Genome sequence of the squalene-degrading bacterium Corynebacterium terpenotabidum type strain Y-11(T) (= DSM 44721(T)).

Corynebacterium terpenotabidum Takeuchi et. al 1999 is a member of the genus Corynebacterium, which contains Gram-positive and non-spore forming bacteria with a high G+C content. C. terpenotabidum was isolated from soil based on its ability to degrade squalene and belongs to the aerobic and non-hemolytic Corynebacteria. It displays tolerance to salts (up to 8%) and is related to Corynebacterium variabile involved in cheese ripening. As this is a type strain of Corynebacterium, this project describing the 2.75 Mbp long chromosome with its 2,369 protein-coding and 72 RNA genes will aid the G enomic E ncyclopedia of Bacteria and Archaea project.

Introduction

Strain Y-11T (= DSM 444721T) is the type strain of the species [1]. It was originally isolated from soil, although the exact source has not been published [2,3]. The genus is comprised of Gram-positive bacteria with a high G+C content. It currently contains over 80 members [4] isolated from diverse backgrounds like human clinical samples [5] and animals [6], but also from soil [7] and ripening cheese [8].

Within this diverse genus, has been proposed to form a subclade together with DSM 20132T and S6-4T, demonstrating 97.4% and 95.9% similarity respectively between the 16S rRNA gene sequences. Information on the strain is scarce. It was isolated for its ability to metabolize the linear triterpene squalene and classified as an species [2,3], but no further information on the strain was supplied. Neither the origin nor the exact isolation procedures were reported. can cleave squalene yielding geranylacetone [2] but also accepts some squalene derivatives [3].

Here we present a summary classification and a set of features for DSM 44721T, together with the description of the genomic sequencing and annotation.

Classification and features

A representative genomic 16S rRNA sequence of DSM 44721T was compared to the Ribosomal Database Project database [9]. shows highest similarity to (97.4%).

Figure 1 shows the phylogenetic neighborhood of in a 16S rRNA based tree. Within the genus forms a distinct subclade together with and .

Figure 1

Phylogenetic tree highlighting the position of relative to type strains of other species within the genus . Species with at least one publicly available genome sequence (not necessarily the type strain) are highlighted in bold face. The tree is based on sequences aligned by the RDP aligner and utilizes the Jukes-Cantor corrected distance model to construct a distance matrix based on alignment model positions without alignment inserts, using a minimum comparable position of 200. The tree is built with RDP Tree Builder, which utilizes the Weighbor method [10] with an alphabet size of 4 and length size of 1,000. The building of the tree also involves a bootstrapping process repeated 100 times to generate a majority consensus tree [11]. (X80614) was used as an outgroup.

Y-11T cells are Gram-positive non acid fast rods (1.0-1.5 μm x 0.5-0.8 μm wide) that grow strictly aerobically in rough, grayish-white colonies without diffusible pigments or aerial mycelia [1], [Table 1]. Cells grow with a wax-like quality on solid medium and tend to clot in liquid culture. Scanning electron micrograph pictures of liquid grown cultures revealed slight morphological differences between free-floating cells and clotted cells (Figure 2).

Table 1

Classification and general features of Y-11T according to the MIGS recommendations [12].

MIGS ID	Property	Term	Evidence codea)
	Current classification	Domain Bacteria	TAS [13]
		Phylum Actinobacteria	TAS [14]
		Class Actinobacteria	TAS [15]
		Order Actinomycetales	TAS [15-18]
		Family Corynebacteriaceae	TAS [15-17,19]
		Genus Corynebacterium	TAS [15-17,20,21]
		Species Corynebacterium terpenotabidum	TAS [1]
		Type-strain Y-11T (=DSM 44721T)	TAS [1]
	Gram stain	positive	TAS [1]
	Cell shape	rod-shaped	TAS [1]
	Motility	non-motile	TAS [1]
	Sporulation	non-sporulating	TAS [1]
	Temperature range	mesophile	TAS [1]
	Optimum temperature	28°C	TAS [1]
	Salinity	0-8% (w/v) NaCl	TAS [1]
MIGS-22	Oxygen requirement	aerobe	TAS [1]
	Carbon source	fructose, galactose, mannose, lactate, ethanol	TAS [1]
	Energy metabolism	chemoorganoheterotrophic	NAS
	Terminal electron acceptor	oxygen	NAS
MIGS-6	Habitat	soil	TAS [2]
MIGS-15	Biotic relationship	free-living	NAS
MIGS-14	Pathogenicity	non-pathogenic	NAS
	Biosafety level	1	NAS
MIGS-23.1	Isolation	not reported
MIGS-4	Geographic location	not reported
MIGS-5	Sample collection time	not reported
MIGS-4.1	Latitude	not reported
MIGS-4.2	Longitude
MIGS-4.3	Depth	not reported
MIGS-4.4	Altitude	not reported

a) 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 of the Gene Ontology project [22].

Figure 2

Scanning electron micrograph of Y-11T. A) Free-floating cells. B) Aggregated cells.

was found to be able to utilize fructose, galactose, mannose, lactate, and ethanol as carbon source, while many others like arginine, aspartate, histidine, methylamine, ethylamine, methanol, galactose, lactose, maltose, sucrose, glycerol, sorbitol, mannitol, inositol, citrate, succinate, malonate, pimelate, m-hydroxybenzoate and p-hydroxybenzoate cannot be used. Optimal growth of strain Y-11T is reported at 28°C. was shown to grow with a salinity between 0 and 8.0% (w/v NaCl), with no growth at 10% [1]. The biochemical characterization revealed positive signals for urease, catalase, and hydrolysis of Tween 80.

Chemotaxonomy

The cell wall of Y-11T contains alanine, glutamic acid, and meso-diaminopimelic acid in a molar ratio of 2.12: 1.00: 0.97. The main components of the cell wall sugars are described to be arabinose, galactose, and mannose in a molar ratio of 2.47: 1.71: 1.00. The glycan moiety of the cell wall was found to contain acetyl residues [1].

In , cellular fatty acids are composed mainly of oleic acid (C18:1ω9c, 31%), palmitic acid (C16:0, 28%), and tuberculostearic acid 10-methyl (C18:0, 21%). The whole-cell methanolysate of strain Y-11 contained mycolic esters [1]. The predominant isoprenoid quinone is menaquinone MK-9(H2).

Genome sequencing and annotation

Genome project history

Y-11T was selected for sequencing as part of a project to define the core genome and pan genome of the non-pathogenic corynebacteria. While not being part of the enomic ncyclopedia of and (GEBA) project [23], sequencing of the type strain will nonetheless aid the GEBA effort. The genome project is deposited in the Genomes OnLine Database [24] and the complete genome sequence is deposited in GenBank. Sequencing, finishing and annotation were performed by the Center of Biotechnology (CeBiTec). 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	Finished
MIGS-28	Libraries used	Two genomic libraries: one 454 pyrosequencing PE library (3.4 kb insert sizes), one Illumina library
MIGS-29	Sequencing platforms	454 GS FLX Titanium, Illumina MiSeq
MIGS-31.2	Sequencing coverage	29.52× Pyrosequencing; 61.71 × SBS
MIGS-30	Assemblers	Newbler version 2.3
MIGS-32	Gene calling method	GeneMark, Glimmer
	INSDC ID	CP003696
	GenBank Date of Release	September 1, 2013 / after publication
	GOLD ID	Gi18852
	NCBI project ID	168617
MIGS-13	Source material identifier	DSM 44721
	Project relevance	Industrial, GEBA

Growth conditions and DNA isolation

strain Y-11T, DSM 44721, was grown aerobically in LB broth (Carl Roth GmbH, Karlsruhe,Germany) at 30 °C. DNA was isolated from ~ 108 cells using the protocol described by Tauch et al. 1995 [25].

Genome sequencing and assembly

The genome was sequenced using a 454 sequencing platform. A standard 3k paired end sequencing library was prepared according to the manufacturers protocol (Roche). The genome was sequenced using the GS-FLX platform with Titanium chemistry, yielding 384,252 total reads, providing 29.52× coverage of the genome. Pyrosequencing reads were assembled using the Newbler assembler v2.3 (Roche). The initial Newbler assembly consisted of 22 contigs in six scaffolds. Analysis of the six scaffolds revealed five that made up the chromosome, while the remaining one contained five copies of the RRN operon that caused the scaffold breaks. The scaffolds were ordered based on alignments to the complete genomes of [26] and subsequent verification by restriction digestion, Southern blotting and hybridization with a 16S rDNA specific probe.

The Phred/Phrap/Consed software package [27-30] was used for sequence assembly and quality assessment in the subsequent finishing process. After the shotgun stage, gaps between contigs were closed by editing in Consed (for repetitive elements) and by PCR with subsequent Sanger sequencing (IIT Biotech GmbH, Bielefeld, Germany). A total of 12 additional reactions were necessary to close gaps not caused by repetitive elements.

To raise the quality of the assembled sequence, Illumina reads were used to correct potential base errors and increase consensus quality. A WGS library was prepared using the Illumina-Compatible Nextera DNA Sample Prep Kit (Epicentre, WI, U.S.A) according to the manufacturer's protocol. The library was sequenced in a 2x 120 bp paired read run on the MiSeq platform, yielding 2,307,926 total reads. Together, the combination of the Illumina and 454 sequencing platforms provided 91.2× coverage of the genome.

Genome annotation

Gene prediction and annotation were done using the PGAAP pipeline [31]. Genes were identified using GeneMark [32], GLIMMER [33], and Prodigal [34]. For annotation, BLAST searches against the NCBI Protein Clusters Database [35] are performed and the annotation is enriched by searches against the Conserved Domain Database [36] and subsequent assignment of coding sequences to COGs. Non-coding genes and miscellaneous features were predicted using tRNAscan-SE [37], Infernal [38], RNAMMer [39], Rfam [40], TMHMM [41], and SignalP [42].

Genome properties

The genome consists of one circular chromosome of 2,751,233 bp (67.02% G+C content) with no additional extrachromosomal elements present. A total of 2,441 genes were predicted, 2,369 of which are protein coding genes. 1,306 (55.13%) of the protein coding genes were assigned to a putative function with the remaining annotated as hypothetical proteins. In addition, 910 protein coding genes belong to 281 paralogous families in this genome, corresponding to a gene content redundancy of 38.41% [Figure 3]. The properties and the statistics of the genome are summarized in Table 3, and Table 4.

Figure 3

Graphical map of the chromosome. From the outside in: Genes on forward strand (colored according to COG categories), Genes on reverse strand (colored according to COG categories), GC content, GC skew.

Table 3

Genome Statistics

Attribute	Value	% of totala
Genome size (bp)	2,751,233	100.00
DNA coding region (bp)	2,441,394	88.74
DNA G+C content (bp)	1,843,810	67.02
Total genes	2,441	100.00
RNA genes	72	2.96
rRNA operons	5
tRNA genes	57	2.34
Protein-coding genes	2,369	97.04
Genes with function prediction (protein)	1,306	55.13
Genes assigned to COGs	1,812	74.23
Genes in paralog clusters	910	38.41
Genes with signal peptides	224	9.54
Genes with transmembrane helices	606	25.58

a) The total is based on either the size of the genome in base pairs or the total number of genes in the annotated genome.

Table 4

Number of genes associated with the general COG functional categories

Code	Value	%age	Description
J	151	6.37	Translation, ribosomal structure and biogenesis
A	1	0.04	RNA processing and modification
K	152	6.42	Transcription
L	136	5.74	Replication, recombination and repair
B	0	0.00	Chromatin structure and dynamics
D	20	0.84	Cell cycle control, cell division, chromosome partitioning
Y	0	0.00	Nuclear structure
V	32	1.35	Defense mechanisms
T	58	2.45	Signal transduction mechanisms
M	81	3.42	Cell wall/membrane biogenesis
N	1	0.04	Cell motility
Z	0	0.00	Cytoskeleton
W	0	0.00	Extracellular structures
U	26	1.10	Intracellular trafficking and secretion, and vesicular transport
O	72	3.04	Posttranslational modification, protein turnover, chaperones
C	127	5.36	Energy production and conversion
G	115	4.85	Carbohydrate transport and metabolism
E	218	9.20	Amino acid transport and metabolism
F	68	2.87	Nucleotide transport and metabolism
H	97	4.09	Coenzyme transport and metabolism
I	121	5.11	Lipid transport and metabolism
P	151	6.37	Inorganic ion transport and metabolism
Q	76	3.21	Secondary metabolites biosynthesis, transport and catabolism
R	274	11.57	General function prediction only
S	138	5.83	Function unknown
-	557	23.51	Not in COGs