International Journal of Systematic Bacteriology (1999), 49, 681–687 Printed in Great Britain Williamsia muralis gen. nov., sp. nov., isolated from the indoor environment of a children’s day care centre Peter Ka$ mpfer,1 Maria A. Andersson,2 Fred A. Rainey,3 Reiner M. Kroppenstedt4 and Mirja Salkinoja-Salonen2 Author for correspondence: Peter Ka$ mpfer. Tel: j49 641 99 37352. Fax: j49 641 99 37359. e-mail: peter.kaempfer!agrar.uni-giessen.de 1 Institut fu$ r Angewandte The taxonomic status of an actinomycete (MA140/96T) isolated from indoor Mikrobiologie, Justus- building materials of a children’s day care centre was studied using the Liebig Universita$ t, Senckenbergstr. 3, D-35390 polyphasic approach. The cell morphology was atypical for an actinomycete, Giessen, Germany electron microscopy revealed a hairy surface, highly unusual for Gram-positive 2 Department of Applied bacteria. The organisms grew at 10–37 mC, no growth was visible at 5 mC and Chemistry and 45 mC in 5 d. The cell wall contained the diamino acid meso-diaminopimelic acid Microbiology, PO Box 56 and the sugars arabinose, galactose, mannose and ribose. The phospholipids (Biocentre), 00014 University of Helsinki, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol and Finland diphosphatidylglycerol were detected. The only menaquinone found was MK- 3 Department of Biological 9(H2). The fatty acid pattern was composed of palmitic acid (236%) palmitoleic Sciences, 508 Life Sciences acid (165%) and another hexadecenoic acid 16:1cis11 (14%), oleic acid Building, Louisiana State (299%), stearic acid (29%) and the 10-methyl-branched tuberculostearic acid University, Baton Rouge, LA 70803, USA (232%). A gas-chromatographic analysis of the mycolic acid revealed a carbon- chain length of C –C . The GMC was 648 mol%. The results of 16S rDNA 4 DSMZ–Deutsche Sammlung 50 56 T von Mikroorganismen und sequence comparisons revealed that strain MA140/96 represents a new Zellkulturen GmbH, lineage in the suborder Corynebacterineae of the order Actinomycetales. Mascheroder Weg 1b, Therefore, it was concluded that strain MA140/96T should be assigned to a new D-38124 Braunschweig, Germany genus and species, for which the name Williamsia muralis gen. nov., sp. nov. is proposed. The type strain of the species is MA140/96T (l DSM 44343T). Keywords: Williamsia muralis gen. nov., sp. nov., actinomycete, indoor contaminant INTRODUCTION detected. One isolate from these samples was subjected to an extensive polyphasic characterization. In this The colonization of indoor building materials with paper we describe the morphological, physiological, bacteria and fungi is a common problem in case of high chemotaxonomic and phylogenetic characteristics of moisture (Maroni et al., 1995). In an extensive study of this organism. On the basis of our results and the the bacteria, moulds and their toxins found in water- unique taxonomic properties of the organism, it can be damaged buildings, Mycobacterium species and Gor- concluded that strain MA140\96T represents a new donia species were detected (Andersson et al., 1997). genus and a new species, for which we propose the Rapidly growing mycobacteria, especially, seem to be name Williamsia muralis gen. nov., sp. nov. the major bacterial colonizers of water-damaged sites; however, it can be expected that several other actino- METHODS mycetes are present, which remain to be described. Isolation. The organism (strain MA140\96T) was isolated In a survey of a children’s day care centre, no myco- from non-water-damaged building material of a children’s day care centre as described elsewhere (Andersson et al., bacteria or gordoniae were found in non-damaged 1997). The strain was isolated from gypsum liner walls of a areas; instead, a different type of actinomycete was children’s sleeping room at 22 mC on tryptone soy agar plates (Difco) incubated for 14 d. ................................................................................................................................................. Morphological characteristics. Cell morphology was The EMBL accession number for the 16S rDNA sequence of strain MA140/96T examined by phase-contrast microscopy with a light micro- is Y17384. scope (Leitz). Motility was studied by the hanging drop 00928 # 1999 IUMS 681 P. Ka$ mpfer and others method. Cell dimensions were measured with an ocular (MIS) conditions were used (Sasser, 1990). The trimethyl- (i10) and an objective (i100\1n25). Gram staining was silylated derivatives of the MAMEs were analysed by high- performed by using Hucker’s modification (Gerhardt et al., temperature gas chromatography with a model HP 5790A 1994). Colony morphology was studied by using a stereo gas chromatograph (Hewlett Packard) equipped with a microscope (Olympus model SZ 11). For electron micro- flame-ionization detector and a 12 m type HT5 column (part scopy the cells were grown on tryptone soy agar for 7 d at no. 051385; SGE, Victoria, Australia), using H as carrier " # 28 C. Thin sections were prepared as described previously gas at a flow rate of 30 ml min− . The oven temperature was m " (Andersson et al., 1995) and negative stainings as described increased from 210 to 400 mC at a rate of 10 mC min− . The by Nohynek et al. (1995). final temperature was kept for 7 min. Peaks of the derivatives were identified by comparing their retention times with those Physiological characteristics. The effects of different tempera- tures on growth were determined on Bacto nutrient agar of known standard mycolic acids (Klatte, 1994). In addition the mycolic acids were analysed by TLC following the incubated at 5, 10, 28, 37, 45 and 50 mC. Physiological tests in microtitre plates were done as described previously procedure of Minnikin et al. (1975). (Ka$ mpfer et al., 1997). Tests were read after 7 d at 30 mC. Base composition of DNA. The base composition of DNA Chemotaxonomy. Strain MA140\96T was grown on and the calculation of the GjC content was determined as solidified glucose\yeast extract\malt extract DSMZ described elsewhere (Nohynek et al., 1995). (Deutsche Sammlung von Mikroorganismen und 16S rDNA sequence determination. Genomic DNA extrac- Zellkulturen) medium no. 65 (DSMZ Catalogue of Strains, tion, PCR-mediated amplification of the 16S rDNA, and 1998) at 28 mC for 3–5 d. Cell material for analyses of fatty purification of PCR products were carried out using pro- acids and mycolic acids were scraped from trypticase soy cedures described previously (Rainey et al., 1996). Purified broth agar plates (DSMZ medium no. 535). For all other PCR products were sequenced using Taq DyeDeoxy Ter- chemotaxonomic analyses the strains were grown in minator Cycle Sequencing Kit (Applied Biosystems) as trypticase soy broth collected by centrifugation washed directed in the manufacturer’s protocol. The Applied Bio- twice with distilled water and freeze-dried. systems 310 DNA Genetic Analyzer was used for the Analysis of cell-wall amino acids and sugars. The amino acid electrophoresis of the sequence reaction products. and sugar analysis of whole-cell hydrolysate followed Phylogenetic analysis. The ae2 editor (Maidak et al., 1994) described procedures (Staneck & Roberts, 1974). was used to align the 16S rDNA sequence of strain T Determination of acyl-type of cell wall. The acyl-type of cell MA140\96 against the 16S rDNA sequences of wall was determined using a modification of the colorimetric representatives of the suborder Corynebacterineae the method of Uchida & Aida (1977). In contrast to the (Stackebrandt et al., 1997) available from the public data- original procedures, the whole-cell hydrolysate was bases. Pairwise evolutionary distances were computed using neutralized by passing it through an ion-exchange column the correction of Jukes & Cantor (1969). The least squares (Analytichem Bond Elut SCX; Varian). distance method of De Soete (1983) was used in the construction of the phylogenetic dendrogram from distance Extraction and analysis of isoprenoid quinones and polar matrices. lipids. Isoprenoid quinones were extracted and purified using the small-scale integrated procedure of Minnikin et al. Nucleotide sequence accession numbers. The strain desig- (1984). Dried preparations were dissolved in 200 µl2- nations and accession numbers of the reference strains used propanol and 1–10 µl amounts separated by HPLC without in the phylogenetic analyses are as follows: Corynebacterium glutamicum DSM 20300T, X80629; Dietzia maris DSM further purification. The menaquinones were separated by T T HPLC on Lichrosorb RP-18 at 40 mC using acetonitrile\2- 43672 , X79290; Gordonia aichiensis DSM 43978 , X80633; Gordonia amarae DSM 43392T, X80635; Gordonia propanol (65:35, v\v) as solvent (Kroppenstedt, 1985; T Kroppenstedt et al., 1981). Polar lipids were extracted, bronchialis DSM 43247 , X79287; Gordonia hirsuta DSM 44140T, X93485; Gordonia hydrophobica DSM 44015T, examined by two-dimensional TLC and identified using T published procedures (Minnikin et al., 1984). X87340; Gordonia rubropertincta DSM 43197 , X80632; Gordonia sputi DSM 43896T, X80634; Gordonia terrae DSM Preparation and analysis of fatty and mycolic acids. The fatty 43249T, X79286; Mycobacterium tuberculosis H37\RvT, acid methyl esters (FAMEs) and mycolic acid methyl esters X55588; Nocardia asteroides DSM 43757T, X80606; (MAMEs) were prepared from 40–80 mg wet cells (Miller & Nocardia brasiliensis DSM 43758T, X80608; Nocardia trans- Berger, 1984). The extracts of the methanolysates were split. valensis DSM 43405T, X80609; Rhodococcus equi