Arthrobacter Chlorophenolicus A6 Research Articles

Improving the catalytic activity of difructose anhydride III hydrolase from Arthrobacter chlorophenolicus A6 by modification of two residues

Inulin is an important fructan in nature, which provides energy to organisms by different metabolic pathways. In one of the metabolic pathways, inulin can be converted to difructose anhydride III (DFA-III) by DFA-III-forming inulin fructotransferase (IFTase-III) and DFA-III can be further hydrolyzed to inulobiose by difructose anhydride III hydrolase (DFA-IIIase). Inulobiose is a kind of fructooligosaccharide that can play a role of prebiotics. However, the low activity of DFA-IIIase is the key factor restricting the production and investigation of inulobiose. Therefore, in this work, the enzymatic activity was attempted to be improved by protein engineering. According to the sequence analysis and homologous modeling, several amino acids involved in the active site and the loop region above the active pocket were modified and eight mutants were constructed. The mutants E389A and E91K obtained significantly increased activities, which were 3.63 and 2.57 times that of the wild-type AcDFA-Ⅲase, respectively. The higher activity of E389A and E91K was possibly ascribed to the higher hydrophobicity of Ala389 and tighter catalytic pocket area formed between Lys91 and His158, respectively. The work explored how to enhance the activity of DFA-IIIase, laying a foundation for mass production, further investigation, and application of inulobiose.

Mass spectral imaging showing the plant growth-promoting rhizobacteria's effect on the Brachypodium awn.

The plant growth-promoting rhizobacteria (PGPR) on the host plant surface play a key role in biological control and pathogenic response in plant functions and growth. However, it is difficult to elucidate the PGPR effect on plants. Such information is important in biomass production and conversion. Brachypodium distachyon (Brachypodium), a genomics model for bioenergy and native grasses, was selected as a C3 plant model; and the Gram-negative Pseudomonas fluorescens SBW25 (P.) and Gram-positive Arthrobacter chlorophenolicus A6 (A.) were chosen as representative PGPR strains. The PGPRs were introduced to the Brachypodium seed's awn prior to germination, and their possible effects on the seeding and growth were studied using different modes of time-of-flight secondary ion mass spectrometry (ToF-SIMS) measurements, including a high mass-resolution spectral collection and delayed image extraction. We observed key plant metabolic products and biomarkers, such as flavonoids, phenolic compounds, fatty acids, and auxin indole-3-acetic acid in the Brachypodium awns. Furthermore, principal component analysis and two-dimensional imaging analysis reveal that the Brachypodium awns are sensitive to the PGPR, leading to chemical composition and morphology changes on the awn surface. Our results show that ToF-SIMS can be an effective tool to probe cell-to-cell interactions at the biointerface. This work provides a new approach to studying the PGPR effects on awn and shows its potential for the research of plant growth in the future.

Linking Increased Isotope Fractionation at Low Concentrations to Enzyme Activity Regulation: 4-Cl Phenol Degradation by Arthrobacter chlorophenolicus A6.

Slow microbial degradation of organic trace chemicals (“micropollutants”) has been attributed to either downregulation of enzymatic turnover or rate-limiting substrate supply at low concentrations. In previous biodegradation studies, a drastic decrease in isotope fractionation of atrazine revealed a transition from rate-limiting enzyme turnover to membrane permeation as a bottleneck when concentrations fell below the Monod constant of microbial growth. With degradation of the pollutant 4-chlorophenol (4-CP) by Arthrobacter chlorophenolicus A6, this study targeted a bacterium which adapts its enzyme activity to concentrations. Unlike with atrazine degradation, isotope fractionation of 4-CP increased at lower concentrations, from ε(C) = −1.0 ± 0.5‰ in chemostats (D = 0.090 h–1, 88 mg L–1) and ε(C) = −2.1 ± 0.5‰ in batch (c0 = 220 mg L–1) to ε(C) = −4.1 ± 0.2‰ in chemostats at 90 μg L–1. Surprisingly, fatty acid composition indicated increased cell wall permeability at high concentrations, while proteomics revealed that catabolic enzymes (CphCI and CphCII) were differentially expressed at D = 0.090 h–1. These observations support regulation on the enzyme activity level—through either a metabolic shift between catabolic pathways or decreased enzymatic turnover at low concentrations—and, hence, reveal an alternative end-member scenario for bacterial adaptation at low concentrations. Including more degrader strains into this multidisciplinary analytical approach offers the perspective to build a knowledge base on bottlenecks of bioremediation at low concentrations that considers bacterial adaptation.

Co-metabolic biodegradation of 4-bromophenol in a mixture of pollutants system by Arthrobacter chlorophenolicus A6.

Brominated phenols are listed as priority pollutants together with nitrophenol and chlorophenol are the key components of paper pulp wastewater. However, the biodegradation of bromophenol in a mixed substrate system is very scanty. In the present investigation, simultaneous biodegradation kinetics of three substituted phenols 4-bromophenol (4-BP), 4-nitrophenol (4-NP), and 4-chlorophenol (4-CP) were investigated using Arthrobacter chlorophenolicus A6. A 23 full factorial design was applied with varying 4-BP and 4-CP from 75-125 mg/L and 4-NP from 50-100 mg/L. Almost complete degradation of this mixture of substituted phenols was achieved at initial concentration combinations of 125, 125, and 100 mg/L of 4-CP, 4-BP, and 4-NP, respectively, in 68 h. Statistical analysis of the results revealed that, among the three variables, 4-NP had the most prominent influence on the degradation of both 4-CP and 4-BP, while the concentration of 4-CP had a strong negative interaction effect on the biodegradation of 4-NP. Irrespective of the concentration levels of these three substrates, 4-NP was preferentially biodegraded over 4-CP and 4-BP. Furthermore, 4-BP biodegradation rates were found to be higher than those of 4-CP, followed by 4-NP. Besides, the variation of the biomass yield coefficient of the culture was investigated at different initial concentration combinations of these substituted phenols. Although the actinomycetes consumed 4-NP at a faster rate, the biomass yield was very poor. This revealed that the microbial cells were more stressed when grown on 4-NP compared to 4-BP and 4-CP. Overall, this study revealed the potential of A. chlorophenolicus A6 for the degradation of 4-BP in mixed substrate systems.

Kinetics of simultaneous degradation of 4-bromophenol and 4-chlorophenol by Arthrobacter chlorophenolicusA6

Biodegradation of p-bromophenol (4-BP) along with p-chlorophenol (4-CP) was investigated in batch shake flasks using a actinomycetes strain of Arthrobacterchlorophenolicus A6. A Two level full factorial design at two different levels (low and high) was applied to perform the biodegradation of 4-CP and 4-BP in the mixed substrate system. Result reveals that the presence of 4-CP in low concentration range in the mixture (20–60 mgl-1) did not inhibit 4-BP biodegradation by the actinomycetes. However, at high concentration range of 4-CP (100-200 mgl-1), the 4-BP biodegradation was inhibited. Further, 4-BP degradation was faster than 4-CP. In order to study variation in the specific degradation rate of these pollutants and their interaction effect exist between them, the experimental data were fitted to a sum kinetics model. The experimental data have been fitted with the model with a high correlation coefficient value (R2> 0.94). The model fitted data reveals a strong negative interaction effect of 4-CP on biodegradation of 4-BP by the A. chlorophenolicusA6.

Kinetic of 4-Chlorophenols Biodegradation by Arthrobacter Chlorophenolicus A6

Chlorophenols are listed as priority pollutants both by European community and US EPA. Biodegradation of pchlorophenol (4-CP) was investigated in batch shake flasks by Arthrobacter chlorophenolicus A6 at initial 4-CP concentrations between 25 to 350 mgl-1. The rate of 4-CP removal decreased with increasing initial 4-CP concentrations due to toxic effects on the microorganisms. The growth and biodegradation kinetic of the culture was evaluated. The batch growth profile of the A chlorophenolicus A6 followed substrate inhibition kinetics with the estimated biokinetic parameters of Ksi = 272 mgl-1, Ks = 65 mgl-1 for 4-CP respectively. High inhibition constant (KSI = 272 mg -l) with a KSI/KS ratio of 4.18 indicates superior 4-CP biodegradation potential of the A chlorophenolicus A6. The maximum rate of 4-CP degradation has been achieved at an optimum substrate concentration of Smax = (KSKSI)1/2 = (65×272)1/2 = 133 mgl−1.

Bioconversion of inulin to difructose anhydride III by a novel inulin fructotransferase from Arthrobacter chlorophenolicus A6

The gene encoding difructose anhydride III (DFA III)-forming inulin fructotransferase (IFTase) from Arthrobacter chlorophenolicus A6 was cloned and overexpressed in Escherichia.coli. The recombinant IFTase (DFA III-forming) was purified using one-step nickel affinity chromatography. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration analyses, the enzyme showed a homotrimeric form composed of three identical subunits, each with an apparent molecular mass of 43 kDa. The maximum catalytic activity was shown at 65 °C and pH 5.5, and the specific activity was measured as 902 U mg−1. The enzyme showed remarkable thermostability and over half of the initial activity was retained after incubation at 80 °C for 1 h. The Km and Vmax were estimated to 12.93 mM and 2.89 μmol min−1 ml−1, respectively. After complete hydrolysis of inulin for DFA III production by the purified enzyme, the produced minor products contained sucrose (GF), 1-kestose (GF2), and nystose (GF3). The smallest substrate was identified to be GF3. When 50, 100, and 200 g l−1 of inulin were hydrolyzed by the purified IFTase, the DFA III yield reached 81%, 72%, and 67%, respectively.

4-Chlorophenol biodegradation facilitator composed of recombinant multi-biocatalysts immobilized onto montmorillonite

A biodegradation facilitator which catalyzes the initial steps of 4-chlorophenol (4-CP) oxidation was prepared by immobilizing multiple enzymes (monooxygenase, CphC-I and dioxygenase, CphA-I) onto a natural inorganic support. The enzymes were obtained via overexpression and purification after cloning the corresponding genes (cphC-I and cphA-I) from Arthrobacter chlorophenolicus A6. Then, the recombinant CphC-I was immobilized onto fulvic acid-activated montmorillonite. The immobilization yield was 60%, and the high enzyme activity (82.6%) was retained after immobilization. Kinetic analysis indicated that the Michaelis-Menten model parameters for the immobilized CphC-I were similar to those for the free enzyme. The enzyme stability was markedly enhanced after immobilization. The immobilized enzyme exhibited a high level of activity even after repetitive use (84.7%) and powdering (65.8%). 4-CP was sequentially oxidized by a multiple enzyme complex, comprising the immobilized CphC-I and CphA-I, via the hydroquinone pathway: oxidative transformation of 4-CP to hydroxyquinol followed by ring fission of hydroxyquinol.

Formulation and stabilization of an Arthrobacter strain with good storage stability and 4-chlorophenol-degradation activity for bioremediation

Chlorophenols are widespread and of environmental concern due to their toxic and carcinogenic properties. Development of less costly and less technically challenging remediation methods are needed; therefore, we developed a formulation based on micronized vermiculite that, when air-dried, resulted in a granular product containing the 4-chlorophenol (4-CP)-degrading Gram-positive bacterium Arthrobacter chlorophenolicus A6. This formulation and stabilization method yielded survival rates of about 60% that remained stable in storage for at least 3 months at 4 °C. The 4-CP degradation by the formulated and desiccated A. chlorophenolicus A6 cells was compared to that of freshly grown cells in controlled-environment soil microcosms. The stabilized cells degraded 4-CP equally efficient as freshly grown cells in two different set-ups using both hygienized and non-treated soils. The desiccated microbial product was successfully employed in an outdoor pot trial showing its effectiveness under more realistic environmental conditions. No significant phytoremediation effects on 4-CP degradation were observed in the outdoor pot experiment. The 4-CP degradation kinetics from both the microcosms and the outdoor pot trial were used to generate a predictive model of 4-CP biodegradation potentially useful for larger-scale operations, enabling better bioremediation set-ups and saving of resources. This study also opens up the possibility of formulating and stabilizing also other Arthrobacter strains possessing different desirable pollutant-degrading capabilities.

Identification of the upstream 4-chlorophenol biodegradation pathway using a recombinant monooxygenase from Arthrobacter chlorophenolicus A6

This study aimed to clarify the initial 4-chlorophenol (4-CP) biodegradation pathway promoted by a two-component flavin-diffusible monooxygenase (TC-FDM) consisting of CphC-I and CphB contained in Arthrobacter chlorophenolicus A6 and the decomposition function of CphC-I. The TC-FDM genes were cloned from A. chlorophenolicus A6, and the corresponding enzymes were overexpressed. Since CphB was expressed in an insoluble form, Fre, a flavin reductase obtained from Escherichia coli, was used. These enzymes were purified using Ni2+-NTA resin. It was confirmed that TC-FDM catalyzes the oxidation of 4-CP and the sequential conversion of 4-CP to benzoquinone (BQN)→hydroquinone (HQN)→HQL. This indicated that CphC-I exhibits substrate specificity for 4-CP, BQN, and HQN. The activity of CphC-I for 4-CP was 63.22U/mg-protein, and the Michaelis-Menten kinetic parameters were vmax=0.21mM/min, KM=0.19mM, and kcat/KM=0.04mM−1min−1. These results would be useful for the development of a novel biochemical treatment technology for 4-CP and phenolic hydrocarbons.

Oxidative biodegradation of 4-chlorophenol by using recombinant monooxygenase cloned and overexpressed from Arthrobacter chlorophenolicus A6

In this study, cphC-I and cphB, encoding a putative two-component flavin-diffusible monooxygenase (TC-FDM) complex, were cloned from Arthrobacter chlorophenolicus A6. The corresponding enzymes were overexpressed to assess the feasibility of their utilization for the oxidative decomposition of 4-chlorophenol (4-CP). Soluble CphC-I was produced at a high level (∼50%), and subsequently purified. Since CphB was expressed in an insoluble form, a flavin reductase, Fre, cloned from Escherichia coli was used as an alternative reductase. CphC-I utilized cofactor FADH2, which was reduced by Fre for the hydroxylation of 4-CP. This recombinant enzyme complex exhibited a higher specific activity for the oxidation of 4-CP (45.34U/mg-protein) than that exhibited by CphC-I contained in cells (0.18U/mg-protein). The Michaelis-Menten kinetic parameters were determined as: vmax=223.3μM·min−1, KM=249.4μM, and kcat/KM=0.052min−1·μM−1. These results could be useful for the development of a new biochemical remediation technique based on enzymatic agents catalyzing the degradation of phenolic contaminants.

Enzymatic degradation of aromatic hydrocarbon intermediates using a recombinant dioxygenase immobilized onto surfactant-activated carbon nanotube

This study examined the enzymatic decomposition of aromatic hydrocarbon intermediates (catechol, 4-chlorocatechol, and 3-methylcatechol) using a dioxygenase immobilized onto single-walled carbon nanotube (SWCNT). The surfaces of SWCNTs were activated with surfactants. The dioxygenase was obtained by recombinant technique: the corresponding gene was cloned from Arthrobacter chlorophenolicus A6, and the enzyme was overexpressed and purified subsequently. The enzyme immobilization yield was 62%, and the high level of enzyme activity was preserved (60–79%) after enzyme immobilization. Kinetic analyses showed that the substrate utilization rates and the catalytic efficiencies of the immobilized enzyme for all substrates (target aromatic hydrocarbon intermediates) tested were similar to those of the free enzyme, indicating that the loss of enzyme activity was minimal during enzyme immobilization. The immobilized enzyme was more stable than the free enzyme against abrupt changes in pH, temperature, and ionic strength. Moreover, it retained high enzyme activity even after repetitive use.

Noncovalent and covalent immobilization of oxygenase on single-walled carbon nanotube for enzymatic decomposition of aromatic hydrocarbon intermediates.

The decomposition of various aromatic hydrocarbon intermediates was examined using a recombinant oxidative enzyme immobilized on single-walled carbon nanotubes (SWCNTs). Hydroxyquinol 1,2-dioxygenase (CphA-I), which catalyzes ring cleavage of catechol and its analogues, was obtained from Arthrobacter chlorophenolicus A6 via cloning, overexpression, and subsequent purification. This recombinant enzyme was immobilized on SWCNTs by physical adsorption and covalent coupling in the absence and presence of N-hydroxysuccinimide. The immobilization yield was as high as 52.1%, and a high level of enzyme activity of up to 64.7% was preserved after immobilization. Kinetic analysis showed that the substrate utilization rates (vmax) and catalytic efficiencies (kcat/KM) of the immobilized enzyme for all substrates evaluated were similar to those of the free enzyme, indicating minimal loss of enzyme activity during immobilization. The immobilized enzyme was more stable toward extreme pH, temperature, and ionic strength conditions than the free enzyme. Thus, the oxidative enzyme immobilized on SWCNTs can be used as an effective and stable biocatalyst for the biochemical remediation process if further investigations would be carried out under field conditions.

Function Analysis of Chlorophenol Monooxygenase for Chlorophenol Degradation

4-Chlorophenol is well-known as an extensively used antiseptic, and it may cause severe damage to the environment and human health. 4-Chlorophenol can be biologically degraded by Arthrobacter chlorophenolicus A6, which can be attributed to the cphC-I and cphB genes of the microorganism that encode for a two-component flavin-diffusible monooxygenase (TC-FDM), composed of oxygenase and reductase components. This study reports the cloning, overexpression, purification, and function analysis of the oxygenase and reductase components from the genes cphC-I and cphB. The genes were cloned into vector pET-24a, and 4 different strains of Escherichia coli were transformed with these recombinant genes. The optimization of expression conditions indicated that cphC-I is best overexpressed in E. coli BL21(DE3) incubated for 24 hours at 15°C with 0.5 mM of isopropyl-β-D-1-thiogalactopyranoside. However, cphB was not expressed into soluble form enzyme in any of the conditions, and therefore fre of E. coli was used instead to analyze the function of CphC-I. CphC-I was able to degrade approximately 13.86% of 4-chlorophenol, indicating that it is indeed a reduced flavin-dependent monooxygenase and utilizes 4-chlorophenol as a substrate. The results of this study are expected to establish the basic understanding of TC-FDM for its application to enzymatic bioremediation of phenol-contaminated environment.

4-Chlorophenol 분해박테리아 Arthrobacter chlorophenolicus A6로부터의 monooxygenase의 복제 및 대량발현과 정제 그리고 기질분해활성도 분석

Arthrobacter chlorophenolicus A6 possesses several monooxygenases (CphC-I, CphC-II, and CphB) that can catalyze the transformation of 4-chlorophenol (4-CP) to hydroxylated intermediates in the initial steps of substrate metabolism. The corresponding genes of the monooxygenases were cloned, and the competent cells were transformed with these recombinant plasmids. Although CphC-II and CphB were expressed as insoluble forms, CphC-I was successfully expressed as a soluble form and isolated by purification. The specific activity of the purified CphC-I was analyzed by using 4-CP, 4-chlorocatechol (4-CC), and catechol (CAT) as substrates. The specific activities for 4-CP, 4-CC, and CAT were determined to be 0.312 U/mg, 0.462 U/mg, 0.246 U/mg, respectively. The results of this study indicated that CphC-I is able to catalyze the degradation of 4-CC and CAT in addition to 4-CP, which is a primary substrate. This research is expected to provide the fundamental information for the development of an eco-friendly biochemical degradation of aromatic hydrocarbons.

Treatment of refinery wastewater usingArthrobacter chlorophenolicusA6 in an upflow packed bed reactor

AbstractIn this study, refinery wastewater collected from Guwahati, India, was treated in an upflow packed bed reactor (PBR) with immobilized Arthrobacter chlorophenolicus A6. The wastewater was initially characterized using standard methods. The toxicity of the wastewater was determined using aerobic mixed microbial consortia following the resazurin toxicity assay. As the chemical oxygen demand (COD) level in the raw refinery wastewater was very low (190 mg l−1), the raw wastewater was spiked with a mixture of substituted phenol at different concentrations. The batch shake flask experiment revealed that the COD removal efficiency was found to be less when the raw refinery wastewater was not spiked with nutrient. On the other hand, when supplemented with nutrients in the form of mineral salt medium, more than 98% COD removal was achieved. The PBR was operated by varying the influent phenolic concentration in the range of 250–350 mg l−1 and at a hydraulic retention time of 12.5 h. Results indicated that mo...

Evaluation of 4-bromophenol biodegradation in mixed pollutants system by Arthrobacter chlorophenolicus A6 in an upflow packed bed reactor.

Bromophenol is listed as priority pollutant by U.S. EPA, however, there is no report so far on its removal in mixed pollutants system by any biological reactor operated in continuous mode. Furthermore, bromophenol along with chlorophenol and nitrophenol are usually the major constituents of paper pulp and pesticide industrial effluent. The present study investigated simultaneous biodegradation of these three pollutants with specially emphasis on substrate competition and crossed inhibition by Arthrobacter chlorophenolicus A6 in an upflow packed bed reactor (UPBR). A 2(3) full factorial design was employed with these pollutants at two different levels by varying their influent concentration in the range of 250-450 mg l(-1). Almost complete removal of all these pollutants and 97 % effluent toxicity removal were achieved in the UPBR at a pollutant loading rate of 1707 mg l(-1) day(-1) or lesser. However, at higher loading rates, the reactor performance deteriorated due to transient accumulation of toxic intermediates. Statistical analysis of the results revealed a strong negative interaction of 4-CP on 4-NP biodegradation. On the other hand, interaction effect between 4-CP and 4-BP was found to be insignificant. Among these three pollutants 4-NP preferentially degraded, however, 4-CP exerted more inhibitory effect on 4-NP biodegradation. This study demonstrated the potential of A. chlorophenolicus A6 for biodegradation of 4-BP in mixed pollutants system by a flow through UPBR system.

Arthrobacter bambusae sp. nov., isolated from soil of a bamboo grove.

A Gram-stain-positive, aerobic, motile by gliding, rod-shaped bacterial strain, THG-GM18(T), was isolated from soil of a bamboo grove. Strain THG-GM18(T) was able to grow in the presence of up to 6.0 % (w/v) NaCl, at 4-37 °C and at pH 7.0-10.0 in R2A medium. Based on 16S rRNA gene sequence similarity, strain THG-GM18(T) was closely related to species of the genus Arthrobacter. The most closely related strains to strain THG-GM18(T) are Arthrobacter ramosus CCM 1646(T) (98.5 % similarity), Arthrobacter nitroguajacolicus G2-1(T) (98.4 %), Arthrobacter nicotinovorans DSM 420(T) (98.2 %), Arthrobacter aurescens DSM 20116(T) (98.1 %) and Arthrobacter chlorophenolicus A6(T) (98.0 %). Strain THG-GM18(T) possessed chemotaxonomic properties consistent with those of members of the genus Arthrobacter, such as peptidoglycan type A3α (l-Lys-l-Ala-l-Thr-l-Ala), MK-9 as major menaquinone and anteiso- and iso-branched compounds (anteiso-C15 : 0, iso-C16 : 0 and anteiso-C17 : 0) as major cellular fatty acids. The polar lipid profile contained diphosphatidylglycerol, phosphatidylglycerol, an unidentified phosphoglycolipid, unidentified phospholipids, unidentified aminolipids, an unidentified glycolipid and unidentified lipids. The G+C content of the genomic DNA was 61.0 mol%. The DNA-DNA relatedness values between strain THG-GM18(T) and its closest phylogenetic neighbours were below 26.0 %. The results of physiological and biochemical tests allowed the differentiation of strain THG-GM18(T) from species of the genus Arthrobacter with validly published names. Arthrobacter bambusae sp. nov. is the proposed name, and the type strain is THG-GM18(T) ( = KACC 17531(T) = JCM 19335(T)).

Decomposition of aromatic hydrocarbon intermediates by recombinant hydroxyquinol 1,2-dioxygenase from Arthrobacter chlorophenolicus A6 and its structure characterization

This study was carried out to characterize hydroxyquinol 1,2-dioxygenase from Arthrobacter chlorophenolicus A6 (Ar 1,2-HQD). The cphA-I gene encoding Ar 1,2-HQD was cloned and the enzyme was overexpressed for subsequent purification of the recombinant protein by Ni2+-NTA affinity chromatography and fast protein liquid chromatography (FPLC). Purification of the enzyme by Ni2+-NTA affinity chromatography increased the substrate activity for various aromatic hydrocarbon intermediates by 9.4- to 12.9-fold. Subsequent purification of the enzyme by FPLC increased the activity by 51.7- to 65.2-fold. The substrate specificity of Ar 1,2-HQD indicated that the catalytic function of this enzyme is similar to that of the type-II catechol 1,2-dioxygenase (1,2-CTD). The deduced 304 amino acid sequence analysis also revealed that Ar 1,2-HQD is an intradiol dioxygenase and it is related closely to 1,2-CTD. Its structure consisted of 5 α-helices in the N-terminal domain and 13 β-sheets in the C-terminal domain. The ferric ion was coordinated to two histidine residues and two tyrosine residues. The results of this study suggest that the highly purified Ar 1,2-HQD can be used as a key enzyme in the biodegradation of aromatic hydrocarbon contaminants.

Transcriptional profiling of Gram‐positive Arthrobacter in the phyllosphere: induction of pollutant degradation genes by natural plant phenolic compounds

Arthrobacter chlorophenolicus A6 is a Gram-positive, 4-chlorophenol-degrading soil bacterium that was recently shown to be an effective colonizer of plant leaf surfaces. The genetic basis for this phyllosphere competency is unknown. In this paper, we describe the genome-wide expression profile of A.chlorophenolicus on leaves of common bean (Phaseolus vulgaris) compared with growth on agar surfaces. In phyllosphere-grown cells, we found elevated expression of several genes known to contribute to epiphytic fitness, for example those involved in nutrient acquisition, attachment, stress response and horizontal gene transfer. A surprising result was the leaf-induced expression of a subset of the so-called cph genes for the degradation of 4-chlorophenol. This subset encodes the conversion of the phenolic compound hydroquinone to 3-oxoadipate, and was shown to be induced not only by 4-chlorophenol but also hydroquinone, its glycosylated derivative arbutin, and phenol. Small amounts of hydroquinone, but not arbutin or phenol, were detected in leaf surface washes of P.vulgaris by gas chromatography-mass spectrometry. Our findings illustrate the utility of genomics approaches for exploration and improved understanding of a microbial habitat. Also, they highlight the potential for phyllosphere-based priming of bacteria to stimulate pollutant degradation, which holds promise for the application of phylloremediation.