Complete genome sequence of the actinomycete Actinoalloteichus hymeniacidonis type strain HPA 177T isolated from a marine sponge

Actinoalloteichus hymeniacidonis HPA 177T is a Gram-positive, strictly aerobic, black pigment producing and spore-forming actinomycete, which forms branching vegetative hyphae and was isolated from the marine sponge Hymeniacidon perlevis. Actinomycete bacteria are prolific producers of secondary metabolites, some of which have been developed into anti-microbial, anti-tumor and immunosuppressive drugs currently used in human therapy. Considering this and the growing interest in natural products as sources of new drugs, actinomycete bacteria from the hitherto poorly explored marine environments may represent promising sources for drug discovery. As A. hymeniacidonis, isolated from the marine sponge, is a type strain of the recently described and rare genus Actinoalloteichus, knowledge of the complete genome sequence enables genome analyses to identify genetic loci for novel bioactive compounds. This project, describing the 6.31 Mbp long chromosome, with its 5346 protein-coding and 73 RNA genes, will aid the Genomic Encyclopedia of Bacteria and Archaea project.


Introduction
Strain HPA 177 T is the type strain of the species Actinoalloteichus hymeniacidonis, it was isolated from the marine sponge Hymeniacidon perlevis at the intertidal beach of Dalian, Yellow Sea, North-China, during investigation of its actinomycete diversity [1].
Members of the diverse order Actinomycetales are a major source of a variety of novel bioactive and possibly pharmaceutically important compounds and drugs, such as anticancer agents [2][3][4], antibiotics [5,6] and also other industrially relevant molecules and enzymes with diverse biological activities [5,7]. Especially marine actinomycetes became a focus of research since they have evolved the greatest genomic and metabolic diversity and are auspicious sources of novel secondary metabolites and enzymes [5,[7][8][9].

Classification and features
The genus Actinoalloteichus was established by Tamura et al. (2000) on the basis of morphological, physiological, chemotaxonomic and phylogenetic criteria. The genus contains Gram-positive, non-acid-fast, aerobic organisms with branching vegetative hyphae [20]. The aerial mycelium of Actinoalloteichus develops straight spore chains [20]. According to 16S rDNA gene sequence analysis Actinoalloteichus is part of the family Pseudonocardiaceae, suborder Pseudonocardineae, order Actinomycetales, class Actinobacteria [20,21] (Table 1). It differs from other genera of its family by its morphological characteristics, fatty acid components and its nonmotility [20].
The genus Actinoalloteichus currently contains only five known species. Besides Actinoalloteichus hymeniacidonis HPA 177 T the other currently known members are the halophilic Actinoalloteichus hoggarensis [22], Actinoalloteichus nanshanensis, isolated from the rhizosphere of a fig tree [23], the soil bacterium Actinoalloteichus spitiensis [24] and Actinoalloteichus cyanogriseus, the type species of the genus isolated from a soil sample collected from the Yunnan province of China [20].
A representative 16S rRNA sequence of A. hymeniacidonis HPA 177 T was compared to the Ribosomal Database Project database [25] confirming the initial taxonomic classification. On the basis of the 16S rDNA, A. hymeniacidonis shows highest similarity to A. hoggarensis AH97 T (99.2%) and A. nanshanensis NEAU119 T (98.3%). Together with A. spitiensis DSM 44848 T (96.8%) and A. cyanogriseus IFO 14455 T (96.4%), they form a distinct clade within the family Pseudonocardiaceae. Figure 1 shows the phylogenetic neighborhood of A. hymeniacidonis in a 16S rRNA gene based tree. Phylum ' Actinobacteria' TAS [48] Class Actinobacteria TAS [21] Order Actinomycetales TAS [49,50] Suborder Pseudonocardianeae TAS [51] Family Pseudonocardiaceae TAS [51,52] Genus Actinoalloteichus TAS [20] Species Evidence codes -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, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project [53] A. hymeniacidonis HPA 177 T forms branching vegetative hyphae (Fig. 2), which are grey to black in color and tend to fragment after 3 weeks of cultivation (1). The aerial hyphae develop spores of a dimension of 0.6 × 0.8 μm [1]. HPA 177 T is strictly aerobic and non-motile [1]. Growth of A. hymeniacidonis was shown at temperatures between 15 and 45°C (optimal growth between 20 and 37°C) [1]. HPA 177 T can utilize fructose, glucose, maltose, mannitol, mannose, xylose, rhamnose, sucrose, sorbitol, sodium citrate, casein, or starch as carbon sources, but not arabinose, inositol, and raffinose [1] ( Table 1). It grows well on yeast extract/malt extract agar or oatmeal agar and produces a black soluble pigment when growing on yeast extract/malt extract agar as well as on peptone/yeast extract/iron agar [1]. It has been shown that the strain grows faster on ISP2 agar media prepared with 50% of artificial sea water, which, considering the source of isolation, probably reflects an adaptation to the marine environment. Urea is not decomposed by A. hymeniacidonis, and this strain shows neither hydrolysis of aesculin or hippurate, nor utilization The tree is built with RDP Tree Builder, which utilizes Weighbor [54] with an alphabet size of 4 and length size of 1000. The building of the tree also involves a bootstrapping process repeated 100 times to generate a majority consensus tree [55]. Streptomyces albus DSM 40313 T was used as the root organism. Species for which a complete or draft genome sequence is available are underlined Fig. 2 Colony of A. hymeniacidonis HPA 177 T grown at 28°C for 8 days on ISP2 agar medium prepared with artificial sea water of calcium malate, sodium oxalate, or sodium succinate nor reduction of nitrate [1].
The phospholipids were shown to be mainly composed of phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside as well as of some other glucosamine containing phospholipids of unknown structure as diagnostic polar lipids [1]. A. hymeniacidonis does not contain mycolic acids [1].

Genome sequencing information
Genome project history Due to the increasing interest in exploiting new and rare actinomycetes as new sources of novel secondary metabolites [5], Actinoalloteichus hymeniacidonis HPA 177 T , a member of the rare genus Actinoalloteichus [20], was selected for sequencing. While not being part of the GEBA project [26], sequencing of the type strain will aid the GEBA effort. The genome project is deposited in the Genomes OnLine Database [27] and the complete genome sequence is deposited in GenBank. A summary of the project information is shown in Table 2.

Growth conditions and DNA isolation
A. hymeniacidonis HPA 177 T was grown aerobically in 50 ml 3% TSB medium (Oxoid, UK) in 250 mL baffled flasks at 28°C, 250 rpm. Genomic DNA was isolated using Wizard Genomic DNA Purification Kit (Promega, USA) from~2 g of mycelium (wet weight) using the manufacturer's protocol with the following modification. The clarified lysate prior to precipitation of DNA with isopropanol was extracted once with ½ volume of a 1:1 mixture of phenol/chloroform (pH 8.0).

Genome sequencing and assembly
Two libraries were prepared: a WGS library using the Illumina-Compatible Nextera DNA Sample Prep Kit (Epicentre, WI, U.S.A.) and a 6 k MatePair library using the Nextera Mate Pair Sample Preparation Kit, both according to the manufacturer's protocol. Both libraries were sequenced in a 2× 250 bp paired read run on the MiSeq platform, yielding 4,594,541 total reads, providing 159.00× coverage of the genome. Reads were assembled using the Newbler assembler v2.8 (Roche). The initial Newbler assembly consisted of 31 contigs in five scaffolds, with a total of 50 contigs larger than 100 bp. Analysis of the five scaffolds revealed three to make up the chromosome and the remaining two containing the three copies of the RRN operon. The Phred/Phrap/Consed software package [28][29][30][31] was used for sequence assembly and quality assessment in the subsequent finishing process, gaps between contigs were closed by manual editing in Consed (for repetitive elements).

Genome properties
The genome includes one circular chromosome of 6,306,386 bp (68.08% G+C content) (Fig. 3). Among a total of 5425 predicted genes, 5346 are protein coding genes. 4068 (74.90%) of the protein coding genes were assigned a putative function, the remaining were annotated as hypothetical proteins. The properties and the statistics of the genome are summarized in Tables 3 and 4, and the circular plot is shown in Fig. 3. The total is based on either the size of the genome in base pairs or the total number of total genes in the annotated genome Insights from the genome sequence

Gene clusters for biosynthesis of secondary metabolites
So far, there have been no reports on isolation of secondary metabolites from A. hymeniacidonis HPA 177 T . However, keeping in mind that all actinomycete genomes sequenced so far contain SMBGCs, the genome of strain HPA 177 T was analyzed for their presence using the online version of software antiSMASH 3.0.4 [44]. The results of the analysis were manually curated to confirm or edit borders of the clusters, identify closest homologues in the databases based on BLAST search (Table 5), and to gain a more detailed insight into the biosynthesis of the corresponding compound. In total, 25 SMBGCs were identified, 11 of which appeared to be unique at the time of analysis and based on the public database searches. This conclusion was based on the unique composition of the core genes in the clusters encoding scaffold-building enzymes, and in some cases, such as stand-alone terpene cyclase or type III polyketide synthase genes, on low (below 60%) identity of their products to proteins in the NCBI database. Based on this analysis, it seems possible that A. hymeniacidonis HPA 177 T has the genetic capacity to produce novel compounds some of which, e.g. peptidepolyketide hybrids, terpenoids, and unique lassopeptides, may represent bioactive metabolites suitable for drug development. Given its habitat, A. hymeniacidonis might be the real source of secondary metabolites that are thought to originate from its host sponge, comparable to. e.g. Theonella swinhoi and Entotheonella sp. [45]. The knowledge on the SMBGCs and their putative products will assist in identification of the corresponding compounds, and may pave the way to biosynthetic engineering toward generation of new analogues.

Conclusion
The genome sequence of A. hymeniacidonis HPA 177 T represents the first genome of the A. hoggarensis/A. hymeniacidonis/A. nanshanensis subgroup, the first complete genome of this genus as well as the first of a marine species of this genus. As such, it will be a useful basis for future genome comparisons. The presence of 25 SMBGCs indicates a great potential for secondary metabolite production, either by heterologous expression in suitable hosts or by activating the clusters by genetic engineering. Authors' contributions LS prepared and wrote the manuscript, AA and AW performed library preparation and sequencing, JK coordinated the study, SZ isolated genomic DNA, analyzed genome for the presence of secondary metabolite biosynthesis gene clusters, and contributed to writing the manuscript, and CR assembled and analyzed the genome sequence. All authors read and approved the final manuscript.

Competing interests
The authors declare that they have no competing interests.