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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 is Crossiella cryophila gen. nov., comb. nov.

Keywords: Pseudonocardineae, Actinosynnemataceae, polyphasic , 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: 0 9·77 cis-9-16: 1 3·11 The recent phylogenetic study of Saccharothrix and 16:0 1·76 related taxa (Labeda et al., 2001) revealed that many of 10-methyl-iso-16: 0 1·09 the described species of Saccharothrix belong to other 9?-methyl-16: 0 11·58 genera. Most of the species were contained within the iso-17: 0 11·36 family Actinosynnemataceae, in the genus Lentzea anteiso-17: 0 1·86 (Lentzea waywayandensis) or in the newly proposed cis-9-17: 1 1·86 genus Lechevalieria (Lechevalieria aerocolonigenes and 17:0 1·21 Lechevalieria flava) , but Saccharothrix cryophilis formed a monophyletic lineage with the genera Actino­ alloteichus, Kutzneria and Streptoalloteichus, which all fell phylogenetically between the Actinosynnemataceae were also observed in the culture (Fig. 1b). Motile and the . Its nearest phylogenetic spores were not detected during extensive observation neighbour is the genus Streptoalloteichus, from which on many different growth media. it can be distinguished chemotaxonomically and morphologically. Actinoalloteichus forms long spore The chemotaxonomic profile of RRL B-16238T chains, while Kutzneria and Streptoalloteichus form compared with related taxa is shown in Table 2 and the true sporangia. Streptoalloteichus has also been fatty acid profile is shown in Table 3. The physiological reported to produce motile spores. The morphological characteristics of RRL B-16238T are listed in the characteristics observed in Saccharothrix cryophilis formal description of Crossiella cryophila gen. nov., T RRL B-16238 , production of sclerotia or pseudo­ comb. nov., given below. The phylogenetic position of sporangia on the substrate mycelium, are quite the proposed genus Crossiella relative to the taxa different. On the basis ofphylogenetic position, chemo­ within or related to the families Actinosynnemataceae taxonomy and novel micromorphology (pseudo­ and Pseudonocardiaceae can be seen in the nearest­ sporangium production), a new genus is proposed for neighbour radial tree shown in Fig. 2. this strain, to be named Crossiella gen. nov. in In their original description of' Nocardiopsis mutabilis recognition of the contributions of Thomas Cross, subsp. cryophilis', Takahashi et al. (1986) noted that formerly at the University of Bradford, UK, to their isolate had novel morphological characteristics actinomycete biology and systematics. The type species but, based on the chemotaxonomic data, felt that it of Crossiella is Crossiella cryophila gen. nov., comb. was best to describe the strain as a subspecies of nov. This genus is clearly within the suborder Pseudo-

International Journal of Systematic and Evolutionary Microbiology 51 1577 D. P. Labeda

PSEUDONOCARDMCEAE

Pseudonocardia

Saccharomonospora Actinopolyspora halophilo.ATCC 27976T004287 Amycolatopsis

T Kihdelosporangium aridum ATCC 39323 / X53191 T Arthrobacter globijormis DSM 20124 / M23411

T Actinoalloteichus cyanogriseus IFO 1445S / AB006178

T Kutmeria viridogrisea DSM 438S0 / X70429 T Kutmeria kofuensis NRRL B-16061 / AFl14801 T Streptoalloteichus hindustanus IFO lS11S / D8S497

T Crossielkz cryophilo. NRRL B-16238 / AF114807 ACTINOSYNNEMATACEAE Saccharothrix Actinokineospora

0.10

Fig. 2. Radial phylogenetic tree of the suborder Pseudonocardineae calculated from 16 rDNA sequences using Kimura's evolutionary distance method (Kimura, 1980) and the neighbour-joining method of Saitou & Nei (1987). This tree shows the relationship of Crossiella cryophila NRRL B-16238T to the genera Actinoalloteichus, Kutzneria and Streptoalloteichus. T The taxa and sequences included in the genus groups are: Actinokineospora diospyrosa NRRL B-24047 , AFl14797; Actinokineospora globicatena NRRL B-24048T, AFl14798; Actinokineospora inagensis NRRL B-24050T, AFl14799; T T Actinokineospora riparia NRRL B-16432 , AFl14802; Actinosynnema mirum DSM 43827 , X84447; Actinosynnema T T pretiosum subsp. pretiosum NRRL B-16060 , AFl14800; Amycolatopsis azurea NRRL 11412 , X53199; Amycolatopsis fastidiosa ATCC 31181 T, X53200; Amycolatopsis mediterranei ATCC 13685T, X76957; Amycolatopsis methanolica NCiB T T T 11946 , X54274; Amycolatopsis orientalis DSM 40040 , X76958; Lechevalieria aerocolonigenes NRRL B-3298 , AFl14804; T T T Lechevalieria flava NRRL B-16131 , AFl14808; Lentzea albida IFO 16102 , AB006176; Lentzea albidocapillata DSM 44073 , T T X84321; Lentzea waywayandensis NRRL B-16159 , AFl14813; alni VKM Ac-901 , X76954; Pseudonocardia T autotrophica DSM 43210, X54288; Pseudonocardia compacta DSM 43592 , X76959; Pseudonocardia halophobica DSM 43089T, Z14111; Pseudonocardia hydrocarbonoxydans DSM 43281, X76955; Pseudonocardia nitrificans IFAM 379T, T T X55609; Pseudonocardia petroleophila ATCC 15777 (= DSM 43193 ), X80596; Pseudonocardia saturnea DSM 43195, T X76956; Pseudonocardia thermophila ATCC 19285 , X53195; 'Saccharomonospora caesia' INMI 19125, X76960; T Saccharomonospora viridis Goodfellow SB-33, X54286; Saccharopolyspora erythraea NRRL 2338 , X53198; Saccharopolyspora gregorii NCiMB 12823T, X76962; Saccharopolyspora hirsuta ATCC 27875T, X53196; Saccharopolyspora T T hordei ATCC 49856 , X53197; Saccharopolyspora rectivirgula ATCC 33515 , X53194; Saccharothrix australiensis NRRL T T T 11239 , AFl14803; Saccharothrix coeruleofusca NRRL B-16115 , AFl14805; Saccharothrix espanaensis NRRL 15764 , T T AFl14807; Saccharothrix longispora NRRL B-16116 , AFl14809; Saccharothrix mutabilis subsp. capreolus DSM 40225 , T T X76965; Saccharothrix mutabilis subsp. mutabilis DSM 43853 , X76966; Saccharothrix syringae NRRL B-16468 , AFl14812; T Saccharothrix texasensis NRRL B-16134 , AFl14814. Bar, 0·1 nucleotide substitutions per site.

nocardineae Stackebrandt et al. 1997, but it cannot be rod-shaped elements and sclerotium-like pseudo­ placed clearly within either of the families Actino­ sporangia may be produced on the substrate mycelium. synnemataceae or Pseudonocardiaceae at this time. Swellings may be produced at or near the tip of aerial hyphae. Nocardiomycolic acids are absent. Catalase­ positive. Contain meso-diaminopimelic acid as the Description of Crossiella gen. nov. diamino acid and acetylated peptidoglycan. The Crossiella (Cross.i.el'la. L. dim. ending -ella; N.L. fern. whole-cell sugar pattern consists of galactose, man­ n. Crossiella named for Thomas Cross, a micro­ nose, rhamnose and ribose. The phospholipid pattern biologist at the University of Bradford, who made consists of phosphatidyl ethanolamine, phosphatidyl many contributions to actinomycete biology and methylethanolamine, phosphatidyl inositol and systematics). phosphatidyl inositol mannosides. The predominant menaquinone is MK-9(H4). Members have a fatty­ Aerobic. Gram-positive, non-acid-fast, non-motile­ acid profile rich in branched-chain and saturated actinomycetes. Branched substrate mycelium (approx. components. Phylogenetically, the nearest neighbour 0·5 11m in diameter) and, on some media, aerial myc'e1ia is the genus Streptoalloteichus. The type species is are produced. Vegetative mycelium mayfragment into Crossiella cryophila.

1578 International Journal of Systematic and Evolutionary Microbiology 51 Crossiella gen. nov.

Description of Crossiella cryophila comb. nov. (1974). Nocardia coeliaca, Nocardia autotrophica, and the nocardin strain. Int J Syst Bacteriol24, 54-63. Crossiella cryophila (cry.o.phira. N.L. adj. cryophila Grund, E. & Kroppenstedt, R. M. (1989). Transfer of five cold-loving, referring to the low permissive tempera­ Nocardiopsis species to the genus Saccharothrix Labeda et al. ture range for growth). 1984. Syst Appl Microbiol12, 267-274. Basonym: Saccharothrix cryophilis Labeda & Kimura, M. (1980). A simple method for estimating evolutionary Lechevalier 1989. rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evo116, 111-120. This description is based on results from this and Kurup, P. V. & Schmitt, J. A. (1973). umerical taxonomy of earlier studies (Labeda & Lechevalier, 1989). Pale­ Nocardia. Can J Microbiol19, 1035-1048. yellow to light-brown substrate mycelium is produced Labeda, D. P. & Kroppenstedt, R. M. (2000). Phylogenetic analysis on most media. White to yellowish-white aerial my­ of Saccharothrix and related taxa: proposal for Actino­ celium is produced, particularly on inorganic salts/ synnemataceae fam. nov. Int J Syst Evol Microbiol50, 331-336. starch agar or glycerol/asparagine agar. Casein, Labeda, D. P. & Lechevalier, M. P. (1989). Amendment of the aesculin, gelatin, starch, tyrosine and urea are hydro­ genus Saccharothrix Labeda et al. 1984 and descriptions of lysed or decomposed. Adenine, allantoin, hippurate, Saccharothrix espanaensis sp. nov., Saccharothrix cryophilis sp. hypoxanthine and xanthine are not hydrolysed or nov., and Saccharothrix mutabilis comb. nov. Int J Syst decomposed. Nitrate is reduced to nitrite. Phosphatase BacterioI39,420-423. is produced. Acetate, oL-lactate, oL-malate, oxalate, Labeda, D. P., Hatano, K., Kroppenstedt, R. M. & Tamura, T. propionate and succinate are assimilated; benzoate, (2001). Revival of the genus Lentzea and proposal for citrate, mucate and oL-tartrate are not assimilated. Lechevalieria gen. nov. Int J Syst Evol Microbiol51, 1045-1050. Acid is produced from o-fructose, o-galactose, Maidak, B. L., Larsen, N., McCaughey, M. J., Overbeek, R., Olsen, o-glucose, glycerol, myo-inositol, lactose, maltose, G. J., Fogel, K., Blandy, J. & Woese, C. R. (1994). The Ribosomal o-mannose and trehalose; no acid is produced Database Project. Nucleic Acids Res 22, 3485-3487. from adonitol, L-arabinose, dulcitol, meso-erythritol, Pridham, T. G. & Lyons, A. J. (1980). Methodologies for Actino­ mannitol, methyl a-o-glucoside, methyl ,B-o-xyloside, mycetales with special reference to streptomycetes and strepto­ L-rhamnose, salicin, o-sorbitol, sucrose or D-xylose; verticillia. In Actinomycete Taxonomy, Special Publication no. acid production is variable from cellobiose, dextrin, 6, pp. 153-224. Edited by A. Dietz & D. W Thayer. Arlington, lactose, melibiose and raffinose. Growth occurs in the VA: Society for Industrial Microbiology. presence of 4 % aCI but not in the presence of 5 % Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new NaCl. The temperature range for growth is 10-33 °C. method for reconstructing phylogenetic trees. Mol BioI Evol 4, The G + C content of the DA is 71-4 mol % (thermal 406-425. denaturation midpoint method). Isolated from soil Shirling, E. B. & Gottlieb, D. (1966). Methods for characterization from Shosenkyo, Yamanashi Prefecture, Japan. of Streptomyces species. Int J Syst Bacteriol16, 313-340. Produces the antibiotic dopsisamine. The type strain Stackebrandt, E., Kroppenstedt, R. M., Jahnke, K.-D., T T of C. cryophila is NRRL B-16238 ( = ATCC 51143 Kemmerling, C. & GUrtler, H. (1994). Transfer of Strepto­ = DSM 44230T = IFO 14475T = Y. Okami TS­ sporangium viridogriseum (Okuda et al. 1966), Strepto­ T 1980 ). sporangium viridogriseum subsp. kofuense (Nonomura and Ohara 1969), and Streptosporangium albidum (Furumai et al. 1968) to Kutzneria gen. nov. as Kutzneria viridogrisea comb. ACKNOWLEDGEMENTS nov., Kutzneria kofuensis comb. nov., and Kutzneria albida comb. nov., respectively, and emendation of the genus Strepto­ The able technical assistance of E. . Hoekstra with the sporangium. Int J Syst Bacteriol44, 265-269. studies ofphysiological properties, isolation and purification Stackebrandt, E., Rainey, F. A. & Ward-Rainey, N. L. (1997). DNA and chemotaxonomic determinations for this study is Proposal for a new hierarchic classification system, Actino­ gratefully acknowledged. c1assis nov. Int J Syst Bacteriol47, 479-491. Takahashi, A., Hotta, K., Saito, N., Morioka, M., Okami, Y. & REFERENCES Umezawa, H. (1986). Production of novel antibiotic, dopsisamine, by a new subspecies of Nocardiopsis mutabilis with Cote, R., Daggett, P.-M., Gantt, M. J., Hay, R., Jong, S.-c. & Pienta, multiple antibiotic resistance. J Antibiot 39, 175-183. P. (1984). ATCC Media Handbook. Rockville, MD: American Tamura, T. & Hatano, K. (1998). Phylogenetic analyses on the Type Culture Collection. strains belonging to invalidated genera of the order Actino­ Galtier, N., Gouy, M. & Gautier, C. (1996). SEAVIEW and mycetales. Actinomycetologia 12, 15-28. PHYLO_WIN: two graphic tools for sequence alignment and Tamura, T., Zhiheng, L., Yamei, Z. & Hatano, K. (2000). Actino­ molecular phylogeny. Comput Appl Biosci 12, 543-548. alloteichus cyanogriseus gen. nov., sp. nov. Int J Syst Evol Gordon, R. E., Barnett, D. A., Handerhan, J... E. & Pang, C. H.-N. Microbiol50, 1435-1440.

Su ppheo by U.S. DeDl of Agricutture National Center for Agncultural Utilization Research, Peon., Illinois

International Journal of Systematic and Evolutionary Microbiology 51 1579