Antonie van Leeuwenhoek (2015) 107:1369–1378 DOI 10.1007/s10482-015-0431-5
ORIGINAL PAPER
Streptomyces gilvigriseus sp. nov., a novel actinobacterium isolated from mangrove forest soil Hooi-Leng Ser . Nurullhudda Zainal . Uma Devi Palanisamy . Bey-Hing Goh . Wai-Fong Yin . Kok-Gan Chan . Learn-Han Lee
Received: 19 January 2015 / Accepted: 11 March 2015 / Published online: 12 April 2015 Ó Springer International Publishing Switzerland 2015
Abstract A novel Streptomyces, strain MUSC 26T, was isolated from mangrove soil at Tanjung Lumpur, Malaysia. The bacterium was observed to be Grampositive and to form grayish yellow aerial and substrate mycelium on ISP 7 agar. A polyphasic approach was used to study the taxonomy of strain MUSC 26T, which shows a range of phylogenetic and chemotaxonomic properties consistent with those of the members of the genus Streptomyces. The cell wall peptidoglycan was determined to contain LL-diaminopimelic acid. The predominant menaquinones were identified as MK-9 (H8) and MK-9(H6). The polar lipids detected were identified as diphosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, hydroxyphosphatidylethanolamine, phosphatidylmethylethanolamine and hydroxyphosphatidylmethylethanolamine. The predominant
Electronic supplementary material The online version of this article (doi:10.1007/s10482-015-0431-5) contains supplementary material, which is available to authorized users. H.-L. Ser N. Zainal U. D. Palanisamy B.-H. Goh L.-H. Lee (&) Jeffrey Cheah School of Medicine and Health Sciences, Monash University Malaysia, 46150 Bandar Sunway, Selangor Darul Ehsan, Malaysia e-mail: [email protected]; [email protected] N. Zainal W.-F. Yin K.-G. Chan Division of Genetics and Molecular Biology, Faculty of Science, Institute of Biological Sciences, University of Malaya, 50603 Kuala Lumpur, Malaysia
cellular fatty acids ([10.0 %) were identified as anteiso-C15:0 (31.4 %), iso-C16:0 (16.3 %), iso-C15:0 (13.9 %) and anteiso-C17:0 (12.6 %). The cell wall sugars were found to be galactose, glucose, mannose, ribose and rhamnose. These results suggest that MUSC 26T should be placed within the genus Streptomyces. Phylogenetic analysis indicated that closely related strains include Streptomyces qinglanensis 172205T (96.5 % sequence similarity), S. sodiiphilus YIM 80305T (96.5 %) and S. rimosus subsp. rimosus ATCC 10970T (96.4 %). DNA– DNA relatedness values between MUSC 26T and closely related type strains ranged from 17.0 ± 2.2 to 33.2 ± 5.3 %. Comparison of BOX-PCR fingerprints indicated MUSC 26T presents a unique DNA profile. The DNA G?C content was determined to be 74.6 mol%. Based on this polyphasic study of MUSC 26T, it is concluded that this strain represents a novel species, for which the name Streptomyces gilvigriseus sp. nov. is proposed. The type strain is MUSC 26T (=DSMZ 42173T = MCCC 1K00504T). Keywords Streptomyces gilvigriseus sp. nov Actinobacteria Mangrove forest
Introduction The mangrove is a unique woody plant community of intertidal coasts in tropical and subtropical coastal regions. The mangrove ecosystem is among the world’s most productive environments and improves
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coastal waters, produces commercial forest products, supports coastal fisheries and protects coastlines. Mangrove ecosystems are habitats of diverse flora and fauna of marine, freshwater and terrestrial species (Jennerjahn and Ittekkot 2002), but comparatively little is known about the diversity of the microbial community in mangrove ecosystems (Hong et al. 2009; Xu et al. 2009). Recently, there has been increasing interest in mangrove microorganism resources (Hong et al. 2009; Lee et al. 2014a) as the constant changes in tidal gradient and salinity in the mangrove ecosystems are thought to be a driving force for metabolic pathway adaptations that might lead to the production of unusual metabolites. Many studies have discovered novel actinobacteria from the poorly explored mangrove environment, for example the isolation of Streptomyces avicenniae (Xiao et al. 2009), S. xiamenensis (Xu et al. 2009), S. sanyensis (Sui et al. 2011), S. qinglanensis (Hu et al. 2012) and S. pluripotens (Lee et al. 2014b). The genus Streptomyces was proposed by Waksman and Henrici (1943) and members of this genus have made significant contributions to mankind due to their abilities to produce various natural products (Be´rdy 2005). At the time of writing (March 2015), the genus Streptomyces is comprised of ca. 600 species with validly published names (http://www.bacterio. cict.fr/). During a systematic investigation of the actinobacterial diversity in a mangrove environment located on the east coast of Peninsular Malaysia, strain MUSC 26T was isolated. A polyphasic taxonomic study showed that strain MUSC 26T represents a novel species of the genus Streptomyces, for which the name S. gilvigriseus sp. nov. is proposed.
Materials and methods Isolation and maintenance of isolate Strain MUSC 26T was isolated from a soil sample collected at site MUSC-TLS1 (3o480 3.200 N o 0 00 103 20 11.0 E), located in the mangrove forest of Tanjung Lumpur in the state of Pahang, Peninsular Malaysia, in December 2012. Topsoil samples of the upper 20-cm layer (after removing the top 2–3 cm) were collected and sampled into sterile plastic bags using an aseptic metal trowel, and stored at -20 °C. Air-dried soil samples were ground with a mortar and pestle. Selective
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Antonie van Leeuwenhoek (2015) 107:1369–1378 Fig. 1 Maximum-likelihood phylogenetic tree based on almost c complete 16S rRNA sequences (1447 nucleotides) showing the relationship between strain MUSC 26T and representatives of some other related taxa. Numbers at nodes indicate percentages of 1000 bootstrap re-samplings, only values above 50 % are shown. Bar 0.005 substitutions per site. Asterisks indicate that the corresponding nodes were also recovered using the neighbour-joining tree-making algorithm
pretreatment of soil samples was performed using wet heat in sterilized water (15 min at 50 °C; Takahashi et al. 1996). Five grams of the pretreated air-dried soil was mixed with 45 ml sterilized water and mill ground, spread onto the isolation medium ISP 2 (Shirling and Gottlieb 1966) supplemented with cycloheximide (25 lg ml-1) and nystatin (10 lg ml-1), and incubated at 28 °C for 14 days. Pure cultures of strain MUSC 26T were isolated and maintained on slants of ISP 2 medium at 28 °C and as glycerol suspensions (20 %, v/v) at -20 °C for long term preservation. Genomic and phylogenetic analyses Genomic DNA extraction for PCR was performed as described by Hong et al. (2009). The 16S rRNA gene was amplified as described by Lee et al. (2014b). The 16S rRNA gene sequence of strain MUSC 26T was aligned with representative sequences of related type strains of the genus Streptomyces retrieved from the GenBank/EMBL/DDBJ databases using CLUSTALX software (Thompson et al. 1997). The alignment was verified manually and adjusted prior to the reconstruction of phylogenetic trees. Phylogenetic trees were constructed with the maximum-likelihood (Felsenstein 1981) (Fig. 1) and neighbour-joining (Saitou and Nei 1987) (Fig. S1) algorithms using MEGA version 5.2 (Tamura et al. 2011). Evolutionary distances for the neighbour-joining algorithm were computed using Kimura’s two-parameter model (Kimura 1980). The EzTaxon-e server (http:// eztaxon-e.ezbiocloud.net/; Kim et al. 2012) was used for calculations of sequence similarity. The stability of the resultant trees topologies were evaluated by using the bootstrap resampling method of Felsenstein (1985). BOX-PCR fingerprint analysis was used to characterize strain MUSC 26T and the closely related strains using the primer BOX-A1R (50 -CTACGGCAAGGC GACGCTGACG-30 ) (Versalovic et al. 1991; Lee et al. 2014c). The PCR condition for BOX-PCR was
Antonie van Leeuwenhoek (2015) 107:1369–1378
1371 T
Streptomyces endus NRRL 2339 (AY999911)
98*
T
Streptomyces demainii NRRL B-1478 (DQ334782) T
Streptomyces sporocinereus NBRC 100766 (AB249933) T
Streptomyces hygroscopicus subsp. hygroscopicus NRRL 2387 (AB231803) 56*
T
Streptomyces yogyakartensis NBRC 100779 (AB249942) T
97*
Streptomyces javensis NBRC 100777 (AB249940) T
Streptomyces violaceusniger NBRC 13459 (AB184420) T
Streptomyces iranensis HM 35 (FJ472862) T
63*
Streptomyces rapamycinicus NRRL B-5491 (EF408733) 100*
T
Streptomyces rhizosphaericus NBRC 100778 (AB249941) T
Streptomyces griseiniger NRRL B-1865 (AJ391818) T
Streptomyces mordarskii NRRL B-1346 (EF408735)
73*
T
Streptomyces castelarensis DSM 40830 (AY508511) T
Streptomyces melanosporofaciens NBRC 13061 (AB184283)
84*
T
Streptomyces yatensis NBRC 101000 (AB249962) T
Streptomyces sporoclivatus NBRC 100767 (AB249934) T
Streptomyces antimycoticus NBRC 12839 (AB184185) 97* 85*
T
Streptomyces hygroscopicus subsp. glebosus NBRC 13786 (AB184479) T
Streptomyces libani subsp. rufus LMG 20087 (AJ781351) T
Streptomyces glebosus CGMCC 4.1873 (HQ244456) T
Streptomyces ramulosus NRRL B-2714 (DQ026662) T
Streptomyces youssoufiensis X4 (FN421338) T
Streptomyces luteireticuli NBRC 13422 (AB249969)
64*
T
Streptomyces thioluteus LMG 20253 (AJ781360)
55
T
Streptomyces varsoviensis NRRL B-3589 (DQ026653) T
Streptomyces coerulescens NBRC 12758 (AB184122) T
Streptomyces mobaraensis NBRC 13819 (AORZ01000256) T
Streptomyces orinoci NBRC 13466 (AB184866) T
Streptomyces daliensis YIM 31724 (AY785161) T
83 Streptomyces sclerotialus DSM 43032 (AJ621608) T
Streptomyces albofaciens JCM 4342 (AB045880) T
Streptomyces rimosus subsp. rimosus ATCC 10970 (ANSJ01000404) T
Streptomyces monomycini NBRC 100769 (AB249936) 94*
T
Streptomyces olivaceiscleroticus DSM 40595 (AJ621606) T
94* Streptomyces niger NBRC 13362 (AB184352)
58
T
Streptomyces thermoviolaceus subsp. apingens DSM 41392 (Z68095)
*
T
Streptomyces somaliensis DSM 40738 (AJ007403) 88*
T
Streptomyces atrovirens NRRL B-16357 (DQ026672) T
88* Streptomyces flavoviridis NBRC 12772 (AB184842) T 94* Streptomyces pilosus NBRC 12807 (AB184161) T
Streptomyces albiaxialis NRRL B-24327 (AY999901) T
Streptomyces gilvigriseus MUSC26 (KJ632660) T
Streptomyces sodiiphilus YIM 80305 (AY236339) T
Streptomyces albus NRRL B-2365 (DQ026669) T
Streptomyces qinglanensis 172205 (HQ660227) T
Streptomyces marinus DSM 41970 (AB473556)
59* 58*
T
Streptomyces panacagri Gsoil 519 (AB245388)
0.005
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performed as described by Lee et al. (2014d). The PCR products were visualized by 2 % agarose gel electrophoresis. The extraction of genomic DNA for DNA–DNA hybridization of strain MUSC 26T, S. qinglanensis DSM 42035T, S. sodiiphilus DSM 41839T (96.49 %) and S. rimosus subsp. rimosus NBRC 12907T were carried out by the Identification Service of the DSMZ, Braunschweig, Germany following the protocol of Cashion et al. (1977). DNA–DNA hybridization was carried out as described by De Ley et al. (1970) under consideration of the modifications described by Huss et al. (1983). The G?C content of strain MUSC 26T was determined by HPLC (Mesbah et al. 1989). Phenotypic characteristics The cultural characteristics of strain MUSC 26T were determined following growth on ISP 2 and ISP 7 media (Shirling and Gottlieb 1966), actinomycetes isolation agar (AIA; Atlas and Parks 1993), Streptomyces agar (SA; Atlas and Parks 1993), starch casein agar (SCA; Ku¨ster and Williams 1964) and nutrient agar (MacFaddin 2000) for 14 days at 28 °C. Light microscopy (80i, Nikon) and scanning electron microscopy (JEOL-JSM 6400) were used to observe the morphology of the strain after incubation on ISP 2 medium at 28 °C for 7–14 days. The designation of colony colour was determined by using the ISCC-NBS colour charts (Kelly 1964). Gram staining was performed by standard Gram reaction and confirmed by using KOH lysis (Cerny 1978). The growth temperature range was tested at 12–48 °C at intervals of 4 °C on ISP 2 medium. The pH range for growth was tested in tryptic soy broth (TSB) between pH 4.0 and 10.0 at intervals of 1 pH unit. The NaCl tolerance was tested in TSB and salt concentrations ranging from 0 to 18 % (w/v) at intervals of 2 %. The responses to temperature, pH and NaCl were observed for 14 days. Production of melanoid pigments was examined using ISP 7 medium. Catalase activity and production of melanoid pigments were determined following protocols described by Lee et al. (2014e). Hemolytic activity was assessed on blood agar medium containing 5 % (w/v) peptone, 3 % (w/v) yeast extract, 5 % (w/v) NaCl and 5 % (v/v) horse blood (Carillo et al. 1996). Plates were examined for hemolysis after incubation at 32 °C for 7–14 days. Amylolytic, cellulase, chitinase, lipase, protease and
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xylanase activities were determined by growing cells on ISP 2 medium and following protocols as described by Meena et al. (2013). The presence of clear zones around colonies was taken to signify the potential of isolates for surfactant production. Antibiotic susceptibility tests were performed by the disc diffusion method as described by Shieh et al. (2003). Antimicrobials used and their concentrations per disc (Oxoid, Basingstoke, UK) were as follows: ampicillin (10 lg), ampicillin sulbactam (30 lg), cefotaxime (30 lg), cefuroxime (30 lg), cephalosporin (30 lg), chloramphenicol (30 lg), ciprofloxacin (10 lg), erythromycin (15 lg), gentamicin (20 lg), nalidixic acid (30 lg), Penicillin G (10 lg), streptomycin (10 lg), tetracycline (30 lg) and vancomycin (30 lg). Carbon-source utilization and chemical sensitivity assays were determined using Biolog GenIII MicroPlates (Biolog, USA) according to the manufacturer’s instructions. All of the phenotypic assays mentioned were performed concurrently for strain MUSC 26T, S. qinglanensis DSM 42035T, S. sodiiphilus DSM 41839T and S. rimosus subsp. rimosus NBRC 12907T. Chemotaxonomic characteristics The analyses of peptidoglycan amino acid composition and sugars of strain MUSC 26T were carried out by the Identification Service of the DSMZ using published protocols (Schumann 2011). Major diagnostic cell wall sugars of strain MUSC 26T were obtained as described by Whiton et al. (1985) and analyzed by TLC on cellulose plates (Staneck and Roberts 1974). Analysis of respiratory quinones, polar lipids (Kates 1986) and fatty acids (Sasser 1990) were carried out by the Identification Service of the DSMZ.
Results and discussion Strain MUSC 26T was observed to grow well on ISP 2 medium, ISP 7 medium, Streptomyces agar, starch casein agar and tryptic soy agar after 7–14 days at 28 °C, and to grow moderately on actinomycetes isolation agar, Luria–Bertani agar and nutrient agar. The colours of the aerial and substrate mycelium were media-dependent (Table S1). The morphological observation of a 15-day-old culture grown on ISP 2 medium revealed a smooth spore surface and abundant growth of both aerial and vegetative hyphae, which
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were well developed and not fragmented. These morphological features are consistent with assignment of the strain to the genus Streptomyces (Williams et al. 1989). Growth was found to occur at pH 5.0–8.0 (optimum pH 6.0–7.0), with 0-6 % NaCl tolerance (optimum 2–4 %) and at 20–36 °C (optimum 28–32 °C). Cells were found to be positive for catalase but negative for melanoid pigment production and hemolytic activity. Hydrolysis of carboxymethylcellulose and soluble starch were found to be positive, but negative for hydrolysis of casein, chitin, tributyrin (lipase) and xylan. Strain MUSC 26T can be differentiated from closely related members of the genus Streptomyces using a range of phenotypic properties (Table 1). In chemical sensitivity assays, cells are resistant to aztreonam, D-serine, fusidic acid, guanine HCL, lincomycin, lithium chloride, minocycline, 8 % NaCl, nalidixic acid, niaproof 4, potassium tellurite, rifamycin RV, sodium bromate, sodium butyrate, 1 % sodium lactate, tetrazolium blue, tetrazolium violet and troleandomycin. The nearly complete 16S rRNA gene sequence was obtained for strain MUSC 26T (1447 bp; GenBank/ EMBL/DDBJ accession number KJ632660) and phylogenetic trees were reconstructed to determine the phylogenetic position of this strain (Fig. 1; S1). Phylogenetic analysis exhibited that strain MUSC 26T is closely related to S. sodiiphilus YIM 80305T, as they formed a distinct clade (Fig. 1). The type strain S. sodiiphilus YIM 80305T was isolated from a muddy sample in Chaka salt lake located in Qinghai Province of China (Li et al. 2005). The 16S rRNA gene sequence analysis of strain MUSC 26T showed the highest similarity to that of S. qinglanensis 172205T (96.5 %), followed by S. sodiiphilus YIM 80305T (96.5 %) and S. rimosus subsp. rimosus ATCC 10970T (96.4 %); sequences similarities of less than 96.1 % were obtained with the type strains of other species of the genus Streptomyces. According to Stackebrandt and Goebel (1994), generally a 16S rRNA gene sequence divergence greater than 3 % is accepted as a criterion for delineating different prokaryotic species. The sequence divergence between strain MUSC 26T and the most closely related type strain S. qinglanensis 172205T was 3.5 %, a value consistent with strain MUSC 26T representing a novel species in the genus Streptomyces. The DNA–DNA relatedness values between strain MUSC 26T and S. qinglanensis DSM 42035T (33.2 ± 5.3 %), S. sodiiphilus DSM 41839T
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(17.0 ± 2.2 %) and S. rimosus subsp. rimosus NBRC 12907T (29.4 ± 3.1 %) were significantly below 70 %, the threshold value for the delineation of bacterial species (Wayne et al. 1987). The BOX-PCR results indicated that strain MUSC 26T yielded a unique BOX-PCR fingerprint compared with the closely related type strains (Fig. S2). These results are in agreement with results of DNA–DNA hybridizations, which indicate that strain MUSC 26T represents a novel species. The polar lipid analysis showed the presence of diphosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, hydroxyphosphatidylethanol amine, phosphatidylmethylethanolamine and hydroxyphosphatidylmethylethanolamine; an unidentified glycolipid and phospholipid were also detected. Differences in polar lipid profiles indicated that MUSC 26T is different from related type strains; for example, strain MUSC 26T (Fig. S3a) was found to contain hydroxyphosphatidylethanolamine, phosphatidylmethylethanolamine and hydroxyphosphatidylmethylethanolamine, lipids that were not detected in S. qinglanensis DSM 42035T (Fig. S3b). The fatty acids profiles of strain MUSC 26T and closely related type strains are shown in Table 2. The major cellular fatty acids in MUSC 26T were identified as anteiso-C15:0 (31.4 %), iso-C16:0 (16.3 %), iso-C15:0 (13.9 %) and anteiso-C17:0 (12.6 %). The fatty acids profile of MUSC 26T is consistent with those of closely related phylogenetic neighbours such as S. qinglanensis DSM 42035T, S. sodiiphilus DSM 41839T and S. rimosus subsp. rimosus NBRC 12907T, which contain anteiso-C15:0 (25.1–45.9 %), iso-C16:0 (12.5–25.6 %) and iso-C15:0 (12.6–17.9 %) as their major fatty acids (Table 2). Nevertheless, the fatty acid profile of MUSC 26T was quantitatively different from those of these type strains; for example, although anteiso-C15:0 (31.4 %) was found to be predominant in strain MUSC 26T, the amount of anteiso-C15:0 was significantly greater (45.9 %) in S. sodiiphilus DSM 41839T (Table 2). Chemotaxonomic analyses showed that the cell wall of strain MUSC 26T is of cell-wall type I (Lechevalier and Lechevalier 1970) as it contains LL-diaminopimelic. The presence of LL-diaminopimelic has been observed in many other species of the genus Streptomyces (Lee et al. 2005; Xu et al. 2009; Hu et al. 2012; Lee et al. 2014b). The predominant menaquinones of strain MUSC 26T were identified as MK-9(H8) (77 %) and MK-9(H6) (18 %). This is in agreement with the report
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Table 1 Differentiation characteristics of strain MUSC 26T and type strains of phylogenetically closely related species of the genus Streptomyces Characteristic
1
2
3
4
Color of aerial mycelium
Grayish yellow
Yellowish white
Pale yellow
Light yellowish brownb
Color of substrate mycelium
Grayish yellow
Greenish yellow
Pale yellow
Moderate olive brownb
24 °C
?
(?)
(?)
(?)
40 °C
-
-c
?
(?)
Morphology (on ISP 7)
Growth at
c
a
pH 5
(?)
-
-
?
pH 9
-
(?)
?a
?
8 % NaCl
-
?c
(?)a
-
Catalase
?
?c
?
?
Hemolytic
-
?
?
?
Casein (protease)
-
-
-
?
Tributyrin (lipase)
-
-
?
-
Starch (amylolytic) Carboxymethylcellulose (cellulase)
? ?
?c ?c
? ?
? ?
Xylan (xylanase)
-
-
-
-
Chitin (chitinase)
-
-
-
-
Hydrolysis of
Carbon source utilization Stachyose
?
-
-
?
D-raffinose
?
-
-a
?b
D-melibiose
?
-
-
?
N-acetyl-b-D-mannosamine
?
-
?
?
N-acetyl-D-galactosamine
-
?
?
?
L-rhamnose
?
-c
-
?
Inosine
-
?
?
?
D-mannitol
-
?
-a
?b
Myo-inositol
-
?
-
?
Gelatin
-
?
?
?
Glycyl-L-proline L-alanine
-
? ?
? ?
? ?
L-arginine
-
?
?
?
-
?
-
?
-
?
-
?
-
?
?
?
-
?
-
?
?
-
?
?
?
-
?
? ?
L-histidine L-pyroglutamic
acid
L-serine D-gluconic
acid
Mucic acid D-saccharic
acid
D-lactic
acid methyl ester
-
?
-
L-lactic
acid
?
-
-
?
D-malic
acid
-
?
?
?
Bromo-succinic acid
-
?
-
?
Acetoacetic acid
-
?
?
?
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Table 1 continued Characteristic
1
2
3
4
-
? ?
-
? ?
Troleandomycin
?
-
-
-
Vancomycin
?
?
-
-
Fusidic acid
?
?
-
?
Rifamycin RV
?
?
-
?
Minocycline
?
?
-
?
Lincomycin
?
?
-
?
Guanidine HCl
?
?
-
?
Niaproof 4
?
?
-
?
Tetrazolium violet
?
?
-
?
Tetrazolium blue
?
?
-
Propionic acid Formic acid Chemical sensitivity assays
T
? T
Strains: 1 Streptomyces gilvigriseus sp. nov. MUSC 26 ; 2 Streptomyces qinglanensis DSM 42035 ; 3, Streptomyces sodiiphilus DSM 41839T; 4, Streptomyces rimosus subsp. rimosus NBRC 12907T. All data were obtained concurrently in this study. ?, Positive; -, negative; (?), weak. All strains are positive for utilization of D-maltose, D-trehalose, D-cellobiose, N-acetyl-D-glucosamine, glycerol, D-glucose-6-phosphate, L-aspartic acid, L-glutamic acid, pectin, citric acid, L-malic acid, tween 40, c-amino-butyric acid, bhydroxy-D,L-butyric acid, a-keto-butyric acid and acetic acid. All strains are negative for assimilation of D-serine and L-galactonic acid lactone a
Results in accordance with that published for Streptomyces sodiiphilus DSM 41839T by Li et al. (2005)
b
Results in accordance with that published for Streptomyces rimosus subsp. rimosus NBRC 12907T by Yan (1992) and Jiang et al. (2011)
c
Results in accordance with that published for Streptomyces qinglanensis DSM 42035T by Hu et al. (2012)
of Kim et al. (2003) that the predominant menaquinones of members of the genus Streptomyces are MK-9(H6) and MK-9(H8). The cell wall sugars detected were galactose, glucose, mannose, ribose and rhamnose. The sugars galactose and glucose were both detected in the type strains S. qinglanensis DSM 42035T (Hu et al. 2012) and S. sodiiphilus DSM 41839T (Li et al. 2005). The G?C content of strain MUSC 26T was determined to be 74. 6 mol%; this is within the range of 67.0–78.0 mol% described for species of the genus Streptomyces (Kim et al. 2003). Based on the results of phenotypic, chemotaxonomic, DNA–DNA hybridization, DNA fingerprinting and phylogenetic analysis, strain MUSC 26T merits assignment to a novel species in the genus Streptomyces, for which the name Streptomyces gilvigriseus sp. nov. is proposed.
Description of Streptomyces gilvigriseus sp. nov Streptomyces gilvigriseus (gil.vi.gri’se.us. L. adj. gilvus, yellow; L. adj. griseus, grey; N.L. masc. adj.
gilvigriseus, yellow-grey, referring to the colour of the mycelium). Cells stain Gram-positive and form grayish yellow aerial and substrate mycelium on ISP 7 medium. The colours of the aerial and substrate mycelium are media-dependent. Grows well on ISP 2 medium, ISP 7 medium, Streptomyces agar, starch casein agar and tryptic soy agar after 1–2 weeks at 28 °C; grows moderately on actinomycetes isolation agar, Luria– Bertani agar and nutrient agar. Grows at pH 5.0–8.0 (optimum pH 6.0–7.0), with 0–6 % NaCl tolerance (optimum 2–4 %) at 20–36 °C (optimum 28–32 °C). Cells are positive for catalase but negative for melanoid pigment production and hemolytic activity. Carboxymethylcellulose and soluble starch are hydrolysed but casein, chitin, tributyrin (lipase) and xylan are not. The following compounds are utilized as sole carbon sources: acetic acid, a-D-glucose, a-Dlactose, a-hydroxy-butyric acid, a-keto-butyric acid, a-keto-glutaric acid, b-hydroxyl-D,L-butyric acid, bmethyl-D-glucoside, citric acid, D-arabitol, D-cellobiose, Dextrin, D-fructose, D-fructose-6-phosphate, D-fucose, D-galactose, D-galacturonic acid, D-glucose-
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Table 2 Cellular fatty acid composition of strain MUSC 26T and its closely related Streptomyces species Fatty acid
1
2
3
4
iso-C13:0
–
0.1
0.2
0.3
anteiso-C13:0
–
0.2
0.2
0.4
iso-C14:0
1.2
5.1
1.8
3.3
anteiso-C14:0
–
–
–
0.3
iso-C15:0
13.9
6.3
12.5
11.5
anteiso-C15:0
31.4
34.5
45.9
25.1
C15:0w6c
–
0.3
0.1
–
C15:0
0.5
1.6
0.6
2.2
iso-C16:1H
1.1
3.0
0.6
3.1
iso-C16:0
16.3
25.6
12.5
19.5
anteiso-C16:0
0.4
–
–
0.4 –
anteiso-C15:02OH
2.6
–
–
C16:0
0.7
1.1
0.5
2.7
C16:0 9-Methyl
0.3
–
–
–
iso-C17:1w9c anteiso-C17:1C
– 2.7
0.3 –
1.3 –
4.2 –
anteiso-C17:1w9c
–
0.8
2.2
4.4
iso-C17:0
3.5
3.1
3.3
4.5
12.6
16.5
17.9
13.4
C17:1w8c
–
–
0.1
–
C17:0CYCLO
–
0.2
–
1.5
anteiso-C16:02OH
5.3
–
–
–
C17:0
0.2
0.5
0.2
0.4
anteiso-C17:0
iso-C18:0
0.3
0.3
–
–
iso-C17:02OH
0.6
–
–
–
anteiso-C17:02OH
3.1
–
–
– T
Strains: 1 Streptomyces gilvigriseus sp. nov. MUSC 26 ; 2 Streptomyces qinglanensis DSM 42035T; 3 Streptomyces sodiiphilus DSM 41839T; 4 Streptomyces rimosus subsp. rimosus NBRC 12907T. – \0.1 % or not detected. All data were obtained concurrently from this study
6-phosphate, D-glucuronic acid, D-maltose, D-mannose, D-melibiose, D-raffinose, D-saccharic acid, Dsalicin, D-trehalose, D-turanose, gentiobiose, glucuronamide, glycerol, L-fucose, L-lactic acid, L-malic acid, L-rhamnose, methyl pyruvate, mucic acid, N-acetyl-bD-mannosamine, N-acetyl-D-glucosamine, pectin, stachyose, Tween 40 and c-amino-butyric acid. The following compounds are not utilized as sole carbon sources: acetoacetic acid, bromo-succinic acid, Daspartic acid, D-gluconic acid, D-lactic acid methyl ester, D-malic acid, D-mannitol, D-serine, D-sorbitol, formic acid, gelatin, glycyl-L-proline, inosine, L-
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galactonic acid lactone, 3-methyl glucose, myoinositol, N-acetyl-D-galactosamine, N-acetyl-neuraminic acid, p-hydroxyl-phenylacetic acid, propionic acid, quinic acid and sucrose. L-aspartic acid and Lglutamic acid are utilized as sole nitrogen sources. Lalanine, L-arginine, L–histidine, L-pyroglutamic acid and L-serine are not utilized as sole nitrogen sources. The G?C content of the genomic DNA of the type strain is 74.6 mol%. The type strain is MUSC 26T (=DSMZ 42173T = MCCC 1K00504T), isolated from mangrove soil collected from the Tanjung Lumpur mangrove forest located in the state of Pahang, Peninsular Malaysia. The 16S rRNA gene sequence of strain MUSC 26T has been deposited in GenBank/EMBL/DDBJ under the accession number KJ632660. Acknowledgments This work was supported by a University of Malaya for High Impact Research Grant (UM-MOHE HIR Nature Microbiome Grant No. H-50001-A000027) awarded to K.-G. C. and an External Industry Grant from Biotek Abadi Sdn Bhd (vote nos. GBA-808138 & GBA-808813) awarded to L.-H. L. The authors are thankful to Professor Bernhard Schink for the support in the Latin etymology of the new species name.
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ORIGINAL PAPER
Streptomyces gilvigriseus sp. nov., a novel actinobacterium isolated from mangrove forest soil Hooi-Leng Ser . Nurullhudda Zainal . Uma Devi Palanisamy . Bey-Hing Goh . Wai-Fong Yin . Kok-Gan Chan . Learn-Han Lee
Received: 19 January 2015 / Accepted: 11 March 2015 / Published online: 12 April 2015 Ó Springer International Publishing Switzerland 2015
Abstract A novel Streptomyces, strain MUSC 26T, was isolated from mangrove soil at Tanjung Lumpur, Malaysia. The bacterium was observed to be Grampositive and to form grayish yellow aerial and substrate mycelium on ISP 7 agar. A polyphasic approach was used to study the taxonomy of strain MUSC 26T, which shows a range of phylogenetic and chemotaxonomic properties consistent with those of the members of the genus Streptomyces. The cell wall peptidoglycan was determined to contain LL-diaminopimelic acid. The predominant menaquinones were identified as MK-9 (H8) and MK-9(H6). The polar lipids detected were identified as diphosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, hydroxyphosphatidylethanolamine, phosphatidylmethylethanolamine and hydroxyphosphatidylmethylethanolamine. The predominant
Electronic supplementary material The online version of this article (doi:10.1007/s10482-015-0431-5) contains supplementary material, which is available to authorized users. H.-L. Ser N. Zainal U. D. Palanisamy B.-H. Goh L.-H. Lee (&) Jeffrey Cheah School of Medicine and Health Sciences, Monash University Malaysia, 46150 Bandar Sunway, Selangor Darul Ehsan, Malaysia e-mail: [email protected]; [email protected] N. Zainal W.-F. Yin K.-G. Chan Division of Genetics and Molecular Biology, Faculty of Science, Institute of Biological Sciences, University of Malaya, 50603 Kuala Lumpur, Malaysia
cellular fatty acids ([10.0 %) were identified as anteiso-C15:0 (31.4 %), iso-C16:0 (16.3 %), iso-C15:0 (13.9 %) and anteiso-C17:0 (12.6 %). The cell wall sugars were found to be galactose, glucose, mannose, ribose and rhamnose. These results suggest that MUSC 26T should be placed within the genus Streptomyces. Phylogenetic analysis indicated that closely related strains include Streptomyces qinglanensis 172205T (96.5 % sequence similarity), S. sodiiphilus YIM 80305T (96.5 %) and S. rimosus subsp. rimosus ATCC 10970T (96.4 %). DNA– DNA relatedness values between MUSC 26T and closely related type strains ranged from 17.0 ± 2.2 to 33.2 ± 5.3 %. Comparison of BOX-PCR fingerprints indicated MUSC 26T presents a unique DNA profile. The DNA G?C content was determined to be 74.6 mol%. Based on this polyphasic study of MUSC 26T, it is concluded that this strain represents a novel species, for which the name Streptomyces gilvigriseus sp. nov. is proposed. The type strain is MUSC 26T (=DSMZ 42173T = MCCC 1K00504T). Keywords Streptomyces gilvigriseus sp. nov Actinobacteria Mangrove forest
Introduction The mangrove is a unique woody plant community of intertidal coasts in tropical and subtropical coastal regions. The mangrove ecosystem is among the world’s most productive environments and improves
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coastal waters, produces commercial forest products, supports coastal fisheries and protects coastlines. Mangrove ecosystems are habitats of diverse flora and fauna of marine, freshwater and terrestrial species (Jennerjahn and Ittekkot 2002), but comparatively little is known about the diversity of the microbial community in mangrove ecosystems (Hong et al. 2009; Xu et al. 2009). Recently, there has been increasing interest in mangrove microorganism resources (Hong et al. 2009; Lee et al. 2014a) as the constant changes in tidal gradient and salinity in the mangrove ecosystems are thought to be a driving force for metabolic pathway adaptations that might lead to the production of unusual metabolites. Many studies have discovered novel actinobacteria from the poorly explored mangrove environment, for example the isolation of Streptomyces avicenniae (Xiao et al. 2009), S. xiamenensis (Xu et al. 2009), S. sanyensis (Sui et al. 2011), S. qinglanensis (Hu et al. 2012) and S. pluripotens (Lee et al. 2014b). The genus Streptomyces was proposed by Waksman and Henrici (1943) and members of this genus have made significant contributions to mankind due to their abilities to produce various natural products (Be´rdy 2005). At the time of writing (March 2015), the genus Streptomyces is comprised of ca. 600 species with validly published names (http://www.bacterio. cict.fr/). During a systematic investigation of the actinobacterial diversity in a mangrove environment located on the east coast of Peninsular Malaysia, strain MUSC 26T was isolated. A polyphasic taxonomic study showed that strain MUSC 26T represents a novel species of the genus Streptomyces, for which the name S. gilvigriseus sp. nov. is proposed.
Materials and methods Isolation and maintenance of isolate Strain MUSC 26T was isolated from a soil sample collected at site MUSC-TLS1 (3o480 3.200 N o 0 00 103 20 11.0 E), located in the mangrove forest of Tanjung Lumpur in the state of Pahang, Peninsular Malaysia, in December 2012. Topsoil samples of the upper 20-cm layer (after removing the top 2–3 cm) were collected and sampled into sterile plastic bags using an aseptic metal trowel, and stored at -20 °C. Air-dried soil samples were ground with a mortar and pestle. Selective
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Antonie van Leeuwenhoek (2015) 107:1369–1378 Fig. 1 Maximum-likelihood phylogenetic tree based on almost c complete 16S rRNA sequences (1447 nucleotides) showing the relationship between strain MUSC 26T and representatives of some other related taxa. Numbers at nodes indicate percentages of 1000 bootstrap re-samplings, only values above 50 % are shown. Bar 0.005 substitutions per site. Asterisks indicate that the corresponding nodes were also recovered using the neighbour-joining tree-making algorithm
pretreatment of soil samples was performed using wet heat in sterilized water (15 min at 50 °C; Takahashi et al. 1996). Five grams of the pretreated air-dried soil was mixed with 45 ml sterilized water and mill ground, spread onto the isolation medium ISP 2 (Shirling and Gottlieb 1966) supplemented with cycloheximide (25 lg ml-1) and nystatin (10 lg ml-1), and incubated at 28 °C for 14 days. Pure cultures of strain MUSC 26T were isolated and maintained on slants of ISP 2 medium at 28 °C and as glycerol suspensions (20 %, v/v) at -20 °C for long term preservation. Genomic and phylogenetic analyses Genomic DNA extraction for PCR was performed as described by Hong et al. (2009). The 16S rRNA gene was amplified as described by Lee et al. (2014b). The 16S rRNA gene sequence of strain MUSC 26T was aligned with representative sequences of related type strains of the genus Streptomyces retrieved from the GenBank/EMBL/DDBJ databases using CLUSTALX software (Thompson et al. 1997). The alignment was verified manually and adjusted prior to the reconstruction of phylogenetic trees. Phylogenetic trees were constructed with the maximum-likelihood (Felsenstein 1981) (Fig. 1) and neighbour-joining (Saitou and Nei 1987) (Fig. S1) algorithms using MEGA version 5.2 (Tamura et al. 2011). Evolutionary distances for the neighbour-joining algorithm were computed using Kimura’s two-parameter model (Kimura 1980). The EzTaxon-e server (http:// eztaxon-e.ezbiocloud.net/; Kim et al. 2012) was used for calculations of sequence similarity. The stability of the resultant trees topologies were evaluated by using the bootstrap resampling method of Felsenstein (1985). BOX-PCR fingerprint analysis was used to characterize strain MUSC 26T and the closely related strains using the primer BOX-A1R (50 -CTACGGCAAGGC GACGCTGACG-30 ) (Versalovic et al. 1991; Lee et al. 2014c). The PCR condition for BOX-PCR was
Antonie van Leeuwenhoek (2015) 107:1369–1378
1371 T
Streptomyces endus NRRL 2339 (AY999911)
98*
T
Streptomyces demainii NRRL B-1478 (DQ334782) T
Streptomyces sporocinereus NBRC 100766 (AB249933) T
Streptomyces hygroscopicus subsp. hygroscopicus NRRL 2387 (AB231803) 56*
T
Streptomyces yogyakartensis NBRC 100779 (AB249942) T
97*
Streptomyces javensis NBRC 100777 (AB249940) T
Streptomyces violaceusniger NBRC 13459 (AB184420) T
Streptomyces iranensis HM 35 (FJ472862) T
63*
Streptomyces rapamycinicus NRRL B-5491 (EF408733) 100*
T
Streptomyces rhizosphaericus NBRC 100778 (AB249941) T
Streptomyces griseiniger NRRL B-1865 (AJ391818) T
Streptomyces mordarskii NRRL B-1346 (EF408735)
73*
T
Streptomyces castelarensis DSM 40830 (AY508511) T
Streptomyces melanosporofaciens NBRC 13061 (AB184283)
84*
T
Streptomyces yatensis NBRC 101000 (AB249962) T
Streptomyces sporoclivatus NBRC 100767 (AB249934) T
Streptomyces antimycoticus NBRC 12839 (AB184185) 97* 85*
T
Streptomyces hygroscopicus subsp. glebosus NBRC 13786 (AB184479) T
Streptomyces libani subsp. rufus LMG 20087 (AJ781351) T
Streptomyces glebosus CGMCC 4.1873 (HQ244456) T
Streptomyces ramulosus NRRL B-2714 (DQ026662) T
Streptomyces youssoufiensis X4 (FN421338) T
Streptomyces luteireticuli NBRC 13422 (AB249969)
64*
T
Streptomyces thioluteus LMG 20253 (AJ781360)
55
T
Streptomyces varsoviensis NRRL B-3589 (DQ026653) T
Streptomyces coerulescens NBRC 12758 (AB184122) T
Streptomyces mobaraensis NBRC 13819 (AORZ01000256) T
Streptomyces orinoci NBRC 13466 (AB184866) T
Streptomyces daliensis YIM 31724 (AY785161) T
83 Streptomyces sclerotialus DSM 43032 (AJ621608) T
Streptomyces albofaciens JCM 4342 (AB045880) T
Streptomyces rimosus subsp. rimosus ATCC 10970 (ANSJ01000404) T
Streptomyces monomycini NBRC 100769 (AB249936) 94*
T
Streptomyces olivaceiscleroticus DSM 40595 (AJ621606) T
94* Streptomyces niger NBRC 13362 (AB184352)
58
T
Streptomyces thermoviolaceus subsp. apingens DSM 41392 (Z68095)
*
T
Streptomyces somaliensis DSM 40738 (AJ007403) 88*
T
Streptomyces atrovirens NRRL B-16357 (DQ026672) T
88* Streptomyces flavoviridis NBRC 12772 (AB184842) T 94* Streptomyces pilosus NBRC 12807 (AB184161) T
Streptomyces albiaxialis NRRL B-24327 (AY999901) T
Streptomyces gilvigriseus MUSC26 (KJ632660) T
Streptomyces sodiiphilus YIM 80305 (AY236339) T
Streptomyces albus NRRL B-2365 (DQ026669) T
Streptomyces qinglanensis 172205 (HQ660227) T
Streptomyces marinus DSM 41970 (AB473556)
59* 58*
T
Streptomyces panacagri Gsoil 519 (AB245388)
0.005
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1372
performed as described by Lee et al. (2014d). The PCR products were visualized by 2 % agarose gel electrophoresis. The extraction of genomic DNA for DNA–DNA hybridization of strain MUSC 26T, S. qinglanensis DSM 42035T, S. sodiiphilus DSM 41839T (96.49 %) and S. rimosus subsp. rimosus NBRC 12907T were carried out by the Identification Service of the DSMZ, Braunschweig, Germany following the protocol of Cashion et al. (1977). DNA–DNA hybridization was carried out as described by De Ley et al. (1970) under consideration of the modifications described by Huss et al. (1983). The G?C content of strain MUSC 26T was determined by HPLC (Mesbah et al. 1989). Phenotypic characteristics The cultural characteristics of strain MUSC 26T were determined following growth on ISP 2 and ISP 7 media (Shirling and Gottlieb 1966), actinomycetes isolation agar (AIA; Atlas and Parks 1993), Streptomyces agar (SA; Atlas and Parks 1993), starch casein agar (SCA; Ku¨ster and Williams 1964) and nutrient agar (MacFaddin 2000) for 14 days at 28 °C. Light microscopy (80i, Nikon) and scanning electron microscopy (JEOL-JSM 6400) were used to observe the morphology of the strain after incubation on ISP 2 medium at 28 °C for 7–14 days. The designation of colony colour was determined by using the ISCC-NBS colour charts (Kelly 1964). Gram staining was performed by standard Gram reaction and confirmed by using KOH lysis (Cerny 1978). The growth temperature range was tested at 12–48 °C at intervals of 4 °C on ISP 2 medium. The pH range for growth was tested in tryptic soy broth (TSB) between pH 4.0 and 10.0 at intervals of 1 pH unit. The NaCl tolerance was tested in TSB and salt concentrations ranging from 0 to 18 % (w/v) at intervals of 2 %. The responses to temperature, pH and NaCl were observed for 14 days. Production of melanoid pigments was examined using ISP 7 medium. Catalase activity and production of melanoid pigments were determined following protocols described by Lee et al. (2014e). Hemolytic activity was assessed on blood agar medium containing 5 % (w/v) peptone, 3 % (w/v) yeast extract, 5 % (w/v) NaCl and 5 % (v/v) horse blood (Carillo et al. 1996). Plates were examined for hemolysis after incubation at 32 °C for 7–14 days. Amylolytic, cellulase, chitinase, lipase, protease and
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xylanase activities were determined by growing cells on ISP 2 medium and following protocols as described by Meena et al. (2013). The presence of clear zones around colonies was taken to signify the potential of isolates for surfactant production. Antibiotic susceptibility tests were performed by the disc diffusion method as described by Shieh et al. (2003). Antimicrobials used and their concentrations per disc (Oxoid, Basingstoke, UK) were as follows: ampicillin (10 lg), ampicillin sulbactam (30 lg), cefotaxime (30 lg), cefuroxime (30 lg), cephalosporin (30 lg), chloramphenicol (30 lg), ciprofloxacin (10 lg), erythromycin (15 lg), gentamicin (20 lg), nalidixic acid (30 lg), Penicillin G (10 lg), streptomycin (10 lg), tetracycline (30 lg) and vancomycin (30 lg). Carbon-source utilization and chemical sensitivity assays were determined using Biolog GenIII MicroPlates (Biolog, USA) according to the manufacturer’s instructions. All of the phenotypic assays mentioned were performed concurrently for strain MUSC 26T, S. qinglanensis DSM 42035T, S. sodiiphilus DSM 41839T and S. rimosus subsp. rimosus NBRC 12907T. Chemotaxonomic characteristics The analyses of peptidoglycan amino acid composition and sugars of strain MUSC 26T were carried out by the Identification Service of the DSMZ using published protocols (Schumann 2011). Major diagnostic cell wall sugars of strain MUSC 26T were obtained as described by Whiton et al. (1985) and analyzed by TLC on cellulose plates (Staneck and Roberts 1974). Analysis of respiratory quinones, polar lipids (Kates 1986) and fatty acids (Sasser 1990) were carried out by the Identification Service of the DSMZ.
Results and discussion Strain MUSC 26T was observed to grow well on ISP 2 medium, ISP 7 medium, Streptomyces agar, starch casein agar and tryptic soy agar after 7–14 days at 28 °C, and to grow moderately on actinomycetes isolation agar, Luria–Bertani agar and nutrient agar. The colours of the aerial and substrate mycelium were media-dependent (Table S1). The morphological observation of a 15-day-old culture grown on ISP 2 medium revealed a smooth spore surface and abundant growth of both aerial and vegetative hyphae, which
Antonie van Leeuwenhoek (2015) 107:1369–1378
were well developed and not fragmented. These morphological features are consistent with assignment of the strain to the genus Streptomyces (Williams et al. 1989). Growth was found to occur at pH 5.0–8.0 (optimum pH 6.0–7.0), with 0-6 % NaCl tolerance (optimum 2–4 %) and at 20–36 °C (optimum 28–32 °C). Cells were found to be positive for catalase but negative for melanoid pigment production and hemolytic activity. Hydrolysis of carboxymethylcellulose and soluble starch were found to be positive, but negative for hydrolysis of casein, chitin, tributyrin (lipase) and xylan. Strain MUSC 26T can be differentiated from closely related members of the genus Streptomyces using a range of phenotypic properties (Table 1). In chemical sensitivity assays, cells are resistant to aztreonam, D-serine, fusidic acid, guanine HCL, lincomycin, lithium chloride, minocycline, 8 % NaCl, nalidixic acid, niaproof 4, potassium tellurite, rifamycin RV, sodium bromate, sodium butyrate, 1 % sodium lactate, tetrazolium blue, tetrazolium violet and troleandomycin. The nearly complete 16S rRNA gene sequence was obtained for strain MUSC 26T (1447 bp; GenBank/ EMBL/DDBJ accession number KJ632660) and phylogenetic trees were reconstructed to determine the phylogenetic position of this strain (Fig. 1; S1). Phylogenetic analysis exhibited that strain MUSC 26T is closely related to S. sodiiphilus YIM 80305T, as they formed a distinct clade (Fig. 1). The type strain S. sodiiphilus YIM 80305T was isolated from a muddy sample in Chaka salt lake located in Qinghai Province of China (Li et al. 2005). The 16S rRNA gene sequence analysis of strain MUSC 26T showed the highest similarity to that of S. qinglanensis 172205T (96.5 %), followed by S. sodiiphilus YIM 80305T (96.5 %) and S. rimosus subsp. rimosus ATCC 10970T (96.4 %); sequences similarities of less than 96.1 % were obtained with the type strains of other species of the genus Streptomyces. According to Stackebrandt and Goebel (1994), generally a 16S rRNA gene sequence divergence greater than 3 % is accepted as a criterion for delineating different prokaryotic species. The sequence divergence between strain MUSC 26T and the most closely related type strain S. qinglanensis 172205T was 3.5 %, a value consistent with strain MUSC 26T representing a novel species in the genus Streptomyces. The DNA–DNA relatedness values between strain MUSC 26T and S. qinglanensis DSM 42035T (33.2 ± 5.3 %), S. sodiiphilus DSM 41839T
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(17.0 ± 2.2 %) and S. rimosus subsp. rimosus NBRC 12907T (29.4 ± 3.1 %) were significantly below 70 %, the threshold value for the delineation of bacterial species (Wayne et al. 1987). The BOX-PCR results indicated that strain MUSC 26T yielded a unique BOX-PCR fingerprint compared with the closely related type strains (Fig. S2). These results are in agreement with results of DNA–DNA hybridizations, which indicate that strain MUSC 26T represents a novel species. The polar lipid analysis showed the presence of diphosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, hydroxyphosphatidylethanol amine, phosphatidylmethylethanolamine and hydroxyphosphatidylmethylethanolamine; an unidentified glycolipid and phospholipid were also detected. Differences in polar lipid profiles indicated that MUSC 26T is different from related type strains; for example, strain MUSC 26T (Fig. S3a) was found to contain hydroxyphosphatidylethanolamine, phosphatidylmethylethanolamine and hydroxyphosphatidylmethylethanolamine, lipids that were not detected in S. qinglanensis DSM 42035T (Fig. S3b). The fatty acids profiles of strain MUSC 26T and closely related type strains are shown in Table 2. The major cellular fatty acids in MUSC 26T were identified as anteiso-C15:0 (31.4 %), iso-C16:0 (16.3 %), iso-C15:0 (13.9 %) and anteiso-C17:0 (12.6 %). The fatty acids profile of MUSC 26T is consistent with those of closely related phylogenetic neighbours such as S. qinglanensis DSM 42035T, S. sodiiphilus DSM 41839T and S. rimosus subsp. rimosus NBRC 12907T, which contain anteiso-C15:0 (25.1–45.9 %), iso-C16:0 (12.5–25.6 %) and iso-C15:0 (12.6–17.9 %) as their major fatty acids (Table 2). Nevertheless, the fatty acid profile of MUSC 26T was quantitatively different from those of these type strains; for example, although anteiso-C15:0 (31.4 %) was found to be predominant in strain MUSC 26T, the amount of anteiso-C15:0 was significantly greater (45.9 %) in S. sodiiphilus DSM 41839T (Table 2). Chemotaxonomic analyses showed that the cell wall of strain MUSC 26T is of cell-wall type I (Lechevalier and Lechevalier 1970) as it contains LL-diaminopimelic. The presence of LL-diaminopimelic has been observed in many other species of the genus Streptomyces (Lee et al. 2005; Xu et al. 2009; Hu et al. 2012; Lee et al. 2014b). The predominant menaquinones of strain MUSC 26T were identified as MK-9(H8) (77 %) and MK-9(H6) (18 %). This is in agreement with the report
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Antonie van Leeuwenhoek (2015) 107:1369–1378
Table 1 Differentiation characteristics of strain MUSC 26T and type strains of phylogenetically closely related species of the genus Streptomyces Characteristic
1
2
3
4
Color of aerial mycelium
Grayish yellow
Yellowish white
Pale yellow
Light yellowish brownb
Color of substrate mycelium
Grayish yellow
Greenish yellow
Pale yellow
Moderate olive brownb
24 °C
?
(?)
(?)
(?)
40 °C
-
-c
?
(?)
Morphology (on ISP 7)
Growth at
c
a
pH 5
(?)
-
-
?
pH 9
-
(?)
?a
?
8 % NaCl
-
?c
(?)a
-
Catalase
?
?c
?
?
Hemolytic
-
?
?
?
Casein (protease)
-
-
-
?
Tributyrin (lipase)
-
-
?
-
Starch (amylolytic) Carboxymethylcellulose (cellulase)
? ?
?c ?c
? ?
? ?
Xylan (xylanase)
-
-
-
-
Chitin (chitinase)
-
-
-
-
Hydrolysis of
Carbon source utilization Stachyose
?
-
-
?
D-raffinose
?
-
-a
?b
D-melibiose
?
-
-
?
N-acetyl-b-D-mannosamine
?
-
?
?
N-acetyl-D-galactosamine
-
?
?
?
L-rhamnose
?
-c
-
?
Inosine
-
?
?
?
D-mannitol
-
?
-a
?b
Myo-inositol
-
?
-
?
Gelatin
-
?
?
?
Glycyl-L-proline L-alanine
-
? ?
? ?
? ?
L-arginine
-
?
?
?
-
?
-
?
-
?
-
?
-
?
?
?
-
?
-
?
?
-
?
?
?
-
?
? ?
L-histidine L-pyroglutamic
acid
L-serine D-gluconic
acid
Mucic acid D-saccharic
acid
D-lactic
acid methyl ester
-
?
-
L-lactic
acid
?
-
-
?
D-malic
acid
-
?
?
?
Bromo-succinic acid
-
?
-
?
Acetoacetic acid
-
?
?
?
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Antonie van Leeuwenhoek (2015) 107:1369–1378
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Table 1 continued Characteristic
1
2
3
4
-
? ?
-
? ?
Troleandomycin
?
-
-
-
Vancomycin
?
?
-
-
Fusidic acid
?
?
-
?
Rifamycin RV
?
?
-
?
Minocycline
?
?
-
?
Lincomycin
?
?
-
?
Guanidine HCl
?
?
-
?
Niaproof 4
?
?
-
?
Tetrazolium violet
?
?
-
?
Tetrazolium blue
?
?
-
Propionic acid Formic acid Chemical sensitivity assays
T
? T
Strains: 1 Streptomyces gilvigriseus sp. nov. MUSC 26 ; 2 Streptomyces qinglanensis DSM 42035 ; 3, Streptomyces sodiiphilus DSM 41839T; 4, Streptomyces rimosus subsp. rimosus NBRC 12907T. All data were obtained concurrently in this study. ?, Positive; -, negative; (?), weak. All strains are positive for utilization of D-maltose, D-trehalose, D-cellobiose, N-acetyl-D-glucosamine, glycerol, D-glucose-6-phosphate, L-aspartic acid, L-glutamic acid, pectin, citric acid, L-malic acid, tween 40, c-amino-butyric acid, bhydroxy-D,L-butyric acid, a-keto-butyric acid and acetic acid. All strains are negative for assimilation of D-serine and L-galactonic acid lactone a
Results in accordance with that published for Streptomyces sodiiphilus DSM 41839T by Li et al. (2005)
b
Results in accordance with that published for Streptomyces rimosus subsp. rimosus NBRC 12907T by Yan (1992) and Jiang et al. (2011)
c
Results in accordance with that published for Streptomyces qinglanensis DSM 42035T by Hu et al. (2012)
of Kim et al. (2003) that the predominant menaquinones of members of the genus Streptomyces are MK-9(H6) and MK-9(H8). The cell wall sugars detected were galactose, glucose, mannose, ribose and rhamnose. The sugars galactose and glucose were both detected in the type strains S. qinglanensis DSM 42035T (Hu et al. 2012) and S. sodiiphilus DSM 41839T (Li et al. 2005). The G?C content of strain MUSC 26T was determined to be 74. 6 mol%; this is within the range of 67.0–78.0 mol% described for species of the genus Streptomyces (Kim et al. 2003). Based on the results of phenotypic, chemotaxonomic, DNA–DNA hybridization, DNA fingerprinting and phylogenetic analysis, strain MUSC 26T merits assignment to a novel species in the genus Streptomyces, for which the name Streptomyces gilvigriseus sp. nov. is proposed.
Description of Streptomyces gilvigriseus sp. nov Streptomyces gilvigriseus (gil.vi.gri’se.us. L. adj. gilvus, yellow; L. adj. griseus, grey; N.L. masc. adj.
gilvigriseus, yellow-grey, referring to the colour of the mycelium). Cells stain Gram-positive and form grayish yellow aerial and substrate mycelium on ISP 7 medium. The colours of the aerial and substrate mycelium are media-dependent. Grows well on ISP 2 medium, ISP 7 medium, Streptomyces agar, starch casein agar and tryptic soy agar after 1–2 weeks at 28 °C; grows moderately on actinomycetes isolation agar, Luria– Bertani agar and nutrient agar. Grows at pH 5.0–8.0 (optimum pH 6.0–7.0), with 0–6 % NaCl tolerance (optimum 2–4 %) at 20–36 °C (optimum 28–32 °C). Cells are positive for catalase but negative for melanoid pigment production and hemolytic activity. Carboxymethylcellulose and soluble starch are hydrolysed but casein, chitin, tributyrin (lipase) and xylan are not. The following compounds are utilized as sole carbon sources: acetic acid, a-D-glucose, a-Dlactose, a-hydroxy-butyric acid, a-keto-butyric acid, a-keto-glutaric acid, b-hydroxyl-D,L-butyric acid, bmethyl-D-glucoside, citric acid, D-arabitol, D-cellobiose, Dextrin, D-fructose, D-fructose-6-phosphate, D-fucose, D-galactose, D-galacturonic acid, D-glucose-
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Antonie van Leeuwenhoek (2015) 107:1369–1378
Table 2 Cellular fatty acid composition of strain MUSC 26T and its closely related Streptomyces species Fatty acid
1
2
3
4
iso-C13:0
–
0.1
0.2
0.3
anteiso-C13:0
–
0.2
0.2
0.4
iso-C14:0
1.2
5.1
1.8
3.3
anteiso-C14:0
–
–
–
0.3
iso-C15:0
13.9
6.3
12.5
11.5
anteiso-C15:0
31.4
34.5
45.9
25.1
C15:0w6c
–
0.3
0.1
–
C15:0
0.5
1.6
0.6
2.2
iso-C16:1H
1.1
3.0
0.6
3.1
iso-C16:0
16.3
25.6
12.5
19.5
anteiso-C16:0
0.4
–
–
0.4 –
anteiso-C15:02OH
2.6
–
–
C16:0
0.7
1.1
0.5
2.7
C16:0 9-Methyl
0.3
–
–
–
iso-C17:1w9c anteiso-C17:1C
– 2.7
0.3 –
1.3 –
4.2 –
anteiso-C17:1w9c
–
0.8
2.2
4.4
iso-C17:0
3.5
3.1
3.3
4.5
12.6
16.5
17.9
13.4
C17:1w8c
–
–
0.1
–
C17:0CYCLO
–
0.2
–
1.5
anteiso-C16:02OH
5.3
–
–
–
C17:0
0.2
0.5
0.2
0.4
anteiso-C17:0
iso-C18:0
0.3
0.3
–
–
iso-C17:02OH
0.6
–
–
–
anteiso-C17:02OH
3.1
–
–
– T
Strains: 1 Streptomyces gilvigriseus sp. nov. MUSC 26 ; 2 Streptomyces qinglanensis DSM 42035T; 3 Streptomyces sodiiphilus DSM 41839T; 4 Streptomyces rimosus subsp. rimosus NBRC 12907T. – \0.1 % or not detected. All data were obtained concurrently from this study
6-phosphate, D-glucuronic acid, D-maltose, D-mannose, D-melibiose, D-raffinose, D-saccharic acid, Dsalicin, D-trehalose, D-turanose, gentiobiose, glucuronamide, glycerol, L-fucose, L-lactic acid, L-malic acid, L-rhamnose, methyl pyruvate, mucic acid, N-acetyl-bD-mannosamine, N-acetyl-D-glucosamine, pectin, stachyose, Tween 40 and c-amino-butyric acid. The following compounds are not utilized as sole carbon sources: acetoacetic acid, bromo-succinic acid, Daspartic acid, D-gluconic acid, D-lactic acid methyl ester, D-malic acid, D-mannitol, D-serine, D-sorbitol, formic acid, gelatin, glycyl-L-proline, inosine, L-
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galactonic acid lactone, 3-methyl glucose, myoinositol, N-acetyl-D-galactosamine, N-acetyl-neuraminic acid, p-hydroxyl-phenylacetic acid, propionic acid, quinic acid and sucrose. L-aspartic acid and Lglutamic acid are utilized as sole nitrogen sources. Lalanine, L-arginine, L–histidine, L-pyroglutamic acid and L-serine are not utilized as sole nitrogen sources. The G?C content of the genomic DNA of the type strain is 74.6 mol%. The type strain is MUSC 26T (=DSMZ 42173T = MCCC 1K00504T), isolated from mangrove soil collected from the Tanjung Lumpur mangrove forest located in the state of Pahang, Peninsular Malaysia. The 16S rRNA gene sequence of strain MUSC 26T has been deposited in GenBank/EMBL/DDBJ under the accession number KJ632660. Acknowledgments This work was supported by a University of Malaya for High Impact Research Grant (UM-MOHE HIR Nature Microbiome Grant No. H-50001-A000027) awarded to K.-G. C. and an External Industry Grant from Biotek Abadi Sdn Bhd (vote nos. GBA-808138 & GBA-808813) awarded to L.-H. L. The authors are thankful to Professor Bernhard Schink for the support in the Latin etymology of the new species name.
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