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Int J Syst Evol Microbiol 57 (2007), 721-724; DOI  10.1099/ijs.0.64805-0
© 2007 International Union of Microbiological Societies

Actinoplanes couchii sp. nov.

Peter Kämpfer1, Birgit Huber2, Kathrin Thummes1, Iris Grün-Wollny3 and Hans-Jürgen Busse2,4

1 Institut für Angewandte Mikrobiologie, Justus-Liebig-Universität Giessen, D-35392 Giessen, Germany
2 Institut für Bakteriologie, Mykologie und Hygiene, Veterinärmedizinische Universität, A-1210 Wien, Austria
3 Labor Grün-Wollny, D-35394 Giessen, Germany
4 Institut für Mikrobiologie und Genetik, Universität Wien, A-1030 Wien, Austria

Correspondence
Peter Kämpfer
peter.kaempfer{at}agrar.uni-giessen.de


   ABSTRACT
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A Gram-positive bacterium, strain GW8-1761T, was isolated from soil close to the Marmore waterfalls, Terni, Italy. 16S rRNA gene sequence similarity studies showed that strain GW8-1761T belonged to the genus Actinoplanes, being most closely related to Actinoplanes italicus JCM 3165T (98.9 %), A. rectilineatus IFO 13941T (98.5 %), A. palleronii JCM 7626T (97.8 %), A. utahensis IFO 13244T (97.6 %) and A. cyaneus DSM 46137T (97.6 %). Strain GW8-1761T could be distinguished from any other Actinoplanes species with validly published names by 16S rRNA gene sequence similarity values of less than 97.5 %. Chemotaxonomic data [major menaquinone MK-9(H4); major polar lipids diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol, with phosphatidylcholine and aminoglycolipids absent; major fatty acids C15 : 0, C16 : 0, C16 : 0 iso, C17 : 1{omega}8c and summed feature 3 (C16 : 1{omega}7c and/or C15 : 0 iso 2-OH)] supported the affiliation of strain GW8-1761T to the genus Actinoplanes. The results of DNA–DNA hybridizations and physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain GW8-1761T from the most closely related species. Strain GW8-1761T therefore merits species status, and we propose the name Actinoplanes couchii sp. nov., with the type strain GW8-1761T (=DSM 45050T=CIP 109316T).

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain GW8-1761T is AM400230.

A neighbour-joining tree based on 16S rRNA gene sequences showing the position of strain GW8-1761T is available as supplementary material in IJSEM Online.


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The genus Actinoplanes Couch 1950 emend. Stackebrandt and Kroppenstedt 1988Go belongs phylogenetically to the family Micromonosporaceae Krassil'nikov 1938 emend. Stackebrandt et al. 1997Go. Members of the genus Actinoplanes are characterized by the presence of spherical, cylindrical or very irregular sporangia. The sporangiospores are motile by tufts of polar flagella. Production of aerial hyphae is scant. The peptidoglycan composition is characterized by meso-diaminopimelic acid, which may be replaced by hydroxydiaminopimelic acid as the major diamino acid. Phosphatidylethanolamine is the diagnostic phospholipid. Iso- and anteiso-branched and monounsaturated fatty acids and/or cis-9-octadecenoic acid (oleic acid) are the predominant fatty acids. The isoprenoid quinone is MK-9(H4). The type species is Actinoplanes philippinensis Couch 1950 and more than 20 species with validly published names have been described to date.

A detailed phenotypic analysis of the genus has been given by Goodfellow et al. (1990)Go, who determined the chemotaxonomic and phenotypic characteristics of species of Actinoplanes and reported that chemical and numerical taxonomic data supported the integrity of the genus. A comprehensive phylogenetic analysis of the genus has been given by Tamura & Hatano (2001)Go.

During the characterization of organisms isolated from different soils, strain GW8-1761T was recovered from 1 g of a soil sample (heated for 1 min at 100 °C) originating from Terni, Italy. The strain was isolated from a 0.1 % (v/v) Tween 80 solution containing 5 mg ampicillin spread on mannitol-rifampicin agar [containing (l–1): mannitol, 10 g; yeast extract, 7 g; Casamino acids, 2 g; peptone (Bacto), 1 g; NaCl, 1 g; CaCO3, 0.2 g; nystatin, 100 mg; rifampicin, 5 mg] incubated for 6 weeks at 27 °C. The strain was maintained on DSMZ medium 65 (http://www.dsmz.de/microorganisms/html/media/medium000065.html) at 25 °C and, on this medium, showed an orange- to red-coloured substrate mycelium that fragmented easily in irregular rod-shaped cells.

Cells of the strain stained Gram-positive with the method of Gerhardt et al. (1994)Go. Cell morphology was observed under a Zeiss light microscope at x1000, with cells grown for 7 days at 25 °C on DSMZ medium 65. The 16S rRNA gene was analysed as described by Kämpfer et al. (2003)Go. Phylogenetic analysis was performed using the software package MEGA version 2.1 (Kumar et al., 2001Go) after multiple alignment of data by CLUSTAL X (Thompson et al., 1997Go). Distances (distance options according to the Kimura-2 model) were determined and clustering with the neighbour-joining and maximum-parsimony methods was performed by using bootstrap values based on 1000 replications (see Supplementary Fig. S1 available in IJSEM Online). The 16S rRNA sequence of strain GW8-1761T was a continuous stretch of 1371 bp. Sequence similarity calculations after a neighbour-joining analysis indicated that the closest relatives of strain GW8-1761T were Actinoplanes italicus JCM 3165T (98.9 %), A. rectilineatus IFO 13941T (98.5 %) and A. palleronii JCM 7626T (97.8 %). Lower sequence similarities (<97.7 %) were found with all other species of the genus Actinoplanes with validly published names.

Results of chemotaxonomic analyses are given in the species description. The following cell components were analysed using the procedures indicated: menaquinones (Tindall, 1990Go), polar lipids (Ventosa et al., 1993Go) and fatty acids (Kämpfer & Kroppenstedt, 1996Go). The quinone system with the predominant compound MK-9(H4) (75 %), with a moderate amount of MK-9(H6) (11 %), minor amounts of MK-9, MK-9(H2), MK-9(H8) and MK-10(H2) (1–4 %) and traces of MK-10 and MK-10(H6) (<1.0 %), supports the affiliation of GW8-1761T to the genus Actinoplanes, where all species examined so far share this major quinone (Goodfellow et al., 1990Go; Matsumoto et al., 2000Go; Wink et al., 2006Go). The polar lipid profile of GW8-1761T was similar to those reported for other Actinoplanes species such as Actinoplanes missouriensis, A. rectilineatus (Goodfellow et al., 1990Go), A. capillaceus (Matsumoto et al., 2000Go), A. liguriensis and A. teichomyceticus (Wink et al., 2006Go), consisting of the major compounds diphosphatidylglycerol and phosphatidylethanolamine and lacking phosphatidylcholine and aminoglycolipids and thus exhibiting phospholipid type PII according to Lechevalier et al. (1977)Go. Additionally, moderate amounts of phosphatidylinositol, two unknown phospholipids, a highly hydrophilic glycolipid and a polar lipid and trace amounts of a single phosphatidylinositol mannoside were detected (Fig. 1Go). The fatty acid profile of strain GW8-1761T was similar to those of the other closely related species and was congruent with the fatty acid profiles reported for other Actinoplanes species. However, some quantitative differences could be detected (Table 1Go).


Figure 1
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  		Fig. 1. Polar lipid profile of strain GW8-1761T after two-dimensional TLC and detection with molybdatophosphoric acid. DPG, Diphosphatidylglycerol; GL, highly hydrophilic lipid; L1, unknown polar lipid; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PIM, phosphatidylinositol mannoside; PL1, PL2, unknown phospholipids.

 

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  		Table 1. Major fatty acid compositions of strain GW8-1761T and related type strains of Actinoplanes species

Strains: 1, GW8-1761T; 2, A. italicus DSM 43146T; 3, A. rectilineatus DSM 43808T; 4, A. philippinensis DSM 43019T. In addition to A. philippinensis DSM 43019T (the type strain of the type species), the type strains included are those that grouped into the same cluster as strain GW8-1761T on the basis of 16S rRNA gene sequence similarity studies (see Supplementary Fig. S1). All strains were grown on trypticase soy broth at 28 °C for 7 days prior to fatty acid analysis. For unsaturated fatty acids, the position of the double bond is located by counting from the methyl ({omega}) end of the carbon chain. cis isomers are indicated by the suffix c.

 
Detailed results of the physiological characterization are given in the species description and in Table 2Go, determined using methods described previously (Kämpfer et al., 1991Go). Degradation of polymeric substances were assessed using standard procedures (Williams et al., 1983Go) and read after 7 days of incubation at 28 °C. Results of DNA–DNA hybridization experiments (values are means from two analyses), performed with labelled DNA from GW8-1761T (first value) and labelled DNA from reference type strains (second value), were 31.3/29.4 % with A. italicus DSM 43146T and 36.9/46.0 % with A. rectilineatus DSM 43808T, using the method described by Ziemke et al. (1998)Go, except that, for nick translation, 2 µg DNA was labelled during a 3 h incubation at 15 °C.


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  		Table 2. Physiological characteristics of strain GW8-1761T and related type strains of Actinoplanes species

Strains: 1, GW8-1761T; 2, A. italicus DSM 43146T; 3, A. rectilineatus DSM 43808T. +, Positive; –, negative; (+), weakly positive. The type strains included are those that grouped into the same cluster as strain GW8-1761T on the basis of 16S rRNA gene sequence similarity studies (see Supplementary Fig. S1). All strains were positive for the hydrolysis of aesculin, p-nitrophenyl (pNP) {alpha}-D-glucopyranoside, pNP beta-D-glucopyranoside and pnp beta-D-xylopyranoside. All strains were negative for the hydrolysis of pNP beta-D-glucuronide, L-glutamate-{gamma}-3-carboxy p-nitroanilide (pna) and L-proline pNA. The following compounds were used by all strains as sole sources of carbon: L-arabinose*, D-fructose*, D-galactose*, gluconate, D-glucose*, D-mannose*, D-maltose*, L-rhamnose*, D-trehalose*, D-xylose*, D-mannitol*, glutarate (weak) and L-malate. The following compounds were not utilized by all strains: N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, {alpha}-D-melibiose, adonitol*, sorbitol*, acetate, propionate, trans-aconitate, adipate, azelate, citrate, itaconate, mesaconate, suberate, phenylacetate*, 3-hydroxybenzoate and 4-hydroxybenzoate. oNP, o-Nitrophenyl.

 
Description of Actinoplanes couchii sp. nov.
Actinoplanes couchii [cou'chi.i. N.L. gen. n. couchii of Couch, named after J. N. Couch (1896–1986), a mycologist, who proposed the genus name Actinoplanes in 1950].

Cells are Gram-positive. Globose to oval sporangia are formed. A rudimentary sterile mycelium is formed. Colour of substrate mycelium is yellow–orange on DSMZ medium 65. A red to brown soluble pigment is formed on this medium. Oxidase-positive, showing an oxidative metabolism. Good growth occurs on nutrient agar and DSMZ medium 65 at 25–30 °C. The quinone system consists of MK-9(H4) (75 %), MK-9(H6) (11 %), MK-9 (1 %), MK-9(H2) (4 %), MK-9(H8) (2 %), MK-10 (0.6 %), MK-10(H2) (2 %) and traces of MK-10(H6) (<0.1 %). The polar lipid profile contains the major compounds diphosphatidylglycerol and phosphatidylethanolamine and lacks phosphatidylcholine and aminoglycolipids (phospholipid type PII). Additionally, moderate amounts of phosphatidylinositol, two unknown phospholipids, a highly hydrophilic glycolipid and a polar lipid are detectable and traces of a single phosphatidylinositol mannoside are present. Major fatty acids are C15 : 0, C16 : 0, C16 : 0 iso, C17 : 1{omega}7c and summed feature 3 (C16 : 1{omega}7c and/or C15 : 0 iso 2-OH). The detailed fatty acid profile is given in Table 1Go. Carbon source utilization and hydrolysis of chromogenic substrates (including differentiating characters) are indicated in Table 2Go. Starch, xylan, tyrosine, casein, hypoxanthine, adenine and xanthine are degraded.

The type strain, GW8-1761T (=DSM 45050T=CIP 109316T), was isolated from soil near to the Marmore waterfalls, Terni, Italy.


   ACKNOWLEDGEMENTS
 
We thank R. M. Kroppenstedt (DSMZ) for supplying Actinoplanes cultures for comparative work.


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

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Palleroni, N. J. (1989). Genus Actinoplanes Couch 1950. In Bergey's Manual of Systematic Bacteriology, vol. 4, pp. 2419–2428. Edited by S. T. Williams, M. E. Sharpe & J. G. Holt. Baltimore: Williams & Wilkins.

Stackebrandt, E. & Kroppenstedt, R. M. (1987). Union of the genera Actinoplanes Couch, Ampullariella Couch, and Amorphosporangium Couch in a redefined genus Actinoplanes. Syst Appl Microbiol 9, 110–114.

Stackebrandt, E., Rainey, F. A. & Ward-Rainey, N. L. (1997). Proposal for a new hierarchic classification system, Actinobacteria classis nov. Int J Syst Bacteriol 47, 479–491.[Abstract/Free Full Text]

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Tindall, B. J. (1990). Lipid composition of Halobacterium lacusprofundi. FEMS Microbiol Lett 66, 199–202.[CrossRef]

Ventosa, A., Marquez, M. C., Kocur, M. & Tindall, B. J. (1993). Comparative study of "Micrococcus sp." strains CCM 168 and CCM 1405 and members of the genus Salinicoccus. Int J Syst Bacteriol 43, 245–248.[Abstract/Free Full Text]

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Ziemke, F., Höfle, M. G., Lalucat, J. & Rosselló-Mora, R. (1998). Reclassification of Shewanella putrefaciens Owen's genomic group II as Shewanella baltica sp. nov. Int J Syst Bacteriol 48, 179–186.[Abstract/Free Full Text]





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