International Journal of Systematic Bacteriology (1 998),48, 53-58 Printed in Great Britain Saccharopolyspora spinosporotrichia sp. nov., a novel actinomycete from soil Zhi-Hong Zhou,'t2 Zhi-Heng Liu,' Yu-Dong Qian,' Seung Bum Kim2 and Michael Goodfellow2 Author for correspondence: Zhi-Heng Liu. Tel: +86 10 62553628. Fax: +86 10 62560912. 1 Institute of Microbiology, The generic position of an aerobic, Gram-positive, non-acid-alcohol-fast Chinese Academy of actinomycete was determined following isolation of the PCR-amplified 16s Sciences, Beijing 100080, People's Republic of China rRNA genes and alignment of the resultant sequence with corresponding sequences from representatives of the family Pseudonocardiaceae.The 2 Department of Microbiology, The Medical assignment of the organism to the genus Saccharopolyspora was strongly School, Framlington Place, supported by chemotaxonomic and morphological data. The strain was Newcastle upon Tyne distinguished from representatives of validly described Saccharopolyspora NE2 4HH, UK species by a number of phenotypic properties. It is proposed that the organism, strain AS4.198T, be classified in the genus Saccharopolyspora as Saccharopolyspora spinosporo trich ia s p. nov . Keywords : Saccharopolyspora spinosporotrichia sp. nov., 16s rDNA sequences INTRODUCTION (10), Saccharopolyspora rectivirgula Korn-Wendisch et al. (1 S), Saccharopolyspora spinosa Mertz and Yao The genus Saccharopolyspora was proposed by Lacey (26), Saccharopolyspora taberi Korn-Wendisch et al. & Goodfellow (20) for actinomycetes isolated from (1 S), and Saccharopolyspora hirsuta Lacey and spontaneously heated, sugar-cane bagasse. The genus Goodfellow (20), the type species. These taxa form a currently encompasses aerobic, non-acid-fast organ- distinct phyletic branch within the evolutionary radi- isms which form extensively branched substrate ation encompassed by the family Pseudonocardiaceae hyphae that fragment into rod-shaped, non-motile (7,33). Members of the genus Saccharopolyspora are a elements, and aerial hyphae which differentiate into potentially rich source of natural products but only bead-like chains of spores. Saccharopolysporae are erythromycin, produced by Saccharopolyspora eryth- also characterized by the presence of rneso-diamino- raea, is currently commercially important (4). pimelic acid, arabinose and galactose in their wall peptidoglycan [wall chemotype IV sensu Lechevalier An actinomycete, strain AS4.1 9ST, isolated during an ecological survey of soil actinomycetes in China, was and Lechevalier (21)], have major amounts of phos- found to have morphological properties consistent phatidylglycerol, phosphatidylcholine, phosphatidyl- ethanolamine and with its assignment to the genus Saccharopolyspora. In phosphatidylmethylethanolamine the present investigation, this organism was examined [phospholipid pattern I11 sensu Lechevalier et al. (23)], for an array of genotypic and phenotypic properties are rich in iso- and anteiso- branched-chain fatty acids and was shown to form a new centre of taxonomic (5, 6, lo), have predominant amounts of tetra-hydr- ogenated menaquinones with nine isoprene units (5, variation within the genus Saccharopolyspora. The name Saccharopolyspora spinosporotrichia is proposed lo), but lack mycolic acids (5, 10). The DNA base composition of members of the genus falls within the for this organism. range 66-77 mol YOG + C (10). The genus Saccharopolyspora contains seven validly METHODS described species, Saccharopolyspora erythraea La- Organisms and culture conditions. Strain AS4. 198T, which beda (19), Saccharopolyspora gregorii Goodfellow et was donated by the Institute of Antibiotics of the Chinese al. (10), Saccharopolyspora hordei Goodfellow et al. Academy of Medical Sciences, was isolated from soil in Jiangxi province, China. The organism was maintained on glucose yeast extract agar (GYEA) (12) at room temperature The GenBanWEMBL accession numbers for the nucleotide sequences and as glycerol suspensions (20 %, v/v) at - 20 "C. Biomass described in this paper are 709571 (strain AS4.198), AF002818 (5. spinosa) for the chemotaxonomic studies was prepared by growing and AF002819 (S. taberd. the strains in shake flasks of nutrient broth (Oxoid) at 28 "C 00496 0 1998 IUMS 53 Z.-H. Zhou and others I-. I-. Kibdelosporangium aridum subsp. aridum ATCC 39323' 4 I-' Pseudonocardia autotrophica DSM 43210' Pseudonocardia saturnea DSM 43 195' hyrdocarbonoxydans DSM 43281' Pseudonocardia petroleophila DSM 43 193' Amycolatopsis mediterranei ATCC 13685' 100 1 Amycolatopsis azurea NRRL 1 14 12' -53 Amycolatopsis orientalis subsp. orientalis DSM 40040' Saccharothrix longispora DSM 43749' 100 Saccharothrix australiensis ATCC 3 1497' Saccharothrix coeruleofusca DSM 43679' "Saccharomonospora caesia" INMI 191 25 Saccharomonospora viridis ATCC 15386' Saccharomonospora sp. A1206 Saccharopolyspora rectivirgula ATCC 335 15T ................. .................................................................................. I-' I-' Saccharopolyspora gregorii NCIMB 12823' Fig. 1. Neighbour-joining tree (15) based on nearly complete 165 rRNA gene sequences b-, Saccharopolyspora taberi DSM 43856' showing relationships between 4 Saccharopolyspora spin osp o rotrichia *- Saccharopolyspora spinosporotrichia AS4.198 AS4.198T and representative strains of the family Pseudonocardiaceaeand related taxa. Saccharopolyspora erythraea NRRL 2338' z 55 Asterisks indicate branches that were recovered using the Fitch-Margoliash method. The numbers at the nodes indicate the levels of bootstrap support based on a neighbour-joining analysis of 1000 resampled data sets; only values greater than 50% are given. The scale bar indicates 001 substitutions per nucleotide position. The arrow indicates the estimated position of -0.0 1 the root organisms. for 5 d. At maximum growth, the broth cultures were agar was detected using lead acetate strips and the ability to checked for purity, killed with formaldehyde (1 O/O, v/v), grow at various salt concentrations (1-5 %, w/v) on oatmeal harvested by centrifugation, washed three times with dis- agar. The organism was also tested for its ability to grow on tilled water and freeze dried, The microbiological tests were a range of sole carbon sources using the basal medium of incubated at 28 "C for 2 weeks unless otherwise stated. Stevenson (31) and at 10,25,30,37,45and 50 "C on oatmeal agar. The ability to degrade cellulose was detected using Cultural and morphological properties. The undisturbed filter-paper strips immersed in a minimal broth (MgSO,, arrangement of hyphae, especially aerial hyphae and spore 0.5 g; NaC1, 0.5 g; K,HPO,, 0.5 g; KNO,, 1-0g; distilled chains, was observed on the seven basal media described by water, 1 l), and incubated at 28 "C for 30 d. The organism Shirling & Gottlieb (30) using the coverslip technique (1 6). was examined for its ability to coagulate and peptonize Growth on coverslips was fixed in 5 aqueous tannic acid defatted milk after incubation at 28 "C for up to 30 d; the for 10-15 min, washed in distilled water and stained in milk was defatted by adding 0.02% (w/v) CaCO, and aqueous crystal violet (0.2 O/O, w/v) for 3 min then examined centrifuging at 4000 r.p.m. for 30 min. at high magnification using a light microscope (Carl Zeiss Chemotaxonomy. Diagnostic isomers of diaminopimelic Universal Microscope). Additional morphological data were acid, sugars and polar lipids were detected using established obtained by examining dehydrated specimens of the or- procedures (22). Isoprenoid quinones were extracted and ganism, coated with gold, using a Hitachi S-570 scanning purified according to Collins (3) and analysed by using electron microscope. HPLC (34). The presence of mycolic acids was tested using Biochemical and physiological properties. The degradation the acid'methanoiysis procedure described by Minnikin et a[ of casein, gelatin, starch and tyrosine, and the reduction of (27). nitrate were determined using established procedures (1 1, DNA base composition. The G + C content of the DNA of 13). The production of hydrogen sulphide from peptone iron the test strain was determined using the thermal de- 54 International Journal of Systematic Bacteriology 48 Saccharopolyspora spinosporotrichia sp. nov. naturation method of Marmur & Doty (25) with Escherichia coli AS1.365 as the control. DNA extraction. Biomass was prepared by centrifuging 7-d- old cultures of the test strain, S. spinosa DSM 44228Tand S. taberi DSM 43856T grown in shake flasks of tryptic soy broth (Difco) at 30 "C. Genomic DNA was isolated from the test strains by using a procedure (2) slightly modified from that of Pitcher et al. (28). Sequencing of 165 rDNA. PCR amplification of 16s rDNA was performed as described by Kim et al. (17). The PCR products were purified using 2-Spin Plus Centrifugal Filters (Gelman Sciences) according to the procedure provided by the manufacturer. The purified products were directly sequenced by using a Taq DyeDeoxy Terminator Cycle Sequencing kit (Applied Biosystems) and previously de- scribed oligonucleotide primers (2). Sequence gel electro- phoresis was carried out and nucleotide sequences were automatically obtained by using an Applied Biosystems DNA sequencer (model 373A) and software provided by the manufacturer. Analysis of sequence data. The 16s rDNA sequences were aligned manually with sequences of Saccharopolyspora and related strains obtained from Ribosomal Database Project (24), EMBL (14) and GenBank databases by using the ALl6S program