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1 Sanofi-Aventis Deutschland GmbH, SM&A, Chemical Sciences, Natural Products, 65926 Frankfurt am Main, Germany
2 DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1B, 38124 Braunschweig, Germany
Correspondence
Joachim M. Wink
Joachim.Wink{at}sanofi-aventis.com
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; Vertesy et al., 1999). Actinoplanes strain AB8327, the producer of the antibiotic gardimycin, was described and named by Parenti and coworkers as ‘Actinoplanes teichomyceticus’, referring to inhibition of cell-wall synthesis (Parenti et al., 1976). Neither name was validly published. In a study on the phylogenetic relationship of Actinoplanes species by comparative 16S rRNA gene sequencing by Tamura & Hatano (2001), the two strains were included. In the resulting tree, derived by the neighbour-joining method, strains A/6353 and AB8327 were found to cluster within the radiation of Actinoplanes species, related sufficiently remotely from validly named species to warrant their description as novel species. This step is presented in this communication by providing sufficient taxonomic evidence to describe Actinoplanes liguriensis sp. nov. with the type strain A/6353T and Actinoplanes teichomyceticus sp. nov. with the type strain AB8327T. The strains under study were A/6353T (=DSM 43865T), AB8327T (=DSM 43866T), Actinoplanes palleronii (DSM 43940T), Actinoplanes regularis (DSM 43151T) and Actinoplanes rectilineatus (DSM 43808T).
The morphological and physiological characteristics of the strains were studied after incubation for 10 days at 28 °C on various media described by Shirling & Gottlieb (1966): yeast extract/malt extract agar (ISP 2), oatmeal agar (ISP 3), inorganic salt/starch agar (ISP 4), glycerol/asparagine agar (ISP 5), peptone/yeast extract/iron agar (ISP 6) and tyrosine agar (ISP 7). For scanning electron microscopy, the strains were grown on different ISP media and prepared according to Grabley et al. (1992). Colours were determined according to the RAL colour code (RAL is the Deutsches Institut für Gütesicherung und Kennzeichnung e.V.-Reichsausschuß für Lieferbedingungen).
Utilization of carbohydrates was investigated on ISP medium 9 (Shirling & Gottlieb, 1966) by using a microtitre-plate technique with 12-well plates. A fingerprint of enzymic activities was obtained by API 20E and API ZYM test strips (bioMérieux).
Analysis of the antimicrobial spectrum was performed as described by Williams et al. (1989). Bacterial strains were grown on Mueller–Hinton agar and fungal strains on Czapek Dox. For metabolite production, the Actinoplanes strains were incubated in four different media: a soybean meal medium, a starch medium and ISP media 2 and 3 for 7 days in shaking-flask cultures at 28 °C and 180 r.p.m. After cultivation, the whole culture broth was extracted in methanol, evaporated to dryness and the residue was dissolved in water for antimicrobial testing.
Analysis of the whole-cell diaminopimelic acid isomers and the sugars was done by the method of Hasegawa et al. (1983). The phospholipids and menaquinones were analysed by the method of Minnikin et al. (1984). Analyses of whole-cell fatty acid pattern followed described methods using the MIDI system (Microbial ID, Inc.) (Kroppenstedt, 1985; Sasser, 1990).
Genomic DNA extraction, PCR-mediated amplification of the 16S rRNA gene and purification of PCR products were carried out as described previously (Rainey et al., 1996). The ae2 editor (Maidak et al., 2001) was used to align the almost-complete 16S rRNA gene sequences of strains A/6353T and AB8327T against the 16S rRNA gene sequences of representatives of the main actinobacterial lineages and subsequently against members of the genus Actinoplanes. Pairwise evolutionary distances were computed by using the correction of Jukes & Cantor (1969). A phylogenetic dendrogram was reconstructed from a distance matrix by using the treeing algorithm of DeSoete (1983). Automated ribotyping of the isolates was accomplished by using the RiboPrinter (Qualicon) system (Bruce, 1996) and PvuII as restriction enzyme for cutting the genomic DNA. The patterns were compared with those included in the DSMZ RiboPrint database.
Formation of characteristic sporangia was observed in both strains (Fig. 1). Fragmentation of substrate mycelium was not found. Strain AB8327T formed a white aerial mycelium on ISP medium 2. The surface of the spores was smooth. Only rudimentary aerial mycelium was observed for strain A/6353T. The substrate mycelium of strain A/6353T was yellow–orange (RAL 2000) on all tested ISP media. Strain AB8327T formed pastel-yellow (RAL 1034) mycelium on ISP media 2, 3, 4 and 7 and beige (RAL 1001) mycelium on ISP media 5 and 6. A yellow, soluble pigment was formed by strain A/6353T on ISP media 4 and 5 and a red one on ISP 7. Strain AB8327T produced a red, soluble pigment on ISP 5 and a brown one on ISP 6 and Suter medium with tyrosine (Table 1).
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). Each strain showed a characteristic pattern with API 20E as well as with API ZYM. In the API 20E system, strains A/6353T and AB8327T showed urease activity, but lacked citrate utilization and indole production. In API ZYM tests, both strains were positive for alkaline phosphatase, esterase (C4), esterase lipase (C8) and phosphohydrolase, but negative for lipase (C14), leucine arylamidase, valine arylamidase, cystine arylamidase and 
-fucosidase.
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. The fatty acid patterns were characteristic for Actinoplanes species, with major amounts of iso-C16 : 0, iso-C17 : 0, anteiso-C17 : 0, iso-C18 : 0 and C18 : 0. A detailed listing of compounds with their quantities is indicated in the species descriptions.
Strains A/6353T and AB8327T share 97.1 % 16S rRNA gene sequence similarity. Whilst A. teichomyceticus AB8327T is related moderately to the type strain of A. rectilineatus, A. liguriensis A/6353T shares 99.3 % similarity with the type strain of A. cyaneus. The binary DNA–DNA similarity value for the latter strain pair was 41.5 % [mean of two values determined in 2x SSC, 12 % formamide at 70 °C, according to Wink et al. (2003)]. 16S rRNA gene sequence similarity values with other type strains of the genus were <98.4 %. Fig. 2 displays the dendrogram of 16S rRNA gene sequence similarities. Except for the branching of strains DSM 43865T (A/6353T) and A. cyaneus DSM 46137T, bootstrap values were <80 %, hence they are not shown. Comparison with sequences deposited in public databases showed the similarity between strain AB8327T and ‘A. teichomyceticus’ IFO 1999 (GenBank accession no. AB047513) to be 100 %, whilst the value between the two strains labelled ‘A. liguriae’ (A/6353T and IFO 13977, GenBank accession no. AB047497) was only 97.3 %. In order to check the authenticity of strain A/6353T, originally obtained as ATCC 31048, we sequenced another culture of ATCC 31048 and confirmed its identity. Results of the sequencing studies were confirmed by RiboPrint analysis of strains A/6353T, AB8327T and the closest phylogenetic relatives. Strains A/6353T and AB8327T showed unique RiboPrint patterns that were clearly separated from the patterns of the other type strains (Fig. 3) and from those of all other Actinoplanes type strains (not shown). Based on the phylogenetic, chemotaxonomic and phenotypic results, it can be concluded that A/6353T and AB8327T represent the type strains of two novel species of the genus Actinoplanes and we therefore propose the names Actinoplanes liguriensis sp. nov. (type strain A/6353T) and Actinoplanes teichomyceticus sp. nov. (type strain AB8327T).
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Cells are Gram-positive. Globose to oval sporangia are formed (see Fig. 1
). Rudimentary sterile mycelium is formed only on ISP medium 6. Colour of substrate mycelium is chrome yellow–orange (RAL 2000) on ISP media 2, 3, 4, 5, 6 and 7. A red, soluble pigment is formed on ISP medium 7. Colonial characteristics, carbohydrate-utilization pattern, enzyme activities and antimicrobial activities are indicated in Tables 1, 2, 3 and 4, respectively. Peptidoglycan contains meso-diaminopimelic acid; diagnostic sugars are xylose and arabinose; mycolic acids are absent; MK-9(H4) and MK-10(H4) are the principal menaquinones; the major phospholipid is phosphatidylethanolamine. Major fatty acid methyl esters are (>10 %): iso-C15 : 0 (26.1 %), anteiso-C15 : 0 (13.1 %), iso-C16 : 0 (12.1 %), anteiso-C17 : 0 (11.0 %); others occur in smaller amounts (
1.0 %): iso-C14 : 0 (1.7 %), C14 : 0 (1.8 %), C15 : 0 (1.5 %), C16 : 1 (5.80 %), C16 : 0 (6.5 %), iso-C17 : 1 (1.9 %), anteiso-C17 : 0 (1.0 %), iso-C17 : 0 (3.0 %), cis-9-C17 : 1 (2.2 %), cis-10-C17 : 1 (3.3 %), C17 : 0 (1.2 %), cis-9-C18 : 1 (2.9 %) and C18 : 0 (1.5 %). The antibiotics gardimycin and Ala(O)-actagardine are produced.
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Description of Actinoplanes teichomyceticus sp. nov.
Actinoplanes teichomyceticus [tei.cho.my.ce'ti.cus. Gr. n. teichos wall; Gr. n. myces fungus; L. adj. suff. -icus belonging to; teichomyceticus belonging to a fungus cell wall (referring to inhibition of cell-wall synthesis by teichomycin, produced by the type strain)].
Cells are Gram-positive. Spherical to oval sporangia are formed (see Fig. 1). Long, sterile hyphae are formed on ISP media 2, 3, 4 and 5. Colour of substrate mycelium is pastel yellow (RAL 1034) on ISP media 2, 3, 4 and 7 and beige (RAL 1001) on ISP media 5 and 6. A red, soluble pigment is formed on ISP medium 5 and a brown one on ISP media 3 and 6. Colonial characteristics, carbohydrate-utilization pattern, enzyme activities and antimicrobial activities are indicated in Tables 1, 2, 3 and 4, respectively. Peptidoglycan contains meso-diaminopimelic acid; diagnostic sugars are xylose and arabinose; mycolic acids are absent; MK-9(H4) and MK-10(H4) are the principal menaquinones; the major phospholipid is phosphatidylethanolamine. Major fatty acid methyl esters are (>10 %): iso-C16 : 0 (20.7 %), iso-C15 : 0 (15.1 %), anteiso-C15 : 0 (8.8 %), anteiso-C17 : 0 (19.8 %); others occur in smaller amounts (1.0): iso-C14 : 0 (1.0 %), C15 : 0 (1.1 %), iso-C16 : 1 (1.6 %), cis-9-C16 : 1 (3.4 %), C16 : 0 (2.5 %), iso-C17 : 1 (2.6 %), anteiso-C17 : 1 (2.0 %), iso-C17 : 0 (3.8 %), cis-9-C17 : 1 (6.7 %), C17 : 0 (3.5 %), cis-9-C18 : 1 (4.3 %) and C18 : 0 (1.8 %). The antibiotics teichomycin A1 and A2 (renamed teicoplanin) are produced.
The type strain is AB8327T (=FH 2149T=DSM 43866T=ATCC 31121T=BCRC 12106T=FERM P-3462T IMSNU 20043T=IMET 9254T=JCM 3252T=KCC A-0252T=KCTC 9543T=NBRC 13999T=NCIMB 12640T=NRRL B-16726T=SANK 60479T). Isolated from Indian soil.
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Sasser, M. (1990). Identification of Bacteria by Gas Chromatography of Cellular Fatty Acids (technical note no. 101). Newark, DE: MIDI Inc. (http://www.midi-inc.com/media/pdfs/TechNote_101.pdf).
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Tamura, T. & Hatano, K. (2001). Phylogenetic analysis of the genus Actinoplanes and transfer of Actinoplanes minutisporangius Ruan et al. 1986 and ‘Actinoplanes aurantiacus’ to Cryptosporangium minutisporangium comb. nov. and Cryptosporangium aurantiacum sp. nov. Int J Syst Evol Microbiol 51, 2119–2125.[Abstract]
Vertesy, L., Aretz, W., Bonnefoy, A. & 7 other authors (1999). Ala(O)-actagardine, a new lantibiotic from cultures of Actinoplanes liguriae ATCC 31048. J Antibiot 52, 730–741.[Medline]
Williams, S. T., Goodfellow, M. & Alderson, G. (1989). Genus Streptomyces. In Bergey's Manual of Systematic Bacteriology, vol. 4, pp. 2452–2492. Edited by S. T. Williams, M. E. Sharpe & J. G. Holt. Baltimore: Williams & Wilkins.
Wink, J. M., Kroppenstedt, R. M., Ganguli, B. N., Nadkarni, S. R., Schumann, P., Seibert, G. & Stackebrandt, E. (2003). Three new antibiotic producing species of the genus Amycolatopsis, Amycolatopsis balhimycina sp. nov., A. tolypomycina sp. nov., A. vancoresmycina sp. nov., and description of Amycolatopsis keratiniphila subsp. keratiniphila subsp. nov. and A. keratiniphila subsp. nogabecina subsp. nov. Syst Appl Microbiol 26, 38–46.[CrossRef][Medline]
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