Genome sequence of the ocean sediment bacterium Saccharomonospora marina type strain (XMU15(T)).

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).

Introduction

Strain XMU15T (= DSM 45390 = KCTC 19701 = CCTCC AA 209048) is the type strain of the species Saccharomonospora marina [1], one of nine species currently in the genus Saccharomonospora [2]. The strain was originally isolated from an ocean sediment sample collected from Zhaoan Bay, East China Sea, in 2005 [1]. 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) [3]; the species epithet was derived from the Latin adjective marina, of the sea, referring to the origin of the strain [1]. 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 [4], one of the recently sequenced GEBA genomes [5]. 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 [6]. Hydrolysis of cellulose and starch were also reported for other members of the genus (that are included in CSP 312), including S. marina [1], S. halophila [7], S. saliphila [8], S. paurometabolica [9], and S. xinjiangensis [10]. 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.

Classification and features

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 [13] and the relative frequencies of taxa and keywords (reduced to their stem [14]) 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).

Figure 1

Phylogenetic tree highlighting the position of S. marina relative to the type strains of the other species within the family Pseudonocardiaceae. The tree was inferred from 1,391 aligned characters [15,16] of the 16S rRNA gene sequence under the maximum likelihood (ML) criterion [17]. Rooting was done initially using the midpoint method [18] and then checked for its agreement with the current classification (Table 1). The branches are scaled in terms of the expected number of substitutions per site. Numbers adjacent to the branches are support values from 600 ML bootstrap replicates [19] (left) and from 1,000 maximum-parsimony bootstrap replicates [20] (right) if larger than 60%. Lineages with type strain genome sequencing projects registered in GOLD [21] are labeled with one asterisk, those also listed as 'Complete and Published' with two asterisks [4,22,23], with S. azurea missing second asterisk but published in this issue [24]. Actinopolyspora iraqiensis Ruan et al. 1994 [25] was ignored in the tree. The species was proposed to be a later heterotypic synonym of S. halophila [26], although the name A. iraqiensis would have had priority over S. halophila. This taxonomic problem will soon be resolved with regard to the genomes of A. iraqiensis and S. halophila, which were both part of CSP 312.

Table 1

Classification and general features of S. marina XMU15T according to the MIGS recommendations [27].

MIGS ID	Property	Term	Evidence code
	Current classification	Domain Bacteria	TAS [28]
		Phylum Actinobacteria	TAS [29]
		Class Actinobacteria	TAS [30]
		Subclass Actinobacteridae	TAS [30,31]
		Order Actinomycetales	TAS [30-33]
		Suborder Pseudonocardineae	TAS [30,31,34]
		Family Pseudonocardiaceae	TAS [30,31,34-36]
		Genus Saccharomonospora	TAS [32,37]
		Species Saccharomonospora marina	TAS [1]
		Type-strain XMU15	TAS [1]
	Gram stain	positive	TAS [1]
	Cell shape	variable, substrate and aerial mycelia	TAS [1]
	Motility	non-motile	TAS [1]
	Sporulation	smooth or wrinkled spores, singly, in pairs or in short chains from aerial mycelium	TAS [1]
	Temperature range	mesophile	TAS [1]
	Optimum temperature	28-37°C	TAS [1]
	Salinity	optimum 0-3% (w/v) NaCl, tolerated up to 5%	TAS [1]
MIGS-22	Oxygen requirement	aerobic	TAS [1]
	Carbon source	D-glucose, manose, melibiose, L-rhamnose, myo-inositol	TAS [1]
	Energy metabolism	chemoheterotrophic	NAS
MIGS-6	Habitat	marine, ocean sediment	TAS [1]
MIGS-15	Biotic relationship	free living	TAS [1]
MIGS-14	Pathogenicity	none	NAS
	Biosafety level	1	NAS
MIGS-23.1	Isolation	ocean sediment	TAS [1]
MIGS-4	Geographic location	Zhaoan Bay, East China Sea	TAS [1]
MIGS-5	Sample collection time	December 2005	NAS
MIGS-4.1	Latitude	24.108	TAS [1]
MIGS-4.2	Longitude	117.294	TAS [1]
MIGS-4.3	Depth	4 m	TAS [1]
MIGS-4.4	Altitude	-4 m	TAS [1]

Evidence codes - IDA: Inferred from Direct Assay (first time in publication); 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. If the evidence code is IDA, then the property was directly observed for a living isolate by one of the authors or an expert mentioned in the acknowledgements [38].

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) [1]. Non-motile, smooth or wrinkled spores were observed on the aerial mycelium, occasionally in short spore chains [1]. 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 [1]. Strain XMU15T grows well in up to 5% NaCl, with an optimum at 0-3% NaCl [1]. Substrates used by the strain are summarized in the strain description [1].

Figure 2

Scanning electron micrograph of S. marina XMU15T

Chemotaxonomy

The cell wall of strain XMU15T contains meso-diaminopimelic acid [1]; arabinose, galactose and ribose are present [1]. 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%) [1]. Main menaquinone is MK-9 H4 (90%) complemented by MK-8 H4 (10%) [1]; phospholipids comprised phosphatidylglycerol, diphosphatidylglycerol, and phosphatidylinisitol with a minor fraction of phosphatidylethanolamine [1].

Genome sequencing and annotation

Genome project history

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 [21] 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.

Table 2

Genome sequencing project information

MIGS ID	Property	Term
MIGS-31	Finishing quality	Permanent draft
MIGS-28	Libraries used	Three genomic libraries: one 454 pyrosequence standard library,      one 454 PE library (10 kb insert size), one Illumina library
MIGS-29	Sequencing platforms	Illumina GAii, 454 GS FLX Titanium
MIGS-31.2	Sequencing coverage	780.0 × Illumina; 8.6 × pyrosequence
MIGS-30	Assemblers	Newbler version 2.3, Velvet version 1.0.13,     phrap version SPS - 4.24
MIGS-32	Gene calling method	Prodigal
	INSDC ID	CM001439
	GenBank Date of Release	February 3, 2012
	GOLD ID	Gi07581
	NCBI project ID	61991
	Database: IMG	2508501012
MIGS-13	Source material identifier	DSM 45390
	Project relevance	Bioenergy and phylogenetic diversity

Growth conditions and DNA isolation

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. [1], 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) [39] 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 [40].

Genome sequencing and assembly

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 [41]. 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 [42] 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 [43] 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 [42] 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 [41], Dupfinisher [44], 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 [45]. 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.

Genome annotation

Genes were identified using Prodigal [46] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline [47]. 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 [48], RNAMMer [49], Rfam [50], TMHMM [51], and signalP [52].

Genome properties

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.

Table 3

Genome Statistics

Attribute	Value	% of Total
Genome size (bp)	5,965,593	100.00%
DNA coding region (bp)	5,364,872	89.93%
DNA G+C content (bp)	4,112,466	68.94%
Number of replicons	1
Extrachromosomal elements	0
Total genes	5,784	100.00%
RNA genes	57	0.99%
rRNA operons	3
tRNA genes	47	0.81%
Protein-coding genes	5,727	99.01%
Pseudo genes	149	2.58%
Genes with function prediction (proteins)	4,341	75.05%
Genes in paralog clusters	3,491	60.36%
Genes assigned to COGs	4,261	73.67%
Genes assigned Pfam domains	4,426	76.52%
Genes with signal peptides	1,159	20.04%
Genes with transmembrane helices	1,256	21.72%
CRISPR repeats	1

Figure 3

Graphical map of the chromosome. From outside to the center: Genes on forward strand (color by COG categories), Genes on reverse strand (color by COG categories), RNA genes (tRNAs green, rRNAs red, other RNAs black), GC content, GC skew.

Table 4

Number of genes associated with the general COG functional categories

Code	value	%age	Description
J	173	3.6	Translation, ribosomal structure and biogenesis
A	3	0.1	RNA processing and modification
K	509	10.7	Transcription
L	226	4.7	Replication, recombination and repair
B	3	0.1	Chromatin structure and dynamics
D	40	0.8	Cell cycle control, cell division, chromosome partitioning
Y	0	0.0	Nuclear structure
V	68	1.4	Defense mechanisms
T	220	4.6	Signal transduction mechanisms
M	191	4.0	Cell wall/membrane biogenesis
N	6	0.1	Cell motility
Z	0	0.0	Cytoskeleton
W	0	0.0	Extracellular structures
U	52	1.1	Intracellular trafficking and secretion, and vesicular transport
O	152	3.2	Posttranslational modification, protein turnover, chaperones
C	369	7.7	Energy production and conversion
G	294	6.2	Carbohydrate transport and metabolism
E	381	8.0	Amino acid transport and metabolism
F	93	2.0	Nucleotide transport and metabolism
H	223	4.7	Coenzyme transport and metabolism
I	291	6.1	Lipid transport and metabolism
P	210	4.4	Inorganic ion transport and metabolism
Q	264	5.5	Secondary metabolites biosynthesis, transport and catabolism
R	649	13.6	General function prediction only
S	364	7.6	Function unknown
-	1,523	26.4	Not in COGs