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LABRYS PORTUCALENSIS, a BACTERIAL Provided by Repositório Institucional Da Universidade Católica Portuguesa STRAIN with the CAPACITY to DEGRADE FLUOROBENZENE M

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LABRYS PORTUCALENSIS, a BACTERIAL Provided by Repositório Institucional Da Universidade Católica Portuguesa STRAIN with the CAPACITY to DEGRADE FLUOROBENZENE M View metadata, citation and similar papers at core.ac.uk brought to you by CORE LABRYS PORTUCALENSIS, A BACTERIAL provided by Repositório Institucional da Universidade Católica Portuguesa STRAIN WITH THE CAPACITY TO DEGRADE FLUOROBENZENE M. F. Carvalho 1, P. De Marco 2, A. F. Duque 1, C. C. Pacheco 2, D. B. Janssen 3 and P. M. L. Castro 1 1CBQF/Escola Superior de Biotecnologia, Universidade Católica Portuguesa, Rua Dr. António Bernardino de Almeida, 4200-072 Porto, Portugal 2IBMC – Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua Campo Alegre, 823, 4150-180 Porto, Portugal 3Biochemical Laboratory, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands INTRODUCTION During the last decades, environmental contamination by fluorinated organic compounds has received increasing attention because of their use as herbicides, fungicides, surfactants, refrigerants, intermediates in organic synthesis, solvents and pharmaceuticals. The physico-chemical characteristics of fluoroaromatics often make them highly recalcitrant (1). During our studies on the microbial degradation of fluorobenzene (FB), a pure bacterial culture with the unique capacity to utilize this compound as a sole carbon and energy source was isolated from a sediment sample collected from an industrially polluted site in northern Portugal (2). The isolated strain, designated F11, was found to belong to subgroup 2 of the class Alphaproteobacteria and to fall within the order Rhizobiales. The present study presents a summary of the main morphological and physiological characteristics of strain F11, including metabolic versatility studies and FB degradation pathway, as well as a detailed classification of this strain based on cellular fatty acid profiling, phylogenetic analysis of the 16S rRNA gene and DNA–DNA hybridization experiments. RESULTS AND DISCUSSION Table 1. Summary of some morphological and physiological characteristics of strain F11 F 1. Fluorobenzene dioxygenase Characteristic Strain F11 + + O2 + H + O2 + H + NADH NADH Origin Contaminated sediments + + 2. Fluorobenzene dihydrodiol dehydrogenase NAD 1 9 NAD Cell morphology Short, thick rods FOH F OH OH White circular mucous H H 3. Fluorocatechol 1,2-dioxygenase H OH Colony description glistening and entire- NAD + 2 10 edged colonies NADH + H + HF 4. Fluoromuconate cycloisomerase F OH OH Catalase/oxidase +/+ 5 and 6. Possible side reactions to cis - Growth pH range 4.0-8.0 OH OH O 2 3 Optimum pH range 6.0-8.0 O dienelactone by fluoromuconate cycloisomerase 2 11 F (activity 5) or by slow spontaneous conversion Growth temperature range (ºC) 16-37 COOH COOH (activity 6) Optimum growth temperature range (ºC) 28-32 COOH HF Fluorescent pigments – 5 4 COOH Nitrate reduction – HOOC HOOC 7. trans -Dienelactone hydrolase F Hydrolysis of: HF 12 O O Gelatin – 6 8. Maleylacetate reductase O O Agarose – O O COOH H2O 9. Fluorobenzenedioxygenase Indol production – 7 HF Voges–Proskauer reaction + HOOC 10. Nonenzymatic defluorination 13 Urease production + O O O N2 fixation + COOH 11. Catechol 1,2-dioxygenase Utilization of: COOH NADH + H + 8 14 12. Muconate cycloisomerase Acetate + H2O NAD + Benzoate + HOOC 13. Muconolactone isomerase Citrate + D-Gluconate + O 14. 3-Oxoadipate enol-lactone hydrolase DL-Lactate + COOH D-Lactose + Figure 1. FB metabolic pathway by strain F11. DL-Malate + Maltose (+) Methanol – Acetobacter aceti JCM 7641 T (D30768) Methylamine + Rhodospirillum rubrum ATCC11170 T (D30778) Toluene – Trimethylamine + Azospirillum lipoferum NCIMB 11861 T (Z29619) Fatty acids (% of total content) Caulobacter vibrioides DSM 9893 T (AJ227754) C16:0 22.7 Labrys methylaminiphilus JLW10 T (AY766152) C18:1 w7 c 18.4 94 Labrys portucalensis F11 T (AY362040) 100 C18:0 3.0 T Labrys monachus VKM B-1479 (AJ535707) C19 : 0 cyclo w8c 48.5 Bradyrhizobium japonicum DSM 30131 T (X87272) Major quinones UQ-10 69 88 DNA G+C content (mol%) 62.9 Methylobacterium organophilum JCM 2833 T (D32226) +, Positive; -, negative; (+), weakly positive Beijerinckia indica ATCC 9039 T (M59060) 83 Methylocystis parvus NCIMB 11129 T (Y18945) Table 1. Utilization of various aromatic Methylorhabdus multivorans VKM B-2030 T (AF004845) DNA–DNA hybridization 99 compounds by strain F11 T 100 Starkeya novella IAM 12100 (D32247) experiments indicated that strain 84 Ancylobacter aquaticus ATCC 25396 T (M62790) Substrate F11 F11 represents a separate Hyphomicrobium vulgare ATCC 27500 T (Y14302) Rhodobium orientis JCM 9337 T (D30792) 4-Fluorobenzoate ++ species, based on the Agrobacterium radiobacter ATCC 19358 T (AJ389904) 2-Fluorobenzoate + recommended minimum value of 92 Trifluoroacetic acid + Brucella melitensis ATCC 23456 T (L26166) 70% for strains of the same 100 Benzoate ++ Bartonella bacilliformis ATCC 35685 T (M65249) Benzene ++ species. Mesorhizobium loti ATCC 70043 T (D14514) Fluorobenzene ++ 97 Phyllobacterium myrsinacearum IAM 13584 T (D12789) Bromobenzene - Iodobenzene - The phenotypic, chemotaxonomic, Chlorobenzene - genotypic and phylogenetic data 0.1 4-Chlorophenol - indicate that strain F11 represents a Figure 2. Phylogenetic tree obtained by neighbour-joining analysis of 16S 3-Chloro-4-fluoroaniline - 4-Chlorobenzoate - novel species of the genus Labrys, rRNA gene sequences. The 16S rRNA gene sequence of Escherichia coli Phenol ++ being named as Labrys MRE600 (GenBank accession number J01859) was used to root the tree (not 4-Fluorophenol ++ portucalensis sp. nov. shown). Only bootstrap values >65% are reported at nodes. Bar, 0.1 -, negative; +, positive; ++, strongly positive substitutions per site. REFERENCES 1. Key BD, Howell RD, Criddle CS (1997) Fluorinated organics in the biosphere. Environ Sci Technol 31:2445-2454. 2. Carvalho MF, Ferreira Jorge R, Pacheco CC, De Marco P, Castro PML (2005) Isolation and properties of a pure bacterial strain capable of fluorobenzene degradation as sole carbon and energy source. Environ Microbiol 7: 294-298. ACKNOWLEDGEMENTS M.F. Carvalho wish to acknowledge a research grant from Fundação para a Ciência e Tecnologia (FCT), Portugal (SFRH/BPD/44670/2008) and Fundo Social Europeu (III Quadro Comunitário de Apoio). This work was supported by the FCT Project -POCI/V.5/A0105/2005..
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