Streptomyces xiamenensis sp. nov., isolated from mangrove sediment

Mangrove forests, important protectors of the coastline, are located in the tidal zones in tropical and subtropical areas. These highly productive ecosystems are home to a diverse flora and fauna of marine, freshwater and terrestrial species (Jennerjahn & Ittekkot, 2002). In contrast to the well-documented species diversity of larger animals and plants in these ecosystems, the microbial community diversity of mangrove forests has been poorly studied.

The regular changes in environmental factors seen in mangrove forests, such as salinity and tidal gradients, are believed to be effective selectors for metabolic pathway adaptations that could generate unusual metabolites. This has led to increasing exploitation of the mangrove micro-organism resources (Long et al., 2005). The micro-organisms in mangrove forest sediments are not only involved in the decomposition and mineralization of litter-fall (Holguin et al., 2001), but are also versatile producers of various enzymes and antibiotics.

In the course of our screening program for new antibiotic producers, groups of actinomycete strains, which contained both type I and type II polyketide biosynthesis pathway genes, were isolated from soil samples collected from the national mangrove reserve in the Fujian Province of China. In this paper, the results of a polyphasic taxonomic study of a novel Streptomyces strain, MCCC 1A01550^T, are presented.

Strain MCCC 1A01550^T was isolated after 2 weeks incubation at 28 °C on ISP 3 agar (Shirling & Gottlieb, 1966). The novel strain was maintained on nutrient agar at 4 °C and stored in glycerol suspensions at −20 °C. For chemical and molecular analyses, biomass was prepared by culturing in trypticase soytone broth (TSB) for 3–5 days at 28 °C in a rotary shaker (180 r.p.m.) and was then harvested by centrifugation.

Morphological observations of spores and mycelia grown on Gause's synthetic agar at 28 °C for 12 days were made by light microscopy (COBER-015; Olympus) and scanning electron microscopy (XL30; ESEM-TMP Philips-FEI). Cultural characteristics were determined after 3 weeks at 28 °C by methods used in the ISP (Shirling & Gottlieb, 1966). Colours were assessed according to colour chips in the ISCC-NBS Color Charts Standard no. 2106 (Kelly, 1964). The temperature range for growth was determined on modified ISP 2 agar at 4, 10, 15, 20, 28, 37 and 45 °C for 1–2 weeks. Tolerance to NaCl was determined by adding 0, 5, 10, 15 or 20 % (w/v) NaCl to the ISP 2 agar medium, followed by incubation at 28 °C in a rotary shaker (180 r.p.m.) for 1 week.

The novel strain developed well on several media. The temperature range for growth was 15–37 °C and NaCl tolerance was 5 %, with an optimal temperature at 28 °C. At maturity, the aerial mycelium formed long, straight to rectiflexibiles spiral spore chains. The spores were non-motile and elliptoid in shape (Fig. 1⇓). Colonies were grey in colour on ISP 2, ISP 3, Czapek's agar and starch agar, and white on ISP 4, ISP 5 and glucose-asparagine agar. Diffusible pigments were produced on most media (Table 1⇓).

The media and procedures used to assess physiological and biochemical features were as described by Shirling & Gottlieb (1966). Carbohydrate utilization was carried out according to the method of Kämpfer et al. (1991). Resistance to various antibiotics was determined on ISP 2 agar supplemented with different antibiotics (Shirling & Gottlieb, 1966). Gelatin liquefaction, starch hydrolysis and reaction with milk were tested according to the methods of Goodfellow (1971) and Gordon et al. (1974).

All physiological and biochemical tests were performed at 28 °C. The novel strain utilized mannose, arabinose, ribose, rhamnose and mannitol, but not raffinose, xylose or galactose. Tests for gelatin liquefaction, nitrate reduction and milk peptonization were positive and tests for H₂S and melanin production were negative. Good growth occurred in the presence of penicillin G (5 μg ml⁻¹), rifampicin (5 μg ml⁻¹), streptomycin (10 μg ml⁻¹) and ampicillin (10 μg ml⁻¹).

Tests to determine the diagnostic isomer of diaminopimelic acid and the whole-cell sugars were conducted using freeze-dried cells that were hydrolysed with 6 M and 0.5 M HCl at 120 °C for 30 min (Lechevalier & Lechevalier, 1980) and then analysed by TLC (Hasegawa et al., 1983). Phospholipid analysis was performed as described by Komagata & Suzuki (1987). Menaquinones were extracted according to the method of Collins et al. (1977) and analysed by HPLC (Kroppenstedt, 1985). Cellular fatty acid composition was determined as described by Sasser (1990) using the Microbial Identification System (MIDI, Inc.) and Sherlock MIS software.

The amino acids in the peptidoglycan layer of the cell wall of strain MCCC 1A01550^T were ll-diaminopimelic acid, alanine, glycine and glutamic acid. The whole-cell sugar was galactose. The predominant menaquinones were MK-9(H₄) (71.2 %), MK-9(H₆) (16.3 %), MK-9(H₂) (4 %) and MK-9(H₈) (1.6 %). The diagnostic phospholipid was phosphatidylethanolamine (PE); other phospholipids detected were phosphatidylglycerol and phosphatidylinositol and phosphatidylinositol mannosides. The major cellular fatty acids were iso-C_16 : 0 (43.82 %), cis9-C_16 : 1 (12.13 %) and C_16 : 0 (11.30 %) (for details see Supplementary Table S1 in IJSEM Online).

Amplification of the 16S rRNA gene sequence was performed as described by Cui et al. (2001). Sequence analysis was then conducted using the blast network service provided by the NCBI (Altschul et al., 1997). The 16S rRNA gene sequences of representative actinomycetes were aligned using clustal_x (Thompson et al., 1997) and similarity values were used to construct a phylogenetic tree by the maximum-parsimony and neighbour-joining methods with mega3.1 software (Saitou & Nei, 1987). The topology of the tree was evaluated by performing a bootstrap analysis (Felsenstein, 1985) using 1000 replications.

Genomic DNA was extracted as described by Weisburg et al. (1991). DNA was digested and dephosphorylated before determination of base composition by HPLC analysis (Tamaoka & Komagata, 1984). Molar G+C content was calculated according to Mesbah et al. (1989). DNA–DNA relatedness studies were performed between strain MCCC 1A01550^T and Streptomyces carpaticus NRRL B-16359^T (Gause et al., 1983), Streptomyces cheonanensis VC-A46^T (Kim et al., 2006) and Streptomyces sodiiphilus YIM 80305^T (Li et al., 2005). Chromosomal DNA of these strains was digested by HaeIII and fragments of 400 bp to 5 kb were collected and labelled as probe. The total chromosomal DNA samples were spotted onto Hybond-N⁺ nylon membrane (Amersham) for hybridization. DNA–DNA hybridization studies were carried out using a DIG-High Prime DNA Labelling and Detection Starter kit II (Roche), as described in the instruction manual.

The G+C content of the DNA was determined to be 71.6 mol%. The almost complete 16S rRNA gene sequence (1493 nt) of strain MCCC 1A01550^T was obtained and was compared with the GenBank/EMBL/DDBJ databases using blast. The phylogenetic tree (Fig. 2⇓) shows that strain MCCC 1A01550^T formed a distinct branch with S. carpaticus NRRL B-16359^T, S. cheonanensis VC-A46^T and S. sodiiphilus YIM 80305^T. The novel strain showed 97.5 % gene sequence similarity to S. carpaticus NRRL B-16359^T, 96.6 % similarity to S. cheonanensis VC-A46^T and 96.1 % similarity to S. sodiiphilus YIM 80305^T. DNA–DNA relatedness values below 80 % have been recommended for the recognition of novel genomic species of Streptomyces (Labeda, 1992). As determined by DNA hybridization, the DNA–DNA relatedness values between strain MCCC 1A01550^T and S. carpaticus NRRL B-16359^T (48.38 %), S. cheonanensis VC-A46^T (21.29 %) and S. sodiiphilus YIM 80305^T (25 %) were all below 70 %, and thus support the conclusion that strain MCCC 1A01550^T represents a separate genomic species.

The phylogenetic distinctiveness and DNA–DNA relatedness data were sufficient to categorize strain MCCC 1A01550^T as distinct from previously recognized species of the genus Streptomyces. This conclusion also was supported by the number of phenotypic differences between the novel strain and phylogenetically related species of the genus Streptomyces (Table 2⇓). It is concluded that strain MCCC 1A01550^T represents a novel member of the genus Streptomyces, for which the name Streptomyces xiamenensis sp. nov. is proposed.