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Int J Syst Evol Microbiol 56 (2006), 2309-2312; DOI  10.1099/ijs.0.64217-0
© 2006 International Union of Microbiological Societies

Actinoalloteichus hymeniacidonis sp. nov., an actinomycete isolated from the marine sponge Hymeniacidon perleve

Haitao Zhang1,2, Wen Zheng3, Jianyu Huang1,2, Hongli Luo3, Yan Jin1, Wei Zhang1,4, Zhiheng Liu3 and Ying Huang3

1 Marine Bioproducts Engineering Group, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China
2 Graduate School of the Chinese Academy of Sciences, Chinese Academy of Sciences, Beijing 100049, China
3 State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, China
4 Department of Medical Biotechnology, School of Medicine, Flinders University, Adelaide, SA 5042, Australia

Correspondence
Wei Zhang
weizhang{at}dicp.ac.cn
Ying Huang
huangy{at}im.ac.cn


   ABSTRACT
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A polyphasic study was undertaken to establish the taxonomic status of a Gram-positive, aerobic actinomycete, strain HPA177T, isolated from a marine sponge, Hymeniacidon perleve. The organism formed branching, non-fragmenting vegetative hyphae and produced black pigment. Chemotaxonomic characteristics were consistent with its assignment to the genus Actinoalloteichus. Analysis of the 16S rRNA gene sequence showed that strain HPA177T formed a robust clade with type strains of the genus Actinoalloteichus, but was distinct from them. A number of phenotypic characteristics also readily distinguished strain HPA177T from species of the genus Actinoalloteichus with validly published names. On the basis of the above data, it is proposed that strain HPA177T represents a novel species, Actinoalloteichus hymeniacidonis sp. nov. The type strain of Actinoalloteichus hymeniacidonis is HPA177T (=CGMCC 4.2500T=JCM 13436T).

Abbreviations: A2pm, diaminopimelic acid

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain HPA177T is DQ144222.


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The genus Actinoalloteichus was established by Tamura et al. (2000)Go after several initial taxonomic studies on its phenotypic and genotypic properties by Liu et al. (1984)Go, Itoh et al. (1987)Go and Tamura & Hatano (1998)Go. The genus currently comprises only two species, Actinoalloteichus cyanogriseus (Tamura et al., 2000Go) and Actinoalloteichus spitiensis (Singla et al., 2005Go). During the investigation of actinomycete diversity of an intertidal marine sponge, Hymeniacidon perleve, a novel actinomycete, strain HPA177T, was isolated. The aim of the present study was to determine the taxonomic status of this strain using a polyphasic approach.

Strain HPA177T was isolated by the following procedure. The marine sponge was collected at the inter-tidal beach of Dalian, on the Chinese Yellow Sea, located in northern China (38° 52' N 121° 41' E). Freshly collected sponge specimens were rinsed five times in sterile seawater to remove transient and loosely attached bacteria and then thoroughly homogenized in a sterile mortar. A 10-fold dilution series of sponge homogenate was made and plated in triplicate on modified arginine/glycerol agar (ISP medium 5) plates (6 ml 100 % glycerol, 1 g arginine, 1 g K2HPO4, 0.5 g MgSO4, 18 g agar and 1 l natural seawater). After incubating the plates at 28 °C for 4 weeks, an isolate, designated HPA177T, was picked and subcultured on TSA plates (Difco) until a pure culture was obtained.

The arrangement of hyphae and spore chains of strain HPA177T were observed on yeast extract/malt extract agar (ISP medium 2) and oatmeal agar (ISP medium 3) after 2–3 weeks at 28 °C using light and scanning electron microscopes. Cultural characteristics were observed on a number of standard media (Table 1Go) after 2 weeks at 28 °C. The strain was examined further for a range of physiological properties using established procedures described by Gordon et al. (1974)Go and Yokota et al. (1993)Go. Biomass for chemical studies was prepared by growing the strain in shake flasks of TSB at 28 °C for 7 days. Cells were harvested by centrifugation, washed with distilled water and freeze-dried. The isomers of diaminopimelic acid (A2pm) and whole-organism sugars were analysed according to the procedures developed by Hasegawa et al. (1983)Go and Lechevalier & Lechevalier (1980)Go. Polar lipids were examined by two-dimensional TLC and identified using the method of Minnikin et al. (1984)Go. Menaquinones were extracted from freeze-dried biomass and purified according to Collins (1985)Go; the purified preparation was analysed using an HPLC procedure (Wu et al., 1989Go). Mycolic acids were checked by the acid methanolysis method as described previously (Minnikin et al., 1980Go). Fatty acids were extracted, methylated and analysed by GC using the standard Sherlock MIDI (Microbial Identification) system (Sasser, 1990Go; Kämpfer & Kroppenstedt, 1996Go).


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  		Table 1. Growth and cultural characteristics of strain HPA177T

 
The morphological characteristics of strain HPA177T were similar to those of A. cyanogriseus, the type species of the genus. It formed grey to black, branched vegetative hyphae that tended to fragment after 3 weeks of cultivation, aerial hyphae with straight spore chains (Fig. 1Go) and a greyish aerial spore mass on several standard media (Table 1Go). The assignment of strain HPA177T to the genus Actinoalloteichus is also supported by its chemotaxonomic profile. This strain contained meso-A2pm as the cell wall diamino acid, galactose, glucose, mannose and ribose as the diagnostic sugars in whole-organism hydrolysates and tetrahydrogenated menaquinone with nine isoprene units [MK-9(H4)] (64 %) as the predominant isoprenoid quinone, with smaller amounts of MK-9(H6) (23 %) and MK-9(H8) (12 %). It also contained phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside and some phospholipids of unknown structure containing glucosamine as diagnostic polar lipids, but lacked mycolic acids (data not shown). The predominant cellular fatty acids were 15 : 0 anteiso (20 %), 17 : 1{omega}8c (19 %), 16 : 0 iso (16 %) and 17 : 0 (11 %). Strain HPA177T differed significantly from A. cyanogriseus (Tamura et al., 2000Go) and A. spitiensis (Singla et al., 2005Go) in having larger amounts of 17 : 1{omega}8c and 17 : 0. The complete fatty acid profile and differences from those of A. cyanogriseus and A. spitiensis are shown in Table 2Go.


Figure 1
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  		Fig. 1. Scanning electron micrograph of strain HPA177T grown on yeast extract/malt extract agar for 14 days at 28 °C. Bar, 5 µm.

 

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  		Table 2. Chemotaxonomic characteristics that distinguish strain HPA177T from A. cyanogriseus IFO 14455T and A. spitiensis DSM 44848T

Strains: 1, HPA177T; 2, A. cyanogriseus IFO 14455T (data from Tamura et al., 2000Go); 3, A. spitiensis DSM 44848T (data from Singla et al., 2005Go). Values less than 2 % are not shown. –, Not detected.

 
For 16S rRNA gene sequencing, genomic DNA of HPA177T was extracted as described previously by Lee et al. (2003)Go and PCR amplification and sequencing of the 16S rRNA gene were performed as described by Webster & Hill (2001)Go. The resultant sequence (1470 bp) was compared with those deposited in public databases. Phylogenetic analysis was performed using the neighbour-joining (Saitou & Nei, 1987Go) and maximum-parsimony (Fitch, 1972Go) methods in the PHYLIP package version 3.6 (Felsenstein, 1985Go), after multiple alignment of data by CLUSTAL X version 1.8 (Thompson et al., 1997Go). Evolutionary distances were calculated according to the two-parameter model of Kimura (1980)Go. Tree topologies were evaluated by bootstrap analyses (Felsenstein, 1985Go) based on 1000 replications. It is clear from Fig. 2Go that strain HPA177T forms a distinct lineage within the evolutionary radiation encompassed by the genus Actinoalloteichus, supported by both tree-making algorithms and by a 99 % bootstrap value recorded using the neighbour-joining method. Sequence similarity calculations after neighbour-joining analysis indicated that the isolate shared relatively low similarities with its closest relatives, A. spitiensis DSM 44848T (97.1 %) and A. cyanogriseus IFO 14455T (96.7 %). Sequence similarities to all other species of related genera with validly published names were lower than 95 %. Strain HPA177T could also be distinguished from members of the genus Actinoalloteichus using a combination of phenotypic properties (Table 3Go).


Figure 2
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  		Fig. 2. Neighbour-joining tree showing the phylogenetic position of strain HPA177T and related taxa based on 16S rRNA gene sequences. Streptomyces ambofaciens ATCC 23877T was used as the root organism. Numbers at nodes represent levels of bootstrap support, based on neighbour-joining analyses of 1000 resampled datasets. Solid circles represent clades that were also recovered in the maximum-parsimony tree. Bar, 0.01 substitutions per nucleotide position.

 

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  		Table 3. Differential physiological characteristics of strain HPA177T, A. cyanogriseus IFO 14455T and A. spitiensis DSM 44848T

Strains: 1, HPA177T; 2, A. cyanogriseus IFO 14455T; 3, A. spitiensis DSM 44848T. ++, Good; +, moderate; +/–, poor; –, none.

 
It is evident from the genotypic and phenotypic data that strain HPA177T should be classified as a representative of a novel Actinoalloteichus species; the name Actinoalloteichus hymeniacidonis sp. nov. is proposed.

Description of Actinoalloteichus hymeniacidonis sp. nov.
Actinoalloteichus hymeniacidonis (hy.me.ni.a.ci'do.nis. N.L. gen. n. hymeniacidonis of Hymeniacidon, the generic name of the marine sponge Hymeniacidon perleve, the source of the type strain).

Gram-positive, strictly aerobic actinomycete with branching hyphae. Non-fragmenting substrate mycelia are present within 3 weeks of cultivation. Aerial mycelia with straight spore (0.6x0.8 µm) chains aggregate. Grows well on yeast extract/malt extract agar and oatmeal agar at 20–37 °C. A black soluble pigment is produced on yeast extract/malt extract agar and peptone/yeast extract/iron agar. Decomposition of urea, growth in Sabouraud's dextrose broth and MacConkey agar, hydrolysis of aesculin and hippurate, utilization of calcium malate, sodium oxalate and sodium succinate and reduction of nitrate are all negative. Fructose, glucose, maltose, mannose, mannitol, rhamnose, sucrose, sorbitol and xylose are utilized as sole carbon sources, but arabinose, inositol and raffinose are not. Grows weakly at 15 °C and does not grow at 45 °C. The cell wall chemotype is III. The major menaquinone is MK-9(H4); small amounts of MK-9(H6) and MK-9(H8) are also present. The phospholipid profile comprises mainly phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol and phosphatidylinositol mannoside. Major fatty acids are 15 : 0 anteiso (20 %), 17 : 1{omega}8c (19 %), 16 : 0 iso (16 %) and 17 : 0 (11 %).

The type strain is HPA177T (=CGMCC 4.2500T=JCM 13436T), isolated from the marine sponge Hymeniacidon perleve in Dalian, China.


   ACKNOWLEDGEMENTS
 
This work was supported by the ‘100 Talent Project’ of the Chinese Academy of Sciences, ‘Innovation Fund’ from Dalian Institute of Chemical Physics and ‘973 Hi-Tech Research and Development Program of China’ (2003CB716001). The authors are grateful to Professor Jin-He Li (Qingdao Institute of Oceanology, Chinese Academy of Sciences) for identification of the sponge and to Professor J. P. Euzéby (Toulouse) for his invaluable help with the nomenclature of the novel species.


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Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791.[CrossRef]

Fitch, W. M. (1971). Toward defining the course of evolution: minimum change for a specific tree topology. Syst Zool 20, 406–416.[CrossRef]

Gordon, R. E., Barnett, D. A., Handerhan, J. E. & Pang, C. H.-N. (1974). Nocardia coeliaca, Nocardia autotrophica, and the nocardin strain. Int J Syst Bacteriol 24, 54–63.[Abstract/Free Full Text]

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

Itoh, T., Kudo, T. & Seino, A. (1987). Chemotaxonomic studies on new genera of actinomycetes proposed in Chinese papers. Actinomycetologica 1, 43–59.

Kämpfer, P. & Kroppenstedt, R. M. (1996). Numerical analysis of fatty acid patterns of coryneform bacteria and related taxa. Can J Microbiol 42, 989–1005.

Kimura, M. (1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16, 111–120.[CrossRef][Medline]

Lechevalier, M. P. & Lechevalier, H. A. (1980). The chemotaxonomy of actinomycetes. In Actinomycete Taxonomy, Special Publication 6, pp. 277–284. Arlington, VA: Society for Industrial Microbiology.

Lee, Y. K., Kim, H. W., Liu, C. L. & Lee, H. K. (2003). A simple method for DNA extraction from marine bacteria that produce extracellular materials. J Microbiol Methods 52, 245–250.[CrossRef][Medline]

Liu, Z., Zhang, Y. & Yan, X. (1984). A new genus of the order Actinomycetales. Acta Microbiol Sin 24, 295–298.

Minnikin, D. E., Hutchinson, I. G., Caldicott, A. B. & Goodfellow, M. (1980). Thin-layer chromatography of methanolysates of mycolic acid-containing bacteria. J Chromatogr 188, 221–233.[CrossRef]

Minnikin, D. E., O'Donnell, A. G., Goodfellow, M., Alderson, G., Athalye, M., Schaal, K. & Parlett, J. H. (1984). An integrated procedure for the extraction of bacterial isoprenoid quinones and polar lipids. J Microbiol Methods 2, 233–241.[CrossRef]

Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.[Abstract]

Sasser, M. (1990). Identification of bacteria by gas chromatography of cellular fatty acids. Technical Note 101. Newark, DE: MIDI.

Singla, A. K., Mayilraj, S., Kudo, T., Krishnamurthi, S., Prasad, G. S. & Vohra, R. M. (2005). Actinoalloteichus spitiensis sp. nov., a novel actinobacterium isolated from a cold desert of the Indian Himalayas. Int J Syst Evol Microbiol 55, 2561–2564.[Abstract/Free Full Text]

Tamura, T. & Hatano, K. (1998). Phylogenetic analyses on the strains belonging to invalidated genera of the order Actinomycetales. Actinomycetologica 12, 15–28.

Tamura, T., Liu, Z., Zhang, Y. & Hatano, K. (2000). Actinoalloteichus cyanogriseus gen. nov., sp. nov. Int J Syst Evol Microbiol 50, 1435–1440.[Abstract]

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