Bacterial communities degrading amino- and hydroxynaphthalene-2-sulfonates

A 6-aminonaphthalene-2-sulfonic acid (6A2NS)-degrading mixed bacterial community was isolated from a sample of river Elbe water. The complete degradation of this xenobiotic compound may be described by a mutualistic interaction of two Pseudomonas strains isolated from this culture. One strain, BN6, could also grow on 6A2NS in monoculture, however, with accumulation of black polymers. This organism effected the initial conversion of 6A2NS into 5-aminosalicylate (5AS) through regioselective attack of the naphthalene skeleton in the 1,2-position. 5AS was totally degraded by another member of the community, strain BN9. After prolonged adaptation of strain BN6 to growth on 6A2NS, this organism readily converted all naphthalene-2-sulfonates with OH- or NH2-substituents in the 5-, 6-, 7-, or 8-position. The corresponding hydroxy- or aminosalicylates were excreted in stoichiometric amounts, with the exception that the metabolite from 5A2NS oxidation was not identical with 6AS.     

Bacterial communities degrading amino- and hydroxynaphthalene-2-sulfonates.

A 6-aminonaphthalene-2-sulfonic acid (6A2NS)-degrading mixed bacterial community was isolated from a sample of river Elbe water. The complete degradation of this xenobiotic compound may be described by a mutualistic interaction of two Pseudomonas strains isolated from this culture. One strain, BN6, could also grow on 6A2NS in monoculture, however, with accumulation of black polymers. This organism effected the initial conversion of 6A2NS into 5-aminosalicylate (5AS) through regioselective attack of the naphthalene skeleton in the 1,2-position. 5AS was totally degraded by another member of the community, strain BN9. After prolonged adaptation of strain BN6 to growth on 6A2NS, this organism readily converted all naphthalene-2-sulfonates with OH- or NH2-substituents in the 5-, 6-, 7-, or 8-position. The corresponding hydroxy- or aminosalicylates were excreted in stoichiometric amounts, with the exception that the metabolite from 5A2NS oxidation was not identical with 6AS.     

Biochemical and molecular characterization of a ring fission dioxygenase with the ability to oxidize (substituted) salicylate(s) from Pseudaminobacter salicylatoxidans

The gene coding for a dioxygenase with the ability to cleave salicylate by a direct ring fission mechanism to 2-oxohepta-3,5-dienedioic acid was cloned from Pseudaminobacter salicylatoxidans strain BN12. The deduced amino acid sequence encoded a protein with a molecular mass of 41,176 Da, which showed 28 and 31% sequence identity, respectively, to a gentisate 1,2-dioxygenase from Pseudomonas alcaligenes NCIMB 9867 and a 1-hydroxy-2-naphthoate 1,2-dioxygenase from Nocardioides sp. KP7. The highest degree of sequence identity (58%) was found to a presumed gentisate 1,2-dioxygenase from Corynebacterium glutamicum. The enzyme from P. salicylatoxidans BN12 was heterologously expressed in Escherichia coli and purified as a His-tagged enzyme variant. The purified enzyme oxidized in addition to salicylate, gentisate, 5-aminosalicylate, and 1-hydroxy-2-naphthoate also 3-amino- and 3- and 4-hydroxysalicylate, 5-fluorosalicylate, 3-, 4-, and 5-chlorosalicylate, 3-, 4-, and 5-bromosalicylate, 3-, 4-, and 5-methylsalicylate, and 3,5-dichlorosalicylate. The reactions were analyzed by high pressure liquid chromatography/mass spectrometry, and the reaction products were tentatively identified. For comparison, the putative gentisate 1,2-dioxygenase from C. glutamicum was functionally expressed in E. coli and shown to convert gentisate but not salicylate or 1-hydroxy-2-naphthoate.     

Direct Ring Fission of Salicylate by a Salicylate 1,2-Dioxygenase Activity from Pseudaminobacter salicylatoxidans

In cell extracts of Pseudaminobacter salicylatoxidans strain BN12, an enzymatic activity was detected which converted salicylate in an oxygen-dependent but NAD(P)H-independent reaction to a product with an absorbance maximum at 283 nm. This metabolite was isolated, purified, and identified by mass spectrometry and (1)H and (13)C nuclear magnetic resonance spectroscopy as 2-oxohepta-3,5-dienedioic acid. This metabolite could be formed only by direct ring fission of salicylate by a 1,2-dioxygenase reaction. Cell extracts from P. salicylatoxidans also oxidized 5-aminosalicylate, 3-, 4-, and 5-chlorosalicylate, 3-, 4-, and 5-methylsalicylate, 3- and 5-hydroxysalicylate (gentisate), and 1-hydroxy-2-naphthoate. The dioxygenase was purified and shown to consist of four identical subunits with a molecular weight of about 45,000. The purified enzyme showed higher catalytic constants with gentisate or 1-hydroxy-2-naphthoate than with salicylate. It was therefore concluded that P. salicylatoxidans synthesized a gentisate 1,2-dioxygenase with an extraordinary substrate range, which also allowed the oxidation of salicylate.     

Utilization of aminoaromatic acids by a methanogenic enrichment culture and by a novel<Emphasis Type="Italic"> Citrobacter freundii</Emphasis> strain

Following incubation of mesophilic methanogenic floccular sludge from a lab-scale upflow anaerobic sludge bed reactor used to treat cattle manure wastewater, a stable 5-aminosalicylate-degrading enrichment culture was obtained. Subsequently, a Citrobacter freundii strain, WA1, was isolated from the 5-aminosalicylate-degrading methanogenic consortium. The methanogenic enrichment culture degraded 5-aminosalicylate completely to CH₄, CO₂ and NH₄ ⁺, while C. freundii strain WA1 reduced 5-aminosalicylate with simultaneous deamination to 2-hydroxybenzyl alcohol during anaerobic growth with electron donors such as pyruvate, glucose or serine. When grown on pyruvate, C. freundii WA1 converted 3-aminobenzoate to benzyl alcohol and also reduced benzaldehyde to benzyl alcohol. Pyruvate was fermented to acetate, CO₂, H₂ and small amounts of lactate, succinate and formate. Less lactate (30%) was produced from pyruvate when C. freundii WA1 grew with 5-aminosalicylate as co-substrate.     

Characterization and evolution of anthranilate 1,2-dioxygenase from Acinetobacter sp. strain ADP1

The two-component anthranilate 1,2-dioxygenase of the bacterium Acinetobacter sp. strain ADP1 was expressed in Escherichia coli and purified to homogeneity. This enzyme converts anthranilate (2-aminobenzoate) to catechol with insertion of both atoms of O(2) and consumption of one NADH. The terminal oxygenase component formed an alpha(3)beta(3) hexamer of 54- and 19-kDa subunits. Biochemical analyses demonstrated one Rieske-type [2Fe-2S] center and one mononuclear nonheme iron center in each large oxygenase subunit. The reductase component, which transfers electrons from NADH to the oxygenase component, was found to contain approximately one flavin adenine dinucleotide and one ferredoxin-type [2Fe-2S] center per 39-kDa monomer. Activities of the combined components were measured as rates and quantities of NADH oxidation, substrate disappearance, product appearance, and O(2) consumption. Anthranilate conversion to catechol was stoichiometrically coupled to NADH oxidation and O(2) consumption. The substrate analog benzoate was converted to a nonaromatic benzoate 1,2-diol with similarly tight coupling. This latter activity is identical to that of the related benzoate 1, 2-dioxygenase. A variant anthranilate 1,2-dioxygenase, previously found to convey temperature sensitivity in vivo because of a methionine-to-lysine change in the large oxygenase subunit, was purified and characterized. The purified M43K variant, however, did not hydroxylate anthranilate or benzoate at either the permissive (23 degrees C) or nonpermissive (39 degrees C) growth temperatures. The wild-type anthranilate 1,2-dioxygenase did not efficiently hydroxylate methylated or halogenated benzoates, despite its sequence similarity to broad-substrate specific dioxygenases that do. Phylogenetic trees of the alpha and beta subunits of these terminal dioxygenases that act on natural and xenobiotic substrates indicated that the subunits of each terminal oxygenase evolved from a common ancestral two-subunit component.     

Nitrobenzoates and aminobenzoates are chemoattractants for Pseudomonas strains

Three Pseudomonas strains were tested for the ability to sense and respond to nitrobenzoate and aminobenzoate isomers in chemotaxis assays. Pseudomonas putida PRS2000, a strain that grows on benzoate and 4-hydroxybenzoate by using the beta-ketoadipate pathway, has a well-characterized beta-ketoadipate-inducible chemotactic response to aromatic acids. PRS2000 was chemotactic to 3- and 4-nitrobenzoate and all three isomers of aminobenzoate when grown under conditions that induce the benzoate chemotactic response. P. putida TW3 and Pseudomonas sp. strain 4NT grow on 4-nitrotoluene and 4-nitrobenzoate by using the ortho (beta-ketoadipate) and meta pathways, respectively, to complete the degradation of protocatechuate derived from 4-nitrotoluene and 4-nitrobenzoate. However, based on results of catechol 1,2-dioxygenase and catechol 2,3-dioxygenase assays, both strains were found to use the beta-ketoadipate pathway for the degradation of benzoate. Both strains were chemotactic to benzoate, 3- and 4-nitrobenzoate, and all three aminobenzoate isomers after growth with benzoate but not succinate. Strain TW3 was chemotactic to the same set of aromatic compounds after growth with 4-nitrotoluene or 4-nitrobenzoate. In contrast, strain 4NT did not respond to any aromatic acids when grown with 4-nitrotoluene or 4-nitrobenzoate, apparently because these substrates are not metabolized to the inducer (beta-ketoadipate) of the chemotaxis system. The results suggest that strains TW3 and 4NT have a beta-ketoadipate-inducible chemotaxis system that responds to a wide range of aromatic acids and is quite similar to that present in PRS2000. The broad specificity of this chemotaxis system works as an advantage in strains TW3 and 4NT because it functions to detect diverse carbon sources, including 4-nitrobenzoate.     

Catabolism of benzoate and monohydroxylated benzoates by Amycolatopsis and Streptomyces spp

Eight actinomycetes of the genera Amycolatopsis and Streptomyces were tested for the degradation of aromatic compounds by growth in a liquid medium containing benzoate, monohydroxylated benzoates, or quinate as the principal carbon source. Benzoate was converted to catechol. The key intermediate in the degradation of salicylate was either catechol or gentisate, while m-hydroxybenzoate was metabolized via gentisate or protocatechuate. p-Hydroxybenzoate and quinate were converted to protocatechuate. Catechol, gentisate, and protocatechuate were cleaved by catechol 1,2-dioxygenase, gentisate 1,2-dioxygenase, and protocatechuate 3,4-dioxygenase, respectively. The requirement for glutathione in the gentisate pathway was dependent on the substrate and the particular strain. The conversion of p-hydroxybenzoate to protocatechuate by p-hydroxybenzoate hydroxylase was gratuitously induced by all substrates that were metabolized via protocatechuate as an intermediate, while protocatechuate 3,4-dioxygenase was gratuitously induced by benzoate and salicylate in two Amycolatopsis strains.     

Catabolism of benzoate and monohydroxylated benzoates by Amycolatopsis and Streptomyces spp.

Eight actinomycetes of the genera Amycolatopsis and Streptomyces were tested for the degradation of aromatic compounds by growth in a liquid medium containing benzoate, monohydroxylated benzoates, or quinate as the principal carbon source. Benzoate was converted to catechol. The key intermediate in the degradation of salicylate was either catechol or gentisate, while m-hydroxybenzoate was metabolized via gentisate or protocatechuate. p-Hydroxybenzoate and quinate were converted to protocatechuate. Catechol, gentisate, and protocatechuate were cleaved by catechol 1,2-dioxygenase, gentisate 1,2-dioxygenase, and protocatechuate 3,4-dioxygenase, respectively. The requirement for glutathione in the gentisate pathway was dependent on the substrate and the particular strain. The conversion of p-hydroxybenzoate to protocatechuate by p-hydroxybenzoate hydroxylase was gratuitously induced by all substrates that were metabolized via protocatechuate as an intermediate, while protocatechuate 3,4-dioxygenase was gratuitously induced by benzoate and salicylate in two Amycolatopsis strains.     

Biological degradation of 4-chlorobenzoic acid by a PCB-metabolizing bacterium through a pathway not involving (chloro)catechol

Cupriavidus sp. strain SK-3, previously isolated on polychlorinated biphenyl mixtures, was found to aerobically utilize a wide spectrum of substituted aromatic compounds including 4-fluoro-, 4-chloro- and 4-bromobenzoic acids as a sole carbon and energy source. Other chlorobenzoic acid (CBA) congeners such as 2-, 3-, 2,3-, 2,5-, 3,4- and 3,5-CBA were all rapidly transformed to respective chlorocatechols (CCs). Under aerobic conditions, strain SK-3 grew readily on 4-CBA to a maximum concentration of 5 mM above which growth became impaired and yielded no biomass. Growth lagged significantly at concentrations above 3 mM, however chloride elimination was stoichiometric and generally mirrored growth and substrate consumption in all incubations. Experiments with resting cells, cell-free extracts and analysis of metabolite pools suggest that 4-CBA was metabolized in a reaction exclusively involving an initial hydrolytic dehalogenation yielding 4-hydroxybenzoic acid, which was then hydroxylated to protocatechuic acid (PCA) and subsequently metabolized via the β-ketoadipate pathway. When strain SK-3 was grown on 4-CBA, there was gratuitous induction of the catechol-1,2-dioxygenase and gentisate-1,2-dioxygenase pathways, even if both were not involved in the metabolism of the acid. While activities of the modified ortho- and meta-cleavage pathways were not detectable in all extracts, activity of PCA-3,4-dioxygenase was over ten-times higher than those of catechol-1,2- and gentisate-1,2-dioxygenases. Therefore, the only reason other congeners were not utilized for growth was the accumulation of CCs, suggesting a narrow spectrum of the activity of enzymes downstream of benzoate-1,2-dioxygenase, which exhibited affinity for a number of substituted analogs, and that the metabolic bottlenecks are either CCs or catabolites of the modified ortho-cleavage metabolic route.     

三氯卡班降解菌株Sphingomonas sp. YL-JM2C中多个邻苯二酚双加氧酶的功能

【目的】研究Sphingomonas sp.YL-JM2C菌株的生长特性,确定以三氯卡班作为碳源的生长情况。挖掘菌株YL-JM2C潜在的邻苯二酚1,2-双加氧酶及邻苯二酚2,3-双加氧酶基因,在大肠杆菌(Escherichia coli)中异源表达邻苯二酚双加氧酶基因并研究其酶学性质。【方法】优化S.sp.YL-JM2C菌株以三氯卡班作为碳源时的培养条件,并利用全自动生长曲线测定仪测定菌株生长情况,绘制生长曲线。通过生物信息学方法挖掘潜在的邻苯二酚双加氧酶基因,并分别在Escherichia coli BL21(DE3)中进行异源表达,通过AKTA快速纯化系统纯化蛋白,分别以邻苯二酚、3-和4-氯邻苯二酚为底物检测重组蛋白的酶学特性。【结果】菌株在pH为7.0-7.5时生长最优。在以浓度为4-8 mg/L的三氯卡班做为底物时,菌株适宜生长。当R2A培养基仅含有0.01%酵母提取物和无机盐时,加入终浓度为4 mg/L的三氯卡班可促进菌株生长。挖掘到6个潜在的邻苯二酚双加氧酶基因stcA1、stcA2、stcA3、stcE1、stcE2和stcE3,表达并通过粗酶液分析证明其中5个基因stcA1、stcA2、stcA3、stcE1和stcE2编码的酶均具有邻苯二酚双加氧酶和氯邻苯二酚双加氧酶的活性;纯化酶的底物范围研究揭示了StcA1、StcA2和StcA3均属于Ⅱ型邻苯二酚1,2-双加氧酶,StcE1和StcE2为两个新型邻苯二酚2,3-双加氧酶;它们酶动力学分析研究证明了5个酶对邻苯二酚的亲和力和催化效率最高,4-氯邻苯二酚次之。【结论】在同一菌株中发现了5个具有功能的邻苯二酚双加氧酶基因,stcA1、stcA2和stcA3编码的酶均属于Ⅱ型邻苯二酚1,2-双加氧酶,stcE1和stcE2为两个新型邻苯二酚2,3-双加氧酶编码基因。5个酶均具有催化邻苯二酚和氯邻苯二酚开环反应的功能,这为更好地理解微生物基因组内代谢邻苯二酚及其衍生物氯代邻苯二酚基因的多样性奠定了基础。     

恶臭假单胞菌ND6菌株catA基因的克隆和表达及其儿茶酚裂解途径探讨

恶臭假单胞菌ND6菌株的萘降解质粒pND6-1中编码儿茶酚1,2-双加氧酶的catA基因在大肠杆菌中进行了克隆和表达,并研究表达产物的酶学性质。结果表明:酶的K_m为0.019μmol/L,V_max为1.434μmol/(min.mg);具有很好的耐热性,在50℃保温45min后仍能够保留酶活力的93.7%;Fe²⁺对酶活性有显著的促进作用,其比活力是对照反应的292%;酶对4-氯儿茶酚的催化活性非常低,属于Ⅰ型儿茶酚1,2-双加氧酶。以萘为底物生长时,ND6菌株的细胞提取液中既存在催化邻位裂解途径的儿茶酚1,2-双加氧酶活性,也存在催化间位裂解途径的儿茶酚2,3-双加氧酶活性。以苯甲酸、对羟基苯甲酸和苯乙酸为唯一碳源生长时,ND6菌株细胞提取液的儿茶酚1,2-双加氧酶活性远远大于儿茶酚2,3-双加氧酶活性。表明ND6菌株既能通过儿茶酚间位裂解途径降解萘,也能通过儿茶酚邻位裂解途径降解萘,而以苯甲酸、对羟基苯甲酸和苯乙酸为诱导物时只利用儿茶酚邻位裂解途径。     

A gene cluster involved in degradation of substituted salicylates via ortho cleavage in Pseudomonas sp. strain MT1 encodes enzymes specifically adapted for transformation of 4-methylcatechol and 3-methylmuconate

Pseudomonas sp. strain MT1 has recently been reported to degrade 4- and 5-chlorosalicylate by a pathway assumed to consist of a patchwork of reactions comprising enzymes of the 3-oxoadipate pathway. Genes encoding the initial steps in the degradation of salicylate and substituted derivatives were now localized and sequenced. One of the gene clusters characterized (sal) showed a novel gene arrangement, with salA, encoding a salicylate 1-hydroxylase, being clustered with salCD genes, encoding muconate cycloisomerase and catechol 1,2-dioxygenase, respectively, and was expressed during growth on salicylate and chlorosalicylate. A second gene cluster (cat), exhibiting the typical catRBCA arrangement of genes of the catechol branch of the 3-oxoadipate pathway in Pseudomonas strains, was expressed during growth on salicylate. Despite their high sequence similarities with isoenzymes encoded by the cat gene cluster, the catechol 1,2-dioxygenase and muconate cycloisomerase encoded by the sal cluster showed unusual kinetic properties. Enzymes were adapted for turnover of 4-chlorocatechol and 3-chloromuconate; however, 4-methylcatechol and 3-methylmuconate were identified as the preferred substrates. Investigation of the substrate spectrum identified 4- and 5-methylsalicylate as growth substrates, which were effectively converted by enzymes of the sal cluster into 4-methylmuconolactone, followed by isomerization to 3-methylmuconolactone. The function of the sal gene cluster is therefore to channel both chlorosubstituted and methylsubstituted salicylates into a catechol ortho cleavage pathway, followed by dismantling of the formed substituted muconolactones through specific pathways.     

Intradiol pathway of para-cresol conversion by Rhodococcus opacus 1CP

The growth of Rhodococcus opacus 1CP in medium with different concentrations of p-cresol as the sole source of carbon and energy was studied. It was shown that the optimal concentration of p-cresol was 600 mg/L. The ability of this strain to transform practically all amounts of p-cresol to 4-methylcatechol followed by its utilization through ortho-pathway was shown. New enzymes (4-methylcatechol 1,2-dioxygenase, catechol 1,2-dioxygenase, and methylmuconate cycloisomerase) were purified to homogeneity and characterized. Based on the data obtained on p-cresol degradation, formation of intermediates, and the enzymes participating in this pathway, we suggest an ortho-pathway of p-cresol degradation by R. opacus 1CP through 4-methylcatechol and 3-methyl-cis, cis-muconate.     

Nitrobenzoates and Aminobenzoates Are Chemoattractants for Pseudomonas Strains

Three Pseudomonas strains were tested for the ability to sense and respond to nitrobenzoate and aminobenzoate isomers in chemotaxis assays. Pseudomonas putida PRS2000, a strain that grows on benzoate and 4-hydroxybenzoate by using the β-ketoadipate pathway, has a well-characterized β-ketoadipate-inducible chemotactic response to aromatic acids. PRS2000 was chemotactic to 3- and 4-nitrobenzoate and all three isomers of aminobenzoate when grown under conditions that induce the benzoate chemotactic response. P. putida TW3 and Pseudomonas sp. strain 4NT grow on 4-nitrotoluene and 4-nitrobenzoate by using the ortho (β-ketoadipate) and meta pathways, respectively, to complete the degradation of protocatechuate derived from 4-nitrotoluene and 4-nitrobenzoate. However, based on results of catechol 1,2-dioxygenase and catechol 2,3-dioxygenase assays, both strains were found to use the β-ketoadipate pathway for the degradation of benzoate. Both strains were chemotactic to benzoate, 3- and 4-nitrobenzoate, and all three aminobenzoate isomers after growth with benzoate but not succinate. Strain TW3 was chemotactic to the same set of aromatic compounds after growth with 4-nitrotoluene or 4-nitrobenzoate. In contrast, strain 4NT did not respond to any aromatic acids when grown with 4-nitrotoluene or 4-nitrobenzoate, apparently because these substrates are not metabolized to the inducer (β-ketoadipate) of the chemotaxis system. The results suggest that strains TW3 and 4NT have a β-ketoadipate-inducible chemotaxis system that responds to a wide range of aromatic acids and is quite similar to that present in PRS2000. The broad specificity of this chemotaxis system works as an advantage in strains TW3 and 4NT because it functions to detect diverse carbon sources, including 4-nitrobenzoate.     

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).     

Degradation of 2-chlorobenzoate by in vivo constructed hybrid pseudomonads

Abstract 5-Chlorosalicylate degrading bacteria were obtained from the mating between Pseudomonas sp. strain WR401 and Pseudomonas sp. strain B13. Further selection of the hybrid organisms for growth on 2-chlorobenzoate allowed the isolation of strains such as JH230. During growth on 2-chlorobenzoate stoichiometric amounts of chloride were released. Steps in the pathway for 2-chlorobenzoate degradation were determined by simultaneous adaptation studies, assays of enzymes in cell extracts and cooxidation of the analogous substrate 2-methylbenzoate. Results indicate that 2-chlorobenzoate was degraded to 3-chlorocatechol. Ring cleavage of 3-chlorocatechol was by a catechol 1,2-dioxygenase to from 2-chloro- cis, cis - muconate. Further degradation runs via 4-carboxymethylenebut-2-en-4-olide.     

Induction of aromatic ring: cleavage dioxygenases in <Emphasis Type="Italic">Stenotrophomonas maltophilia</Emphasis> strain KB2 in cometabolic systems

Stenotrophomonas maltophilia KB2 is known to produce different enzymes of dioxygenase family. The aim of our studies was to determine activity of these enzymes after induction by benzoic acids in cometabolic systems with nitrophenols. We have shown that under cometabolic conditions KB2 strain degraded 0.25–0.4 mM of nitrophenols after 14 days of incubation. Simultaneously degradation of 3 mM of growth substrate during 1–3 days was observed depending on substrate as well as cometabolite used. From cometabolic systems with nitrophenols as cometabolites and 3,4-dihydroxybenzoate as a growth substrate, dioxygenases with the highest activity of protocatechuate 3,4-dioxygenase were isolated. Activity of catechol 1,2- dioxygenase and protocatechuate 4,5-dioxygenase was not observed. Catechol 2,3-dioxygenase was active only in cultures with 4-nitrophenol. Ability of KB2 strain to induce and synthesize various dioxygenases depending on substrate present in medium makes this strain useful in bioremediation of sites contaminated with different aromatic compounds.     

Microbial metabolism of chlorosalicylates: accelerated evolution by natural genetic exchange

Methylsalicylate-grown cells of Pseudomonas sp. WR 401 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. B 13 which are necessary for the assimilation of chlorocatechols. This derivative (WR 4011) was unable to use 4- or 5-chlorosalicylates. Derivatives able to use these compounds were obtained by plating WR 4011 on 5-chlorosalicylate minimal medium; one such derivative was designated WR 4016. 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 WR 4016 in the presence of 3-chlorosalicylate produced variants (WR 4016-1) which grew on all three isomers.Abbreviations CS chlorosalicylate - MS methylsalicylate - 3CB 3-chlorobenzoate - nal^r nalidixin-resistant - str^r streptomycin-resistant - C230 catechol-2,3-dioxygenase - C120 catechol-1,2-dioxygenase - HMSH 2-hydroxymuconic semialdehyde hydrolase or 2-hydroxy-6-oxo-hexa-2,4-dienoic acid-hydrolase - HMSD 2-hydroxymuconic semialdehyde dehydrogenase - Dienlacton hydrolase 4-carboxymethylenebut-2-en-4-olide hydrolase