Introduction 1997 1999 3 3 1997 1996 1989 1980 1971 1990 Rhodococcus 1998 2001 1998 1999 2000 Rhizobiales Rhodococcus ortho 1998 Rhodococcus opacus 2000 Exophiala jeanselmei 1998 Penicillium frequentans meta para 1993 1994 To our knowledge, studies on the metabolism of fluorophenols by a bacterial culture that is capable of using such as compound as a sole source of carbon and energy have not been reported. In the present paper, we describe the isolation and characterization of a bacterial strain growing on 4-FP as the sole source of carbon and energy. Based on the identification of several intermediates, a metabolic route for the degradation of 4-FP by this strain is proposed. Materials and methods Media and growth conditions 2 4 2 2 4 4 2 4 2 4 1985 E. coli Enrichment and isolation of 4-FP-degrading cultures Sequencing of the 16S rRNA gene 1998 Taq 2 Taq E. coli Phylogenetic analysis 2005 1997 1999 2000 1 Fig. 1 scale bar a b Arthrobacter values in parenthesis  Preparation of cell extracts g g Enzyme assays The 4-FP monooxygenase activity was measured spectrophotometrically by following the consumption of NADH at 340 nm in a reaction mixture containing cell-free extract (0.1 mg protein), 0.1 mM 4-FP, 0.1 mM NADH, and buffer in a total volume of 1 ml. The observed rates were corrected for substrate-independent NADH oxidation. Pseudomonas putida 1998 Hydroquinone dioxygenase was assayed spectrophotometrically by monitoring the change in absorbance between 230 and 330 nm in a reaction mixture of 1 ml final volume containing 0.1 mM hydroquinone, TD buffer, and cell-free extract (0.1 mg of protein). Hydroquinone hydroxylase and hydroxyquinol dioxygenase were assayed by using a fiber optic oxygen sensor. Reaction mixtures contained 1 mM of substrate, MM, and cell suspension (0.5 mg/ml protein) in a final volume of 1.25 ml. Oxygen uptake measurements g 2 Analytical methods Liquid chromatography (LC)-MS was carried out with a ZMD Micromass spectrometer, equipped with a XTerra MS, Symmetry Shield C8 column (4.6 × 150 mm), a Waters 996 photodiode array detector, and a Waters 2690 separations module. Samples of 20 μl were analyzed, and compounds were isocratically eluted at a flow rate of 1 ml/min with a solution of water/acetonitrile (80:20) and 10 mM formic acid. Concentrations of free fluoride in the culture supernatants were measured with a fluoride electrode (model 96-09, Thermo Russell, Scotland). Fresh sodium fluoride standards were prepared for calibration curves. Chemicals Nucleotide sequence accession numbers Results Isolation of a 4-FP-degrading bacterium 600 600 Microbiological characterization Arthrobacter Arthrobacter 2005 Arthrobacter nitroguajacolicus 2004 Actinobacteria Arthrobacter 1 Arthrobacter Arthrobacter Arthrobacter Arthrobacter Growth and substrate removal were found when catechol, hydroquinone, hydroxyquinol, benzoate, phenol, 4-fluorocinnamic acid, and 4-nitrophenol were used as substrates. The organism did not grow on 2-fluorophenol, 3-fluorophenol, 4-chlorophenol, 4-bromophenol, 4-iodophenol, fluoroacetate, trifluoroacetate, fluoroacetamide, trifluoroethanol, or on 2-bromoethanol. The fact that strain IF1 is capable of growth on catechol, hydroquinone, and hydroxyquinol but not on 4-fluorocatechol indicates that 4-fluorocatechol is not the most likely intermediate in the 4-FP pathway, although toxic effects could also play a role. 2 2 Fig. 2 a squares circles b triangles circles diamonds squares  Growth on 4-FP and formation of metabolites 3 Fig. 3 triangles diamonds squares  1 m z m z Table 1 Retention times in GC and HPLC analyses of metabolic intermediates formed by cells of strain IF1 growing on 4-FP and of some authentic standards Compound GC retention time (min) a m z Metabolites I 20.7 1.3 110, 81, 54 II 24.3 1.1 126, 80, 52 III c 0.75 – IV – 2.6 – V 16.5 – – VI 19.4 – – VII – b d Authentic standards 4-FP 15.7 3 112, 83, 57 4-Fluorocatechol 14.4 2 128, 82, 51 Hydroquinone 20.7 1.3 110, 81, 54 Hydroxyquinol 24.3 1.1 126, 80, 52 Catechol 19.1 – – a b c d 1 4 m z m z Fig. 4 diamonds squares x marks  The above results suggest that 4-FP is initially converted to hydroquinone. The most likely enzyme involved in such a conversion is a 4-FP monooxygenase. Enzyme activities Arthrobacter 2 Table 2 Arthrobacter a Assay substrate 2 Fluorophenol Glucose 4-Fluorophenol 0.356 <0.001 Hydroquinone 0.313 <0.001 Hydroxyquinol 0.195 0.156 4-Fluorocatechol <0.001 <0.001 Catechol <0.001 <0.001 a 3 Table 3 Specific activities of enzymes in crude extracts of strain IF1 pregrown in 4-FP or glucose Assay substrate Enzyme tested Specific activity (U/mg of protein) after growth on 4-Fluorophenol Glucose Catechol 1,2-Dioxygenase <0.01 <0.01 2,3-Dioxygenase <0.01 <0.01 4-Fluorocatechol Dioxygenase <0.01 <0.01 4-Fluorophenol Monooxygenase 0.12 <0.01 Hydroquinone Dioxygenase <0.01 <0.01 Monooxygenase 0.313 <0.01 Hydroxymuconic semialdehyde Dehydrogenase <0.01 <0.01 Hydroxyquinol Dioxygenase 0.195 0.156 When hydroquinone was used as an assay substrate, its typical spectrophotometric peak at 288 nm was not replaced by a peak absorbing at 290 to 320 nm, which would have pointed to formation of hydroxymuconic semialdehyde. Thus, no hydroquinone dioxygenase was induced, or its product was rapidly further converted. The latter was ruled out by the observation that added hydroxymuconic semialdehyde was not converted by cell extracts of strain IF1, indicating that no hydroxymuconic semialdehyde dehydrogenase was induced during growth in the presence of 4-FP. These results make it unlikely that hydroxymuconic semialdehyde is an intermediate in the 4-FP pathway by strain IF1. When cell extracts were assayed for hydroxyquinol degradation, complete conversion of the substrate was observed with HPLC. When the reaction was monitored spectrophotometrically, the peak at 287 nm, typical of hydroxyquinol, was substituted by a peak at 245 nm, typical of maleylacetate. This indicates the involvement of a hydroxyquinol oxygenase that converts hydroxyquinol into maleylacetate, which is further converted into 3-oxoadipate. Discussion Arthrobacter ortho 5 ortho Fig. 5 Arthrobacter  ortho 1992 1997 1994 1992 1992 1998 2005 2007 p para 1980 Ralstonia eutropha 2004 5 Pseudomonas pickettii 1992 Sphingobium 1991 Arthrobacter 1996 Arthrobacter 2007 2005 2001 1999 2000 1998 1998 Arthrobacter