International Journal of Systematic and Evolutionary Microbiology (2002), 52, 1815–1820 DOI: 10.1099/ijs.0.02204-0 Description of Lentzea flaviverrucosa sp. nov. and transfer of the type strain of Saccharothrix aerocolonigenes subsp. staurosporea to Lentzea albida 1 Institute of Space Qiong Xie,1 Yimin Wang,2 Ying Huang,2 Yuanliang Wu,1 Fusen Ba1 Medico-Engineering, 2 Beijing 100094, and Zhiheng Liu People’s Republic of China 2 State Key Laboratory of Author for correspondence: Zhiheng Liu. Tel: j86 10 6255 3628. Fax: j86 10 6255 3628. Microbial Resources, e-mail: zhliu!sun.im.ac.cn Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, People’s Republic of China A distinct actinomycete isolated from soil was subjected to polyphasic taxonomic analysis. It is demonstrated by comparative 16S rDNA gene sequencing that the organism, designated strain AS 4.0578T, represents a novel species of the genus Lentzea. The phylogenetic results also showed that it formed a monophyletic lineage distinct from the available members of the genera Lentzea, Lechevalieria and Saccharothrix. The organism was distinguished from all the validly described type strains of the genus Lentzea by a combination of phenotypic features and DNA–DNA hybridization. It is proposed, therefore, that strain AS 4.0578T (l JCM 11373T) be classified in the genus Lentzea as Lentzea flaviverrucosa sp. nov. In addition, it is proposed that Saccharothrix aerocolonigenes subsp. staurosporea NRRL 11184T be transferred to Lentzea albida on the basis of phylogenetic analysis, DNA–DNA homology, nucleotide signatures and phenotypic properties. Keywords: Lentzea flaviverrucosa sp. nov., polyphasic taxonomy, 16S rDNA sequencing INTRODUCTION Lentzea, created several novel species (Lentzea albida NRRL B-24073T, Lentzea californiensis NRRL B- The genus Lentzea (Yassin et al., 1995) was proposed 16137T, Lentzea violacea IMSNU 50388T and Lentzea for aerobic actinomycetes that form abundant aerial waywayandensis NRRL B-16159T) and also proposed hyphae that fragment into rod-shaped elements. Lee et Lechevalieria gen. nov. according to an analysis of al. (2000) proposed that Lentzea albidocapillata, the chemotaxonomic properties, almost-complete 16S type and only species of the genus Lentzea, should be rDNA sequences and diagnostic nucleotide signatures. transferred to the genus Saccharothrix. However, The present investigation was designed to establish the chemotaxonomic, morphological and physiological taxonomic position of a soil isolate, AS 4.0578T, that properties and phylogenetic analysis demonstrated had been invalidly described as ‘Streptomyces that the genus Saccharothrix was heterogeneous flavoverrucosus’ on the basis of a few biochemical, (Labeda et al., 1984; Labeda, 1986; Labeda & morphological and physiological properties (Yan & Lechevalier, 1989; Labeda & Lyons, 1989; Grund & Deng, 1966). Genotypic and phenotypic data showed Kroppenstedt, 1989; Warwick et al., 1994; Kinoshita that ‘Streptomyces flavoverrucosus’ strain AS 4.0578T et al., 1999; Labeda & Kroppenstedt, 2000). In a recent should be recognized as a novel species of Lentzea, for proposal, Labeda et al. (2001) revived the genus which the name Lentzea flaviverrucosa sp. nov. is ................................................................................................................................................. proposed. A recent proposal (Labeda et al., 2001) Published online ahead of print on 5 July 2002 as DOI 10.1099/ revealed that several species of Saccharothrix had been misclassified, but the type strain of Saccharothrix ijs.0.02204-0. T Abbreviations: DAP, diaminopimelic acid; GYM, glucose/yeast extract/ aerocolonigenes subsp. staurosporea, NRRL 11184 , malt extract; ISP, International Streptomyces Project. was not mentioned. In this study, physiological, The GenBank accession number for the 16S rDNA sequence of strain AS chemotaxonomic and phylogenetic properties, DNA– 4.0578T (l JCM 11373T) is AF183957. DNA relatedness and nucleotide signatures in the 16S 02204 # 2002 IUMS Printed in Great Britain 1815 Q. Xie and others rDNA sequence were used to compare this actino- carried out by measurement of DNA–DNA hybridization mycete to Lentzea albida NRRL B-24073T in order to from renaturation rates (De Ley et al., 1970). clarify its taxonomic position. 16S rDNA sequencing. PCR amplification of the 16S rDNA was performed as described by Kim et al. (1996) and the METHODS resultant PCR products were purified using the Wizard PCR purification system (Promega) according to the procedures Micro-organisms and culture conditions. Strain AS 4.0578T provided by the manufacturer. The purified products were was isolated from soil in Wutaishan Mountain, Shanxi sequenced directly using a Taq DyeDeoxy Terminator cycle Province, China, by Yan & Deng (1966). The isolate was sequencing kit (Applied Biosystems) and universal primers maintained on modified Sauton’s agar (Mordarska et al., (Huang et al., 2001). The nucleotide sequence was obtained 1972) slants at 4 mC and as a glycerol suspension (20%, v\v) automatically by using an Applied Biosystems 373A DNA at k20 mC. Biomass for chemotaxonomic and molecular- sequencer according to the manufacturer’s protocols. systematic studies was prepared by growing the strain in Phylogenetic analysis. The 16S rDNA sequence of strain AS shake flasks of modified Sauton’s broth at 28 mC for 7 days. 4.0578T was aligned manually against corresponding Cultures were then checked for purity, harvested by centri- sequences of type species of the genera Saccharothrix, fugation, washed twice with distilled water and freeze-dried. Lentzea and Lechevalieria retrieved by search from Cultural and morphological properties. Gross morphological EMBL\GenBank using 1.8 (Thompson et al., observations were made by using cultures grown at 28 mC for 1997). Phylogenetic trees were inferred by using three tree- 14 days on the standard media suggested by the International making algorithms, the neighbour-joining (Saitou & Nei, Streptomyces Project (ISP) (Shirling & Gottlieb, 1966) and 1987), Fitch–Margoliash (Fitch & Margoliash, 1967) and glucose\yeast extract\malt extract (GYM) agar. Melanin maximum-likelihood (Felsenstein, 1981) treeing algorithms, pigment production was determined using peptone\yeast from the package (Felsenstein, 1993). Evolutionary- extract\iron agar (ISP medium 6) and tyrosine agar (ISP distance matrices were generated as described by Kimura medium 7). Micromorphology was examined using the (1980). Tree topologies were evaluated by carrying out cover-slip technique. Growth on cover slips was fixed and bootstrap analysis based on 1000 resamplings of the examined according to the procedures described by Zhou neighbour-joining dataset using the programs and et al. (1998). Additional morphological characters were provided in the package (Felsenstein, examined by using a Hitachi S-570 scanning electron 1993). microscope. Phenotypic tests. The test strains were examined for a range RESULTS AND DISCUSSION of phenotypic properties following the procedures of Lee et An almost-complete 16S rDNA sequence was de- al. (2000), Yassin et al. (1995) and Gordon et al. (1974). To T determine lysozyme sensitivity, a 0n05% (w\v) solution of termined for strain AS 4.0578 (1426 nt). Comparison lysozyme was sterilized by membrane filtration and added to of this sequence with sequences of all validly published sterilized Sauton’s broth (McCarthy & Cross, 1984) to give type strains of the genera Lentzea, Lechevalieria and T a final concentration of 0n0025% (w\v). Test tubes of the Saccharothrix showed that strain AS 4.0578 , Lentzea basal medium with and without lysozyme were inoculated violacea IMSNU 50388T, Lentzea albidocapillata with fresh cultures and examined for growth after 10 days at NRRL B-24057T, Lentzea waywayandensis NRRL B- either 28 or 50 mC. Antibiotic-sensitivity tests were carried 16159T, Lentzea albida NRRL B-24073T and Lentzea out by placing impregnated filter-paper discs (Goodfellow & californiensis NRRL B-16137T formed a lineage that Orchard, 1974) over Sauton’s agar and incubating the also contains Saccharothrix aerocolonigenes subsp. samples for 7–10 days at the incubation temperatures cited T above. Growth was tested at temperatures of 37, 42 and staurosporea NRRL 11184 (Fig. 1). Although pair- wise comparison of 16S rDNA sequences indicated 45 mC. For comparative purposes, Lentzea violacea IMSNU T 50388T and Lentzea albidocapillata NRRL B-24057T were that strain AS 4.0578 shared the highest 16S rDNA T used as reference strains. similarity with Lentzea violacea IMSNU 50388 T Chemotaxonomy. The isomeric form of diaminopimelic acid (98n3%), Lentzea albidocapillata NRRL B-24057 (97n7%) and Lentzea waywayandensis NRRL B- (DAP), predominant whole-organism sugars and the T T phospholipid pattern of the test strain were detected fol- 16159 (97n5%), strain AS 4.0578 showed low DNA– lowing standard procedures developed by Lechevalier et al. DNA relatedness with Lentzea violacea IMSNU (1977), Lechevalier & Lechevalier (1980) and Hasegawa et 50388T (34%), Lentzea albidocapillata NRRL B- al. (1983). Menaquinones were extracted and purified 24057T (43%) and Lentzea waywayandensis NRRL B- according to Collins (1985) and were analysed by HPLC 16159T (12%). The 16S rDNA similarity of strain AS (Wu et al., 1989), with Streptomyces griseus as the control. 4.0578T to the remaining validly published type strains Standard TLC protocols were also used for the extraction of the genera Lentzea,