International Journal of Systematic and Evolutionary Microbiology (2000), 50, 1731–1739 Printed in Great Britain Subtercola boreus gen. nov., sp. nov. and Subtercola frigoramans sp. nov., two new psychrophilic actinobacteria isolated from boreal groundwater M. K. Ma$ nnisto$ ,1 P. Schumann,2 F. A. Rainey,3 P. Ka$ mpfer,4 I. Tsitko,5 M. A. Tiirola6 and M. S. Salkinoja-Salonen5 Author for correspondence: M. Ma$ nnisto$ . Tel: j358 3 365 2947. Fax: j358 3 365 2869. e-mail: mannisto!cc.tut.fi 1 Institute of Water and Psychrophilic actinobacterial isolates from permanently cold groundwater in Environmental Finland were characterized using a polyphasic approach. Growth on agar plates Engineering, Tampere University of Technology, was observed at temperatures down to N2 mC, with an optimum at 15–17 mC, PO Box 541, 33101 but no growth was observed at 30 mC. The peptidoglycan type was B2γ and the Tampere, Finland characteristic diamino acid was diaminobutyric acid. The cell wall sugars of 2 DSMZ-Deutsche Sammlung strain K265T were rhamnose, ribose, xylose and mannose and those of strain von Mikroorganismen und K300T were glucose, rhamnose and xylose. The polar lipids included Zellkulturen GmbH, Beutenbergstraße 11, phosphatidylglycerol, diphosphatidylglycerol, one unknown phospholipid and 07745 Jena, Germany two glycolipids. The main whole-cell fatty acids were 12-methyltetradecanoic 3 Department of Biological acid, 14-methylpentadecanoic acid and 14-methylhexadecanoic acid. Large Sciences, 508 Life Sciences amounts of anteiso-1,1-dimethoxy-pentadecane and also iso-1,1-dimethoxy- Building, Louisiana State hexadecane were present as diagnostic markers. The predominant University, Baton Rouge, LA 70803, USA menaquinones were MK-9 and MK-10. The GMC content of the DNA of strains K265T and K300T was 644 and 678 mol%, respectively. Comparison of 16S rRNA 4 Institut fur Angewandte $ T T Mikrobiologie, Justus- gene sequences revealed that strains K265 and K300 represent a new lineage Liebig Universita$ t, among the type-B-peptidoglycan actinomycetes. The closest relatives were Senckenbergstraße 3, Clavibacter michiganensis, Frigoribacterium faeni and Rathayibacter rathayi. D-35390 Giessen, Germany On the basis of 16S rDNA sequence, GMC content and chemotaxonomical and 5 Department of Applied physiological characteristics, K265T and K300T clearly represent a new genus. Chemistry and Microbiology, PO Box 56 The genus Subtercola gen. nov. is described, together with two species, namely (Biocentre), 00014 Subtercola boreus sp. nov. (type strain K300T l DSM 13056T l CCUG 43135T ) and University of Helsinki, Subtercola frigoramans sp. nov (type strain K265T l DSM 13057T l CCUG Finland 43136T ). 6 Department of Bio and Environmental Sciences, University of Jyva$ skyla$ , PO Box 35, Jyva$ skyla$ , Keywords: Subtercola boreus gen. nov., sp. nov., Subtercola frigoramans sp. nov., Finland psychrophilic actinobacteria, 1,1-dimethyl acetal INTRODUCTION isolates from Finnish groundwater in which the tem- perature is stable at 7 mC(Ma$ nnisto$ et al., 1999; The few psychrophilic or psychrotolerant actino- unpublished results). This paper is a polyphasic des- bacterial species so far described belong to the genera cription of two novel actinomycetes, isolated from the Cryobacterium (Suzuki et al., 1997) and the recently groundwater, capable of growth on solid medium at described Frigoribacterium (Ka$ mpfer et al., 2000). temperatures down to k2 mC. The phylogenetic, Analysis of whole-cell fatty acid composition and chemotaxonomic and physiological data show that the partial 16S rRNA gene sequencing indicated that two isolates represent a new genus within B-type- several taxa of actinomycetes were present among 190 peptidoglycan-containing members of the family Microbacteriaceae. We propose a new genus, Subter- ................................................................................................................................................. cola gen. nov., and the species Subtercola boreus sp. The EMBL accession numbers for the 16S rRNA gene sequences determined nov. and Subtercola frigoramans sp. nov. for these in this study are AF224723 (strain K265T ) and AF224722 (strain K300T ). strains. 01378 # 2000 IUMS 1731 M. K. Ma$ nnisto$ and others METHODS chloroform-isoamyl alcohol extractions, 2-propanol pre- T T cipitation and caesium chloride gradient purification as Isolation. Strains K265 and K300 were isolated from the described by Wilson (1994). Hydrolysis, dephosphorylation groundwater of a shallow aquifer located under a glacial and HPLC measurement were performed as described by gravel ridge in Southern Finland. The water was pumped Johnson (1994). The HPLC column was a Purospher RP-18 from a subsurface depth of 18 m. It was highly humic (with " endcapped reversed-phase column (250 mmi4n0 mm i.d., 4–13 mg dissolved organic carbon lV ), rich in iron (15 mg V" 5n0 µm particle size; Merck). The mobile phase was 20 mM l ) and had a stable temperature of 7p1 mC. The strains triethylamine phosphate (pH 5 1) in 12% methanol. The " n were isolated on PYGV agar plates (Staley, 1968) at 7p1 mC. flow rate was 1 ml minV . Hydrolysed lambda phage DNA The cultures for characterization were grown on PYGV, was used as a standard. TSA (BBL Microbiology Systems) or R2A (BBL Micro- biology Systems) agar. 16S rRNA gene sequence determination and phylogenetic analyses. The extraction of genomic DNA, PCR ampli- Morphology. The cultures were studied by phase-contrast fication of the 16S rRNA gene and sequencing of the purified microscopy (8, 24, 48 and 72 h at 20 mC) using an Olympus PCR products were carried out as described previously BH-2 light microscope. Gram-staining was performed using (Rainey et al., 1996). Sequence reaction products were the Hucker method (Gerhardt et al., 1994). For the prep- purified by ethanol precipitation and electrophoresed with a aration of thin sections, the cultures were grown for 7 d on model 310 Genetic Analyzer (Applied Biosystems). The 16S TSA agar at 8 mC. The cells were prefixed with 4% (v\v) rRNA gene sequences obtained in this study were aligned glutaraldehyde in 0n1 M sodium phosphate buffer (pH 7n2) against the previously determined actinobacterial sequences for 2 h at room temperature and washed three times in the available from the public databases, using the ae2 editor same buffer. Thin sections were prepared and examined as (Maidak et al., 1999). The programs of the package, described elsewhere (Va$ isa$ nen et al., 1994). including and , were used for the phylo- Physiological characteristics. Growth on TSA plates at k2, genetic analyses (Felsenstein, 1993). The method of Jukes & 0, 2, 4, 8, 10, 15, 20, 25, 28 and 30 mC was observed and Cantor (1969) was used to calculate evolutionary distances. recorded after 14 d and the growth rates on PYGV and The tree topology was re-analysed using 1000 bootstrapped trypticase soy broth were determined at 13, 15, 17, 20 and data sets and the programs , and 23 mC by automated kinetic turbidometry (Bioscreen; Lab- of the package (Felsenstein, 1993). Systems). The inoculum (50 µl) was grown on PYGV plates Nucleotide sequence accession numbers. The accession for 3–4 d and suspended in PYGV broth to an optical numbers and strain designations of the reference 16S rRNA density (600 nm) of 0n3; 300 µl PYGV or TSA was pipetted gene sequences used in the phylogenetic analyses are as into each well. Growth, with medium shaking, was measured follows: Agrococcus jenensis DSM 9580T (X92492), Agro- as turbidity using a wide-band filter (450–580 nm) and myces ramosus DSM 43045T (X77447), Agromyces medio- (Labsystems) software. Physiological tests were lanus DSM 20152T (X77449), Arthrobacter globiformis DSM performed on microtitre plates as described elsewhere 20124T (M23411), ‘Brevibacterium helvolum’ DSM 20419 (Ka$ mpfer et al., 1991) and read after 3 d at 20 mC. (X77440), Clavibacter michiganense subsp. michiganense Chemotaxonomic analyses. For whole-cell fatty acid analy- DSM 46364T (X77435), ‘Corynebacterium aquaticum’ DSM sis, the strains were grown on TSA at 4, 10, 15, 20 and 25 mC 20146T (X77450), Cryobacterium psychrophilum IAM12024T for 3–12 d. Fatty acid methyl esters were prepared and (D45058), Curtobacterium citreum DSM 20528T (X77436), analysed as described previously (Va$ isa$ nen et al., 1994). Curtobacterium luteum DSM 20542T (X77437), Frigori- Fatty acid methyl esters were identified using the MIDI bacterium faeni DSM 46346T (Y18807), Leucobacter koma- aerobic library (TSBA, version 3.9; MIDI). 1,1-Dimethyl gatae IFO 15245T (D17751), Microbacterium aurum IFO acetals were identified as described by Ka$ mpfer et al. (2000). 15204T (D21340), Microbacterium barkeri DSM 20145T Peaks in the whole-cell methanolysate not identifed with the (X77446), Microbacterium imperiale DSM 20530T (X77442), MIDI aerobic library version 3.9, were rerun using the Microbacterium lacticum DSM 20427T (X77441), Micro- BHIBLA anaerobic library and also analysed by GC-MS bacterium liquefaciens DSM 20638T (X77437), Rathayibacter using the Wiley 138K and NIST mass-spectral libraries. rathayi DSM 7458T (X77439), Rathayibacter toxicus JCM Methanolysates of Propionibacterium freudenreichii DSMZ 9669T (D84127). 20271T and Propionibacterium jensenii DSMZ 20535T were used as references for 1,1-dimethyl acetals. RESULTS Purified cell wall preparations were obtained as described by Schleifer & Kandler (1972). Amino acids and peptides in cell Morphological and physiological characteristics wall hydrolysates were