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Crossiella Gen. Nov., a New Genus Related to Streptoalloteichus

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Crossiella Gen. Nov., a New Genus Related to Streptoalloteichus 8559 International Journal ofSystematic and Evolutionary Microbiology (2001),51, 1575-1579 Printed in Great Britain Crossiella gen. nov., a new genus related to Streptoalloteichus Microbial Properties D. P. Labeda Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Tel: + 1 309 681 6397. Fax: + 1 309 681 6672. e-mail: [email protected] Service, US Department of Agriculture, 1815 N. University Street, Peoria, Phylogenetic analysis of the genera within the suborder Pseudonocardineae IL 61604, USA based on almost complete sequences of 165 rONA showed that Saccharothrix cryophilis NRRL B-16238T was misplaced within the genus Saccharothrix. Saccharothrix cryophilis NRRL B-16238T appeared to be phylogenetically closest to Streptoalloteichus, but is morphologically distinct from this genus because sporangia with motile spores are not observed. The aerial mycelium fragments into rod-shaped elements and sclerotium-like bodies are observed occasionally in the substrate mycelium. The cell wall contains meso-diaminopimelic acid, whole-cell hydrolysates contain galactose, rhamnose and ribose, the phospholipid pattern is type PIV and the principal menaquinone is MK-9(H 4). A new genus to accommodate Saccharothrix cryophilis is proposed, Crossiella gen. nov., in recognition of the contributions of Thomas Cross, a distinguished actinomycete biologist at the University of Bradford, UK. The type species is Crossiella cryophila gen. nov., comb. nov. Keywords: Pseudonocardineae, Actinosynnemataceae, polyphasic taxonomy, Saccharothrix INTRODUCTION Saccharothrix cryophilis NRRL B-16238 T was not a member of the genus Saccharothrix, or even of the 'Nocardiopsis mutabilis subsp. cryophilis' was family Actinosynnemataceae, but exhibited a close described by Takahashi et al. (1986) for a novel soil relationship to the genus Streptoalloteichus. A poly­ isolate that produced the antibiotic dopsisamine. The phasic study was undertaken to confirm and expand authors noted that this strain exhibited some mor­ on the published characteristics of this strain in phological characteristics that were different from support of the proposal to transfer it to a new genus members of the genus Nocardiopsis but, based on within the suborder Pseudonocardineae (Stackebrandt chemotaxonomic properties, they felt that this genus et al., 1997). was the closest fit. It was subsequently proposed in two independent studies that Nocardiopsis mutabilis be METHODS transferred to the genus Saccharothrix (Grund & Kroppenstedt, 1989; Labeda & Lechevalier, 1989), Strains, cultivation and maintenance. Primary storage of and an evaluation of DNA relatedness between the strains was as lyophilized ampoules of mycelial and spore type strain of 'Nocardiopsis mutabilis subsp. cryo­ suspensions in sterile beefserum held at 4 0c. Working stock T cultures were maintained on slants of ATCC medium 172 phi/is', NRRL B-16238 , and the other taxa of the (Cote et al., 1984) and stored at 4 °C until needed. Biomass genus Saccharothrix demonstrated that this strain for extraction of DA was grown as 7-d streak cultures on represented a distinct species within Saccharothrix ATCC medium 172 agar plates. (Labeda & Lechevalier, 1989). A recent phylogenetic study of Saccharothrix and related genera based on Morphological observations. Gross morphological obser­ almost complete 16S rDNA sequences resulted in the vations were made using cultures grown for 14 d at 28°C on the standard media suggested by the International Strepto­ creation of the new family Actinosynnemataceae myces Project (Shirling & Gottlieb, 1966) and Czapek's (Labeda & Kroppenstedt, 2000), as well as some sucrose agar (Pridham & Lyons, 1980). Micromorphology taxonomic reorganization among species included and sporulation were observed by light microscopy and within the genus Saccharothrix (Labeda et al., 2001). scanning electron microscopy (SEM). Samples for SEM This phylogenetic analysis indicated strongly that observation were 14-d cultures on agar media fixed overnight 01824 1575 D. P. Labeda with osmium tetroxide vapours, post-fixed for 1 h in 1 % osmium tetroxide in 0·1 M sodium cacodylate buffer, pH 7·3, dehydrated through a graded acetone series and then critical­ point dried from liquid CO2 and sputter-coated with gold/palladium. The samples were observed using a lEOL model 15M 6400 V scanning electron microscope. Chemotaxonomy. Chemotaxonomic analysis of strains for menaquinones, fatty acids and whole cell sugars was performed using methods described previously (Grund & Kroppenstedt, 1989). Physiological tests. Physiological tests, including production of acid from carbohydrates, utilization of organic acids and hydrolysis and decomposition of adenine, guanine, hypo­ xanthine, tyrosine, xanthine, casein, aesculin, urea and hippurate, were evaluated by using the media of Gordon et al. (1974). Allantoin hydrolysis was evaluated in the basal medium suggested by Gordon et al. (1974) for aesculin hydrolysis. Phosphatase activity was evaluated by using the method of Kurup & Schmitt (1973). The temperature range for growth was determined on slants of ATCC medium 172 agar (Cote et al., 1984). DNA isolation, 165 rONA gene amplification and sequencing. Genomic DNA was isolated, purified and sequenced following procedures described previously (Labeda & Kroppenstedt, 2000). Phylogenetic analysis. The 16S rDNA sequences obtained in this study were aligned manually with actinomycete ref­ erence sequences obtained from the Ribosomal Database Project (Maidak et al., 1994) and GenBank in the ARB software environment for sequence data developed by Wolfgang Ludwig and Oliver Strunk (Lehrstuhl fUr Mikro­ biologie, University of Munich, Germany). The program PHYLO_WIN (Galtier et al., 1996) was used to calculate evolutionary distances by the method of Kimura (1980) and linkages by the neighbour-joining method of Saitou & Nei (1987) and to perform maximum-parsimony and maximum­ likelihood analyses. The topographies of the trees resulting from neighbour-joining and maximum-parsimony analyses Fig. 1. SEM of Crossiella cryophila NRRL B-16238T. (a) were evaluated by bootstrap analysis of the data with 500 Pseudosporangium on substrate mycelium. (b) Swellings resamplings. typically observed on aerial mycelium. Bars, 111m. RESULTS AND DISCUSSION addition to demonstrating the fragmentation of sub­ Strain NRRL B-16238T grows well on all of the media strate mycelium into rod-shaped elements, showed the evaluated and the gross morphological characteristics presence of sclerotia or pseudosporangium-like bodies on various standard media are shown in Table 1. on the colony surface, as had been noted by Takahashi Soluble pigments were not produced on any of the et at. (1986) in their original description of this micro­ media tested. Observation of the strain by SEM, in organism (Fig. la). Swellings near the tips of mycelia Table 1. Gross morphological characteristics of Crossiella cryophila NRRL B-16238T Soluble pigments were not produced on any of the media listed. Medium Colour of substrate mycelium Colour of aerial mycelium ATCC medium 172 Light-orange to light-brown White to pale-yellowish-pink Czapek's sucrose agar Yellowish-white White Glycerol/asparagine agar (ISP-5) Pale-yellow to medium-yellow Yellowish-white Inorganic salts/starch agar (ISP-4) Light-yellow to brilliant-yellow White Yeast extract/malt extract agar (ISP-2) Light-yellow to light-brown White 1576 International Journal of Systematic and Evolutionary Microbiology 51 Crossiella gen. nov. Table 2. Chemotaxonomic characteristics of Crossiella and related taxa All genera listed contain meso-diaminopimelic acid as the cell wall diamino acid. Abbreviations: DPG, diphosphatidyl glycerol; PE, phosphatidyl ethanolamine; PG, phosphatidyl glycerol; PI, phosphatidyl inositol; PIM, phosphatidyl inositol mannosides; PME, phosphatidyl methylethanolamine. Data were taken from this study and from Tamura et at. (2000) (Actinoalloteichus), Stackebrandt et at. (1994) (Kutzneria) and Tamura & Hatano (1998) (Streptoalloteichus). Taxon Whole-cell sugar pattern Phospholipids Predominant menaquinone(s) ) Crossiella Galactose, mannose, rhamnose, ribose PE, DPG, PI, PIM, PME MK-9(H4 Actinoalloteichus Galactose, mannose, ribose PE, DPG, PME, PG, PIM MK-9(H4) Kutzneria Galactose, trace rhamnose PE, HO-PE, PI, DPG MK-9(H4) ), ) Streptoalloteichus Galactose, mannose, ribose PE MK-1O(H4 MK-IO(H6 Saccharothrix Galactose, rhamnose, mannose PE, HO-PE, PIM, PI, DPG, PG MK-9(H4), MK-I0(H4) Table 3. Fatty acid profile of Crossiella cryophila NRRL Nocardiopsis mutabilis. This strain was subsequently 8-16238T transferred to the genus Saccharothrix as Saccharo­ thrix cryophilis as part ofa study in which Nocardiopsis Fatty acids are listed as determined by the Microbial mutabilis was transferred to the genus Saccharothrix Identification System software (MIDI Inc.) peak naming (Labeda & Lechevalier, 1989). D A-relatedness com­ table. parisons made at that time demonstrated conclusively that this strain was not a subspecies of Saccharothrix Fatty acid Content (% of total) mutabilis because it only showed 9 % DA relatedness to the type strain of this species. It was noted that iso-14: 0 1·02 Saccharothrix cryophilis exhibited extremely low DA iso-IS: 0 44·28 relatedness to all of the other species of Saccharothrix anteiso-15: 0 2·29 tested, but there were insufficient additional data 15: 1 B 3·06 available at that time to create a new genus with any iso-H-16: 1 3·16 confidence. iso-16:
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