SGM Journals
Actinobacteria

Streptomyces avicenniae sp. nov., a novel actinomycete isolated from the rhizosphere of the mangrove plant Avicennia mariana

Jing Xiao, Yin Wang, Yingxue Luo, Shu-Jie Xie, Ji-Sheng Ruan, Jun Xu

  • 1Key Laboratory of Marine Biogenetic Resources, The Third Institute of Oceanography SOA, Xiamen, Fujian, 361005, PR China
  • 2School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, PR China
  • Correspondence
    Jun Xu
    xu.junn{at}gmail.com

    • International Journal of Systematic and Evolutionary Microbiology 2009; 59(10):2624–2628 · https://doi.org/10.1099/ijs.0.009357-0

    View at publisher PubMed

    Abstract

    A novel isolate, designated strain MCCC 1A01535T, belonging to the genus Streptomyces was isolated from the rhizosphere of the mangrove plant Avicennia mariana from Fujian Province, south China. Characterization of the isolate was based on a polyphasic approach. Pairwise comparison of 16S rRNA gene sequences revealed that strain MCCC 1A01535T shared 97.7 and 97.5 % sequence similarities to Streptomyces specialis GW41-1564T and Streptomyces hainanensis YIM47672T, respectively, its most closely related neighbours, whereas the DNA–DNA relatedness value between strains MCCC 1A01535T and GW41-1564T was 50.2 %. The major fatty acids of strain MCCC 1A01535T were iso-C16 : 0, C16 : 0 and anteiso-C15 : 0. These differed from the fatty acid compositions of related strains. Strain MCCC 1A01535T exhibited an unusual menaquinone system that comprised MK-10(H6) as the predominant component and moderate amounts of MK-9(H6), MK-9(H8) and MK-10(H8); minor amounts of MK-9(H4), MK-10(H4), MK-9(H10) and MK-10(H10) were also present. Based on its chemotaxonomic, phenotypic and genotypic characteristics, strain MCCC 1A01535T is considered to represent a novel species of the genus Streptomyces, for which the name Streptomyces avicenniae sp. nov. is proposed. The type strain is MCCC 1A01535T (=DSM 41943T=CGMCC 4.5510T).

    • The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain MCCC 1A01535T is EU399234.

    • Tables showing major quinone and major fatty acid compositions are available as supplementary material with the online version of this paper.

    Members of the genus Streptomyces, proposed by Waksman & Henrici (1943), are a rich source of novel bioactive, commercially significant compounds. Although Streptomyces strains were frequently isolated from terrestrial environments, they have also been recovered from aquatic and symbiotic environments. Marine actinomycetes have been recognized as a resource for biotechnology in recent years (Bull et al., 2000). Analysis of the diversity of actinomycetes isolated from Challenger-Deep sediment from the Mariana Trench has proven the presence of Streptomyces in the deep-sea environment (Pathom-Aree et al., 2006).

    Mangrove forests are highly productive ecosystems that comprise diverse flora and fauna of marine, freshwater and terrestrial species (Jennerjahn & Ittekkot, 2002). The exploitation of the profusion of mangrove micro-organism resources has been expanded in many aspects (Long et al., 2005), including the screening of novel actinomycetes that could be a dormant source of new natural products. A systematic investigation of the actinomycete diversity in the National mangrove reserve in Fujian Province, China was performed, during which strain MCCC 1A01535T was isolated. Here, we report the results of a polyphasic taxonomic investigation on strain MCCC 1A01535T and propose that it represents the type strain of a novel species of the genus Streptomyces that exhibits an unusual menaquinone system.

    Strain MCCC 1A01535T was isolated after 2 weeks incubation at 28 °C on ISP 2 agar (Shirling & Gottlieb, 1966). Morphological observations of spores and mycelia on Gause's synthetic agar at 28 °C for 12 days were made by using light microscopy (COBER-015; Olympus) and scanning electron microscopy (XL30 ESEM-TMP; Philips-FEI). Cultural characteristics were determined after 3 weeks incubation at 28 °C by using methods in the ISP (Shirling & Gottlieb, 1966). Colours were assessed according to colour chips in the ISCC-NBS Colour Charts Standard no. 2106 (Kelly, 1964). Biomass of strain MCCC 1A01535T for chemical and molecular analyses was grown in ISP 2 liquid broth for 3–5 days at 28 °C in a rotary shaker (180 r.p.m.) and harvested by centrifugation.

    The isolate grew well on ISP 3 and 4 agar media with moderate growth occurring on other media. The colours of the aerial and substrate mycelium were media-dependent (Table 1⇓). A deep-brown soluble pigment was produced on ISP 2 media. At maturity, the aerial mycelium developed well on Gause's synthetic agar. The spore chains were compact spiral and contained about eight helixes per chain. The spores were elliptical and non-motile with smooth surfaces (Fig. 1⇓).

    Figure image not available in archive
    Fig. 1.

    Scanning electron micrograph of spore chains of strain MCCC 1A01535T grown on Gause's synthetic agar for 10 days at 28 °C. Bar, 5 μm.

    Table 1.

    Cultural characteristics of strain MCCC 1A01535T on various media at 28 °C after 7 days

    −, Not detected.

    The temperature range for growth was determined on modified ISP 2 agar at 4, 10, 15, 20, 28, 37 and 45 °C for 1 to 2 weeks. Carbohydrate utilization was carried out according to the method of Kämpfer et al. (1991). Gelatin liquefaction, starch hydrolysis, milk coagulation and peptonization, melanin pigment production, nitrate reduction and H2S production were tested using the methods of Goodfellow (1971) and Gordon et al. (1974). Resistance to various antibiotics was determined with antimicrobial susceptibility discs (Oxoid). Phenotypic characteristics that differentiate strain MCCC 1A01535T from related Streptomyces species are given in Table 2⇓.

    Table 2.

    Phenotypic characteristics that differentiate strain MCCC 1A01535T from related Streptomyces species

    Strains: 1, MCCC 1A01535T (S. avicenniae sp. nov.); 2, S. specialis GW41-1564T; 3, S. hainanensis YIM 47672T. Data for S. specialis and S. hainanensis were from Kämpfer et al. (2008) and Jiang et al. (2007). All strains do not utilize d-mannose. +, Positive; −, negative; nd, not determined.

    Analyses of the diagnostic isomer of the diaminopimelic acid (A2pm) and whole-cell sugars were done using freeze-dried cells that were hydrolysed with 6 and 0.5 M HCl at 120 °C for 30 min (Lechevalier & Lechevalier, 1980), and analysed by TLC (Hasegawa et al., 1983). Phospholipid analysis was carried out as described by Komagata & Suzuki (1987). The cellular fatty acid composition was determined as described by Sasser (1990) using the Microbial Identification System (MIDI, Inc.) and Sherlock MIS Software.

    Amino acids in the peptidoglycan layer of strain MCCC 1A01535T were ll-A2pm, alanine, glycine and glutamic acid. Whole-cell hydrolysates contained ribose, xylose, arabinose, galactose and glucose. Diphosphatidylglycerol was the predominant polar lipid. Phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol and an unknown phospholipid were present in minor amounts. The major cellular fatty acids were iso-C16 : 0 (32.0 %), C16 : 0 (15.1 %) and anteiso-C15 : 0 (10.7 %).

    Menaquinones were extracted by using the method of Collins et al. (1977) and analysed by HPLC (Kroppenstedt, 1985). The quinone system of strain MCCC 1A01535T consisted of the predominant menaquinone MK-10(H6) (29 %) and moderate amounts of MK-9(H6) (16.8 %), MK-9(H8) (16.3 %) and MK-10(H8) (14.8 %). Minor amounts (<10 %) of MK-9(H4) (6.7 %), MK-10(H4) (9.4 %), MK-9(H10) (3.0 %) and MK-10(H10) (4.0 %) were also present.

    Genomic DNA was extracted as described by Weisburg et al. (1991). DNA was digested and dephosphorylated prior to the determination of the base composition by using HPLC (Tamaoka & Komagata, 1984). The G+C content of the DNA was calculated according to Mesbah et al. (1989). The G+C content of the DNA strain MCCC 1A01535T was determined to be 73.3 mol%.

    Amplification of the 16S rRNA gene sequence was performed as described by Cui et al. (2001). An almost-complete 16S rRNA gene sequence (1491 nt) of strain MCCC 1A01535T was obtained and compared against the GenBank and EzTaxon databases using blast (Altschul et al., 1997; Chun et al., 2007). The 16S rRNA gene sequences of representative related actinomycetes were aligned using clustal_x (Thompson et al., 1997) and a phylogenetic tree was constructed by using maximum-parsimony and neighbour-joining with mega 3.1 software (Kumar et al., 2004; Saitou & Nei, 1987). The topology of the tree was evaluated by using a bootstrap analysis (Felsenstein, 1985) with 1000 replications. Strain MCCC 1A01535T formed a distinct branch with Streptomyces specialis GW41-1564T in the phylogenetic tree (Fig. 2⇓). The highest 16S rRNA sequence similarities between the isolate and type strains of recognized species in the databases were 97.7 % to S. specialis GW41-1564T (Kämpfer et al., 2008) and 97.5 % to Streptomyces hainanensis YIM 47672T (Jiang et al., 2007).

    Figure image not available in archive
    Fig. 2.

    Phylogenetic dendrogram obtained by using neighbour-joining analysis based on 16S rRNA gene sequences (1458 bp, by omitting unaligned regions), showing the positions of strain MCCC 1A01535T and related strains. The sequence of the type strain of Micrococcus psychrophilum was used as an outgroup. Only bootstrap values above 50 %, expressed as percentages of 1000 replications, are shown at branch points. Bar, 0.01 substitutions per nucleotide position.

    DNA–DNA relatedness studies were carried out between strain MCCC 1A01535T and the closely related strain GW41-1564T using DIG-High prime DNA labelling and detection starter kit II (Roche), as described in the instruction manual. Chromosomal DNA of the strains was digested by HaeIII. Fragments of 400 bp to 5 kb were collected and labelled as a probe. The total chromosomal DNA was spotted onto Hybond-N+ nylon membrane (Amersham) for hybridization. DNA–DNA relatedness values were 50.2 %, below the recommended criterion of 80 % for species delineation of the genus Streptomyces (Labeda, 1992).

    According to Kim et al. (2003), the major menaquinones of the genus Streptomyces are MK-9(H6) and MK-9(H8). It was interesting that strains GW41-1564T and YIM 47672T both contained unusual quinone systems, but strain MCCC 1A01535T exhibited a distinct quinone system that was different from both of them (see Supplementary Table S1, available in IJSEM Online). The detailed profile of the cellular fatty acids given in Supplementary Table S2 (in IJSEM Online) and the sugar pattern gave additional support for the separation of strain MCCC 1A01535T from the related strains GW41-1564T and YIM 47672T.

    Strain MCCC 1A01535T showed a notable difference in the production of diffusible pigment from its closest relatives. The three strains could be discriminated based on the predominant menaquinones. Based on morphology, chemotaxonomy and 16S rRNA phylogeny, it can be concluded that strain MCCC 1A01535T represents a novel species of the genus Streptomyces, for which we propose the name Streptomyces avicenniae sp. nov.

    Description of Streptomyces avicenniae sp. nov.

    Streptomyces avicenniae (a.vi.cen′ni.ae. N.L. fem. n. Avicennia scientific name of genus of tree; N.L. fem. gen. n. avicenniae of Avicennia, isolated from the rhizosphere of the plant Avicennia mariana).

    Aerobic, Gram-positive actinomycete with branching aerial mycelium and substrate mycelium. Non-motile and ellipsoid spores with smooth surfaces are borne in compact spiral spore chains with up to eight helixes per chain. Colony colour is medium-dependent and diffusible pigments are produced on ISP 2 media. Aerial mycelia are light-yellow on Czapek's agar and Starch agar, yellow–brown on glucose-asparagine agar and ISP 3, olive on ISP 4 and ISP 5, and deep orange–red on ISP 2. Substrate mycelia are white, grey to light-yellow and deep orange–red on different media. Growth occurs at temperatures ranging from 15 to 37 °C, with optimum growth at 28 °C. Raffinose, d-xylose, d-galactose, l-arabinose, l-rhamnose, mannitol and sucrose are utilized, but d-mannose, inositol and d-ribose are not. Tests for gelatin liquefaction, milk coagulation and peptonization are positive and tests for nitrate reduction, H2S production and melanin production are negative. Cell wall contains ll-diaminopimelic acid. Whole-cell hydrolysates contain ribose, xylose, arabinose, galactose and glucose. Resistant to ciprofloxacin (5 μg), clindamycin (2 μg), furazolidone (15 μg), metronidazole (5 μg), lincomycin (2 μg) and norfloxacin (10 μg). Sensitive to ampicillin (10 μg), chloromycetin (30 μg), rocephin (30 μg), erythromycin (15 μg), kanamycin (30 μg), cefazolin (30 μg), neomycin (10 μg), penicillin G (10 μg), rifampicin (5 μg) and vancomycin (30 μg). Major menaquinone is MK-10(H6) with moderate amounts of MK-9(H6), MK-9(H8), MK-10(H8), MK-9(H4), MK-10(H4), MK-9(H10) and MK-10(H10). Diagnostic phospholipids are diphosphatidylglycerol with minor amounts of phosphatidylethanolamine, phosphatidylcholine and phosphatidylinositol and an unknown phospholipid. Major fatty acid components are iso-C16 : 0, C16 : 0 and anteiso-C15 : 0. The G+C content of the DNA of the type strain is 73.3 mol%.

    The type strain, MCCC 1A01535T (=DSM 41943T=CGMCC 4.5510T), was isolated from the rhizosphere of the mangrove plant Avicennia mariana from Fujian Province, south China.

    Acknowledgments

    J. X. is indebted to the National Science Foundation of China (40606031), the National High Technology R&D Program of China (2006AA09Z437), the National Basic Research Program of China (2006CB708200) and the National Infrastructure Program of Natural Resources of S&T of China (2005DKA21209). J. X. gratefully acknowledges Professor Wen-Jun Li for technical supervision of some chemotaxonomic analyses in the Yunnan Institute of Microbiology.

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