J. Gen. Appl. Microbiol., 48, 109–115 (2002)
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J. Gen. Appl. Microbiol., 48, 109–115 (2002) Full Paper Haliangium ochraceum gen. nov., sp. nov. and Haliangium tepidum sp. nov.: Novel moderately halophilic myxobacteria isolated from coastal saline environments Ryosuke Fudou,* Yasuko Jojima, Takashi Iizuka, and Shigeru Yamanaka† Central Research Laboratories, Ajinomoto Co., Inc., Kawasaki 210–8681, Japan (Received November 21, 2001; Accepted January 31, 2002) Phenotypic and phylogenetic studies were performed on two myxobacterial strains, SMP-2 and SMP-10, isolated from coastal regions. The two strains are morphologically similar, in that both produce yellow fruiting bodies, comprising several sessile sporangioles in dense packs. They are differentiated from known terrestrial myxobacteria on the basis of salt requirements (2–3% NaCl) and the presence of anteiso-branched fatty acids. Comparative 16S rRNA gene sequencing studies revealed that SMP-2 and SMP-10 are genetically related, and constitute a new cluster within the myxobacteria group, together with the Polyangium vitellinum Pl vt1 strain as the clos- est neighbor. The sequence similarity between the two strains is 95.6%. Based on phenotypic and phylogenetic evidence, it is proposed that these two strains be assigned to a new genus, JCM؍) .Haliangium gen. nov., with SMP-2 designated as Haliangium ochraceum sp. nov .(DSM 14436T؍JCM 11304T؍) .DSM 14365T), and SMP-10 as Haliangium tepidum sp. nov؍11303T Key Words——Haliangium gen. nov.; Haliangium ochraceum sp. nov.; Haliangium tepidum sp. nov.; marine myxobacteria Introduction terized as Gram-negative, rod-shaped gliding bacteria with high GϩC content. Analyses of 16S rRNA gene Myxobacteria are unique in their complex life cycle. sequences reveal that they form a relatively homoge- Under starvation conditions, bacterial cells gather to- neous cluster within the d-subclass of Proteobacteria gether to form aggregates that subsequently differenti- (Shimkets and Woese, 1992). ate into fruiting bodies. Mature fruiting bodies contain The primary habitat of myxobacteria is soil, from numerous myxospores, resistant to environmental desert sand to black chernozem rich in organic matter. stress factors, like desiccation and UV irradiation. Mor- These bacteria are strictly aerobic, and usually live in phological features of fruiting bodies are diverse within the surface layers of the soil. They additionally inhabit myxobacteria, and are therefore employed as primary decaying plant material, including rotting wood and criteria for the classification of groups (McCurdy, 1989; bark (David, 2000; Reichenbach, 1999; Reichenbach Reichenbach, 1993). They are taxonomically charac- and Dworkin, 1992). Surprisingly, isolates of myxobac- teria from marine samples are unable to tolerate the salt concentrations in seawater, and are accordingly * Address reprint requests to: Dr. Ryosuke Fudou, Central mainly portrayed as terrestrial bacteria (Brochman, Research Laboratories, Ajinomoto Co., Inc., 1–1 Suzuki-cho, Kawasaki 210–8681, Japan. 1963; Rückert, 1984). Recently, however, we isolated E-mail: ryosuke_fudou@ajinomoto.com true marine myxobacteria that require 2–3% NaCl for † Present address: Faculty of Textile and Technology, Shinshu optimum growth (Iizuka et al., 1998a). Although these University, Ueda 386–8567, Japan. isolates are phylogenetically placed in the group 110 FUDOU et al. Vol. 48 ‘myxobacteria,’ they are distinct from any known agar as a basal medium. The medium consists of myxobacterial species, suggesting that they be as- 0.1% Casitone (Difco Laboratories, Detroit, MI, USA), signed to a new taxon. In this study, we describe in de- 0.03% yeast extract, 1.5% agar, 1.5% NaCl and half- tail the taxonomic properties of the marine isolate strength SWS solution (Iizuka et al., 1998a). SMP-2, and the newly discovered SMP-10, which has Chemotaxonomy. Cellular fatty acid composition similar characteristics, but grows at elevated tempera- was determined with GLC (Model GC353B, GL Sci- tures. Based on the phenotypic and phylogenetic char- ence Inc., Tokyo, Japan) and GC-MS (HP 5972, acteristics of these two strains, we propose a novel Hewlett Packard Co., Palo Alto, CA, USA), using a genus Haliangium, with two new species, H. capillary column (TC-70, 0.25 mm I.D.ϫ30 m, GL Sci- ochraceum and H. tepidum, attributed to SMP-2 and ence Inc.). Respiratory quinones were extracted with SMP-10, respectively. acetone, and purified and analyzed by TLC and HPLC, according to the method of Hiraishi et al. (1984). DNA Materials and Methods was extracted according to the method of Saito and Miura (1963), and purified using a DNA purification kit Bacterial strains and media. Isolation and cultiva- (Qiagen GmbH, Hilden, Germany) according to the tion were performed as described previously (Iizuka et manufacturer’s instructions. The DNA base composi- al., 1998a). Strains SMP-2 and SMP-10 were isolated tion was determined by HPLC (Tamaoka and Koma- from seaweed and sea grass samples, respectively, gata, 1984). that were collected from a sandy beach on the Miura 16S rDNA analysis. Amplification, sequencing Peninsula, Kanagawa, Japan. In spite of the repeated and phylogenetic analysis of 16S rRNA gene was efforts at isolation from more than 500 samples col- carried out as described previously (Iizuka et al., lected at 24 sites of coastal environments in Japan, no 1998a, b). Accession numbers of the 16S rRNA other halophilic myxobacteria with pale-yellow fruiting gene sequences determined in this study and those bodies could be obtained. Strains were routinely culti- of the reference strains from DDBJ are as follows: vated on Vy2-ASW agar and incubated at 30°C. To ob- SMP-2, AB016469 (Iizuka et al., 1988a); SMP-10, tain cell mass for biochemical analyses, SMP-2 and AB062751 (this study); Polyangium vitellinum Plvt1, SMP-10 were cultivated on 1/5 Cy-SWS broth (Iizuka AJ233944; Nannocystis exedens, M94279; “Nanno- et al., 1998a) at 28°C and 37°C, respectively. Both cystis aggregans” Na a1, AJ233945; “Sorangium cellu- strains have been deposited at JCM and DSMZ (JCM losum” M94282; Polyangium sp. Pl4943, M94280; 11303TϭDSM 14365TϭSMP-2, and JCM 11304Tϭ Chondromyces apiculatus M94274; Chondromyces DSM 14436TϭSMP-10, respectively). crocatus, M94275; Melittangium lichenicola, M94277; Phenotypic characteristics. Morphological studies Corallococcus coralloides, M94278; Myxococcus xan- were performed on fruiting bodies that developed on thus, M34114; Angiococcus disciformis, M94374; Vy2-ASW agar at 30°C. Enzyme activities were inves- Archangium gephyra, M94273; Stigmatella aurantiaca, tigated, using the API-ZYM system (API bióMerieux, M94281; Cystobacter fuscus, M94276; Escherichia Marcy l’Etoile, France) according to the manufacturer’s coli, J01859. instructions, with a slight modification. Small blocks (approx. 3-mm cubes) cut from a swarm area of Vy2- Results and Discussion ASW agar cultures were used as inoculum, and incu- bated for 6 h at 37°C. Catalase and oxidase activities Morphological characteristics were tested with a commercial kit (BBL, Becton Dickin- The two marine isolates demonstrate similar cell son Diagnostic Systems, Sparks, MD, USA). Hydroly- morphology and fruiting structures. The morphological sis of cellulose, starch, yeast cells and bacterial cells features of SMP-2 have been reported in a previous were investigated, principally following the procedure paper (Iizuka et al., 1998a). Yellow colonies of both of Reichenbach (1970). Degradation of DNA was strains gradually spread by gliding as a film-like layer tested according to the method of Jeffries et al. (1957). and were slightly sunk into the agar surface (Fig. 1a): Hydrolysis of casein and chitin was investigated by the neither radial veins nor tiny waves were observed. The method of Santavy et al. (1990). These hydrolysis fruiting bodies of the two isolates usually formed on tests were done at 27°C using the diluted Cy-SWS the agar surfaces but sometimes within the gels. They 2002 H. ochraceum gen. nov., sp. nov. and H. tepidum sp. nov. 111 (a) (b) (c) (d) Fig. 1. Photographs of the marine myxobacteria, SMP-10. a. Swarm colony on Vy/2-SWS agar. Scale bar is 1 cm. b. Fruiting body consisting of a number of densely packed sporangioles. Scale bar is 100 mm. c. Vegetative cells. Scale bar is 10 mm. d. Crushed sporangioles releasing small oval- to sphere-shaped myxospores. Some myxospores are indicated by arrowheads. Scale bar is 10 mm. were variable in shape and size. SMP-2 formed light Nannocystis, rather than Polyangium-Sorangium in yellow to yellowish-brown fruiting bodies (50–200 mm), appearance, in that they were small in size (0.5–0.7 comprising one or more sessile oval-shaped sporangi- mm) and oval to spherical in shape (Fig. 1d). oles, each measuring 20 to 60 mm (Iizuka et al., 1998a). Fruiting bodies of SMP-10 (Fig. 1b) resembled Physiological characteristics those of SMP-2, except that they usually contained The two strains require NaCl for growth, as reported more sporangioles (15 to 60 per fruiting body), and previously for SMP-2 (Iizuka et al., 1998a). The two were slightly polyhedral rather than rounded and strains grow in 0.5–6.0% NaCl (optimum at 1–3% smaller in size (15–70 mm). Both strains had cylindrical NaCl). Upon transfer to distilled water from 2% NaCl- vegetative cells with blunt ends, measuring 0.5–0.6 by SWS solution, most cells from both isolates lysed, 3.0–8.0 mm for SMP-2, and 0.5–0.6 by 3.5–7.0 mm for while the rest remained as ghost-like cells. The tem- SMP-10 (Fig. 1c). These morphological characteristics perature range for growth differed slightly between the of the marine isolate mentioned above were similar to two strains. While SMP-2 grew at 20–40°C (optimum those of the genus Polyangium-Sorangium (Reichen- at 30–34°C), SMP-10 showed growth at an elevated bach and Dworkin, 1992). However, myxospores temperature range of 27–45°C (optimum at 37–40°C). within the fruiting bodies resembled those of the genus Neither strain grew on tryptic soy broth supple- 112 FUDOU et al. Vol. 48 mented with 2% NaCl.