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Int J Syst Evol Microbiol 53 (2003), 721-724; DOI  10.1099/ijs.0.02286-0
© 2003 International Union of Microbiological Societies

Actinomadura namibiensis sp. nov.

Joachim Wink1, Reiner M. Kroppenstedt2, Gerhard Seibert1 and Erko Stackebrandt2

1 Aventis Pharma Deutschland, Drug Innovation and Approval, Natural Products, 65926 Frankfurt, Germany
2 DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen, Mascheroder Weg 1b, 38124 Braunschweig, Germany

Correspondence
Joachim Wink
Joachim.Wink{at}aventis.com


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Strain HAG 010767T was isolated from desert soil from Namibia during a screening programme. On the basis of analysis of 16S rDNA, the principal amino acid of the peptidoglycan, cell-wall sugars, fatty acids and polar lipids, it was possible to identify this strain as a member of the genus Actinomadura. Although DNA–DNA reassociation experiments revealed 72 % DNA similarity between strain HAG 010767T and Actinomadura kijaniata DSM 43764T, significant differences in the colour of the mycelium and physiological properties indicate that strain HAG 010767T represents a novel species of this genus, for which the name Actinomadura namibiensis sp. nov. is proposed. The type strain is strain HAG 010767T (=DSM 44197T=NRRL B-24153T).

Published online ahead of print on 9 September 2002 as DOI 10.1099/ijs.0.02286-0.

The EMBL accession numbers for the 16S rDNA sequences of A. namibiensis sp. nov. DSM 44197T, A. echinospora DSM 43163T, A. hibisca DSM 44148T, A. fulvescens DSM 43923T, A. atramentaria DSM 43919T, A. citrea DSM 43461T, A. formosensis DSM 43997T and A. viridis DSM 20745T are respectively AJ420134–AJ420141.


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Strain HAG 010767T was isolated from the Namib Desert, Namibia, in 1988, using Actinomycete isolation agar. This salmon-pink strain produced aerial mycelium with spiral spore chains. The strain was compared with the type strains of all validly named Actinomadura species. Morphological and physiological characteristics were observed by using agar cultures of the various media described by Shirling & Gottlieb (1966)Go, e.g. yeast extract/malt extract agar (ISP medium 2), oatmeal agar (ISP 3), inorganic salts/starch agar (ISP 4), glycerol/asparagine agar (ISP 5), peptone/yeast extract/iron agar (ISP 6) and tyrosine agar (ISP 7), incubated for 10 days at 28 °C. For scanning electron microscopy, the strain was grown on ISP 4 agar. Microscopy was done as described by Grabley et al. (1992)Go.

Vegetative mycelium developed well on all ISP media tested. Aerial mycelium was formed only on media ISP 4, ISP 5 and ISP 7. No soluble pigment was produced on any of the ISP media tested. After 7–10 days on ISP 4, ISP 5 and ISP 7, the formation of spore chains could be observed. These spores exhibited a smooth surface, and the chains formed spirals that could be observed in both light and scanning electron micrographs (Fig. 1Go).



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  		Fig. 1. Spore-chain formation in Actinomadura namibiensis sp. nov. HAG 010767T grown on ISP 4 for 10 days at 28 °C, viewed using light microscopy (a) and scanning electron microscopy (b, c). Original magnification: x620 (a) and x10 000 (b, c).

 
The vegetative mycelium was salmon pink [according to the RAL colour code (Deutsches Institut für Gütesicherung und Kennzeichnung e.V. – Reichsausschuß für Lieferbedingungen)] on all the ISP media used except medium ISP 6. The smooth spores in the aerial mycelium are in characteristic long chains that are arranged in spirals (Fig. 1Go), in contrast with other Actinomadura species such as Actinomadura kijaniata, which have straight spore chains (Fig. 2Go).



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  		Fig. 2. Spore-chain formation in Actinomadura kijaniata FH 2339 grown on ISP 4 for 10 days at 28 °C, viewed using scanning electron microscopy. Original magnification: x5000 (a) and x7500 (b).

 
Carbohydrate utilization was investigated on ISP 9 medium (Shirling & Gottlieb, 1966Go) by using a microtitre plate technique with 12-well plates. Sodium chloride tolerance was tested on microtitre plates (6-well) using a technique based on the method of Kutzner (1986)Go. Enzymic activities were determined by using API 20E and API ZYM test strips (bioMérieux) (Humble et al., 1977Go; Kilian, 1978Go; Smith et al., 1972Go). Tests were done with type strains of all validly described Actinomadura species. The carbohydrate-utilization pattern and the enzymic activity pattern of strain HAG 010767T were unique among the test strains. Table 1Go displays these results in comparison with the phylogenetically neighbouring type strains and the type strain of the type species of Actinomadura, Actinomadura madurae DSM 43067T.


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  		Table 1. Utilization of carbohydrates and enzymic reactions

Strains are listed as: 1, HAG 010767T; 2, Actinomadura kijaniata ATCC 31588T; 3, Actinomadura atramentaria DSM 43919T; 4, Actinomadura fibrosa ATCC 49459T; 5, Actinomadura fulvescens DSM 43923T; 6, Actinomadura madurae CBS 3315T; 7, Actinomadura hibisca IFO 15585T; 8, Actinomadura nitritigenes DSM 44137T. For utilization tests, growth is scored as: +, like positive control (basal medium with glucose); (+), better than negative control but not like positive control; -, not better than negative control (basal medium with water). All strains are positive for acid phosphatase, leucine arylamidase, naphthol-AS-BI-phosphohydrolase and {beta}-glucosidase and negative for {beta}-glucuronidase.

 
Antimicrobial testing (Williams et al., 1983Go) indicated that none of the test strains, grown either on Mueller–Hinton agar or on Czapek–Dox agar, was affected by isolate HAG 010767T.

Analyses of whole-cell diaminopimelic acid isomers and sugars were done using the method of Hasegawa et al. (1983)Go. Phospholipids and menaquinones were analysed using the method of Kutzner et al. (1986)Go. Fatty acid patterns were determined as described previously (Aretz et al., 2000Go). DL-Diaminopimelic acid is the diagnostic amino acid of the peptidoglycan; the cell wall sugars are galactose, glucose, ribose and madurose (chemotype III according to Lechevalier & Lechevalier, 1970Go). Polar lipids are of type IV, according to the phospholipid classification of Lechevalier et al. (1977)Go, and include diphosphatidylglycerol, phosphatidylinositol, phosphatidylglycerol and phosphatidylinositol mannoside. The principal menaquinones are MK-9(H4), MK-9(H6) and MK-9(H8) (quinone type 4a according to Kroppenstedt & Goodfellow, 1991Go). The fatty acid methyl esters mainly comprised branched saturated and unsaturated fatty acids and 10-methyl C18 : 0 (tuberculostearic acid) (type 3a of Kroppenstedt & Goodfellow, 1991Go). Mycolic acids could not be detected.

Analysis of the almost complete 16S rDNA sequence (Rainey et al., 1996Go) of strain HAG 010767T in comparison with the sequences of type strains of all 27 validly described Actinomadura species revealed a high degree of relatedness (96–99·6 % similarity). The highest value (99·6 %) was found to A. kijaniata DSM 43764T. A relationship dendrogram, based on neighbour-joining analysis (Felsenstein, 1993Go), is depicted in Fig. 3Go. Discrimination at a higher taxonomic level by DNA–DNA hybridization (Escara & Hutton, 1980Go; Huß et al., 1983Go; Jahnke, 1992Go) revealed that these two strains constitute two closely related genomic taxa (72 % similarity, mean of duplicates).



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  		Fig. 3. 16S rDNA dendrogram showing the positions of strain HAG 010767T and type strains of phylogenetically closely related Actinomadura species. Scale bar, 2 inferred nucleotide substitutions per 100 nucleotides.

 
On the basis of the presence of genus-specific chemotaxonomic properties, and supported by its phylogenetic position, strain HAG 010767T is a member of the genus Actinomadura. Though phylogenetically highly related to A. kijaniata, the presence of diagnostic carbon-utilization and enzyme patterns (using APY ZYM) and the colour of the substrate mycelium (HAG 010767T, salmon; A. kijaniata, green) indicate that strain HAG 010767T represents a taxon worthy of species status. We therefore propose the name Actinomadura namibiensis sp. nov., with strain HAG 010767T as the type strain.

Description of Actinomadura namibiensis sp. nov.
Actinomadura namibiensis (na.mi.bi.en'sis. N.L. gen. n. namibiensis of Namibia, where the type strain was isolated).

Gram-positive. White aerial mycelium develops only on ISP 4, ISP 5 and ISP 7 media. The substrate mycelium is salmon pink on ISP 2, ISP 3, ISP 4, ISP 5 and ISP 7 media and is colourless on ISP 6. After about 10 days, spiral spore chains, each composed of about 20 spores, are observed in the aerial mycelium. DL-Diaminopimelic acid is the diagnostic amino acid of the peptidoglycan. Cell hydrolysates contain galactose, glucose, ribose and madurose, whereas arabinose and xylose are absent. Phospholipids include diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol mannoside and phosphatidylinositol. The principal menaquinones are MK-9(H4), MK-9(H6) and MK-9(H8). The fatty acid methyl esters mainly comprise iso C14 : 0 and 10-methyl C18 : 0 (tuberculostearic acid); iso C17 : 0, 10-methyl C17 : 0 and 2-OH C15 : 0 occur in smaller amounts. Physiological reactions are indicated in Table 1Go. Has no antibacterial effect on Micrococcus luteus, Streptomyces murinus, Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Saccharomyces cerevisiae, Aspergillus niger or Candida albicans. Phylogenetically related to Actinomadura kijaniata.

The type strain, strain HAG 010767T (=DSM 44197T =NRRL B-24153T), was isolated from soil from the Namib Desert, Namibia.


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Aretz, W., Meiwes, J., Seibert, G., Vobis, G. & Wink, J. (2000). Friulimicins: novel lipopeptide antibiotics with peptidoglycan synthesis inhibiting activity from Actinoplanes friuliensis sp. nov. I. Taxonomic studies of the producing microorganism and fermentation. J Antibiot 53, 807–815.[Medline]

Escara, J. F. & Hutton, J. R. (1980). Thermal stability and renaturation of DNA in dimethyl sulfoxide solutions: acceleration of the renaturation rate. Biopolymers 19, 1315–1327.[CrossRef][Medline]

Felsenstein, J. (1993). PHYLIP – Phylogeny inference package, version 3.5.1. Distributed by the author. Department of Genetics, University of Washington, Seattle, USA.

Grabley, S., Granzer, E., Hütter, K. & 7 other authors (1992). Secondary metabolites by chemical screening. 8. Decarestrictines, a new family of inhibitors of cholesterol biosynthesis from Penicillium. I. Strain description, fermentation, isolation and properties. J Antibiot 45, 56–65.[Medline]

Hasegawa, T., Takizawa, M. & Tanida, S. (1983). A rapid analysis for chemical grouping of aerobic Actinomycetes. J Gen Appl Microbiol 29, 319–322.[CrossRef]

Humble, M. W., King, A. & Phillips, I. (1977). API ZYM: a simple rapid system for the detection of bacterial enzymes. J Clin Pathol 30, 275–277.[Abstract/Free Full Text]

Huß, V. A. R., Festl, H. & Schleifer, K. H. (1983). Studies on the spectrophotometric determination of DNA hybridization from renaturation rates. Syst Appl Microbiol 4, 184–192.

Jahnke, K.-D. (1992). BASIC computer program for evaluation of spectroscopic DNA renaturation data from Gilford System 2600 spectrophotometer on a PC/XT/AT type personal computer. J Microbiol Methods 15, 61–73.

Kilian, M. (1978). Rapid identification of Actinomycetaceae and related bacteria. J Clin Microbiol 8, 127–133.[Abstract/Free Full Text]

Kroppenstedt, R. M. & Goodfellow, G. (1991). The family Thermomonosporaceae. In The Prokaryotes, 2nd edn, pp. 1085–1114. Edited by A. Balows, H. G. Trüper, M. Dworkin, W. Harder & K. H. Schleifer. New York: Springer.

Kutzner, H. J., Kroppenstedt, R. M. & Korn-Wendisch, F. (1986). Methoden zur Untersuchung von Streptomyceten und einigen anderen Actinomyceten, 4th edn. Report of the Institute for Microbiology, Technical University, Darmstadt and German Culture Collection (DSMZ), Braunschweig. Braunschweig: DSMZ.

Lechevalier, M. P. & Lechevalier, H. A. (1970). Composition of whole cell hydrolysates as a criterion in the classification of aerobic actinomycetes. In The Actinomycetales, pp. 311–316. Edited by H. Prauser. Jena: Gustav Fischer.

Lechevalier, M. P., de Bièvre, C. & Lechevalier, H. A. (1977). Chemotaxonomy of aerobic actinomycetes: phospholipid composition. Biochem Ecol Syst 5, 249–260.[CrossRef]

Rainey, F. A., Ward-Rainey, N., Kroppenstedt, R. M. & Stackebrandt, E. (1996). The genus Nocardiopsis represents a phylogenetically coherent taxon and a distinct actinomycete lineage: proposal of Nocardiopsaceae fam. nov. Int J Syst Bacteriol 46, 1088–1092.[Abstract/Free Full Text]

Shirling, E. B. & Gottlieb, D. (1966). Methods for characterization of Streptomyces species. Int J Syst Bacteriol 16, 313–340.[Abstract/Free Full Text]

Smith, P. B., Thomfohrde, K. M., Rhoden, D. L. & Balows, A. (1972). API system: a multitube micromethod for identification of Enterobacteriaceae. Appl Microbiol 24, 449–452.[Medline]

Williams, S. T., Goodfellow, M., Alderson, G., Wellington, E. M. H., Sneath, P. H. A. & Sackin, M. J. (1983). Numerical classification of Streptomyces and related genera. J Gen Microbiol 129, 1743–1813.[Medline]





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