Enzymatic dehalogenation of pentachlorophenol by extracts from Arthrobacter sp. strain ATCC 33790.

Arthrobacter sp. strain ATCC 33790 was grown with pentachlorophenol (PCP) as the sole source of carbon and energy. Crude extracts, which were prepared by disruption of the bacteria with a French pressure cell, showed no dehalogenating activity with PCP as the substrate. After sucrose density ultracentrifugation of the crude extract at 145,000 x g, various layers were found in the gradient. One yellow layer showed enzymatic conversion of PCP. One chloride ion was released per molecule of PCP. The product of the enzymatic conversion was tetrachlorohydroquinone. NADPH and oxygen were essential for this reaction. EDTA stimulated the enzymatic activity by 67%. The optimum pH for the enzyme activity was 7.5, and the temperature optimum was 25 degrees C. Enzymatic activity was also detected with 2,4,5-trichlorophenol, 2,3,4-trichlorophenol, 2,4,6-trichlorophenol, and 2,3,4,5-tetrachlorophenol as substrates, whereas 3,4,5-trichlorophenol, 2,4-dichlorophenol, 3,4-dichlorophenol, and 4-chlorophenol did not serve as substrates.     

Pentachlorophenol removal from slightly acidic mineral salts,commercial sand,and clay soil by recovered Arthrobacter strain ATCC 33790

Arthrobacter strain ATCC 33790, a pentachlorophenol (PCP)-metabolizer isolated by the author, has been recovered after 10 years of storage. The freeze-dried preparation grown on half-strength Trypticase Soy Broth adapted to utilize PCP within 1 week. Cultures grown on PCP-nutrient agar were found to utilize PCP in mineral salts medium within 2–3 days. The culture was prepared for continuous growth at pH 6.5 by successive feeding of 100–110 mg solid aliquots of PCP to a 1-l culture initially grown at pH 7.4. Continuous culture growth at pH 6.5 was possible on a mineral salts feed containing 1800 ppm PCP. Continuous cultures grown at pH 6.7 on mineral salts feeds containing 500 and 340 mg PCP/l were especially efficient in removing PCP. Less than 4 mg PCP/l were detected in the effluent at dilution rates near washout. In batch culture studies at pH 6.5 the PCP utilization kinetics were found to be similar at low PCP concentration to those at pH 7.4 for the approximately same inoculum size. Utilization of 35 mg PCP/l was very slow at pH 6.0. Growth rates at pH 6.5 at controlled PCP concentration ranges of 5–35 and 75–115 mg/l were 0.09 h^–1 and 0.05 h^–1, respectively. The ability of strain ATCC 33790 to utilize PCP in mineral salts media containing naphthalene, methylnaphthalenes, and cresols was examined. Naphthalene, 1-, and 2-methylnaphthalenes at their solubility limit, and o- and m-cresols at 900–1000 mg/l prevented utilization of 80–90 mg PCP/l. PCP was rapidly removed from both commercial sand at 30°C and from clay soil at room temperature. Estimated inoculum sizes of 6.6 × 10⁶, 6.6 × 10⁴, and 656 cells/g were found to be effective in removing approximately half the starting amount of PCP from sand in 3, 19, and 42 h, respectively. Nearly complete disappearance of extractable PCP was observed after 1 day in clay soil inoculated with 6 × 10⁶ cells/g.     

Cloning and sequence analysis of genes for dehalogenation of 4-chlorobenzoate from Arthrobacter sp. strain SU.

Strains of Arthrobacter catalyze a hydrolytic dehalogenation of 4-chlorobenzoate (4-CBA) to p-hydroxybenzoate. The reaction requires ATP and coenzyme A (CoA), indicating activation of the substrate via a thioester, like that reported for Pseudomonas sp. strain CBS3 (J. D. Scholten, K.-H. Chang, P. C. Babbit, H. Charest, M. Sylvestre, and D. Dunaway-Mariano, Science 253:182-185, 1991). The dehalogenase genes of Arthrobacter sp. strain SU were cloned and expressed in Escherichia coli. Analyses of deletions indicate that dehalogenation depends on three open reading frames (ORFs) which are organized in an operon. There is extensive sequence homology to corresponding gene products in Pseudomonas sp. strain CBS3, suggesting that ORF1 and ORF2 encode a 4-CBA-CoA-ligase and a 4-CBA-CoA dehalogenase, respectively. ORF3 possibly represents a thioesterase, although no homology to the enzyme from Pseudomonas sp. strain CBS3 exists.     

Cloning and sequence analysis of genes for dehalogenation of 4-chlorobenzoate from Arthrobacter sp. strain SU.

Strains of Arthrobacter catalyze a hydrolytic dehalogenation of 4-chlorobenzoate (4-CBA) to p-hydroxybenzoate. The reaction requires ATP and coenzyme A (CoA), indicating activation of the substrate via a thioester, like that reported for Pseudomonas sp. strain CBS3 (J. D. Scholten, K.-H. Chang, P. C. Babbit, H. Charest, M. Sylvestre, and D. Dunaway-Mariano, Science 253:182-185, 1991). The dehalogenase genes of Arthrobacter sp. strain SU were cloned and expressed in Escherichia coli. Analyses of deletions indicate that dehalogenation depends on three open reading frames (ORFs) which are organized in an operon. There is extensive sequence homology to corresponding gene products in Pseudomonas sp. strain CBS3, suggesting that ORF1 and ORF2 encode a 4-CBA-CoA-ligase and a 4-CBA-CoA dehalogenase, respectively. ORF3 possibly represents a thioesterase, although no homology to the enzyme from Pseudomonas sp. strain CBS3 exists.     

Enzymatic dehalogenation of 4-chlorobenzoate by extracts from Arthrobacter sp. SU DSM 20407

In extracts from Arthrobacter sp. SU DSM 20407 an enzyme was detectable, that converted 4-chlorobenzoate into 4-hydroxybenzoate. This conversion was also observed when no oxygen was present in the reaction mixture. Boiling for 5 min destroyed the enzyme activity. 4-Bromo- and 4-iodobenzoate were substrates for the enzyme too, but not 4-fluorobenzoate, 4-chlorophenylacetate and 4-chlorocinnamic acid. The enzyme showed optimum activity at 16 degrees C and at pH 7-7.5. The specific activity in the extracts varied between 0.5 and 5 mU/mg of protein. Zn2+ and Cu2+ inhibited the enzyme, while H2O2 slightly activated. In contrast to all other 4-chlorobenzoate dehalogenases described before the enzyme was not inhibited by EDTA, nor was it activated by Mn2+. Other divalent ions also had no effect. The molecular mass of the enzyme was 45,000 +/- 5,000 Da as judged by gel-filtration.     

Purification and properties of pentachlorophenol hydroxylase, a flavoprotein from Flavobacterium sp. strain ATCC 39723.

A pentachlorophenol (PCP) hydroxylase which catalyzed the conversion of PCP to 2,3,5,6-tetrachlorohydroquinone and released iodide from triiodophenol in the presence of NADPH and oxygen was identified. The enzyme was purified by protamine sulfate precipitation, ammonium sulfate precipitation, hydrophobic chromatography, anion-exchange chromatography, gel filtration chromatography, and crystallization. The enzyme was a monomer with a molecular weight of 63,000. Under certain conditions, dimer and multimer conformations were also observed. The pI of the enzyme was pH 4.3. The optimal conditions for activity were a pH of 7.5 to 8.5 and a temperature of 40 degrees C. Each enzyme molecule contained one flavin adenine dinucleotide molecule. The Km for PCP was 30 microM and the Vmax was 16 mumol/min/mg of protein. The enzymatic reaction required 2 mol of NADPH per mol of halogenated substrate. On the basis of the data we present, it is likely that PCP hydroxylase is a flavoprotein monooxygenase. The addition of flavins to the reaction mixture did not stimulate the enzymatic reaction; however, we identified the photodegradation of triiodophenol and tribromophenol, but not PCP, by flavin mononucleotide or riboflavin and light.     

Mechanism of Nitrification by Arthrobacter sp

Resting cells of Arthrobacter sp. excrete as much as 60 mug of hydroxylamine-nitrogen per ml when supplied with ammonium. An organic carbon source in abundant supply is necessary for the oxidation. Resting cells oxidize hydroxylamine to nitrite and 1-nitrosoethanol, the former accumulating only when an exogenous carbon source is available. Cell-free extracts contain an enzyme catalyzing the formation of hydroxylamine from acetohydroxamic acid, a hydroxylamine-nitrite oxido-reductase, and an enzyme producing nitrite and nitrate from various primary nitro compounds. Nitrite is not produced from hydroxylamine by the extracts, but 1-nitrosoethanol is formed from hydroxylamine in the presence of acetate. 1-Nitrosoethanol is also produced from acetohydroxamic acid by these preparations. Nitrite was formed from hydroxylamine, however, by extracellular enzymes excreted by the bacterium.     

Confirmation of oxidative dehalogenation of pentachlorophenol by a Flavobacterium pentachlorophenol hydroxylase.

Pentachlorophenol (PCP) hydroxylase purified from Flavobacterium sp. strain ATCC 39723 converted PCP or 2,3,5,6-tetrachlorophenol to tetrachloro-p-hydroquinone (TeCH) with the co-consumption of O2 and NADPH. The purified enzyme incorporated 18O from 18O2 but not from H218O into the reaction end product TeCH. The results clearly demonstrate that PCP is oxidatively converted to TeCH by a monooxygenase-type enzyme from Flavobacterium sp. strain ATCC 39723.     

A new lipopeptide biosurfactant produced by Arthrobacter sp. strain MIS38.

A biosurfactant termed arthrofactin produced by Arthrobacter species strain MIS38 was purified and chemically characterized as 3-hydroxydecanoyl-D-leucyl-D-asparagyl-D-threonyl-D- leucyl-D-leucyl-D-seryl-L-leucyl-D-seryl-L-isoleucyl-L-isoleucyl-L-as paragyl lactone. Surface activity of arthrofactin was examined, with surfactin as a control. Critical micelle concentration values of arthrofactin and surfactin were around 1.0 x 10(-5) M and 7.0 x 10(-5) M at 25 degrees C, respectively. Arthrofactin was found to be five to seven times more effective than surfactin. The minimum surface tension value of arthrofactin was 24 mN/m at a concentration higher than the critical micelle concentration. According to the oil displacement assay, arthrofactin was a better oil remover than synthetic surfactants, such as Triton X-100 and sodium dodecyl sulfate. Arthrofactin is one of the most effective lipopeptide biosurfactants.     

3,4-Dihydroxyxanthone dioxygenase from Arthrobacter sp. strain GFB100.

Bacterial extradiol ring-fission dioxygenases play a critical role in the transformation of multiring aromatic compounds to more readily biodegradable aromatic or aliphatic intermediates. Arthrobacter sp. strain GFB100 utilizes an extradiol meta-fission dioxygenase, 3,4-dihydroxyxanthone dioxygenase (DHXD), in the catabolism of the three-ring oxygen heterocyclic compound xanthone. In this paper, we show that DHXD is a cytosolic enzyme, induced by growth on xanthone and maximally expressed during the stationary phase of growth. In addition, we characterize the DHXD activity in terms of its basic enzymological properties. 1,10-Phenanthroline and H2O2 treatments eliminated DHXD activity, indicating that the enzyme required Fe2+ ions for activity. Other divalent cations were either inhibitory or had no effect on activity. DHXD had a temperature optimum of 30 degrees C and a pH optimum of 7.0. DHXD followed typical saturation kinetics and had an apparent Km of 10 microM for 3,4-dihydroxyxanthone. The dye celestine blue served as a noncompetitive DHXD inhibitor (Ki, 5 microM). Several other structural analogs served neither as substrates nor inhibitors. DHXD was thermally labile at temperatures above 40 degrees C. The half-life for thermal DHXD inactivation was 5 min at 40 degrees C. DHXD activity was completely stable through one freeze-thaw cycle, and about 80% of the DHXD activity remained after 2 days of incubation at 0 degree C. The apparent tight binding of the Fe2+ cofactor to DHXD may be a factor contributing to the stability of this extradiol dioxygenase when it is stored.     

3,4-Dihydroxyxanthone dioxygenase from Arthrobacter sp. strain GFB100.

Bacterial extradiol ring-fission dioxygenases play a critical role in the transformation of multiring aromatic compounds to more readily biodegradable aromatic or aliphatic intermediates. Arthrobacter sp. strain GFB100 utilizes an extradiol meta-fission dioxygenase, 3,4-dihydroxyxanthone dioxygenase (DHXD), in the catabolism of the three-ring oxygen heterocyclic compound xanthone. In this paper, we show that DHXD is a cytosolic enzyme, induced by growth on xanthone and maximally expressed during the stationary phase of growth. In addition, we characterize the DHXD activity in terms of its basic enzymological properties. 1,10-Phenanthroline and H2O2 treatments eliminated DHXD activity, indicating that the enzyme required Fe2+ ions for activity. Other divalent cations were either inhibitory or had no effect on activity. DHXD had a temperature optimum of 30 degrees C and a pH optimum of 7.0. DHXD followed typical saturation kinetics and had an apparent Km of 10 microM for 3,4-dihydroxyxanthone. The dye celestine blue served as a noncompetitive DHXD inhibitor (Ki, 5 microM). Several other structural analogs served neither as substrates nor inhibitors. DHXD was thermally labile at temperatures above 40 degrees C. The half-life for thermal DHXD inactivation was 5 min at 40 degrees C. DHXD activity was completely stable through one freeze-thaw cycle, and about 80% of the DHXD activity remained after 2 days of incubation at 0 degree C. The apparent tight binding of the Fe2+ cofactor to DHXD may be a factor contributing to the stability of this extradiol dioxygenase when it is stored.     

Metabolism of cyclohexaneacetic acid and cyclohexanebutyric acid by Arthrobacter sp. strain CA1.

A strain of Arthrobacter was isolated by enrichment culture with cyclohexaneacetate as the sole source of carbon and grew with a doubling time of 4.2 h. In addition to growing with cyclohexaneacetate, the organism also grew with cyclohexanebutyrate at concentrations not above 0.05%, and with a variety of alicyclic ketones and alcohols. Oxidation of cyclohexaneacetate proceeded through formation of the coenzyme A (CoA) ester followed by initiation of a beta-oxidation cycle. beta-Oxidation was blocked before the second dehydrogenation step due to the formation of a tertiary alcohol, and the side chain was eliminated as acetyl-CoA by the action of (1-hydroxycyclohexan-1-yl)acetyl-CoA lyase. The cyclohexanone thus formed was degraded by a well-described route that involves ring-oxygen insertion by a biological Baeyer-Villiger oxygenase. All enzymes of the proposed metabolic sequence were demonstrated in cell-free extracts. Arthrobacter sp. strain CA1 synthesized constitutive beta-oxidative enzymes, but further induction of enzymes active toward cyclohexaneacetate and its metabolites could occur during growth with the alicyclic acid. Other enzymes of the sequence, (1-hydroxycyclohexan-1-yl)acetyl-CoA lyase and enzymes of cyclohexanone oxidation, were present at negligible levels in succinate-grown cells but induced by growth with cyclohexaneacetate. The oxidation of cyclohexanebutyrate was integrated into the pathway for cyclohexaneacetate oxidation by a single beta-oxidation cycle. Oxidation of the compound could be divided into two phases. Initial oxidation to (1-hydroxycyclohexan-1-yl)acetate could be catalyzed by constitutive enzymes, whereas the further degradation of (1-hydroxycyclohexan-1-yl)acetate was dependent on induced enzyme synthesis which could be inhibited by chloramphenicol with the consequent accumulation of cyclohexaneacetate and (1-hydroxycyclohexan-1-yl)acetate.     

New metabolites in the degradation of fluorene by Arthrobacter sp. strain F101.

Identification of new metabolites and demonstration of key enzyme activities support and extend the pathways previously reported for fluorene metabolism by Arthrobacter sp. strain F101. Washed-cell suspensions of strain F101 with fluorene accumulated 9-fluorenone, 4-hydroxy-9-fluorenone, 3-hydroxy-1-indanone, 1-indanone, 2-indanone, 3-(2-hydroxyphenyl) propionate, and a compound tentatively identified as a formyl indanone. Incubations with 2-indanone produced 3-isochromanone. The growth yield with fluorene as a sole source of carbon and energy corresponded to an assimilation of about 34% of fluorene carbon. About 7.4% was transformed into 9-fluorenol, 9-fluorenone, and 4-hydroxy-9-fluorenone. Crude extracts from fluorene-induced cells showed 3,4-dihydrocoumarin hydrolase and catechol 2,3-dioxygenase activities. These results and biodegradation experiments with the identified metabolites indicate that metabolism of fluorene by Arthrobacter sp. strain F101 proceeds through three independent pathways. Two productive routes are initiated by dioxygenation at positions 1,2 and 3,4, respectively. meta cleavage followed by an aldolase reaction and loss of C-1 yield the detected indanones. Subsequent biological Baeyer-Villiger reactions produce the aromatic lactones 3,4-dihydrocoumarin and 3-isochromanone. Enzymatic hydrolysis of the former gives 3-(2-hydroxyphenyl) propionate, which could be a substrate for a beta oxidation cycle, to give salicylate. Further oxidation of the latter via catechol and 2-hydroxymuconic semialdehyde connects with the central metabolism, allowing the utilization of all fluorene carbons. Identification of 4-hydroxy-9-fluorenone is consistent with an alternative pathway initiated by monooxygenation at C-9 to give 9-fluorenol and then 9-fluorenone. Although dioxygenation at 3,4 positions of the ketone apparently occurs, this reaction fails to furnish a subsequent productive oxidation of this compound.     

Physiological factors affecting carbon tetrachloride dehalogenation by the denitrifying bacterium Pseudomonas sp. strain KC.

Pseudomonas sp. strain KC was grown on a medium with a low content of transition metals in order to examine the conditions for carbon tetrachloride (CT) transformation. Several carbon sources, including acetate, glucose, glycerol, and glutamate, were able to support CT transformation. The chelators 2,2'-dipyridyl and 1,10-phenanthroline stimulated CT transformation in a rich medium that otherwise did not support this activity. Low (< 10 microM) additions of dissolved iron(II), iron(III), and cobalt(II), as well as an insoluble iron(III) compound, ferric oxyhydroxide, inhibited CT transformation. The addition of 50 microM iron to actively growing cultures resulted in delayed inhibition of CT transformation. CT transformation was seen in aerobic cultures of KC, but with reduced efficiency compared with denitrifying cultures. Inhibition of CT transformation by iron was also seen in aerobically grown cultures. Optimal conditions were used in searching for effective CT transformation activity among denitrifying enrichments grown from samples of aquifer material. No activity comparable to that of Pseudomonas sp. strain KC was found among 16 samples tested.     

Complete biodegradation of 4-fluorocinnamic acid by a consortium comprising Arthrobacter sp. strain G1 and Ralstonia sp. strain H1

A consortium of the newly isolated bacterial strains Arthrobacter sp. strain G1 and Ralstonia sp. strain H1 utilized 4-fluorocinnamic acid for growth under aerobic conditions. Strain G1 converted 4-fluorocinnamic acid into 4-fluorobenzoic acid and used the two-carbon side chain for growth, with some formation of 4-fluoroacetophenone as a dead-end side product. In the presence of strain H1, complete mineralization of 4-fluorocinnamic acid and release of fluoride were obtained. Degradation of 4-fluorocinnamic acid by strain G1 occurred through a β-oxidation mechanism and started with the formation of 4-fluorocinnamoyl-coenzyme A (CoA), as indicated by the presence of 4-fluorocinnamoyl-CoA ligase. Enzymes for further transformation were detected in cell extract, i.e., 4-fluorocinnamoyl-CoA hydratase, 4-fluorophenyl-β-hydroxy propionyl-CoA dehydrogenase, and 4-fluorophenyl-β-keto propionyl-CoA thiolase. Degradation of 4-fluorobenzoic acid by strain H1 proceeded via 4-fluorocatechol, which was converted by an ortho-cleavage pathway.     

Production of surfactant by Arthrobacter paraffineus ATCC 19558

A. paraffineus ATCC 19558 grown in MMSM (modified mineral salts medium) containing hydrocarbon produced surfactant, with a maximum CMC(-1) value obtained by using hexadecane as the carbon source. No activity of surface active agent in whole broth was observed when glucose was used in the MMSM instead of hexadecane. The biomass concentration obtained with glucose was about 40% of that obtained with hexadecane. Glucose (4%) in the medium contaning hexadecane caused a 27 and 21% decrease of biomass and surfactant concentrations, respectively. In the process of surfactant production, glucose can be used as a carbon source for growth, and hexadecane added later can serve for production of the surface active agent. The optimum temperature for production of surfactant is 27 degrees C.     

Genome sequence of the curdlan-producing Agrobacterium sp. strain ATCC 31749

Agrobacterium sp. ATCC 31749 is an industrial strain for the commercial production of curdlan, an important exopolysaccharide with food and medical applications. Here we report the genome sequence of the curdlan-producing strain ATCC 31749. Genome sequencing is the first step toward the understanding of regulation of curdlan biosynthesis.     

Cloning of Pseudomonas sp. strain CBS3 genes specifying dehalogenation of 4-chlorobenzoate.

The degradation of 4-chlorobenzoate (4-CBA) by Pseudomonas sp. strain CBS3 is thought to proceed first by the dehalogenation of 4-CBA to 4-hydroxybenzoate (4-HBA), which is then metabolized following the protocatechuate branch of the beta-ketoadipate pathway. The cloning of the 4-CBA dehalogenation system was carried out by constructing a gene bank of Pseudomonas sp. strain CBS3 in Pseudomonas putida. Hybrid plasmid pPSA843 contains a 9.5-kilobase-pair fragment derived from the chromosome of Pseudomonas sp. strain CBS3. This plasmid confers on P. putida the ability to dehalogenate 4-CBA and grow on 4-CBA as the only source of carbon. However, pPSA843 did not complement mutants of P. putida unable to grow on 4-HBA (POB-), showing that the genes involved in the metabolism of 4-HBA were not cloned. Subcloning of Pseudomonas sp. strain CBS3 genes revealed that most of the insert is required for the dehalogenation of 4-CBA, suggesting that more than one gene product is involved in this dehalogenation.     

节杆菌(Arthrobacter sp.)降解阿魏酸的效能

[背景]蓄积在土壤中的阿魏酸类化感自毒物质对农作物生长产生危害,利用有益微生物分解该类物质是一项有效的治理措施.[目的]从自然界土壤分离获得能高效降解阿魏酸的菌株,并评估典型环境因子对降解效能的影响,以期为该菌在阿魏酸类自毒物质降解领域中的应用提供理论依据.[方法]采用一次性投加高浓度化合物的驯化方法分离筛选得到能有效...