggests the use of inexpensive electron donor and carbon sources, with subsequent efforts to minimize waste generation. Such an early-stage analysis highlights the main bottlenecks that need to be solved in developing a sustainable bioprocess., (Copyright © 2020 Elsevier B.V. All rights reserved.)

A total of 11 bacterial strains capable of completely degrading 2-butoxyethanol (2-BE) were isolated from forest soil, a biotrickling filter, a bioscrubber, and activated sludge, and identified by 16S rRNA gene sequence analysis. Eight of these strains belong to the genus Pseudomonas; the remaining three strains are Hydrogenophaga pseudoflava BOE3, Gordonia terrae BOE5, and Cupriavidus oxalaticus BOE300. In addition to 2-BE, all isolated strains were able to grow on 2-ethoxyethanol and 2-propoxyethanol, ethanol, n-hexanol, ethyl acetate, 2-butoxyacetic acid (2-BAA), glyoxylic acid, and n-butanol. Apart from the only gram-positive strain isolated, BOE5, none of the strains were able to grow on the nonpolar ethers diethyl ether, di-n-butyl ether, n-butyl vinyl ether, and dibenzyl ether, as well as on 1-butoxy-2-propanol. Strains H. pseudoflava BOE3 and two of the isolated pseudomonads, Pseudomonas putida BOE100 and P. vancouverensis BOE200, were studied in more detail. The maximum growth rates of strains BOE3, BOE100, and BOE200 at 30 °C were 0.204 h -1 at 4 mM, 0.645 h -1 at 5 mM, and 0.395 h -1 at 6 mM 2-BE, respectively. 2-BAA, n-butanol, and butanoic acid were detected as potential metabolites during the degradation of 2-BE. These findings indicate that the degradation of 2-BE by the isolated gram-negative strains proceeds via oxidation to 2-BAA with subsequent cleavage of the ether bond yielding glyoxylate and n-butanol. Since Gordonia terrae BOE5 was the only strain able to degrade nonpolar ethers like diethyl ether, the degradation pathway of 2-BE may be different for this strain.

About 60,000-70,000 tons of 2-chlorotoluene, which shows high toxicity in aquatic ecosystems, are produced worldwide and used in a tremendous field of applications. However, clear proofs of biodegradation were only presented for Comamonas testosteroni KT5 and Rhodococcus sp. OCT10. Hence, this study aims on the isolation of additional strains and their characterization in pilot-scale biotrickling filters. Three strains named OCT2, OCT9, and OCT14 of the genus Rhodococcus were isolated, able to mineralize gaseous 2-chlorotoluene like the previously isolated strain Rhodococcus sp. OCT10. The performance levels of these strains were tested in four biotrickling filters each containing 18.8 L of polyurethane foam package, showing elimination capacities of carbon (C) of 30.9 (OCT2), 30.1 (OCT9), 32.2 (OCT10), and 3.9 g C·m -3 ·h -1 (OCT14) at an average crude gas level of 397.6 mg C·m -3 and an empty bed residence time (EBRT) of 22.6 s. Since OCT10 showed the highest performance levels, this strain was characterized in a second biotrickling filter configuration at long-term conditions of 985 days, varying crude gas levels, EBRT and nutrient supply. Chloride balancing showed a recovery of 94.4% of 2-chlorotoluene eliminated out of the gas phase, pointing out mineralization of 2-chlorotoluene. German emission limit values were met at crude gas levels up to 750 mg C·m -3 at EBRTs of 120 s or higher. The maximum elimination capacity was 51.2 g C·m -3 ·h -1 at a specific freight of 51.9 g C·m -3 ·h -1 and an EBRT of 254 s. Performance levels were strongly boosted by addition of ammonia as nutrient and stabilized at efficiency levels higher than 90% at a feed rate of 4 g ammonium sulfate per week and 100 L of package volume. Repetitive monitoring of the established 2-chlorotoluene degrading community by BOX-PCR fingerprinting revealed a high long-term stability of OCT10, underlining its suitability in this kind of application., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The removal of cyclohexane from gaseous emissions was studied using a biotrickling filter packed with polyurethane foam. Acivodorax sp. CHX100 was chosen as inoculum due to its ability to use cyclohexane as carbon source. Performance was evaluated by means of different resident times from 18 s to 37 s and concentration levels of 60, 90, 120, 160, 320, 480 and 720 mg C m -3 , respectively. Removal efficiencies of 80%-99% and elimination capacities in the range of 5.4 g C m -3  h -1 -38 g C m -3  h -1 were achieved for concentrations among 60 mg C m -3 -480 mg C m -3 . The removal efficiency decreased to 40% at concentrations of cyclohexane of 720 mg C m -3 . The dynamics of the microbial population showed the strain CHX100 as predominant during the different operational process of biotrickling filter. The results of this study propose a novel approach for cleaning waste air containing cyclohexane by means of a biotrickling filter., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The removal of 2-butoxyethanol from gaseous emissions was studied using two biotrickling filters (BTF1 and BTF2) packed with polyurethane foam. Two different inoculum sources were used: a pure culture of Pseudomonas sp. BOE200 (BTF1) and activated sludge from a municipal wastewater treatment plant (BTF2). The bioreactors were operated at inlet loads (ILs) of 130 and 195 g m(-3) hour(-1) and at an empty bed residence time (EBRT) of 12.5s. Under an IL of ∼130 g m(-3) hour(-1), BTF1 presented higher elimination capacities (ECs) than BTF2, with average values of 106±7 and 68±8 g m(-3) hour(-1), respectively. However, differences in ECs between BTFs were decreased by reducing the irrigation intervals from 1 min every 12 min to 1 min every 2 hours in BTF2. Average values of EC were 111±25 and 90±7 g m(-3) hour(-1) for BTF1 and BTF2, respectively, when working at an IL of ∼195 g m(-3) hour(-1). Microbial analysis revealed a significant shift in the microbial community of BTF1 inoculated with Pseudomonas sp. BOE200. At the end of the experiment, the species Microbacterium sp., Chryseobacterium sp., Acinetobacter sp., Pseudomonas sp. and Mycobacterium sp. were detected. In BTF2 inoculated with activated sludge, the denaturing gradient gel electrophoresis (DGGE) technique showed a diverse microbial community including species that was able to use 2-butoxyethanol as its carbon source, such as Pseudomonas aeruginosa and Pseudomonas putida as representative species. Although BTF1 inoculated with Pseudomonas sp. BOE200 and higher gas velocity (probably greater gas/liquid mass transfer rate) showed a slight improvement in performance, the use of activated sludge as inoculum seems to be a more feasible option for the industrial application of this technology., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The applications of biocatalysts in chemical industries are characterized by activity, selectivity, and stability. One key strategy to achieve high biocatalytic activity is the identification of novel enzymes with kinetics optimized for organic synthesis by Nature. The isolation of novel cytochrome P450 monooxygenase genes from Acidovorax sp. CHX100 and their functional expression in recombinant Pseudomonas taiwanensis VLB120 enabled efficient oxidation of cyclohexane to cyclohexanol. Although initial resting cell activities of 20 U gCDW (-1) were achieved, the rapid decrease in catalytic activity due to the toxicity of cyclohexane prevented synthetic applications. Cyclohexane toxicity was reduced and cellular activities stabilized over the reaction time by delivering the toxic substrate through the vapor phase and by balancing the aqueous phase mass transfer with the cellular conversion rate. The potential of this novel CYP enzyme was exploited by transferring the shake flask reaction to an aqueous-air segmented flow biofilm membrane reactor for maximizing productivity. Cyclohexane was continuously delivered via the silicone membrane. This ensured lower reactant toxicity and continuous product formation at an average volumetric productivity of 0.4 g L tube (-1) h(-1) for several days. This highlights the potential of combining a powerful catalyst with a beneficial reactor design to overcome critical issues of cyclohexane oxidation to cyclohexanol. It opens new opportunities for biocatalytic transformations of compounds which are toxic, volatile, and have low solubility in water., (© 2015 Wiley Periodicals, Inc.)

Fluoroaromatics are widely and--in recent years--increasingly used as agrochemicals, starting materials for chemical syntheses and especially pharmaceuticals. This originates from the special properties the carbon-fluorine bond is imposing on organic molecules. Hence, fluoro-substituted compounds more and more are considered to be important potential environmental contaminants. On the other hand, the microbial potentials for their transformation and mineralization have received less attention in comparison to other haloaromatics. Due to the high electronegativity of the fluorine atom, its small size, and the extraordinary strength of the C-F bond, enzymes and mechanisms known to facilitate the degradation of chloro- or bromoarenes are not necessarily equally active with fluoroaromatics. Here, we review the literature on the microbial degradation of ring and side-chain fluorinated aromatic compounds under aerobic and anaerobic conditions, with particular emphasis being placed on the mechanisms of defluorination reactions.

Anthropogenic Trace Compounds (ATCs) that continuously grow in numbers and concentrations are an emerging issue for water quality in both natural and technical environments. The complex web of exposure pathways as well as the variety in the chemical structure and potency of ATCs represents immense challenges for future research and policy initiatives. This review summarizes current trends and identifies knowledge gaps in innovative, effective monitoring and management strategies while addressing the research questions concerning ATC occurrence, fate, detection and toxicity. We highlight the progressing sensitivity of chemical analytics and the challenges in harmonization of sampling protocols and methods, as well as the need for ATC indicator substances to enable cross-national valid monitoring routine. Secondly, the status quo in ecotoxicology is described to advocate for a better implementation of long-term tests, to address toxicity on community and environmental as well as on human-health levels, and to adapt various test levels and endpoints. Moreover, we discuss potential sources of ATCs and the current removal efficiency of wastewater treatment plants (WWTPs) to indicate the most effective places and elimination strategies. Knowledge gaps in transport and/or detainment of ATCs through their passage in surface waters and groundwaters are further emphasized in relation to their physico-chemical properties, abiotic conditions and biological interactions in order to highlight fundamental research needs. Finally, we demonstrate the importance and remaining challenges of an appropriate ATC risk assessment since this will greatly assist in identifying the most urgent calls for action, in selecting the most promising measures, and in evaluating the success of implemented management strategies., (Copyright © 2015. Published by Elsevier Ltd.)

Burkholderia fungorum FLU100 simultaneously oxidized any mixture of toluene, benzene and mono-halogen benzenes to (3-substituted) catechols with a selectivity of nearly 100%. Further metabolism occurred via enzymes of ortho cleavage pathways with complete mineralization. During the transformation of 3-methylcatechol, 4-carboxymethyl-2-methylbut-2-en-4-olide (2-methyl-2-enelactone, 2-ML) accumulated transiently, being further mineralized only after a lag phase of 2 h in case of cells pre-grown on benzene or mono-halogen benzenes. No lag phase, however, occurred after growth on toluene. Cultures inhibited by chloramphenicol after growth on benzene or mono-halogen benzenes were unable to metabolize 2-ML supplied externally, even after prolonged incubation. A control culture grown with toluene did not show any lag phase and used 2-ML as a substrate. This means that 2-ML is an intermediate of toluene degradation and converted by specific enzymes. The conversion of 4-methylcatechol as a very minor by-product of toluene degradation in strain FLU100 resulted in the accumulation of 4-carboxymethyl-4-methylbut-2-en-4-olide (4-methyl-2-enelactone, 4-ML) as a dead-end product, excluding its nature as a possible intermediate. Thus, 3-methylcyclohexa-3,5-diene-1,2-diol, 3-methylcatechol, 2-methyl muconate and 2-ML were identified as central intermediates of productive ortho cleavage pathways for toluene metabolism in B. fungorum FLU100., (© 2014 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

Eight mixed cultures able to degrade benzoic acid under saline conditions were established and kinetic parameters were determined in batch processes with cultures SBM002 (0.5 g d(-1)·g oDM(-1)), SBM003 (0.7 g d(-1)·g oDM(-1)) and SBM007 (2.2 g d(-1)·g oDM(-1)) showing the highest degradation rates. Treatability of an industrial waste water (12 g L(-1) benzoic acid, 82 g L(-1) NaCl) by these cultures was proven in a fed-batch system (SBM002 & SBM003) and a continuous flow reactor (SBM007). The performance of the continuous flow reactor was 15-times higher compared to the fed-batch system due to the change of inocula, higher concentration of ammonia as nutrient and less accumulation of possibly toxic catecholic compounds. Average DOC reduction was found to be 98% at 100 g L(-1) NaCl and 1.2 g L(-1) benzoic acid under these conditions. Pre-treatment of the waste water via chemical precipitation by acidification to pH 3.5 diminished the concentration of benzoic acid to 2.1 g L(-1). In a combined chemical-biological process the volume of the bioreactor is reduced to 15% compared to a pure biological process. A comparison of operational costs for these three alternatives is presented., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Two strains capable of degrading cyclohexane were isolated from the soil and sludge of the wastewater treatment plant of the University of Stuttgart and a biotrickling filter system. The strains were classified as gram negative and identified as Acidovorax sp. CHX100 and Chelatococcus sp. CHX1100. Both strains have demonstrated the capability to degrade cycloalkanes (C5-C8), while only strain CHX1100 used as well short linear n-alkanes (C5-C8) as the sole source of carbon and energy. The growth of Acidovorax sp. CHX100 using cyclohexane was much faster compared to Chelatococcus sp. CHX1100. Degenerated primers were optimized from a set sequences of cyclohexanol dehydrogenase genes (chnA) as well as cyclohexanone monooxygenases (chnB) and used to amplify the gene cluster, which encodes the conversion of cyclohexanol to caprolactone. Phylogenetic analysis has indicated that the two gene clusters belong to different groups. The cyclohexane monooxygenase-induced activity which oxidizes also indole to 5-hydroxyindole has indicated the presence of a CYP-type system monooxygenase involved in the transformation of cyclohexane to cyclohexanol.

A halobenzene-degrading bacterium, Burkholderia fungorum FLU100 (DSM 23736), was isolated due to its outstanding trait to degrade fluorobenzene. Besides fluorobenzene, it utilizes, even in random mixtures, chlorobenzene, bromobenzene, iodobenzene, benzene, and toluene as sole sources of carbon and energy. FLU100 mineralizes mono-halogenated benzenes almost stoichiometrically (according to halide balance); after a lag phase, it also degrades 3-fluorophenol and 3-chlorophenol completely. The FLU100-derived transposon Tn5-mutant FLU100-P14R22 revealed 3-halocatechol to be a central metabolite of this new halobenzene degradation pathway. In FLU100, halocatechols are-as expected-strictly subject to ortho-cleavage of the catechol ring, with meta-cleavage never been observed. The strain is able to completely mineralize 3-fluorocatechol, the principal catecholic metabolite being nearly exclusively formed from fluorobenzene. The temporarily excreted 2-fluoromuconate formed thereof in turn is subsequently metabolized completely. This important finding falsifies the customary opinion of the persistence of 2-fluoromuconate valid up to now. The degradation of 4-fluorocatechol, however, being a very minor intermediate in FLU100, is substantially slower and incomplete and leads to the accumulation of uncharacterized derivatives of muconic acid and muconolactone in the medium. This branch therefore does not seem to be productive. To our knowledge, this represents the first example of the complete metabolism of 3-fluorocatechol via 2-fluoromuconate, a metabolite hitherto described as a dead-end metabolite in fluoroaromatic degradation.

A strain Rhodococcus sp. OCT 10 DSM 45596(T), exhibiting 99.9% of 16S rDNA identity with Rhodococcus wratislaviensis NCIMB 13082, was isolated from a soil sample. The strain completely mineralised 2-chlorotoluene, 2-bromotoluene, o-xylene, benzyl alcohol and benzoate. In contrast, 2-fluorotoluene was only partially mineralised. By GC-MS and (1)H-NMR analyses, 4-chloro-3-methylcatechol was identified as the central intermediate in the degradation pathway of 2-chlorotoluene. It was further degraded by enzymes of the meta cleavage pathway. Catechol 1,2-dioxygenase and chlorocatechol 1,2-dioxygenase as the initial enzymes of the ortho cleavage pathways were not detectable under these conditions. Furthermore, neither formation nor oxidation of 2-chlorobenzylic alcohol, 2-chlorobenzaldehyde, or 2-chlorobenzoate was observed, thereby excluding side chain oxidation activity.

A previously uncultured Propionibacterium was isolated from a highly diluted sample (10(-6)mL) of activated sludge of paper mill effluent. The isolate MOB600 was able to grow on anisole, phenetole, benzene, toluene, phenol, styrene and biphenyl, although it used only limited carbon sources in the minimal media. The partial DNA sequence of 16S ribosomal RNA gene was 93% identical to Luteococcus peritoni CCUG38120 as the closest neighborhood in the family Propionibacteriaceae. Strain MOB600 produced 2-methoxyphenol and 2-ethoxyphenol seemingly in an unproductive pathway from the degradation of anisole and phenetole, respectively. It had a substrate preference to favor 3-alkoxyphenols over 2-alkoxyphenols. Formation of 3-hydroxylated O-aryl alkyl ether was substantially proved by the nearly 1:1 biotransformation of substrate-analogous 1,2-methylenedioxybenzene to 3,4-methylenedioxyphenol (sesamol) showing end-product inhibition. The strain converted 2-/3-methoxyphenols to 3-methoxycatechol. The extradiol ring fission of 3-methoxycatechol appeared to take place in the production of a yellow-colored 2-hydroxymuconate derivative, thereby being able to release methanol spontaneously. High specificity polymerase chain reaction screening for bacterial dioxygenases revealed that the genomic DNA encoded at least three ring-hydroxylating dioxygenase large subunits. Being consistent with substrate availability for this strain, the obtained sequences were closely related to large subunits of an isopropylbenzene 2,3-dioxygenase, a benzene 1,2-dioxygenase, a biphenyl 2,3-dioxygenase, a benzoate 1,2-dioxygenase and a putative dioxygenase in Rhodococcus strains. Our results demonstrate that strain MOB600 may play a major role in the degradation of lignin-like O-aryl alkyl ethers and various aromatic hydrocarbon pollutants in activated sludge of paper mill effluent.

Various substrate specificity groups of alkyl ether (AE)-degrading Actinobacteria coexisted in activated sewage sludge of a mixed wastewater treatment. There were substrate niche overlaps including diethyl ether between linear AE- and cyclic AE-degrading strains and phenetole between monoalkoxybenzene- and linear AE-degrading strains. Representatives of each group showed different substrate specificities and degradation pathways for the preferred substrates. Determining the rates of initial reactions and the initial metabolite(s) from whole cell biotransformation helped us to get information about the degradation pathways. Rhodococcus sp. strain DEE5311 and Rhodococcus rhodochrous strain 117 both were able to degrade anisole and phenetole through aromatic 2-monooxygenation to form 2-alkoxyphenols. In contrast, diethyl ether-oxidizing strain DEE5311 capable of degrading a broad range of linear AE, dibenzyl ether and monoalkoxybenzenes initially transformed anisole and phenetole to phenol via direct O-dealkylation. Compared to this, cyclic AE-degrading Rhodococcus sp. strain THF100 preferred tetrahydrofuran (265 ± 35 nmol min(-1)mg(-1) protein) to diethyl ether (<30), but it cannot oxidize bulkier AE than diethyl ether. Otherwise, 1,4-diethoxybenzene-degrading Rhodococcus sp. strain DEOB100 and Gordonia sp. strain DEOB200 transformed 1,3-/1,4-dialkoxybenzenes to 3-/4-alkoxyphenols by similar manners in the order of rates (nmol min(-1) mg(-1) protein): 1,4-diethoxybenzene (11.1 vs. 3.9)>1,4-dimethoxybenzene (1.6 vs. 2.6)>1,3-dimethoxybenzene (0.6 vs. 0.6). This study suggests that the AE-degrading Actinobacteria can orchestrate various substrate specificity responses to the degradation of various categories of AE pollutants in activated sludge communities.

Twenty-seven Gram-positive strains were characterized physiologically and numerically and classified them into four groups according to their specific activities for utilization of linear alkyl ethers (AEs), cyclic AEs, monoalkoxybenzenes and 1,4-diethoxybenzene. The comparative analysis of the 16S ribosomal RNA gene and 16S-23S intergenic spacer region showed that they belonged to the genera Rhodococcus and Gordonia. Alkyl ether-utilizing rhodococci appeared to involve various and diverse cytochromes P450 of the families CYP116 (25 positive strains from 27), CYP153 (5/27), CYP249 (1/27) and a new family P450RR1 (27/27). The presence of P450RR1 was strongly related to the specific activity for utilization of 2-methoxyphenol and 2-ethoxyphenol. In addition, 26 of 27 strains contained multiple alkB genes coding for probable non-haem iron containing alkane monooxygenases and hydroxylases. Similar DNA fragments coding for a tetrahydrofuran monooxygenase A subunit (ThmA) were found in all cyclic AE-utilizing strains and nearly identical DNA fragments coding for likely orthologues of a propane monooxygenase A subunit (PrmA) in all linear AE-utilizing strains. The substrate availability in the degradation of aryl AEs, cyclic AEs and linear AEs agreed with the molecular probing of the respective genes encoding cytochrome P450RR1, ThmA and PrmA.

Pseudomonas veronii MEK700 was isolated from a biotrickling filter cleaning 2-butanone-loaded waste air. The strain is able to grow on 2-butanone and 2-hexanol. The genes for degradation of short chain alkyl methyl ketones were identified by transposon mutagenesis using a newly designed transposon, mini-Tn5495, and cloned in Escherichia coli. DNA sequence analysis of a 15-kb fragment revealed three genes involved in methyl ketone degradation. The deduced amino acid sequence of the first gene, mekA, had high similarity to Baeyer-Villiger monooxygenases; the protein of the second gene, mekB, had similarity to homoserine acetyltransferases; the third gene, mekR, encoded a putative transcriptional activator of the AraC/XylS family. The three genes were located between two gene groups: one comprising a putative phosphoenolpyruvate synthase and glycogen synthase, and the other eight genes for the subunits of an ATPase. Inactivation of mekA and mekB by insertion of the mini-transposon abolished growth of P. veronii MEK700 on 2-butanone and 2-hexanol. The involvement of mekR in methyl ketone degradation was observed by heterologous expression of mekA and mekB in Pseudomonas putida. A fragment containing mekA and mekB on a plasmid was not sufficient to allow P. putida KT2440 to grow on 2-butanone. Not until all three genes were assembled in the recombinant P. putida was it able to use 2-butanone as carbon source. The Baeyer-Villiger monooxygenase activity of MekA was clearly demonstrated by incubating a mekB transposon insertion mutant of P. veronii with 2-butanone. Hereby, ethyl acetate was accumulated. To our knowledge, this is the first time that ethyl acetate by gas chromatographic analysis has been definitely demonstrated to be an intermediate of MEK degradation. The mekB-encoded protein was heterologously expressed in E. coli and purified by immobilized metal affinity chromatography. The protein exhibited high esterase activity towards short chain esters like ethyl acetate and 4-nitrophenyl acetate.

Mycobacterium sp. strain THO100 was isolated from a morpholine-containing culture of activated sewage sludge. This strain was able to utilize pyrrolidine, morpholine, piperidine, piperazine, and 1,2,3,6-tetrahydropyridine as the sole sources of carbon, nitrogen, and energy. The degradation pathway of pyrrolidine as the best substrate for cellular growth was proposed based on the assays of substrate-induced cytochrome P450 and constitutive enzyme activities toward 4-aminobutyric acid (GABA) and succinic semialdehyde (SSA). Its 16S ribosomal RNA gene sequence (16S rDNA) was identical to that of Mycobacterium tokaiense ATCC 27282(T). The morABC genes responsible for alicyclic amine degradation were nearly identical among different species of Mycobacteria. Remarkably, repetitive sequences at the intergenic spacer (IGS) region between morC and orf1' were detected by comparison of the nearly identical mor gene cluster regions. Considering the strain activity for alicyclic amine degradation, the deleted 65-bp DNA segment did not significantly alter the open reading frames, and the expression and functions of the P450(mor) system remained unaltered. In addition, we found a spontaneous deletion of P450(mor) from another strain HE5 containing the archetypal mor gene cluster, which indicated a possible occurrence of DNA recombination to rearrange the DNA.

Ability to degrade high molecular weight polycyclic aromatic hydrocarbons (PAHs) has been found in diverse species of fast-growing mycobacteria. This study included several PAH-degrading mycobacteria from heavily contaminated sites and an uncontaminated humus soil in the Natural Park, Schwäbische Alb, Germany. The numerical analysis with a total of 131 tests showed that isolates from humus soil and contaminated sites had similar substrate utilization patterns for primary alcohols from ethanol to pentanol, 1,4-butanediol, benzyl alcohol, hexadecane, ethyl acetate, fluoranthene, phenanthrene, and pyrene as the sole carbon and energy (C/E) sources. Significant differences between the two subgroups isolated from humus soil and contaminated sites were observed in the utilization of polyalcoholic sugars, including adonitol, D: -arabitol, L: -arabitol, erythritol, inositol, rhamnose, sorbitol, and xylitol. Among isolates from humus soil, strain PYR100 showed high similarity in 16S rDNA sequence with M. vanbaalenii strain PYR-1 (=DSM 7251, 100%) and M. austroafricanum ATCC 33464 (99.9%). In addition to the numerical analysis, the 16S-23S intergenic spacer sequence was useful for discriminating between the closely related strains PYR100 and PYR-1 (98% similarity). The patterns of the variable V2 and V3 regions in the ribosomal RNA gene corresponding to Escherichia coli positions 179 to 197 and 1006 to 1023, respectively, were useful for dividing fast-growing and thermosensitive PAH-degrading mycobacteria into ten subgroups consistent with the phylogenetic positions.

The effects of pH on the growth of Mycobacterium vanbaalenii PYR-1 and its degradation of phenanthrene and pyrene were compared at pH 6.5 and pH 7.5. Various degradation pathways were proposed in this study, based on the identification of metabolites from mass and NMR spectral analyses. In tryptic soy broth, M. vanbaalenii PYR-1 grew more rapidly at pH 7.5 (mu'=0.058 h(-1)) than at pH 6.5 (mu'=0.028 h(-1)). However, resting cells suspended in phosphate buffers with the same pH values displayed a shorter lag time for the degradation of phenanthrene and pyrene at pH 6.5 (6 h) than at pH 7.5 (48 h). The one-unit pH drop increased the degradation rates four-fold. Higher levels of both compounds were detected in the cytosol fractions obtained at pH 6.5. An acidic pH seemed to render the mycobacterial cells more permeable to hydrophobic substrates. The major pathways for the metabolism of phenanthrene and pyrene were initiated by oxidation at the K-regions. Phenanthrene-9,10- and pyrene-4,5-dihydrodiols were metabolized via transient catechols to the ring fission products, 2,2'-diphenic acid and 4,5-dicarboxyphenanthrene, respectively. The metabolic pathways converged to form phthalic acid. At pH 6.5, M. vanbaalenii PYR-1 produced higher levels of the O-methylated derivatives of non-K-region phenanthrene- and pyrene-diols. Other non-K-region products, such as cis-4-(1-hydroxynaphth-2-yl)-2-oxobut-3-enoic acid, 1,2-dicarboxynaphthalene and benzocoumarin-like compounds, were also detected in the culture fluids. The non-K-region polycyclic aromatic hydrocarbon oxidation might be a significant burden to the cell due to the accumulation of toxic metabolites.

Alkyl ether-degrading Rhodococcus sp. strain DEE5151, isolated from activated sewage sludge, has an activity for the oxidation of a variety of alkyl ethers, aralkyl ethers and dibenzyl ether. The whole cell activity for diethyl ether oxidation was effectively inhibited by 2,3-dihydrofurane, ethyl vinyl ether and glutaraldehyde. Glutaraldehyde of less than 30 microM inhibited the activity by a competitive manner with the inhibition constant, K(I) of 7.07+/-1.36 microM. The inhibition type became mixed at higher glutaraldehyde concentrations >30 microM, probably due to the inactivation of the cell activity by the Schiff-base formation. Structurally analogous ethyl vinyl ether inhibited the diethyl ether oxidation activity in a mixed manner with decreasing the apparent maximum oxidation rate, v(max)(app), and increasing the apparent Michaelis-Menten constant, K(M)(app). The mixed type inhibition by ethyl vinyl ether seemed to be introduced not only by the structure similarity with diethyl ether, but also by the reactivity of the vinyl ether with cellular components in the whole cell system.

Polycyclic aromatic hydrocarbon (PAH) quinone reductase (PQR) and catechol-O-methyltransferase (COMT), from the PAH-degrading Mycobacterium vanbaalenii PYR-1, were demonstrated to be constitutive enzymes located in the soluble fraction of cell extracts. PQR activities for the reduction of 9,10-phenanthrenequinone and 4,5-pyrene- quinone were 1.40+/-0.13 and 0.12+/-0.01 micromol min(-1) mg-protein(-1), respectively. The exogenous catechols alizarin, anthrarobin, 2,3-dihydroxynaphthalene and esculetin inhibited PQR activity. Anthrarobin (100 microM) and esculetin (100 microM) inhibited 4,5-pyrenequinone reduction by 64-92%. COMT was involved in the O-methylation of 1,2-dihydroxyphenanthrene to form 1-methoxy-2-hydroxyphenanthrene and 1,2-dimethoxyphenanthrene. Both pyrene and 1-hydroxypyrene were metabolized by M. vanbaalenii PYR-1 to form 1-methoxypyrene, 1-methoxy-2-hydroxypyrene, 1-hydroxy-2-methoxypyrene and 1,2-dimethoxypyrene. Among the catechols tested, anthrarobin showed the highest COMT activity (1.06+/-0.04 nmol/30 min(-1) mg-protein(-1)). These results suggest that the PQR and COMT activities of M. vanbaalenii PYR-1 may play an important role in the detoxification of PAH catechols.

Twenty strains isolated from sewage sludge were found to degrade various ethers, including alkyl ethers, aralkyl ethers, and dibenzyl ether. In Rhodococcus strain DEE5151, induction of ether degradation needed substrates exhibiting at least one unsubstituted Calpha-methylene moiety as the main structural prerequisite. The cleavage reaction observed with anisole, phenetole, and dibenzyl ether indicates that the initial oxidation occurs at such respective Calpha positions. Diethyl ether-induced strain DEE5151 degraded dibenzyl ether via intermediately accumulated benzoic acid. Phenetole seems to be subject also to another ether-cleaving enzyme. Other strains of this group showed different enzymatic activities towards the substrate classes investigated.

Strain CLN100 was isolated after enrichment on mineral medium with chloronaphthalene as the only carbon and energy source. It was able to use simultaneously and productively chloro- and methyl-derivatives of naphthalene and salicylate through a chromosomally encoded meta pathway. Phenotypic, chemotaxonomic and genotypic characterization classified strain CLN100 as a member of the species Pseudomonas stutzeri. DNA-DNA hybridizations, 16S rDNA, gyrB, rpoD sequences, and molecular fingerprinting indicate that strain CLN100 is a representative of a new genomovar (genomovar 10) within the species.

Polycyclic aromatic hydrocarbon (PAH) o-quinone reductase (PQR) plays a crucial role in the detoxification of PAH o-quinones by reducing them to catechols. Two constitutive PQRs were found in cell extracts of a pyrene-degrading Mycobacterium sp. strain PYR100. The enzymes had an activity towards 9,10-phenanthrenequinone (PQ) and/or 4,5-pyrenequinone (PyQ), and the relative amounts varied with the pH of the culture media. PQR1, containing an FAD cofactor, was a monomer (20.1 kDa), and PQR2, with no flavin cofactor, was a homodimer (26.5 kDa subunits). There was no homology between the N-terminal sequences of PQR1 and PQR2. Dicumarol and quercetin inhibited PQR2 more strongly than PQR1. PQR1 had much lower specificity constants (k(cat)/K(m), 10(5)M(-1)s(-1)) for menadione (0.80) and PQ (5.19) than PQR2 (13.9 for menadione and 176 for PQ). Additionally, PQR2 exhibited a broad substrate specificity with high specificity constants for 1,4-naphthalenequinone, 1,2-naphthalenequinone, and PyQ.

We isolated Gram-positive alkane-degraders from soil and a tricking-bed reactor, and show using polymerase chain reaction (PCR) with degenerate alkane hydroxylase primers and Southern blots that most Rhodococcus isolates contain three to five quite divergent homologues of the Pseudomonas putida GPo1 alkB gene. Two Mycobacterium isolates each contain one homologue, however there is no evidence for the presence of alkB homologues in the remaining strains.

[reaction: see text] Hydroxylation of N-substituted azetidines 11 and 12 and piperidines 15-19 with Sphingomonas sp. HXN-200 gave 91-98% of the corresponding 3-hydroxyazetidines 13 and 14 and 4-hydroxypiperidines 20-24, respectively, with high activity and excellent regioselectivity. High yields and high product concentrations (2 g/L) were achieved with frozen/thawed cells as biocatalyst. For the first time, rehydrated lyophilized cells were successfully used for the biohydroxylation.

1,1-Dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE) is the peri-chlorinated derivative of 1,1-diphenylethylene (DPE). Biodegradation of DDE and DPE by bacteria has so far not been shown. Pure cultures of aerobic bacteria involved in biodegradation of styrene and polychlorinated biphenyls (PCB) were therefore screened for their ability to degrade or cometabolize DPE and DDE. Styrene-metabolizing bacteria (Rho-dococcus strains S5 and VLB150) grew with DPE as their sole source of carbon and energy. Polychlorinated-biphenyl-degrading bacteria (Pseudomonas fluorescens and Rhodococcus globerulus) were unable to degrade DPE even in the presence of an easily utilizable cosubstrate, biphenyl. This is the first report of the utilization of DPE as sole carbon and energy source by bacteria. All the tested bacteria failed to degrade DDE when it was provided as the sole carbon source or in the presence of the respective degradable cosubstrates. DPE transformation could also be detected in cell-free extracts of Rhodococcus S5 and VLB150, but DDE was not transformed, indicating that cell wall and membrane diffusion barriers were not limiting biodegradation. The results of the present study show that, at least for some bacteria, the chlorination of DDE is the main reason for its resistance to biodegradation by styrene and DPE-degrading bacteria.

The dibenzofuran-degrading bacterial strain DPO360 represents a new species of the genus Terrabacter together with the previously described dibenzofuran-mineralizing bacterial strain DPO1361 (K.-H. Engesser, V. Strubel, K. Christoglou, P. Fischer, and H. G. Rast, FEMS Microbiol. Lett. 65:205-210, 1989; V. Strubel, Ph.D. thesis, University of Stuttgart, Stuttgart, Germany, 1991; V. Strubel, H. G. Rast, W. Fietz, H.-J. Knackmuss, and K.-H. Engesser, FEMS Microbiol. Lett. 58:233-238, 1989). Two 2,3-dihydroxybiphenyl-1,2-dioxygenases (BphC1 and BphC2) and one catechol-2,3-dioxygenase (C23O) were shown to be expressed in Terrabacter sp. strain DPO360 growing with dibenzofuran as a sole source of carbon and energy. These enzymes exhibited strong sensitivity to oxygen. They were purified to apparent homogeneity as homodimers (BphC and BphC2) and as a homotetrameric catechol-2,3-dioxygenase (C23O). According to their specificity constants kcat/Km, both BphC1 and BphC2 were shown to be responsible for the cleavage of 2,2',3-trihydroxybiphenyl, the first metabolite in dibenzofuran mineralization along the angular dioxygenation pathway. With this substrate, BphC2 exhibited a considerably higher kcat/Km, value (183 microM/min) than BphC1 (29 microM/min). Catechol-2,3-dioxygenase was recognized to be not involved in the ring cleavage of 2,2',3-trihydroxybiphenyl (kcat/Km, 1 microM/min). Analysis of deduced amino acid sequence data of bphC1 revealed 36% sequence identity to nahC from Pseudomonas putida PpG7 (S. Harayama and M. Rekik, J. Biol. Chem. 264:15328-15333, 1989) and about 40% sequence identity to various bphC genes from different Pseudomonas and Rhodococcus strains. In addition, another 2,3-dihydroxybiphenyl-1,2-dioxygenase gene (bphC3) was cloned from the genome of Terrabacter sp. strain DPO360. Expression of this gene, however, could not be detected in Terrabacter sp. strain DPO360 after growth with dibenzofuran.

Pseudomonas pseudoalcaligenes strain POB310 degrades 3- and 4-carboxydiphenyl ether. The initial reaction involves an angular dioxygenation yielding an unstable hemiacetal that spontaneously decays to phenol and protocatechuate. We cloned a DNA fragment containing the gene encoding the initial dioxygenase from an unstable, self-transmissible plasmid. Sequence analysis revealed two open reading frames encoding proteins with putative molecular masses of 46.3 and 33.6 kDa. The deduced amino acid sequences showed homologies to oxygenase and reductase subunits of aromatic ring-activating dioxygenases, and contained regions identical to consensus sequences that bind chloroplast-like and Rieske-type [2Fe2S] clusters, suggesting that the initial dioxygenase is a class IA aromatic ring-activating dioxygenase system. Initial dioxygenase activity was induced in bacteria grown in M9 minimal medium containing 3- or 4-carboxydiphenyl ether or phenol as carbon source, indicating that the regulation is dependent on the phenol pathway. The maximal specific activity was measured at the beginning of the exponential growth phase.

Angular dioxygenation has been established as the crucial step in dibenzofuran degradation by Brevibacterium sp. strain DPO 1361 (V. Strubel, K. H. Engesser, P. Fischer, and H.-J. Knackmuss, J. Bacteriol. 173:1932-1937, 1991). The same strain utilizes biphenyl and fluorene as sole sources of carbon and energy. The fluorene degradation sequence is proposed to be initiated by oxidation of the fluorene methylene group to 9-fluorenol. Cells grown on fluorene exhibit pronounced 9-fluorenol dehydrogenase activity. Angular dioxygenation of the 9-fluorenone thus formed yields 1,10-dihydro-1,10-dihydroxyfluoren-9-one (DDF). A mechanistic model is presented for the subsequent C-C bond cleavage by an NAD(+)-dependent DDF dehydrogenase, acting on the angular dihydrodiol. This enzyme was purified and characterized as a tetramer of four identical 40-kDa subunits. The following Km values were determined: 13 microM for DDF and 65 microM for 2,3-dihydro-2,3-dihydroxybiphenyl. The enzyme also catalyzes the production of 3-(2'-carboxyphenyl)catechol, which was isolated, and structurally characterized, in the form of the corresponding lactone, 4-hydroxydibenzo-(b,d)-pyran-6-one. Stoichiometry analysis unequivocally demonstrates that angular dioxygenation constitutes the principal pathway in Brevibacterium sp. strain DPO 1361.

2-Chloro-4-methylphenoxyacetate is not a growth substrate for Alcaligenes eutrophus JMP 134 and JMP 134-1. It is, however, being transformed by enzymes of 2,4-dichlorophenoxyacetic acid metabolism to 2-chloro-4-methyl-cis,cis-muconate, which is converted by enzymatic 1,4-cycloisomerization to 4-carboxymethyl-2-chloro-4-methylmuconolactone as a dead end metabolite. Chemically, only 3,6-cycloisomerization occurs, giving rise to both diastereomers of 4-carboxychloromethyl-3-methylbut-2-en-4-olide. Those lactones harboring a chlorosubstituent on the 4-carboxymethyl side chain were surprisingly stable under physiological as well as acidic conditions.

Brevibacterium sp. strain DPO 1361 oxygenates dibenzofuran in the unusual angular position. The 3-(2-hydroxyphenyl)catechol thus generated is subject to meta ring cleavage in the proximal position, yielding 2-hydroxy-6-(2-hydroxyphenyl)-6-oxo-2,4-hexadienoic acid, which is hydrolyzed to 2-oxo-4-pentenoate and salicylate by 2-hydroxy-6-oxo-6-phenyl-2,4-hexadienoic acid hydrolase. The proximal mode of ring cleavage is definitely established by isolation and unequivocal structural characterization of a cyclization product of 2-hydroxy-6-(2-hydroxyphenyl)-6-oxo-2,4-hexadienoic acid, i.e., 3-(chroman-4-on-2-yl)pyruvate.

4-Carboxymethyl-4-methylbut-2-en-4-olide (4-methyl-2-enelactone) isomerase, transforming 4-methyl-2-enelactone to 3-methyl-2-enelactone, was purified from a derivative strain of Pseudomonas sp. B13, named B13 FR1, carrying the plasmid pFRC2OP. This plasmid contained the isomerase gene cloned from Alcaligenes eutrophus JMP 134, which uses 4-methyl-2-enelactone as a carbon source. The enzyme consists of a single peptide chain of Mr 40,000 as judged by SDS/PAGE. In addition to 4-methyl-2-enelactone, the putative reaction intermediate, 1-methyl-3,7-dioxo-2,6-dioxy-bicyclo[3.3.0]octane (1-methylbislactone), was a substrate for the enzyme, but kinetic data presented did not favour its role as a reaction intermediate. Isomeric methyl-substituted 4-carboxymethylbut-2-en-4-olides were neither substrates nor inhibitors. Possible reaction mechanisms are discussed.

A bacterial strain, Pseudomonas sp. POB 310, was enriched with 4-carboxy biphenyl ether as sole source of carbon and energy. Resting cells of POB 310 co-oxidize a substrate analogue, 4-carboxybenzophenone, yielding 1,2-dihydro-1,2-dihydroxy-4-carboxy-benzophenone. The ether bond of 3- and 4-carboxy biphenyl ether is cleaved analogously by initial 1,2-dioxygenation, yielding a hemiacetal which is hydrolysed to protocatechuate and phenol. These intermediates are degraded via an ortho and meta pathway, respectively. Alternative 2,3- and 3,4-dioxygenation can be ruled out as triggering steps in carboxy biphenyl ether degradation.

Sixteen bacterial strains capable of degrading alkylbenzenes and alkylphenols were directly isolated from soil and water. The degradation pathways are discussed. Alkylcatechols are almost exclusively cleaved via meta-ring fission. Meta-cleavage of 3-trifluoromethyl-(TFM)-catechol was observed with all strains at different rates although the reaction rates compared to catechol as a substrate varied considerably. All 2-hydroxy-6-oxohepta-2,4-dienoic acid hydrolases investigated showed strong binding of 7,7,7-trifluoro-2-hydroxy-6-oxohepta-2,4-dienoic acid, the ring fission product of 3-TFM-catechol. Turnover rates, however, were negligible indicating this compound to be a general dead-end metabolite during metabolism of TFM-substituted compounds via meta-cleavage pathways.

Genetic engineering is a powerful means of accelerating the evolution of new biological activities and has considerable potential for constructing microorganisms that can degrade environmental pollutants. Critical enzymes from five different catabolic pathways of three distinct soil bacteria have been combined in patchwork fashion into a functional ortho cleavage route for the degradation of methylphenols and methylbenzoates. The new bacterium thereby evolved was able to degrade and grow on mixtures of chloro- and methylaromatics that were toxic even for the bacteria that could degrade the individual components of the mixtures. Except for one enzymatic step, the pathway was fully regulated and its component enzymes were only synthesized in response to the presence of pathway substrates.

The hybrid strain Pseudomonas sp. WR4016 was subcultivated with increasing concentrations of 5-chlorosalicylate (5----10 mM) as sole carbon source over a period of 9 months. At intervals of approximately 3 months derivative strains WR4017, WR4018 and WR4019 were isolated which exhibited higher growth rates and increased substrate tolerance. Comparative analysis of the turnover rates of the key enzymes in chlorosalicylate degradation showed that the adaptation process did not result from structural modifications of these proteins. Instead, balanced overproduction of the salicylate hydroxylase and catechol 1,2-dioxygenase prevented the accumulation of toxic chlorocatechols and accounted for the reduction of the doubling times with 4- or 5-chlorosalicylate. A comparative analysis of a genetically engineered chlorosalicylate degrader PL300-1 showed similar regulatory patterns as the most advanced isolate WR4019 from the adaptation series.

Dibenzofuran degrading bacteria were enriched from various environmental sources. A mutualistic mixed culture of strain DPO 220 and strain DPO 230 was characterized. Strain DPO 220 alone showed limited growth with dibenzofuran as sole source of carbon and energy (td greater than or equal to 4.5 h). A labile degradation product, C12H10O5, and salicylate were isolated from the culture fluid. Salicylate was found to be a central intermediate of DBF-degradation. Strain DPO 220 co-metabolized a wide range of anellated aromatics as well as heteroaromatics. High rates of co-oxidation of dibenzodioxin demonstrate analogue-enrichment to be a powerful technique for selecting enzymatic activities for otherwise non-degradable substrates.

Two dibenzofuran degrading bacteria, Brevibacterium strain DPO 1361 and strain DPO 220, were found to utilize fluorene as sole source of carbon and energy. Cells which were grown on dibenzofuran, transformed fluorene into a number of products. For five of the seven metabolites isolated, the structure could be established unequivocally. Accumulation of one metabolite, 1,10-dihydroxy-1,10-dihydrofluoren-9-one, indicated the presence of a novel type of dioxygenase, attacking polynuclear aromatic systems in the unusual angular position. Debenzofuran degradation is proposed to likewise proceed via initial angular dioxygenation. One aryl oxygen ether bond, which normally is extremely stable, is thus transformed to a hemiacetal. After spontaneous cleavage and subsequent rearomatization by dehydration, 2,2',3-trihydroxybiphenyl [3-(2-hydroxyphenyl)-catechol] thus results as the immediate product of the first enzymatic reaction in the degradation sequence.

Pseudomonas putida strain CLB 250 (DSM 5232) utilized 2-bromo-, 2-chloro- and 2-fluorobenzoate as sole source of carbon and energy. Degradation is suggested to be initiated by a dioxygenase liberating halide in the first catabolic step. After decarboxylation and rearomatization catechol is produced as a central metabolite which is degraded via the ortho-pathway. After inhibition of ring cleavage activities with 3-chlorocatechol, 2-chlorobenzoate was transformed to catechol in nearly stoichiometric amounts. Other ortho-substituted benzoates like anthranilate and 2-methoxybenzoate seem to be metabolized via the same route.

The TOL plasmid-encoded enzymes of the methylbenzoate pathway in Pseudomonas putida mt-2 cometabolized 3-trifluoromethyl (TFM)-benzoate. Two products, 3-TFM-1,2-dihydroxy-2-hydrobenzoate (3-TFM-DHB) and 2-hydroxy-6-oxo-7,7,7-trifluoro-hepta-2,4-dienoate (7-TFHOD) were identified chemically and by spectroscopic properties. TFM-substituted analogues of the metabolites of the methylbenzoate pathway were generally converted at drastically reduced rates. The catechol-2,3-dioxygenase from Pseudomonas putida showed moderate turnover rates with 3-TFM-catechol. The catechol-1,2-dioxygenase of Rhodococcus rubropertinctus N657 was totally inhibited by 3-TFM-catechol and did not cleave this substrate. Hammett-type analysis showed the catechol-1,2-dioxygenase reaction to be strongly dependent on the electronic nature of the substituents. Electronegative substituents strongly inhibited catechol cleavage. The catechol-2,3-dioxygenase reaction, however, was only moderately sensitive to electronegative substituents.

Alcaligenes eutrophus B9 and Pseudomonas sp. B13 could be adapted to 2-fluorobenzoate as the sole source of carbon and energy. The ability of the A. eutrophus B9 to use this new substrate is clearly based on the defective dihydrodihydroxybenzoate dehydrogenase. Nontoxic 6-fluoro-3,5-cyclohexadiene-1,2-diol-1-carboxylic acid is accumulated instead of 3-fluorocatechol. About 84% of the substrate is dioxygenated to catechol and utilized via the 3-oxoadipate pathway. During continuous adaptation of Pseudomonas sp. B13 regioselectivity of dioxygenation of 2-fluorobenzoate is drastically changed in favor of a 1,2-attack. Consequently, approximately 97% of the substrate is utilized via catechol. A three- to fourfold overproduction of key enzymes of the 3-oxoadipate pathway compensates for the slower turnover rates of the fluorinated substrates.

Methylsalicylate-grown cells of Pseudomonas sp. WR401 cometabolized 3-, 4- and 5-substituted halosalicylates to the corresponding halocatechols. Further degradation was unproductive due to the presence of high levels of catechol 2,3-dioxygenase. This strain acquired the ability to utilize 3-chlorobenzoate following acquisition of genes from Pseudomonas sp. B13 which are necessary for the assimilation of chlorocatechols. This derivative (WR4011) was unable to use 4- or 5-chlorosalicylates. Derivatives able to use these compounds were obtained by plating WR4011 on 5-chlorosalicylate minimal medium; one such derivative was designated WR4016. The acquisition of this property was accompanied by concomitant loss of the methylsalicylate phenotype. During growth on 4- or 5-chlorosalicylate the typical enzymes of chlorocatechol assimilation were detected in cell free extracts, whereas catechol 2,3-dioxygenase activity was not induced. Repeated subcultivation of WR4016 in the presence of 3-chlorosalicylate produced variants (WR4016-1) which grew on all three isomers.

Enzymes of the p-cymene pathway in Pseudomonas putida strains cometabolized the intermediate analogue 4-trifluoromethyl(TFM)benzoate. Three products, 4-TFM-2,3-dihydro-2,3-dihydroxybenzoate, 4-TFM-2,3-dihydroxybenzoate and 2-hydroxy-6-oxo-7,7,7-trifluorohepta-2,4-dienoate (7-TFHOD) were identified chemically and by spectroscopic properties. Certain TFM-substituted analogue metabolites of the p-cymene pathway were transformed at drastically reduced rates. Hammett type analysis of ring cleavage reactions of 4-substituted 2,3-dihydroxybenzoates revealed the negative inductive and especially mesomeric effect of substituents to be rate determining. Whereas decarboxylation of 3-carboxy-7-TFHOD was not affected by fluorine substitution the subsequent hydrolysis of 7-TFHOD proceeded very slowly. The negative inductive effect of the TFM-group probably inhibited heterolysis of the carbon bond between C5 and C6 of 7-TFHOD.

The novel enzyme 4-methyl-2-enelactone methyl-isomerase was detected in, and purified to electrophoretic homogeneity from, p-toluate-grown cells of Rhodococcus rhodocrous N75, a nocardioform actinomycete. The enzyme was very thermostable and had a native Mr of 75,500; as the monomer had an Mr of 17,000, the enzyme is probably tetrameric. The new isomerase is highly specific with respect to its lactone substrate, only accepting (+)-(4S)-4-methylmuconolactone (4-carboxymethyl-4-methylbut-2-en-1,4-olide), and the putative isomerization reaction intermediate 1-methylbislactone ((-)-1-methyl-3,7-dioxo-2,6-dioxabicyclo-[3.3.0]octane) as substrates, and yielding (-)-(4S)-3-methylmuconolactone (4-carboxymethyl-3-methylbut-2-en-1,4-olide) as product. Some other lactone analogues acted as competitive inhibitors. Our data suggest that the isomerization does not involve actual methyl migration, but proceeds via the 1-methybislactone.