Komaki et al. Standards in Genomic Sciences (2016) 11:84 DOI 10.1186/s40793-016-0206-2 SHORTGENOMEREPORT Open Access Draft genome sequence of Micromonospora sp. DSW705 and distribution of biosynthetic gene clusters for depsipeptides bearing 4-amino-2,4- pentadienoate in actinomycetes Hisayuki Komaki1*, Natsuko Ichikawa2, Akira Hosoyama2, Moriyuki Hamada1, Enjuro Harunari3, Arisa Ishikawa3 and Yasuhiro Igarashi3 Abstract Here, we report the draft genome sequence of Micromonospora sp. DSW705 (=NBRC 110037), a producer of antitumor cyclic depsipeptides rakicidins A and B, together with the features of this strain and generation, annotation, and analysis of the genome sequence. The 6.8 Mb genome of Micromonospora sp. DSW705 encodes 6,219 putative ORFs, of which 4,846 are assigned with COG categories. The genome harbors at least three type I polyketide synthase (PKS) gene clusters, one nonribosomal peptide synthetase (NRPS) gene clusters, and three hybrid PKS/NRPS gene clusters. A hybrid PKS/NRPS gene cluster encoded in scaffold 2 is responsible for rakicidin synthesis. DNA database search indicated that the biosynthetic gene clusters for depsipeptides bearing 4-amino-2,4-pentadienoate are widely present in taxonomically diverse actinomycetes. Keywords: Actinomycete, BE-43547, Micromonospora, Nonribosomal peptide synthetase, Polyketide synthase, Rakicidin, Taxonomy, Vinylamycin Introduction biosynthetic gene (rak) cluster for rakicidin D through In our screening of antitumor compounds from rare the genome analysis of Streptomyces sp. MWW064 and actinomycetes, Micromonospora sp. DSW705 collected proposed its biosynthetic pathway [8]. In this study, the from deep seawater was found to produce rakicidins A whole genome shotgun sequencing of Micromonospora and B. Rakicidins are fifteen-membered cyclic depsipep- sp. DSW705 was conducted to assess its potential in tides comprising three amino acids and a modified fatty secondary metabolism, to identify the biosynthetic genes acid. The most intriguing feature of rakicidins is the for rakicidins A and B, and to make a comparative analysis presence of a rare unusual amino acid, 4-amino-2,4-pen- with the gene cluster of rakicidin D in Streptomyces sp. tadienoate (APDA) in their cyclic structures, which is MWW064. We here report the draft genome sequence of present only in a limited range of secondary metabolites Micromonospora sp. DSW705, together with the taxonom- of actinomycetes [1–3]. To date, five rakicidin congeners ical identification of the strain, description of its genome have been reported; rakicidins A, B, and E were isolated properties, and annotation of the rakicidin gene cluster. from Micromonospora, and rakicidins C and D from Furthermore, we investigated distribution of the rak–like Streptomyces [4–7]. Recently, we disclosed the clusters in other bacterial strains to evaluate the gene distri- bution in taxonomically diverse actinomycetes. * Correspondence: [email protected] 1Biological Resource Center, National Institute of Technology and Evaluation, Chiba, Japan Full list of author information is available at the end of the article © The Author(s). 2016 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Komaki et al. Standards in Genomic Sciences (2016) 11:84 Page 2 of 10 Organism information On ISP 7 agars, the growth was poor. No growth was Classification and features observed on ISP 5 agar. No aerial mycelia were ob- In the screening of antitumor compounds from rare served. Substrate mycelium was orange, turning dark actinomycetes, Micromonospora sp. DSW705 was iso- brown on sporulation on ISP 2 agar. No diffusible lated from deep seawater collected in Toyama Bay, Japan pigment was observed on ISP 2, ISP 3, ISP 4, ISP 5, and found to produce BU-4664 L and rakicidins A and B ISP 6, and ISP 7 agar media. The strain bored single (unpublished). The general feature of this strain is shown spore on short sporophore. The spores were spherical in Table 1. This strain grew well on ISP 2 and ISP 4 agars. (0.7–0.8 μm in diameter) with wrinkle surface. A scanning electron micrograph of the strain is shown Table 1 Classification and general features of Micromonospora in Fig. 1. Growth occurred at 20–45 °C (optimum sp. DSW705 [16] 37 °C) and pH 5–8 (optimum pH 7). Strain DSW705 MIGS ID Property Term Evidence codea exhibited growth with 0–3 % (w/v) NaCl (optimum Classification Domain Bacteria TAS [23] 0 % NaCl). Strain DSW705 utilized arabinose, Phylum Actinobacteria TAS [24] fructose, glucose, raffinose, sucrose, and xylose for Class Actinobacteria TAS [25] growth. This strain was deposited in the NBRC culture collection with the registration number of Order Actinomycetales TAS [25–28] NBRC 110037. The genes encoding 16S rRNA were Suborder TAS [25, 28] amplified by PCR using two universal primers, 9 F Micromonosporineae and 1541R. After purification of the PCR product by Family Micromonosporaceae TAS [25, 27–30] AMPure (Beckman Coulter), the sequencing was Genus Micromonospora TAS [27, 31] carried out according to an established method [9]. Species undetermined - Homology search of the sequence by EzTaxon-e [10] Strain DSW705 IDA indicated the highest similarity (99.66 %, 1448/1453) T Gram stain Not tested, likely positive NAS to Micromonospora chalcea DSM 43026 (X92594) as the closest type strain. A phylogenetic tree was recon- Cell shape Branched mycelia IDA structed using ClustalX2 [11] and NJPlot [12] on the Motility Not reported basis of the 16S rRNA gene sequence together with Sporulation Sporulating IDA those of taxonomically close type strains showing over Temperature Grows from 20 °C to 45 °C IDA 98.5 % similarities. Evolutionary distances were range calculated using Kimura’s two-parameter model [13]. Optimum 37 °C IDA The tree has been deposited into TreeBase (http:// temperature purl.org/phylo/treebase/phylows/study/TB2:S19405). In pH range; 5 to 8; 7 IDA the phylogenetic tree, strain DSW705 and M. chalcea Optimum DSM 43026T (X92594) formed a monophyletic cluster Carbon Arabinose, fructose, glucose, IDA with a bootstrap resampling value of 100 % (Fig. 2). source raffinose, sucrose, xylose MIGS-6 Habitat Sea water NAS MIGS-6.3 Salinity Grows from 0 % to 3 % NaCl IDA MIGS-22 Oxygen Aerobic IDA requirement MIGS-15 Biotic Free-living IDA relationship MIGS-14 Pathogenicity Not reported MIGS-4 Geographic Toyama Bay, Japan NAS location MIGS-5 Sample October 10, 2005 NAS collection MIGS-4.1 Latitude Not reported MIGS-4.2 Longitude Not reported MIGS-4.4 Altitude Not reported aEvidence codes - IDA Inferred from Direct Assay, TAS Traceable Author Statement (i.e., a direct report exists in the literature), NAS Non-traceable Author Statement (i.e., not directly observed for the living, isolated sample, Fig. 1 Scanning electron micrograph of Micromonospora sp. DSW705 but based on a generally accepted property for the species, or anecdotal grown on 1/2 ISP 2 agar for 7 days at 28 °C. Bar, 2 μm evidence). These evidence codes are from the Gene Ontology project [32] Komaki et al. Standards in Genomic Sciences (2016) 11:84 Page 3 of 10 Fig. 2 Phylogenetic tree of Micromonospora sp. DSW705 and phylogenetically close type strains showing over 98.5 % similarity to strain DSW705 based on 16S rRNA gene sequences. The accession numbers for 16S rRNA genes are shown in parentheses. The tree was reconstructed by the neighbor-joining method [34] using sequences aligned by ClustalX2 [11]. All positions containing gaps were eliminated. The building of the tree also involves a bootstrapping process repeated 1,000 times to generate a majority consensus tree, and only bootstrap values above 50 % are shown at T branching points. Actinoplanes teichomyceticus NBRC 13999 was used as an outgroup. Bar, 0.005 Knuc substitutions per nucleotide position Chemotaxonomic data Growth conditions and genomic DNA preparation The isomer of diaminopimelic acid in the whole-cell Micromonospora sp. DSW705 was deposited in the NBRC hydrolysate was analyzed according to the method culture collection with the registration number of NBRC described by Hasegawa et al. [14]. Isoprenoid quinones and 110037. The monoisolate of strain DSW705 was grown on cellular fatty acids were analyzed as described previously a polycarbonate membrane filter (Advantec) on double [15]. The whole-cell hydrolysate of strain DSW705 con- tained meso-diaminopimelic acid as its diagnostic peptido- Table 2 Project information glycan diamino acid. The predominant menaquinone was MIGS ID Property Term identified as MK-10(H4); MK-9(H4), MK-10(H2), and MK- MIGS 31 Finishing quality Improved-high-quality draft 10(H6) were also detected as minor components. The MIGS-28 Libraries used 454 shotgun library, Illumina major cellular fatty acids were found to be iso-C16:0, iso- paired-end library C15:0 and anteiso-C17:0. MIGS 29 Sequencing platforms 454 GS FLX+, Illumina HiSeq1000 MIGS 31.2 Fold coverage 5 ×, 100 ×, respectively MIGS 30 Assemblers Newbler v2.6, GenoFinisher Genome sequencing information MIGS 32 Gene calling method Progidal Genome project history Locus tag MSP03 In collaboration between Toyama Prefectural University and NBRC, the organism was selected for genome se- GenBank ID BBVA00000000 quencing to elucidate the rakicidin biosynthetic pathway. GenBank date of release March 30, 2016 The draft genome sequences have been deposited in the GOLD ID Not registered INSDC database under the accession number BioProject PRJDB3540 BBVA01000001-BBVA01000024. The project informa- MIGS 13 Source material identifier NBRC 110037 tion and its association with MIGS version 2.0 compli- Project relevance Industrial ance are summarized in Table 2 [16].