DOI: 10.15468/9spdnt
title: Streptomyces reniochalinae sp. nov. and Streptomyces diacarni sp. nov., from marine sponges

description: This dataset contains the digitized treatments in Plazi based on the original journal article Li, Lei, Wang, Jie, Zhou, Yong-jun, Lin, Hou-wen, Lu, Yan-hua (2019): Streptomyces reniochalinae sp. nov. and Streptomyces diacarni sp. nov., from marine sponges. International Journal of Systematic and Evolutionary Microbiology 69 (1): 99-104, DOI: 10.1099/ijsem.0.003109, URL: http://dx.doi.org/10.1099/ijsem.0.003109AbstractTwo marine actinomycete strains, LHW50302 T and LHW51701 T, were isolated from marine sponges collected in Sansha, Hainan Province, China. The morphological, chemotaxonomic and phylogenetic characteristics were consistent with their classification in the genus Streptomyces. The strains formed hooked and looped chains of arthrospores with smooth surfaces. The cell-wall hydrolysates of the strains contained LL- diaminopimelic acid as the diagnostic diamino acid. MK-9(H 8) was the predominant menaquinone. The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol. Major fatty acids of the strains were iso-C 16: 0, anteiso-C 15: 0 and anteiso-C 17: 0. The 16S rRNA gene sequences indicated that the strains clustered together with Streptomyces albus CGMCC 4.1640 T and Streptomyces qinglanensis CGMCC 4.6825 T. Multilocus sequence analysis (MLSA) confirmed their relationship. Genome relatedness in forms of average nucleotide identity, digital DNA–DNA hybridization value and MLSA evolutionary distance between each of the strains and its closest relatives showed that they belonged to distinct species. On the basis of these results, strains LHW50302 T and LHW51701 T belong to two novel species in the genus Streptomyces, for which the names Streptomyces reniochalinae sp. nov. (type strain LHW50302 T =CCTCC AA 2018013 T =DSM 106194 T) and Streptomyces diacarni sp. nov. (type strain LHW51701 T =CCTCC AA 2018017 T =DSM 106126 T) are proposed, respectively.The genus Streptomyces was proposed by Waksman and Henrici [1] to describe Gram-stain-positive, aerobic, filamentous and spore-forming actinomycetes. As the largest group in the phylum Actinobacteria, the genus Streptomyces encompasses about 800 species at the time of writing (www.bacterio.net/ streptomyces.html). Streptomyces strains are well known for their versatile metabolic ability to produce various natural products of considerable commercial value [2].Sponges (phylum Porifera), the most ancient complex symbionts, are a rich source for diverse, new and rare actinomycetes [3]. As the largest genus amongst marine sponge-associated actinomycetes, Streptomyces is distributed widely in various sponges without any pattern of host-specific [3]. In recent years, a number of novel sponge-associated Streptomycetes were reported such as Streptomyces atlanticus [4], Streptomyces axinellae [5], Streptomyces haliclonae [6], Streptomyces marinus [6], Streptomyces spongiicola [7] and Streptomyces tateyamensis [6].During investigations on diversity and natural products producing potentially cultivable actinomycetes from marine sponges in Sansha, Hainan Province, China, strains LHW50302 T and LHW51701 T were isolated from marine sponges Reniochalina stalagmitis and Diacarnus megaspinorhabdosa, respectively, collected in the South China Sea, on Streptomyces Isolation Medium agar plates [8] containing 3 % sea salt (w/v), 50 mg l ‒1 cycloheximide and 25 mg l ‒1 nalidixic acid. Colony was purified and maintained on International Streptomyces Project (ISP) 2 agar with 3 % sea salt (w/v), and preserved as a suspension of mycelia fragments in glycerol (20 %, v/v) at ‒80 Ǫ C. Biomass for chemical and molecular studies was obtained by cultivation in shake flasks (180 r.p.m.) with ISP 2 broth at 28 Ǫ C for 5 days.Cultural characteristics of strains were determined using ISP 1–7 and 9 [9], ATCC 172, Czapek’ s agar plates [10], M8 agar [11] and modified Bennett (MB) agar [12], at 28 Ǫ C for 1–3 weeks. ISCC–NBS colour charts [13] were used to determine the colours of mycelia and soluble pigments. Morphological characteristics of strains were observed by scanning electron microscopy (S4800, Hitachi) after 2 weeks at 28 Ǫ C on ISP 2.Author affiliations: 1 State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, PR China; 2 Marine Drugs Research Center, Department of Pharmacy, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, PR China.*Correspondence: Hou-wen Lin, franklin67@126.com; Yan-hua Lu, luyanhua@ecust.edu.cnKeywords: Streptomyces reniochalinae; Streptomyces diacarni; marine sponge; new species.Abbreviations: ANI, average nucleotide identity; CMC, carboxymethylcellulose; dDDH, digital DNA–DNA hybridization; ISP, International Streptomyces Project; MB, modified Bennett; MLSA, multilocus sequence analysis; TLC, thin-layer chromatography.The GenBank accession number for the 16S rRNA gene sequence and the draft genome sequence of strains LHW50302 T and LHW51701 T are KX347891, KX347890 and QOIM00000000, QOIN00000000, respectively.Three supplementary figures and seven supplementary tables are available with the online version of this article.Growth was tested at 4, 18, 20, 28, 30, 32, 34, 37, 40, 45 and 50 Ǫ C on ISP 2. Other physiological and biochemical tests were performed at 28 Ǫ C. The pH growth range was examined at pH 4–11 (at intervals of 1.0 pH unit) using the buffer system described by Xu et al. [14]. Tolerance to different NaCl concentrations (0–10 %, at intervals of 1 %, w/v) were determined in ISP 2 broth. Gelatin liquefaction and milk peptonization were determined as described by Arai [15] and Williams and Cross [16]. Carboxymethylcellulose (CMC) hydrolysis was tested by Congo red [17] on ISP 2 supplemented with 1 % CMC. Production of lipase was tested on ISP 2 supplemented with 1 % Tweens 20, 40, 60 and 80. Carbon source utilization was tested by using ISP 9 broth [9] supplemented with 0.1–1.0% (w/v) of each carbon source. Sole nitrogen sources were determined by using the basic media [18] containing 0.05–0.10 % (w/v) of each nitrogen source. Production of melanoid pigments was examined using ISP 6 and ISP 7 media [9]. Other physiological and biochemical characteristics were tested by following the procedures of Williams et al. [19] and K̈ampfer et al. [20].The whole-cell sugar and cell-wall diaminopimelic acid patterns were determined using TLC according to the procedure of Lechevalier and Lechevalier [21]. Menaquinones were extracted [22] and analysed by HPLC. Analysis of phospholipids was carried out by TLC according to Minnikin et al. [22]. Fatty acids analyses were performed by following the method of Sasser [23] and the composition was determined by comparing with the database of fatty acids (TSBA 6.1) in the Microbial Identification System (MIDI Sherlock).DNA template for PCR was obtained from colonies with FastPre-24 instrument (MP Biomedicals). Whole-genome DNA was extracted using the procedures described by Pospiech and Neumann [24]. PCR amplification and sequencing of the 16S rRNA gene were carried out as described by Nakajima et al. [25]. Genomic DNA was sequenced on HiSeq 2500 Sequencing System (Illumina) according to the User Guide and assembling on MiSeq plateform [26]. The obtained sequences were compared with available 16S rRNA gene sequences of validly published species on EzTaxon server (www.ezbiocloud.net) [27] to determine an approximate phylogenetic affiliation. The sequencs of five housekeeping genes, atpD, gyrB, recA, rpoB and trpB were drawn from draft genome sequences by BLAST, and concatenated head to tail in-frame. Multiple alignments with sequences of closely related taxa were carried out using CLUSTAL_X [28]. The alignment was manually verified and adjusted to mask the sequences length discrepancy, and the sequence similarity was determined by counting the identical and total nucleotides. MEGA 7 [29] was used for calculation of Kimura’ s two-parameter distance [30], multilocus sequence analysis (MLSA) and phylogenetic tree construction with the neighbour-joining [31], maximum-parsimony [32] and maximum-likelihood [33] tree-making algorithms. The topology of the clades in the tree was determined using bootstrap analyses [34] based on 1000 replications. The average nucleotide identity (ANI) between the genomes of the strains was calculated using the online plateform Ezbiocloud [27]. The digital DNA–DNA hybridization (dDDH) values between of the genome of the strains were calculated using the genome-to-genome distance calculator, GGDC 2.1 [35, 36].Strains LHW50302 T and LHW51701 T grew well on ISP 2, and formed extensively branched substrate mycelia and white aerial hyphae. The hooked and looped aerial hyphae differentiated into chains of smooth-surfaced spores, as shown in Fig. 1. Most of the spores were size of 0.6×0.9 µm and cylindrical in shape. Diffusible pigments were produced on a number of media, and the detailed colony appearances were provided in Table S1 (available in the online version of this article). Growth was observed at 20–40 Ǫ C and pH 6.0–11.0 on ISP 2 with 0–15% NaCl. The detailed physiological properties of strains are showed in the species description.Fig. 1. Scanning electron micrograph of strain LHW50302 T (a) and strain LHW51701 T (b) showing a hooked and looped spore arrangement and smooth spore ornamentation following growth on ISP 2 at 28 Ǫ C for 7 days. Bar, 1 µm.Fig. 2. Neighbour-joining phylogenetic tree, based on almost-complete 16S rRNA gene sequences, showing the phylogenetic relationships of strains LHW63021 T, LHW51701 T and related members of the genus Streptomyces. Kitasatospora aburaviensis NRRL B-2218 T was used as an outgroup. Numbers at nodes indicate levels of bootstrap support (%) based on neighbour-joining analysis of 1000 resampled datasets; only values above 50 % are shown. Asterisks (*) and crosses (‡) indicate branches that were also found using maximum-likelihood method and the maximum-parsimony method, respectively. Bar, 0.005 substitutions per nucleotide position.Strains LHW50302 T and LHW51701 T shared similar chemotaxonomic characteristics, which were typical of the genus Streptomyces. The strains contained LL- diaminopimelic acid (Fig. S1), and whole-cell sugars were glucose, ribose and galactose. Diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol were the predominant polar lipids, and unidentified phospholipids were also detected (Fig. S2). The menaquinone system contained MK-9(H 6) (76.0 %, 74.3 %) as major components, and MK-9(H 4) (11.6 %, 13.9 %) and MK-9(H 8) (6.4 %, 4.8 %) as minorities (Fig. S3). The major fatty acids were iso-C 16: 0, anteiso-C 15: 0 and anteiso-C 17: 0 (Table S2), which corresponded to fatty acid type 2c [37].The almost-complete 16S rRNA gene sequences of strains LHW50302 T (1488 bp) and LHW51701 T (1487 bp) were obtained. The draft genome assemblies are publicly available in GenBank (accession nos: QOIM00000000 and QOIN00000000) and the statistics including the genome size, DNA G+C ratio, the number of contigs, N50 and the sequencing depth of coverage are provided in Table S3. Heterogeneities of four nucleotides, namely 1007 (C/A), 1010 (T/C), 1023 (C/G) and 1024 (T/G) (according to the E. coli numbering) in the V6 zone of the 16S rRNA gene of strain LHW50302 T was observed in sequencing with primers 1492R (5′-GGTTACCTTGTTACGACTT-3′) and 888F (5′- AGTACGGCCGCAAGGCTAAAACTC-3′), selected for covering and bridging the second half of the gene. In the draft genome of strain LHW50302 T, only one 16S rRNA gene sequence (KX347891) was found and confirmed. The strains shared a 16S rRNA gene sequence similarity of 98.5 %, a value which corresponded to a 22 nt difference over 1487 locations. Strain LHW50302 T shared maximum similarity to Streptomyces flocculus NRRL B-2465 T (98.6 %), Streptomyces gibsonii NRRL B-1335 T (98.4 %), Streptomyces almquistii NRRL B-1685 T (98.4 %) and Streptomyces albus NBRC 13014 T (98.4 %) amongst the close relatives with validly published names. Proposed by Labeda et al. [38], S. flocculus, S. gibsonii and S. almquistii were later heterotypic synonyms of S. albus; therefore, from its group, only the type strain of S. albus was taken into further taxonomic analyses. Strain LHW51701 T shared a maximum similarity of 98.4 % to Streptomyces sclerotialus NRRL ISP- 5269 T amongst valid Streptomyces species. In the neighbour-joining phylogenetic tree (Fig. 2) based on the 16S rRNA gene sequences, strain LHW50302 T clustered with S. albus CGMCC 4.1640 T and Streptomyces qinglanensis CGMCC 4.6825 T (98.1 %); strain LHW51701 T formed an adjacent but distinct monophyletic lineage to the clade of strain LHW50302 T, and was far away from S. sclerotialus NRRL ISP- 5269 T.The close association of strains LHW50302 T, LHW51701 T, S. albus CGMCC 4.1640 T and S. qinglanensis CGMCC 4.6825 T was supported by the MLSA tree with different tree-making algorithms (Fig. 3), based on concatenated partial sequences of the five protein-coding genes (Table S4) available for the related species in the 16S rRNA gene tree. Strains LHW50302 T and LHW51701 T formed a distinct clade in the MLSA tree, supported by a 100 % bootstrap value and with 0.018 MLSA evolutionary distance between one another. The MLSA distance between the strains and closely related Streptomyces species was 0.162 –0.064 (Table S5), which was well above the species level threshold of 0.007 recommended by Rong and Huang [39], indicating the strains LHW50302 T and LHW51701 T formed the nuclei of two novel Streptomyces species.The G+C contents of the genomes of strains LHW50302 T and LHW51701 T were 71.9 and 72.1 mol%, respectively. The ANI and dDDH values between the whole genome sequences of strains LHW50302 T (QOIM00000000) and LHW51701 T (QOIN00000000) to S. albus CGMCC 4.1640 T (BBQG00000000), S. qinglanensis CGMCC 4.6825 T (FOGO00000000) and S. sclerotialus NRRL ISP- 5269 T (JOBC00000000) were 77.20–92.96 and 22.00–50.20 %, respectively, which were well below the 95 and 70 % cut-off point recommended for delineating species (Table S6) [40 – 42].Strains LHW50302 T and LHW51701 T showed a combination of morphological and chemotaxonomic properties typical of members of the genus Streptomyces. However, genotypic and phenotypic of strains evidently distinguish them from their closest phylogenetic relatives (Tables S5, S6 and S7). Based on the results of the polyphasic taxonomic study, strains LHW50302 T and LHW51701 T represent two novel species in the genus Streptomyces, for which the name Streptomyces reniochalinae sp. nov. and Streptomyces diacarni sp. nov. are proposed, respectively.