Non-contiguous finished genome sequence of Corynebacterium timonense type strain 5401744T
© The Author(s) 2014
Published: 15 June 2014
Corynebacterium timonense strain 5401744T is a member of the genus Corynebacterium which contains Gram-positive bacteria with a high G+C content. It was isolated from the blood of a patient with endocarditis. In this work, we describe a set of features of this organism, together with the complete genome sequence and annotation. The 2,553,575 bp long genome contains 2,401 protein-coding genes and 55 RNA genes, including between 5 and 6 rRNA operons.
KeywordsCorynebacterium timonense Actinobacteria
Corynebacterium timonense strain 5401744T(CSUR P20T=CIP 109424T= CCUG 53856T) is the type strain of C. timonense. This bacterium was isolated from the blood of a patient with endocarditis . The genus Corynebacterium is comprised of Gram-positive facultatively anaerobic bacteria with a high G+C content. It currently contains over 80 members . The combination of chemotaxonomic markers [3,4] and a molecular approach based on 16S rRNA and rpoB gene sequence analyses improved the identification of members of this genus [5–7]. Corynebacterium species have been isolated from human clinical sources [8–14], animal sources [15–18] and the environment [19–21].
Here we present a summary classification and a set of features for C. timonense, together with the description of the non-contiguous finished genomic sequencing and annotation.
Classification and features
The bacterium was first characterized in July 2005, in a 56-year-old man with a history of infective endocarditis. It was isolated from blood culture in the Timone Hospital microbiology laboratory.
Classification and general features of Corynebacterium timonense strain 5501744T
Species Corynebacterium timonense
Aerobic and facultatively anaerobic
Human blood sample
Genome sequencing and annotation
Genome project history
One paired end 3-kb library and one Shotgun library
454 GS FLX Titanium
Newbler version 2.5.3
Gene calling method
EMBL Date of Release
February, 2, 2013
Study of new species isolated in the URMITE
Growth conditions and DNA isolation
C. timonense strain 5401744T, was grown aerobically on 5% sheep blood-enriched Columbia agar at 37°C. Five petri dishes were spread and colonies scraped and resuspended in 3 ml of TE buffer. Three hundred µl of 10% SDS and 150 µl of proteinase K were then added and incubation was performed over-night at 56°C. The DNA was then extracted using the phenol/chloroform method. The yield and the concentration was measured by the Quant-it Picogreen kit (Invitrogen) on the Genios Tecan fluorometer at 182 ng/µl.
Genome sequencing and assembly
Shotgun and 3-kb paired-end sequencing strategies were performed. The shotgun library was constructed with 500 ng of DNA with the GS Rapid library Prep kit (Roche). For the paired-end sequencing, 5 µg of DNA was mechanically fragmented on a Hydroshear device (Digilab) with an enrichment size at 3–4 kb. The DNA fragmentation was visualized using the 2100 BioAnalyzer (Agilent) on a DNA labchip 7500 with an optimal size of 3.5 kb. The library was constructed according to the 454 GS FLX Titanium paired-end protocol. Circularization and nebulization were performed and generated a pattern with an optimal size of 501 bp. After PCR amplification through 15 cycles followed by double size selection, the single stranded paired-end library was then quantified using the Genios fluorometer (Tecan) at 2,540 pg/µL. The library concentration equivalence was calculated as 9.30E+09 molecules/µL. The library was stored at −20°C until further use.
The shotgun and paired-end libraries were clonally-amplified with 2 cpb and 1 cpb in 3 SV-emPCR reactions with the GS Titanium SV emPCR Kit (Lib-L) v2 (Roche). The yields of the emPCR were 11.5% and 7.92%, respectively, in the 5 to 20% range from the Roche procedure. Approximately 790,000 beads for the shotgun application and for the 3kb paired end were loaded on the GS Titanium PicoTiterPlate PTP Kit 70x75 and sequenced with the GS FLX Titanium Sequencing Kit XLR70 (Roche). The run was performed overnight and then analyzed on the cluster through the gsRunBrowser and Newbler assembler (Roche). A total of 252,118 passed filter wells were obtained and generated 37.19 Mb with a length average of 366.5 bp. The passed filter sequences were assembled using Newbler with 90% identity and 40 bp as overlap. The final assembly identified 10 scaffolds and 46 large contigs (>1,500 bp).
Open Reading Frames (ORFs) were predicted using Prodigal  with default parameters but the predicted ORFs were excluded if they spanned a sequencing GAP region. The predicted bacterial protein sequences were searched against the GenBank database  and the Clusters of Orthologous Groups (COG) database  using BLASTP. The tRNAscan-SE tool  was used to find tRNA genes, whereas ribosomal RNAs were found by using RNAmmer .
Transmembrane domains and signal peptides were predicted using TMHMM  and SignalP , respectively. ORFans were identified if their BLASTp E-value was lower than 1e-03 for alignment length greater than 80 amino acids. If alignment lengths were smaller than 80 amino acids, we used an E-value of 1e-05. Such parameter thresholds have been used in previous works to define ORFans.
To estimate the mean level of nucleotide sequence similarity at the genome level between C. timonense and the corynebacterium genomes available to date, we compared the ORFs only using comparison sequence based in the server RAST  at a query coverage of ≥60% and a minimum nucleotide length of 100 bp.
Nucleotide content and gene count levels of the genome
% of totala
Genome size (bp)
DNA coding region (bp)
DNA G+C content (bp)
Genes with function prediction
Genes assigned to COGs
Genes with peptide signals
Genes with transmembrane helices
Number of genes associated with the 25 general COG functional categories
RNA processing and modification
Replication, recombination and repair
Chromatin structure and dynamics
Cell cycle control, mitosis and meiosis
Signal transduction mechanisms
Cell wall/membrane biogenesis
Intracellular trafficking and secretion
Posttranslational modification, protein turnover, chaperones
Energy production and conversion
Carbohydrate transport and metabolism
Amino acid transport and metabolism
Nucleotide transport and metabolism
Coenzyme transport and metabolism
Lipid transport and metabolism
Inorganic ion transport and metabolism
Secondary metabolites biosynthesis, transport and catabolism
General function prediction only
Not in COGs
Comparison with other Corynebacterium genomes
Comparison of C. timonense characteristics with Corynebacterium whole genome characteristics.
Genome size (Mb)
Number of predicted genes
Prophage genome properties
Prophage Finder  and PHAST  were used to identify potential proviruses in C. timonense strain 5401744T genome. The bacteria contains at least one genetic element of around 40.3 kb (with a GC content of 64.9%), we named CT1, on contigs 6–7. A total of 53 open reading frames (ORFs) were recovered from CT1, that were longer than 55 amino acids and most of them (44) encode proteins sharing a high identity with proteins found in Actinomycetales order viruses. The preliminary annotation of CT1 was performed and the majority of the putative genes (41) encode hypothetical proteins. The ORFs with an attributed function (12) encode proteins involved in DNA packaging, cell lysis, tail structural components and assembly, head structural components and assembly, lysogeny control, DNA replication, recombination and modification. 47 of the ORFs are located on one strand and 6 on the opposite strand.
The authors thank Mr. Julien Paganini at Xegen Company (www.xegen.fr) for automating the genomic annotation process and Laetitia Pizzo for her technical assistance.
- Merhej V, Falsen E, Raoult D, Roux V. Corynebacterium timonense sp. nov. and Corynebacterium massiliense sp. nov., isolated from human blood and human articular hip fluid. Int J Syst Evol Microbiol 2009; 59:1953–1959. PubMed http://dx.doi.org/10.1099/ijs.0.005827-0View ArticlePubMedGoogle Scholar
- Euzéby JP. List of Bacterial Names with Standing in Nomenclature: a folder available on the Internet. Int J Syst Bacteriol 1997; 47:590–592. PubMed http://dx.doi.org/10.1099/00207713-472-590View ArticlePubMedGoogle Scholar
- Collins MD, Goodfellow M, Minnikin DE. A survey of the structures of mycolic acids in Corynebacterium and related taxa. J Gen Microbiol 1982; 128:129–149. PubMedPubMedGoogle Scholar
- von Graevenitz A, Punter V, Gruner E, Pfyffer GE, Funke G. Identification of coryneform and other gram-positive rods with several methods. APMIS 1994; 102:381–389. PubMed http://dx.doi.org/10.1111/j.1699-0463.1994.tb04887.xView ArticleGoogle Scholar
- Khamis A, Raoult D, La Scola B. rpoB gene sequencing for identification of Corynebacterium species. J Clin Microbiol 2004; 42:3925–3931. PubMed http://dx.doi.org/10.1128/JCM.42.9.3925-3931.2004PubMed CentralView ArticlePubMedGoogle Scholar
- Pascual C, Lawson PA, Farrow JA, Gimenez MN, Collins MD. Phylogenetic analysis of the genus Corynebacterium based on 16S rRNA gene sequences. Int J Syst Bacteriol 1995; 45:724–728. PubMed http://dx.doi.org/10.1099/00207713-45-4-724View ArticlePubMedGoogle Scholar
- Rainy R, Riegel P, Boiron P, Monteil H, Christen R. Phylogeny of the genus Corynebacterium deduced from analyses of small-subunit ribosomal DNA sequences. Int J Syst Bacteriol 1995; 45:740–746. PubMed http://dx.doi.org/10.1099/00207713-45-4-740View ArticleGoogle Scholar
- Feurer C, Clermont D, Bimet F, Candrea A, Jackson M, Glaser P, Bizet C, Dauga C. Taxonomic characterization of nine strains isolated from clinical and environmental specimens, and proposal of Corynebacterium tuberculostearicum sp. nov. Int J Syst Evol Microbiol 2004; 54:1055–1061. PubMed http://dx.doi.org/10.1099/ijs.0.02907-0View ArticlePubMedGoogle Scholar
- Funke G, Lawson PA, Collins MD. Heterogeneity within human-derived centers for disease control and prevention (CDC) coryneform group ANF-1-like bacteria and description of Corynebacterium auris sp. nov. Int J Syst Bacteriol 1995; 45:735–739. PubMed http://dx.doi.org/10.1099/00207713-45-4-735View ArticlePubMedGoogle Scholar
- Funke G, Hutson RA, Hilleringmann M, Heizmann WR, Collins MD. Corynebacterium lipophiloflavum sp. nov. isolated from a patient with bacterial vaginosis. FEMS Microbiol Lett 1997; 150:219–224. PubMed http://dx.doi.org/10.1016/S0378-1097(97)001183View ArticlePubMedGoogle Scholar
- Funke G, Lawson PA, Collins MD. Corynebacterium mucifaciens sp. nov., an unusual species from human clinical material. Int J Syst Bacteriol 1997; 47:952–957. PubMed http://dx.doi.org/10.1099/00207713-47-4-952View ArticlePubMedGoogle Scholar
- Funke G, Osorio CR, Frei R, Riegel P, Collins MD. Corynebacterium confusum sp. nov., isolated from human clinical specimens. Int J Syst Bacteriol 1998; 48:1291–1296. PubMed http://dx.doi.org/10.1099/00207713-48-4-1291View ArticlePubMedGoogle Scholar
- Riegel P, Creti R, Mattei R, Nieri A, von Hunolstein C. Isolation of Corynebacterium tuscaniae sp. nov. from blood cultures of a patient with endocarditis. J Clin Microbiol 2006; 44:307–312. PubMed http://dx.doi.org/10.1128/JCM.44.2.307-312.2006PubMed CentralView ArticlePubMedGoogle Scholar
- Yassin AF. Corynebacterium ureicelerivorans sp. nov., a lipophilic bacterium islated from blood culture. Int J Syst Evol Microbiol 2007; 57:1200–1203. PubMed http://dx.doi.org/10.1099/ijs.0.64832-0View ArticlePubMedGoogle Scholar
- Collins MD, Hoyles L, Foster G, Sjoden B, Falsen E. Corynebacterium capitovis sp. nov., from a sheep. Int J Syst Evol Microbiol 2001; 51:857–860. PubMed http://dx.doi.org/10.1099/00207713-513-857View ArticlePubMedGoogle Scholar
- Collins MD, Hoyles L, Foster G, Falsen E. Corynebacterium caspium sp. nov., from a Caspian seal (Phoca caspica). Int J Syst Evol Microbiol 2004; 54:925–928. PubMed http://dx.doi.org/10.1099/ijs.0.02950-0View ArticlePubMedGoogle Scholar
- Fernández-Garayzábal M, Collins MD. Corynebacterium ciconiae sp. nov., isolated from the trachea of black storks (Ciconia nigra). Int J Syst Evol Microbiol 2004; 54:2191–2195. PubMed http://dx.doi.org/10.1099/ijs.0.63165-0View ArticlePubMedGoogle Scholar
- Vela AI, Mateos A, Collins MD, Briones V, Hutson RA, Dominguez L, Fernandez-Garayzabal JF. Corynebacterium suicordis sp. nov., from pigs. Int J Syst Evol Microbiol 2003; 53:2027–2031. PubMed http://dx.doi.org/10.1099/ijs.0.02645-0View ArticlePubMedGoogle Scholar
- Chen HH, Li WJ, Tang SK, Kroppenstedt RM, Stackebrandt E, Xu LH, Jiang CL. Corynebacterium halotolerans sp. nov., isolated from saline soil in the west of China. Int J Syst Evol Microbiol 2004; 54:779–782. PubMed http://dx.doi.org/10.1099/ijs.0.02919-0View ArticlePubMedGoogle Scholar
- Fudou R, Jojima Y, Seto A, Yamada K, Kimura E, Nakamatsu T, Hiraishi A, Yamanaka S. Corynebacterium efficiens sp. nov., a glutamicacid-producing species from soil and vegetables. Int J Syst Evol Microbiol 2002; 52:1127–1131. PubMed http://dx.doi.org/10.1099/ijs.0.02086-0PubMedGoogle Scholar
- Zhou Z, Yuan M, Tang R, Chen M, Lin M, Zhang W. Corynebacterium deserti sp. nov., isolated from desert sand. Int J Syst Evol Microbiol 2012; 62:791–794. PubMed http://dx.doi.org/10.1099/ijs.0.030429-0View ArticlePubMedGoogle Scholar
- Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: Molecular Evolutionary Genetics Analysis using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Mol Biol Evol 2011; 28:2731–2739. PubMed http://dx.doi.org/10.1093/molbev/msr121PubMed CentralView ArticlePubMedGoogle Scholar
- Woese CR, Kandler O, Wheelis ML. Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc Natl Acad Sci USA 1990; 87:4576–4579. PubMed http://dx.doi.org/10.1073/pnas.87.12.4576PubMed CentralView ArticlePubMedGoogle Scholar
- Garrity GM, Holt JG. The Road Map to the Manual. In: Garrity GM, Boone DR, Castenholz RW (eds), Bergey’s Manual of Systematic Bacteriology, Second Edition, Volume 1, Springer, New York, 2001, p.119–169.View ArticleGoogle Scholar