University of Groningen Labrys portucalensis sp nov., a fluorobenzene-degrading bacterium isolated from an industrially contaminated sediment in northern Portugal Carvalho, Maria F.; De Marco, Paolo; Duque, Anouk F.; Pacheco, Catarina C.; Janssen, Dick; Castro, Paula M. L. Published in: International Journal of Systematic and Evolutionary Microbiology DOI: 10.1099/ijs.0.65472-0 IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2008 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Carvalho, M. F., De Marco, P., Duque, A. F., Pacheco, C. C., Janssen, D. B., & Castro, P. M. L. (2008). Labrys portucalensis sp nov., a fluorobenzene-degrading bacterium isolated from an industrially contaminated sediment in northern Portugal. International Journal of Systematic and Evolutionary Microbiology, 58(3), 692-698. DOI: 10.1099/ijs.0.65472-0 Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 10-02-2018 International Journal of Systematic and Evolutionary Microbiology (2008), 58, 692–698 DOI 10.1099/ijs.0.65472-0 Labrys portucalensis sp. nov., a fluorobenzene- degrading bacterium isolated from an industrially contaminated sediment in northern Portugal Maria F. Carvalho,13 Paolo De Marco,23 Anouk F. Duque,1 Catarina C. Pacheco,2 Dick B. Janssen3 and Paula M. L. Castro1 Correspondence 1Escola Superior de Biotecnologia, Universidade Cato´lica Portuguesa, Rua Dr. Anto´nio Bernardino Paula M. L. Castro de Almeida, 4200-072 Porto, Portugal [email protected] 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 A detailed classification of a novel bacterial strain, designated F11T, capable of degrading fluorobenzene as a sole carbon and energy source, was performed by using a polyphasic approach. This Gram-negative, rod-shaped, non-motile, non-spore-forming, aerobic bacterium was isolated from a sediment sample collected from an industrially contaminated site in northern Portugal. The predominant whole-cell fatty acids were C19 : 0 cyclo v8c,C16 : 0,C18 : 1v7c,C18 : 0, C18 : 0 3-OH and C16 : 0 3-OH. The G+C content of the DNA was 62.9 mol% and the major respiratory quinone was ubiquinone 10 (UQ-10). 16S rRNA gene sequence analysis revealed that strain F11T was a member of the class Alphaproteobacteria and was phylogenetically related to the genus Labrys, having sequence similarities of 95.6 and 93.1 % to the type strains of Labrys monachus and Labrys methylaminiphilus, respectively. DNA–DNA hybridization experiments revealed levels of relatedness of ,70 % between strain F11T and the type strains of L. monachus and L. methylaminiphilus (38.6 and 34.1 %, respectively), justifying the classification of strain F11T as representing a novel species of the genus Labrys. The name Labrys portucalensis sp. nov. is proposed for this organism. The type strain is F11T (5LMG 23412T5DSM 17916T). The genus Labrys was erected by Vasil’eva & Semenov a crucial role in the detoxification of haloaromatics and (1984) to accommodate a strain of budding prosthecate they can be applied in various bioremediation strategies. bacteria that was isolated from silt samples collected from During our studies on the microbial degradation of Lake Mustija¨rv located in the former Estonian SSR. This fluorobenzene (FB), a pure bacterial culture with the organism, designated strain VKM B-1479T, was subse- unique capacity to utilize this compound as a sole carbon quently named Labrys monachus (Vasil’eva & Semenov, and energy source was isolated from a sediment sample 1985). A second species of the genus, Labrys methylami- collected from an industrially polluted site in northern niphilus, was described by Miller et al. (2005) to Portugal (Carvalho et al., 2005). The isolated strain, accommodate a facultatively methylotrophic bacterium designated F11T, was found to belong to subgroup 2 of (strain JLW10T) derived from Lake Washington sediment. the class Alphaproteobacteria (Woese et al., 1984) and to fall At the time of writing, the genus Labrys comprised these within the order Rhizobiales. In the present study, a more two recognized species. detailed classification of this strain is provided by using a The ability to degrade haloaromatic compounds of polyphasic approach, which included a detailed analysis of environmental relevance has been observed in a wide its morphological and physiological characteristics, cellular phylogenetic diversity of micro-organisms (Key et al., 1997; fatty acid profiling, phylogenetic analysis of the 16S rRNA van Pe´e & Unversucht, 2003). These micro-organisms play gene and DNA–DNA hybridization experiments. On the basis of the data presented, we suggest that strain F11T 3These authors contributed equally to this work. represents a novel species of the genus Labrys. Abbreviation: FB, fluorobenzene. Strain F11T was isolated, as described previously (Carvalho The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene et al., 2005), from industrially contaminated sediment sequence of strain F11T is AY362040. located at Estarreja, northern Portugal. For taxonomic 692 65472 G 2008 IUMS Printed in Great Britain Labrys portucalensis sp. nov. studies, the strain was routinely cultivated at 25 uCina F11T in MM without a nitrogen source but supplemented minimal salts liquid medium (MM) (Caldeira et al., 1999) with 0.2 % glycerol. Cultures were incubated and mon- supplemented with 0.2 % glycerol (v/v) or on nutrient agar itored for growth as described above. To test for the ability (NA) plates. For preservation purposes, cultures of strain to utilize nitrate as an electron acceptor, cultures of strain F11T growing exponentially in MM containing 1 mM FB as F11T were grown in triplicate in MM supplemented with the carbon source were supplemented with 20 % (v/v) 0.2 % glycerol and 10 mM KNO3 under anaerobic condi- glycerol and frozen at 280 uC. tions. To ensure that cultures were free of oxygen, cysteine was added at a concentration of 50 mg l21 to reduce all the Light microscopy was used to examine cell morphology oxygen remaining in the cultures, and resazurin (1 mg l21) and motility by analysing wet mounts of 72 h cultures of was used as an oxygen indicator. Control cultures, grown strain F11T. Confirmation of cell morphology and aerobically, were also established. determination of cell dimensions were accomplished in a Zeiss EM C10 electron microscope. Aliquots from pure When grown on NA plates incubated for 3–4 days at 25 uC, cultures were deposited onto Formvar/carbon 400 mm strain F11T formed white, circular, convex, entire-edged, mesh, 3 mm diameter grids and contrasted with 2 % (w/v) mucous, glistening colonies 1–2 mm in diameter. Light uranyl acetate. The presence of a capsule and endospores microscopy revealed rod-shaped, non-motile, capsulated, was assessed according to Creager et al. (1990). Gram stain, non-spore-forming cells. The Gram-stain was negative. oxidase and catalase tests were performed as described by Under the electron microscope, cells of strain F11T were Smibert & Krieg (1994). Motility was tested as described by short, thick rods, 0.8–1.4 mm in width and 1.6–2.4 mmin Alexander & Strete (2001). length, with no flagella. The ability of strain F11T to utilize various carbon sources Strain F11T was able to grow at temperatures of between 16 was tested, in duplicate, in MM supplemented with the and 37 uC and had an optimum temperature range of 28– following single test substrates: FB, 4-fluorobenzoate, 32 uC. The pH range found to support growth was between 2-fluorobenzoate, benzoate, benzene, bromobenzene, iodo- 4.0 and 8.0, with optimum pH values of between 6.0 and benzene, chlorobenzene, 3-chloro-4-fluoroaniline, 4-chloro- 8.0. benzoate, 4-chlorophenol, phenol, 4-fluorophenol and Tests for catalase and cytochrome oxidase were positive. toluene (all at 0.5 mM); acetate, citrate, DL-lactate, D- The organism did not produce fluorescent pigments on glucose, D-lactose, D-mannose, maltose, methylamine and MM supplemented with 0.2 % glycerol, while accumula- trimethylamine (20 mM); ethanol and methanol (0.1 %, tion of poly-b-hydroxy alkanoate granules was observed in v/v); glycerol (0.2 %, v/v), and yeast extract (0.2 %, w/v). the same medium. The Voges–Proskauer test was positive. Growth was monitored by measuring cell density at 600 nm Indole production and D-glucose acidification tests were of cultures incubated aerobically at 25 uC with shaking at negative. The strain was able to hydrolyse aesculin but not 150 r.p.m. Negative controls, consisting of MM without the gelatin, starch or agarose. It was positive for urease activity, addition of a carbon source and inoculated with strain F11T, weakly positive for b-galactosidase activity but negative for were established for all metabolic experiments. L-arginine dihydrolase activity. The utilization of additional carbon sources was tested by The following substrates were found to support growth of using the API 20NE test kit (bioMe´rieux) according to the strain F11T as sole carbon and energy sources: FB, manufacturer’s instructions.