Introduction

Strain HPA 177T 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 [2–4], 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, 7–9].

The comparison of the complete genome sequences of members of the rare genus Actinoalloteichus might unravel unknown gene clusters dedicated to the biosynthesis of such molecules as bioactive secondary metabolites and enzymes. This has already been demonstrated for the genomes of strains belonging to closely related genera, such as Kutzneria , Saccharomonospora , Crossiella , Kibdelosporangium, and Streptoalloteichus [10–19].

Organism information

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 non-motility [20].

Table 1 Classification and general features of Actinoalloteichus hymeniacidonis HPA 177T according to the MIGS recommendations [46]
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The genus Actinoalloteichus currently contains only five known species. Besides Actinoalloteichus hymeniacidonis HPA 177T 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 177T 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 AH97T (99.2%) and A. nanshanensis NEAU119T (98.3%). Together with A. spitiensis DSM 44848 T (96.8%) and A. cyanogriseus IFO 14455T (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.

Fig. 1
figure 1

Phylogenetic tree highlighting the position of A. hymeniacidonis HPA 177T (given in bold) relative to type strains of other species within the genus Actinoalloteichus and related genera of the family Pseudonocardiaceae. The tree uses sequences aligned by the RDP aligner. Using the Jukes-Cantor corrected distance model, a distance matrix is constructed based on alignment model positions without the use of alignment inserts, using a minimum comparable position of 200. 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 40313T was used as the root organism. Species for which a complete or draft genome sequence is available are underlined

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A. hymeniacidonis HPA 177T 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 177T 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 177T 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 of calcium malate, sodium oxalate, or sodium succinate nor reduction of nitrate [1].

Fig. 2
figure 2

Colony of A. hymeniacidonis HPA 177T grown at 28 °C for 8 days on ISP2 agar medium prepared with artificial sea water

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Chemotaxonomic data

The cell wall of A. hymeniacidonis contains diaminopimelic acids (A2pm) [1]. The major menaquinone is MK-9(H4) (64%), followed by MK-9(H6) (23%) and MK-9(H8) (12%).

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].

The cellular fatty acids are mainly composed of anteiso pentadecanoic acid (C15:0 anteiso) (20%), cis-8-heptadecenoic acid (C17:1 ω8c) (19%), isopalmitic acid (C16:0 iso) (16%), heptadecanoic acid (C17:0) (11%) and other fatty acids occurring in lower amounts [1]. Galactose, glucose, mannose, and ribose are whole cell sugars of HPA 177T [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 177T, 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.

Table 2 Genome sequencing project information
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Growth conditions and DNA isolation

A. hymeniacidonis HPA 177T 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 [28–31] 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 annotation

Gene prediction and primary annotation were done using the IMG ER pipeline [32]. Additionally, genes were identified using GeneMark [33], GLIMMER [34], and Prodigal [35]. For annotation, BLAST searches against the NCBI Protein Clusters Database [36] were performed and the annotation was enriched by searches against the Conserved Domain Database [37] and subsequent assignment of coding sequences to COGs. Non-coding genes and miscellaneous features were predicted using tRNAscan-SE [38], Infernal [39], RNAMMer [40], Rfam [41], TMHMM [42], and SignalP [43].

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.

Fig. 3
figure 3

Graphical map of the chromosome of A. hymeniacidonis HPA 177T. From the outside to the center: Genes on forward strand (colored by COG categories), genes on reverse strand (colored by COG categories), RNA genes (tRNAs green, rRNAs red, other RNAs black), G+C content, G+C skew

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Table 3 Genome Statistics
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Table 4 Number of genes associated with the general COG functional categories
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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 177T. However, keeping in mind that all actinomycete genomes sequenced so far contain SMBGCs, the genome of strain HPA 177T 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 177T has the genetic capacity to produce novel compounds some of which, e.g. peptide-polyketide 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.

Table 5 Secondary metabolite biosynthesis gene clusters identified in the genome of Actinoalloteichus hymeniacidonis DSM 45092 using antiSMASH 3.0.4 software followed by manual curation
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Conclusion

The genome sequence of A. hymeniacidonis HPA 177T 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.