This is an author manuscript that has been accepted for publication in International Journal of Systematic and Evolutionary Microbiology, copyright Society for General Microbiology, but has not been copy-edited, formatted or proofed. Cite this article as appearing in International Journal of Systematic and Evolutionary Microbiology. This version of the manuscript may not be duplicated or reproduced, other than for personal use or within the rule of ‘Fair Use of Copyrighted Materials’ (section 17, Title 17, US Code), without permission from the copyright owner, Society for General Microbiology. The View metadata, citation and similar papers at core.ac.uk brought to you by CORE Society for General Microbiology disclaims any responsibility or liability for errors or omissions in this version of the manuscript or in any version derived from it by any other parties. The final copy-edited, published article, which is the version of record, can be found at http://ijs.sgmjournals.org,provided by Giessener Elektronische and is freely Bibliothek available without a subscription 24 months after publication. First published in: Int J Syst Evol Microbiol, 2009. 60(4) 909-913. doi:10.1099/ijs.0.014100-0 Brevibacterium sandarakinum sp. nov., isolated from a wall of an indoor environment Peter Ka¨mpfer,1 Jenny Scha¨fer,1 Nicole Lodders1 and Hans-Ju¨rgen Busse2 Correspondence 1Institut fu¨r Angewandte Mikrobiologie, Justus-Liebig-Universita¨t Giessen, D-35392 Giessen, Peter Ka¨mpfer Germany [email protected] 2Institut fu¨r Bakteriologie, Mykologie und Hygiene, Veterina¨rmedizinische Universita¨t, A-1210 Wien, giessen.de Austria A Gram-stain-positive, rod-shaped, non-endospore-forming, orange-pigmented (coloured) actinobacterium (01-Je-003T) was isolated from the wall of an indoor environment primarily colonized with moulds. On the basis of 16S rRNA gene sequence similarity studies, strain 01-Je- 003T was shown to belong to the genus Brevibacterium and was most similar to the type strains of Brevibacterium picturae (98.8 % similarity), Brevibacterium marinum (97.3 %) and Brevibacterium aurantiacum (97.2 %). Chemotaxonomic data [predominant quinone menaquinone MK-8(H2); polar lipid profile consisting of major compounds diphosphatidylglycerol, phosphatidylglycerol and an unidentified glycolipid; characteristic cell-wall diamino acid meso- diaminopimelic acid; polyamine pattern showing major compounds putrescine and cadaverine; ] T major fatty acids anteiso-C15 : 0 and anteiso-C17 : 0 supported the affiliation of strain 01-Je-003 to the genus Brevibacterium. The results of DNA–DNA hybridizations and physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain 01-Je-003T from the two most closely related species, B. picturae and B. marinum. Strain 01-Je-003T therefore represents a novel species, for which the name Brevibacterium sandarakinum sp. nov. is proposed, with the type strain 01-Je-003T (5DSM 22082T 5CCM 7649T). The genus Brevibacterium, originally proposed by Breed performed with a commercialized DNA extraction kit (1953), served for a long time as a depository for various (GenElute plant genomic DNA kit; Sigma) after disruption Gram-positive rods. Collins et al. (1980) emended the for a 1 min bead-beating step with 1 g of 0.1 mm diameter genus description and, at present, the genus comprises zirconia beads at maximum speed. more than 20 species listed or described by Collins et al. Multiple alignment of the sequence data was performed (1983), McBride et al. (1993), Pascual et al. (1996), Pascual using the software package MEGA version 4 (Tamura et al. & Collins (1999), Gavrish et al. (2004), Ivanova et al. 2007) and ARB (version December 2007; Ludwig et al., (2004), Heyrman et al. (2004), Wauters et al. (2001, 2003, 2004) with the corresponding SILVA SSURef 95 database 2004), Lee (2006), Bhadra et al. (2008), Tang et al. (2008) and Mages et al. (2008). (version July 2008; Pruesse et al., 2007). For the MEGA software, multiple alignment of sequences was done with T Strain 01-Je-003 was enriched and recovered from a wall CLUSTAL W (Thompson et al., 1994) and the alignment was colonized by moulds. After extraction of 1 g sample for edited manually, if necessary. In the ARB software package, 15 min in 10 ml 0.9 % NaCl solution containing 0.01 % the sequence was aligned with the SILVA SSURef 95 database (v/v) Tween 80 and dilution on M79 agar [containing 10 g by the integrated aligner and the alignment was again glucose, 10 g peptone (Bacto), 2 g casein hydrolysate, 2 g edited manually, if necessary. Distances (distance options yeast extract, 6 g NaCl, 15 g agar] for 2 weeks at 28 uC, the according to the Kimura-2 model) were calculated and strain was maintained on M79 agar at 28 uC and showed clustering with the neighbour-joining and maximum- orange-pigmented colonies. parsimony (results not shown) methods was performed Gram-staining behaviour and cell morphology were with MEGA 4 by using bootstrap values based on 1000 observed by phase-contrast microscopy as described by replications. Tree reconstruction using the maximum- Ka¨mpfer & Kroppenstedt (2004). Isolation of the DNA was likelihood method with fastDNAml (Olsen et al., 1994) and 30 % conservation filter (only alignment columns in which The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene the frequency of the most abundant nucleotide is ¢30 % sequence of strain 01-Je-003T is FN293377. are included in the calculation) was performed with the ARB P. Ka¨mpfer and others software package (Fig. 1). Tree topology was further tested genus. Since it has been shown for other bacteria (Munro without filters. No significant differences could be detected et al., 1972; Yamamoto et al., 1986) that increased medium between the two trees. The 16S rRNA gene sequence of osmolarity significantly reduces the intracellular polyamine strain 01-Je-003T was a continuous stretch of 1424 bp. content, relatively low polyamine contents compared with Distance calculations indicated that the closest relatives other brevibacteria might be explained by the fact that our of strain 01-Je-003T were Brevibacterium picturae DSM strain was grown in a salt-supplemented medium, whereas 16132T (98.8 % sequence similarity) and Brevibacterium the other brevibacteria subjected to polyamine analyses marinum DSM 18964T (97.3 %). Adjacent to this sub- were grown without salt supplementation (Altenburger cluster, the type strains of Brevibacterium aurantiacum et al., 1997). The characteristic cell-wall diamino acid was (97.2 % sequence similarity) and Brevibacterium antiquum meso-diaminopimelic acid. The quinone system exhibited (96.6 %) were grouped in the maximum-likelihood tree the major compound MK-8(H ) (89 %), moderate , 2 (Fig. 1). Lower sequence similarities ( 97 %) were found amounts of MK-7(H2) (10 %) and small amounts of to 16S rRNA gene sequences from all other species of the MK-9(H2) (1 %). The presence of meso-diaminopimelic genus Brevibacterium. acid is common to all brevibacteria examined so far. Also, a For analyses of polyamines, cell-wall diamino acid, qui- quinone system with MK-8(H2) predominating has been nones and polar lipids, cells were grown in PYE (0.3 % reported for representatives of this genus including peptone from casein, 0.3 % yeast extract, pH 7.2 supple- Brevibacterium samyangense (Lee, 2006), Brevibacterium mented with 3 % salts used for seawater aquarium). Polar marinum (Lee, 2008), Brevibacterium album (Tang et al., lipids and quinones were extracted and analysed as 2008), Brevibacterium oceani (Bhadra et al., 2008) and reported previously (Tindall, 1990a, b; Altenburger et al., B. picturae (Heyrman et al., 2004). The polar lipid profile T 1996; Stolz et al., 2007). Polyamines were analysed of strain 01-Je-003 consisted of the major components according to Busse & Auling (1988) and Altenburger diphosphatidylglycerol, phosphatidylglycerol and an un- et al. (1997) using the instrumentation described by Stolz identified glycolipid, moderate amounts of an unidentified et al. (2007). The diamino acid was analysed as described aminophospholipid and minor amounts of three phos- by Schleifer (1985). The polyamine pattern consisted of the pholipids and a polar lipid (Fig. 2). The presence of the 2 major compounds putrescine [0.24 mmol (g dry weight) 1] predominant lipids diphosphatidylglycerol, phosphatidyl- 2 and cadaverine [0.24 mmol (g dry weight) 1] and minor glycerol and an unidentified glycolipid was also reported 2 amounts of spermidine [0.02 mmol (g dry weight) 1]. This for B. picturae (Heyrman et al., 2004), and B. album, type of polyamine pattern with the major compounds B. marinum and B. oceani have also been shown to contain putrescine and cadaverine has been reported rarely for the major lipids diphosphatidylglycerol and phosphatidyl- actinobacteria and shown to characterize species of the glycerol (Bhadra et al., 2008; Lee, 2008; Tang et al., 2008). genus Brevibacterium (Altenburger et al., 1997), dem- Phosphatidylinositol, shown to be present in some onstrating the affiliation of strain 01-Je-003T with this Brevibacterium species, could not be detected, but the Fig. 1. Phylogenetic analysis based on 16S rRNA gene sequences available from the EMBL database (accession numbers in T parentheses) showing the position of strain 01-Je-003 . The phylogenetic tree was constructed using the ARB software package and the corresponding SILVA SSURef 95 database as detailed in the text. Tree building was performed using the maximum-likelihood method with fastDNAml (Olsen et al., 1994) and 30 % conservation filter. Bar, 0.05 substitutions per nucleotide position. Brevibacterium sandarakinum sp. nov. Table 2. Physiological characteristics of strain 01-Je-003T and its most closely related type strains Strains: 1, 01-Je-003T;2,B. picturae DSM 16132T;3,B. marinum DSM 18964T. +, Positive; 2, negative; (+), weakly positive. Data were obtained in this study. All three strains were positive for the assimilation of D-glucose, D-sorbitol, acetate (weak), propionate, cis- aconitate, citrate, fumarate (weak), glutarate, DL-3-hydroxybutyrate, 2-oxoglutarate and pyruvate.