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Saccharomonospora marina Liu et al. 2010 is a member of the genus Saccharomonospora, in the family Pseudonocardiaceae that is poorly characterized at the genome level thus far. Members of the genus Saccharomonospora are of interest because they originate from diverse habitats, such as leaf litter, manure, compost, surface of peat, moist, over-heated grain, and ocean sediment, where they might play a role in the primary degradation of plant material by attacking hemicellulose. Organisms belonging to the genus are usually Gram-positive staining, non-acid fast, and classify among the actinomycetes. Here we describe the features of this organism, together with the complete genome sequence (permanent draft status), and annotation. The 5,965,593 bp long chromosome with its 5,727 protein-coding and 57 RNA genes was sequenced as part of the DOE funded Community Sequencing Program (CSP) 2010 at the Joint Genome Institute (JGI).
Strain XMU15T (= DSM 45390 = KCTC 19701 = CCTCC AA 209048) is the type strain of the species Saccharomonospora marina , one of nine species currently in the genus Saccharomonospora . The strain was originally isolated from an ocean sediment sample collected from Zhaoan Bay, East China Sea, in 2005 . The genus name Saccharomonospora was derived from the Greek words for sakchâr, sugar, monos, single or solitary, and spore, a seed or spore, meaning the sugar (-containing) single-spored (organism) ; the species epithet was derived from the Latin adjective marina, of the sea, referring to the origin of the strain . S. marina and the other type strains of the genus Saccharomonospora were selected for genome sequencing in one of the DOE Community Sequencing Projects (CSP 312) at Joint Genome Institute (JGI), because members of the genus (which originate from diverse habitats, such as leaf litter, manure, compost, surface of peat, moist, over-heated grain and ocean sediment) might play a role in the primary degradation of plant material by attacking hemicellulose. This expectation was underpinned by the results of the analysis of the genome of S. viridis , one of the recently sequenced GEBA genomes . The S. viridis genome, the first sequenced genome from the genus Saccharomonospora, contained an unusually large number (24 in total) genes for glycosyl hydrolases (GH) belonging to 14 GH families, which were identified in the Carbon Active Enzyme Database . Hydrolysis of cellulose and starch were also reported for other members of the genus (that are included in CSP 312), including S. marina , S. halophila , S. saliphila , S. paurometabolica , and S. xinjiangensis . Here we present a summary classification and a set of features for S. marina XMU15T, together with the description of the genomic sequencing and annotation.
A representative genomic 16S rRNA sequence of S. marina XMU15T was compared using NCBI BLAST [11,12] under default settings (e.g., considering only the high-scoring segment pairs (HSPs) from the best 250 hits) with the most recent release of the Greengenes database  and the relative frequencies of taxa and keywords (reduced to their stem ) were determined, weighted by BLAST scores. The most frequently occurring genera were Gordonia (63.5%), Saccharomonospora (24.1%), Actinomycetospora (4.5%), Actinopolyspora (1.8%) and Pseudonocardia (1.4%) (195 hits in total). Regarding the single hit to sequences from members of the species, the average identity within HSPs was 99.7%, whereas the average coverage by HSPs was 100.1%. Regarding the 23 hits to sequences from other members of the genus, the average identity within HSPs was 96.1%, whereas the average coverage by HSPs was 98.3%. Among all other species, the one yielding the highest score was Saccharomonospora saliphila (HM368568), which corresponded to an identity of 99.9% and an HSP coverage of 92.1%. (Note that the Greengenes database uses the INSDC (= EMBL/NCBI/DDBJ) annotation, which is not an authoritative source for nomenclature or classification. For instance, the Gordonia hits are likely to be caused by mis-annotations in INSDC). The highest-scoring environmental sequence was FN667533 ('stages composting process pilot scale municipal drum compost clone PS3734'), which showed an identity of 96.0% and a HSP coverage of 97.9%. The most frequently occurring keywords within the labels of all environmental samples which yielded hits were 'skin' (6.3%), 'forearm' (2.8%), 'soil' (2.6%), 'fossa' (2.5%) and 'volar' (2.3%) (55 hits in total). These keywords do not fit to the known habitat of strain XMU15T, because Saccharomonospora rarely occurs in environmental samples so that more distant relatives (here from human skin) distort the automatically generated list of keywords. Environmental samples which yielded hits of a higher score than the highest scoring species were not found.
Figure 1 shows the phylogenetic neighborhood of S. marina in a 16S rRNA based tree. The sequences of the three 16S rRNA gene copies in the genome differ from each other by up to 13 nucleotides, and differ by up to 15 nucleotides from the previously published 16S rRNA sequence (FJ812357).
Cells of S. marina XMU15T are non-acid fast, stain Gram-positive and form an irregularly branched vegetative mycelium of 0.3 to 0.4 μm diameter (Figure 2) . Non-motile, smooth or wrinkled spores were observed on the aerial mycelium, occasionally in short spore chains . The growth range of strain XMU15T spans from 28-37°C, with an optimum at 28°C, and pH 7.0 on ISP 2 medium . Strain XMU15T grows well in up to 5% NaCl, with an optimum at 0-3% NaCl . Substrates used by the strain are summarized in the strain description .
The cell wall of strain XMU15T contains meso-diaminopimelic acid ; arabinose, galactose and ribose are present . The fatty acids spectrum is dominated by penta- to heptadecanoic acids: iso-C16:0 (26.4%), C17:1 ω6c (16.8%), C16:0 (palmitic acid, 8.9%), C15:0 (16.2%), C17:1 ω8c (7.7%), iso-C16:1 H (6.0%) . Main menaquinone is MK-9 H4 (90%) complemented by MK-8 H4 (10%) ; phospholipids comprised phosphatidylglycerol, diphosphatidylglycerol, and phosphatidylinisitol with a minor fraction of phosphatidylethanolamine .
This organism was selected for sequencing as part of the DOE Joint Genome Institute Community Sequencing Program (CSP) 2010, CSP 312, “Whole genome type strain sequences of the genus Saccharomonospora – a taxonomically troubled genus with bioenergetic potential”. The genome project is deposited in the Genomes On Line Database  and the complete genome sequence is deposited in GenBank. Sequencing, finishing and annotation were performed by the DOE Joint Genome Institute (JGI). A summary of the project information is shown in Table 2.
The history of strain XMU15T starts in 2005 with an isolate from ocean sediment collected from Zhaoan Bay in the East China Sea, followed by a detailed chemotaxonomic description by Liu et al. , and deposit of the strain in three collections in 2009: Korean Collection for Type Cultures (accession 19701), Chinese Centre for Type Cultures Collections (accession 209048) and German Collection of Microorganisms and Cell cultures, DSMZ (accession 45390). Strain XMU15T, DSM 45390, was grown in DSMZ medium 83 (Czapek Peptone Medium)  at 28°C. DNA was isolated from 0.5-1 g of cell paste using Jetflex Genomic DNA Purification Kit (GENOMED 600100) following the standard protocol as recommended by the manufacturer with the following modifications: extended cell lysis time (60 min.) with additional 30µl achromopeptidase, lysostaphin, mutanolysin; proteinase K was added at 6-fold the supplier recommended amount for 60 min. at 58°C. The purity, quality and size of the bulk gDNA preparation were assessed by JGI according to DOE-JGI guidelines. DNA is available through the DNA Bank Network .
The genome was sequenced using a combination of Illumina and 454 sequencing platforms. All general aspects of library construction and sequencing can be found at the JGI website . Pyrosequencing reads were assembled using the Newbler assembler (Roche). The initial Newbler assembly consisting of 185 contigs in one scaffold was converted into a phrap  assembly by making fake reads from the consensus, to collect the read pairs in the 454 paired end library. Illumina GAii sequencing data (5,096.2 Mb) was assembled with Velvet  and the consensus sequences were shredded into 1.5 kb overlapped fake reads and assembled together with the 454 data. The 454 draft assembly was based on 95.6 Mb 454 draft data and all of the 454 paired end data. Newbler parameters are -consed -a 50 -l 350 -g -m -ml 20. The Phred/Phrap/Consed software package  was used for sequence assembly and quality assessment in the subsequent finishing process. After the shotgun stage, reads were assembled with parallel phrap (High Performance Software, LLC). Possible mis-assemblies were corrected with gapResolution , Dupfinisher , or sequencing cloned bridging PCR fragments with subcloning. Gaps between contigs were closed by editing in Consed, by PCR and by Bubble PCR primer walks (J.-F. Chang, unpublished). A total of 233 additional reactions were necessary to close gaps and to raise the quality of the finished sequence. Illumina reads were also used to correct potential base errors and increase consensus quality using a software Polisher developed at JGI . The error rate of the completed genome sequence is less than 1 in 100,000. Together, the combination of the Illumina and 454 sequencing platforms provided 788.6 x coverage of the genome. The final assembly contained 397,729 pyrosequence and 61,582,867 Illumina reads.
Genes were identified using Prodigal  as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline . The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) non-redundant database, UniProt, TIGRFam, Pfam, PRIAM, KEGG, COG, and InterPro databases. These data sources were combined to assert a product description for each predicted protein. Non-coding genes and miscellaneous features were predicted using tRNAscan-SE , RNAMMer , Rfam , TMHMM , and signalP .
The genome consists of a 5,965,593 bp long circular chromosome with a 68.9% G+C content (Table 3 and Figure 3). Of the 5,784 genes predicted, 5,727 were protein-coding genes, and 57 RNAs; 149 pseudogenes were also identified. The majority of the protein-coding genes (75.0%) were assigned a putative function while the remaining ones were annotated as hypothetical proteins. The distribution of genes into COGs functional categories is presented in Table 4.
The work conducted by the U.S. Department of Energy Joint Genome Institute was supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231.