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International Journal of Systematic and Evolutionary Microbiology (2000), 50, 355–363 Printed in Great Britain Frigoribacterium faeni gen. nov., sp. nov., a novel psychrophilic genus of the family Microbacteriaceae P. Ka$ mpfer,1 F. A. Rainey,2,6 M. A. Andersson,3 E.-L. Nurmiaho Lassila,4 U. Ulrych,5 H.-J. Busse,5 N. Weiss,6 R. Mikkola3 and M. Salkinoja-Salonen3 Author for correspondence: P. Ka$ mpfer. Tel: ­49 641 99 37352. Fax: ­49 641 99 37359. e-mail: peter.kaempfer!agrar.uni-giessen.de 1 Institut fu$ r Angewandte The taxonomic position of five actinobacterial strains isolated from dust, an Mikrobiologie, Justus- animal shed, the air inside a museum and soil was investigated using a Liebig Universita$ t, Senckenbergstr. 3, D-35390 polyphasic approach. The growth characteristics were unusual for Giessen, Germany actinomycetes. Optimal growth was at temperatures ranging from 2 to 10 SC. 2 Department of Biological After small-step adaptation (5 SC steps) to higher temperatures, the strains Sciences, 508 Life Sciences were also able to grow at 20 SC. Cell wall analyses revealed that the organisms Building, Louisiana State showed a hitherto undescribed, new group B-type peptidoglycan [type B2β University, Baton Rouge, LA 70803, USA according to Schleifer & Kandler (1972), but with lysine instead of ornithine]. All strains contained menaquinone MK-9. Mycolic acids were not detected. 3 Department of Applied Chemistry and Diphosphatidylglycerol, phosphatidylglycerol and an unknown glycolipid were Microbiology, PO Box 56 detected in the polar lipid extracts. The main fatty acids were 12-methyl- (Biocentre) 00014 tetradecanoic acid (15:0anteiso), 12-methyl-tetradecenoic acid (15:1anteiso), University of Helsinki, Finland 14-methyl-pentadecanoic acid (16:0iso) and 14-methyl-hexadecanoic acid (17:0iso), as well as an unusual compound identified as 1,1-dimethoxy-anteiso- 4 Department of Bioscience, PO Box 56 (Biocentre) pentadecane (15:0anteiso-DMA). The GMC content of DNA was approximately 00014 University of 71 mol%. The results of 16S rRNA gene sequence comparisons revealed that Helsinki, Finland the strains represent a new lineage in the suborder Micrococcineae and the 5 Institut fu$ r Bakteriologie, family Microbacteriaceae of the order Actinomycetales. On the basis of these Mykologie und Hygiene, results the new genus Frigoribacterium gen. nov. is proposed, harbouring the Veterinarmedizinische $ T T Universita$ t Wien, new species Frigoribacterium faeni sp. nov. (type strain ¯ 801 ¯ DSM 10309 ). Veterina$ rplatz 1, A-1210 Wien, Austria 6 DSMZ-Deutsche Sammlung Keywords: Frigoribacterium gen. nov., psychrophilic genus of the family von Mikroorganismen und Microbacteriaceae, 16S rDNA, chemotaxonomy Zellkulturen GmbH, D-38124 Braunschweig, Germany INTRODUCTION 1996) and Cryobacterium (Suzuki et al., 1997) were also shown to represent new branches within the The family Microbacteriaceae was proposed by Park et family Microbacteriaceae. The type genus Micro- al. (1993) to accommodate the Gram-positive genera bacterium was redefined by Takeuchi & Hatano (1998), Agromyces, Aureobacterium, Clavibacter, Curtobac- who proposed the unification of the genera Micro- terium and Microbacterium. On the basis of 16S rRNA bacterium and Aureobacterium on the basis of 16S sequence data, Stackebrandt et al. (1997) redefined the rRNA sequence data and chemotaxonomic data. A family to accommodate the above-mentioned genera common feature of the eight currently recognized in addition to the genera Agrococcus and Rathayi- genera within the family Microbacteriaceae is the bacter. The new genera Leucobacter (Takeuchi et al., group B-type peptidoglycan. In this type the di- carboxyl amino acid (in most cases -glutamate or - ................................................................................................................................................. hydroxyglutamate) at position 2 is linked with the Abbreviations: DMA, dimethyl acetal; ECL, equivalent chain length; FAME, fatty acid methyl ester. amino acid at position 4 (-alanine) via an interpep- The EMBL accession numbers for the 16S rRNA gene sequences of strains tide bridge containing a -amino acid. Very recently, 801, NS 4, 227, 301 and 312 are Y18807, AJ243012, AF157478, AF157479 the occurrence of psychrophilic organisms within and AF157480, respectively. the family Microbacteriaceae has been shown with the 01194 # 2000 IUMS 355 P. Ka$ mpfer and others description of the genus Cryobacterium (Suzuki et al., microtitre plates were done as described earlier (Ka$ mpfer et 1997). In an extensive study of bacteria collected from al., 1991, 1997). Tests were read after 7 d at 20 mC. dust samples and animal sheds (Andersson et al., 1999) Chemotaxonomy. Preparation of cell walls and determi- four isolates were obtained which revealed atypical nation of peptidoglycan structure were carried out by the growth characteristics. This was also found for a strain methods described by Schleifer & Kandler (1972) with the isolated from the Sainsbury Center for Visual Arts in modification that TLC on cellulose sheets was used instead Norwich, UK. All of them showed initially good of paper chromatography. Briefly, 1 mg freeze-dried cell growth at 2–10 mC but only moderate growth at walls were hydrolysed in 0±2 ml 4 M HCl at 100 mC for 16 h 25 mC. A detailed study of the cell wall type revealed an (total hydrolysate) or 45 min (partial hydrolysate). Di- interesting hitherto undescribed B-type. amino acids were identified from total hydrolysate by one- dimensional chromatography in the solvent system meth- In this paper we describe the morphological, physio- anol}pyridine}water}10 M HCl (320:40:70:10 by vol.). logical, chemotaxonomic and phylogenetic charac- Amino acids and peptides from partial hydrolysate were teristics of these organisms. On the basis of our results identified after two-dimensional chromatography in the and the unique taxonomic properties of the organisms, systems described by Schleifer & Kandler (1972) by their it can be concluded that they represent a new genus for mobilities and staining characteristics with ninhydrin spray. The resulting ‘fingerprints’ could be compared with known which we propose the genus name Frigoribacterium peptidoglycan structures. The configuration of lysine was gen. nov. and the new species Frigoribacterium faeni determined by using -lysine decarboxylase (Sigma; L 0882). sp. nov. Cell wall acyl type was determined by the method of Uchida & Aida (1977). METHODS Extraction and analysis of isoprenoid quinones and polar lipids. T Menaquinones were extracted and analysed as de- Sampling and isolation. Strains 301, 312 and 801 were scribed by Tindall (1990). Polar lipids were extracted and isolated from airborne dust in a cattle barn in Southern analysed by TLC according to Ventosa et al. (1993). Finland. Dusts aerosolized during the distribution and handling of feed and bedding were collected on nuclepore Preparation and analysis of fatty acids and fatty acetals. filters as described by Palmgren et al. (1986). The cow barn Fatty acid methyl esters (FAMEs) were determined ac- aerosols were collected for 10 min on nuclepore filters with cording to Ka$ mpfer et al. (1997), except that the cells were a nominal pore size of 0 2 µm, using a low flow personal grown at 28 mC. DMAs (dimethyl acetals) were preliminarily ± " sampling pump precalibrated to a flow of 2 l min− . Sam- identified from the extracts prepared for FAME analysis pling was carried out at a distance of 0±4 m from the bales of using the MIDI anaerobic library (BHIBLA, Version 3.8). hay and straw opened while the air contained clouds of The identity of the DMA in Frigoribacterium strains was visible dust. Strain 227 was isolated from settled dust. Settled confirmed by GC-MS analysis using an HP 5MS capillary dusts were collected from horizontal surfaces in the animal column, an HP 6890 gas chromatograph and an HP 6890 sheds " 2 m above the floor. Strain NS 4 was isolated during mass selective detector set at the ionization energy of 70 eV. The temperature was increased from 170 to 270 mC at a rate a sampling campaign for airborne bacteria in the Sainsbury −" Center for Visual Arts in Norwich, UK. It was cultivated on of 5 mC min ; the inlet temperature was 250 mC. The Wiley casein minimal medium (CAS MM; Altenburger et al., 138K and NIST}EPA}NIH mass spectral libraries were 1996) supplemented with cycloheximide and incubated at used for reference. room temperature. Subcultivation was done on PYES Base composition of DNA and DNA–DNA reassociation medium (Altenburger et al., 1996). studies. DNA was isolated by chromatography on hydroxy- Bacteria in settled and airborne dusts were resuscitated by a apatite by the procedure of Cashion et al. (1977). The base method described previously (Andersson et al., 1999) and composition of DNA and the calculation of the G­C ratio cultured on tryptic soy agar at 13–16 mC. The medium and was determined as described by Mesbah et al. (1989) and diluent for serial plating were obtained from Difco unless Tamaoka & Komagata (1984). DNA–DNA reassociation otherwise stated. experiments were performed as described by De Ley et al. (1970), with the modifications described by Huss et al. Morphological characteristics. Cell morphology was ex- (1983), by using a Gilford System model 2600 spectro- amined by phase-contrast microscopy with a light micro- photometer equipped with a model 2527-R thermo- scope (Leitz). Motility was studied by the hanging drop programmer and plotter (Gilford Instruments).

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