Open Access

Non-contiguous finished genome sequence of Corynebacterium timonense type strain 5401744T

  • Véronique Roux
  • , Catherine Robert
  • and Didier Raoult

DOI: 10.4056/sigs.4277954

Received: 25 April 2014

Accepted: 25 April 2014

Published: 15 June 2014

Abstract

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.

Keywords:

Corynebacterium timonenseActinobacteria

Introduction

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 [1]. The genus Corynebacterium is comprised of Gram-positive facultatively anaerobic bacteria with a high G+C content. It currently contains over 80 members [2]. 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 16S rRNA gene sequence of C. timonense strain 5401744T was compared with sequences deposited in the Genbank database, confirming the initial taxonomic classification. Figure 1 shows the phylogenetic neighborhood of C. timonense in a 16S rRNA based tree. 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.

Figure 1

Part of phylogenetic tree highlighting the position of Corynebacterium timonense strain 5401744T relative to other type strains within the Corynebacterium genus by comparison of 16S rRNA gene sequences. GenBank accession numbers are indicated in parentheses. Sequences were aligned using CLUSTALX, and phylogenetic inferences obtained using the neighbor joining method within the MEGA 5 software [22]. Numbers at the nodes are percentages of bootstrap values (≥ 50%) obtained by repeating the analysis 1,000 times to generate a majority consensus tree. Solibacillus silvestris was used as outgroup. The scale bar represents 0.005 nucleotide change per nucleotide position.

Cells are rod-shaped that occur as single cells, in pairs or in small clusters, 0.6-2.1 µm long and 0.4-0.6 µm wide. Optimal growth of strain 5401744T occurs at 37°C with range for growth between 25 and 50 °C. After 24 hours growth on blood sheep agar at 37°C, surface colonies are circular, yellow colored, glistening and up to 1-2 mm in diameter. Carbon sources utilized include glucose and ribose. Activities of catalase, pyrazinamidase, alkaline phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase and acid phosphatase are detected. The fatty acid profile is characterized by the predominance of C18:1 ω9c (36.4%), C17:1 ω9c (27.1%), C16:0 (10.9%) and C18:0 (6.1%). Tuberculostearic acid is not detected. The size and ultrastructure of cells were determined by negative staining transmission electron microscopy. The rods were 0.6-2.1 μm long and 0.4-0.6 μm wide (Figure 2). Table 1 presents the classification and features of the organism.

Figure 2

Transmission electron micrograph of C. timonense strain 5401744T, using a Morgani 268D (Philips) at an operating voltage of 60kV. The scale bar represents 500 nm.

Table 1

Classification and general features of Corynebacterium timonense strain 5501744T

MIGS ID

      Property

       Term

       Evidence codea

       Domain Bacteria

       TAS [23]

       Phylum Actinobacteria

       TAS [24]

       Class Actinobacteria

       TAS [25]

      Current classification

       Order Actinomycetales

       TAS [25-28]

       Family Corynebacteriaceae

       TAS [25,26,28,29]

       Genus Corynebacterium

       TAS [26,30,31]

       Species Corynebacterium timonense

       TAS [1]

       Strain 5401744T

       TAS [1]

      Gram stain

       Positive

       IDA

      Cell shape

       Pleomorphic forms

       IDA

      Motility

       Non-motile

       IDA

      Sporulation

       Non-sporulating

       IDA

      Temperature range

       Mesophile

       IDA

      Optimum temperature

       37°C

       IDA

MIGS-6.3

      Salinity

       Not reported

       IDA

MIGS-22

      Oxygen requirement

       Aerobic and facultatively anaerobic

       IDA

      Carbon source

       Glucose, ribose

       NAS

      Energy source

       Chemoorganotroph

       NAS

MIGS-6

      Habitat

       Host

       IDA

MIGS-15

      Biotic relationship

       Free living

       IDA

MIGS-14

      Pathogenicity      Biosafety level      Isolation

       Unknown       2       Human blood sample

       NAS

MIGS-4

      Geographic location

       Marseille, France

       IDA

MIGS-5

      Sample collection time

       July 2005

       IDA

MIGS-4.1

      Latitude

       43°18 N

       IDA

MIGS-4.1

      Longitude

       5°23 E

       IDA

MIGS-4.3

      Depth

       Surface

       IDA

MIGS-4.4

      Altitude

       21 m above sea level

       IDA

Evidence codes - IDA: Inferred from Direct Assay; 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 [32]. If the evidence is IDA, then the property was directly observed for a live isolate by one of the authors or an expert mentioned in the acknowledgements.

Genome sequencing and annotation

Genome project history

The organism was selected for sequencing on the basis of its phylogenetic position and 16S rRNA similarity to other members of the genus Corynebacterium, and is part of a study of the new species characterized in our laboratory. A summary of the project information is shown in Table 2. The EMBL accession number is CAJP01000000 and consists of 58 contigs (≥ 500 bp) and 10 scaffolds (> 4,375 bp). Table 2 shows the project information and its association with MIGS version 2.0 compliance.

Table 2

Project information

MIGS ID

      Property

       Term

MIGS-31

      Finishing quality

       High-quality draft

MIGS-28

      Libraries used

       One paired end 3-kb library and one Shotgun library

MIGS-29

      Sequencing platforms

       454 GS FLX Titanium

MIGS-31.2

      Fold coverage

       37.2×

MIGS-30

      Assemblers

       Newbler version 2.5.3

MIGS-32

      Gene calling method

       Prodigal

      EMBL ID

       CAJP01000000

      EMBL Date of Release

       February, 2, 2013

      Project relevance

       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).

Genome annotation

Open Reading Frames (ORFs) were predicted using Prodigal [33] 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 [34] and the Clusters of Orthologous Groups (COG) database [35] using BLASTP. The tRNAscan-SE tool [36] was used to find tRNA genes, whereas ribosomal RNAs were found by using RNAmmer [37].

Transmembrane domains and signal peptides were predicted using TMHMM [38] and SignalP [39], 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 [40] at a query coverage of ≥60% and a minimum nucleotide length of 100 bp.

Genome properties

The genome is 2,553,575 bp long with a 66.85% GC content (Table 3, Figure 3). Of the 2,456 predicted genes, 2,401 were protein-coding genes, and 55 were RNAs. A total of 1,779 genes (74.09%) were assigned a putative function,and 116 genes were identified as ORFans (4,83%). The remaining genes were annotated as hypothetical proteins (369 genes (15,37%)). The remaining genes were annotated as either hypothetical proteins or proteins of unknown functions. The distribution of genes into COGs functional categories is presented in Table 4. The properties and the statistics of the genome are summarized in Tables 3 and 4.

Table 3

Nucleotide content and gene count levels of the genome

Attribute

      Value

     % of totala

Genome size (bp)

      2,553,575

     100

DNA coding region (bp)

      2,289,384

     89.65

DNA G+C content (bp)

      1,707,056

     66.85

Total genes

      2,456

     100

RNA genes

      55

     2.24

Protein-coding genes

      2,401

     97.76

Genes with function prediction

      1,779

     74.09

Genes assigned to COGs

      1,753

     73.01

Genes with peptide signals

      353

     14.7

Genes with transmembrane helices

      550

     22.91

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

Figure 3

Graphical circular map of Corynebacterium timonense genome. From outside to the center: Contigs (red / grey), COG category of genes on the forward strand (three circles), genes on forward strand (blue circle), genes on the reverse strand (red circle), COG category on the reverse strand (three circles), GC content.

Table 4

Number of genes associated with the 25 general COG functional categories

Code

       Value

       %age

       Description

J

       148

       6.16

       Translation

A

       1

       0.04

       RNA processing and modification

K

       136

       5.66

       Transcription

L

       179

       7.46

       Replication, recombination and repair

B

       0

       0

       Chromatin structure and dynamics

D

       17

       0.71

       Cell cycle control, mitosis and meiosis

Y

       0

       0

       Nuclear structure

V

       45

       1.87

       Defense mechanisms

T

       62

       2.58

       Signal transduction mechanisms

M

       89

       3.71

       Cell wall/membrane biogenesis

N

       2

       0.08

       Cell motility

Z

       0

       0

       Cytoskeleton

W

       0

       0

       Extracellular structures

U

       27

       1.12

       Intracellular trafficking and secretion

O

       60

       2.50

       Posttranslational modification, protein turnover, chaperones

C

       97

       4.04

       Energy production and conversion

G

       121

       5.04

       Carbohydrate transport and metabolism

E

       205

       8.54

       Amino acid transport and metabolism

F

       65

       2.71

       Nucleotide transport and metabolism

H

       100

       4.16

       Coenzyme transport and metabolism

I

       78

       3.25

       Lipid transport and metabolism

P

       176

       7.33

       Inorganic ion transport and metabolism

Q

       46

       1.92

       Secondary metabolites biosynthesis, transport and catabolism

R

       233

       9.7

       General function prediction only

S

       137

       5.71

       Function unknown

X

       648

       26.99

       Not in COGs

The total is based on the total number of protein coding genes in the annotated genome.

Comparison with other Corynebacterium genomes

To date, 13 genome of species belonging to the genus Corynebacterium were sequenced. The size of the whole genome was between 2.32 Mb and 3.43 Mb (Table 5). The gene number was correlated with the genome size and was between 2,187 and 3,131. The G+C content of the genome was less than 60% for C. diphteriae, C. glutamicum, C. kroppenstedtii, C. pseudotuberculosis, C. resistens and C. ulcerans but was more than 60% for C. aurimucosum, C. efficiens, C. genitalium, C. halotolerans, C. jeikeium, C. timonense, C. urealyticum and C. variabile. C. timonense shared a mean sequence similarity of 72.05% (60-99.01%), 72.15% (60.09-97.54%), 74.63% (60-98.37%), 71.83% (60-98.85%), 72.34% (60-98.02%) and 71.70% (60-97.03%) with C. diphteriae, C. efficiens, C. genitalium, C. glutamicum, C. jeikeium and C. urealyticum, respectively.

Table 5

Comparison of C. timonense characteristics with Corynebacterium whole genome characteristics.

Species

      Genome size (Mb)

      G+C%

      Number of predicted genes

C. arimucosumC. diphteriaeC. efficiensC. genitaliumC. glutamicumC. halotoleransC. jeikeiumC. kroppenstedtiiC. pseudotuberculosisC. resistensC. timonenseC. ulceransC. urealyticumC. variabile

      2.82      2.48      3.22      2.35      3.31      3.22      2.48      2.45      2.32      2.60      2.55      2.56      2.36      3.43

              60.5              53.5              62.9              62.7              53.9              68.3              61.4              57.5              52.2              57.1              66.7              53.4              64.2              67.1

2,6302,3923,0642,2903,1222,9302,1812,0832,1872,2302,4562,3552,0453,131

Prophage genome properties

Prophage Finder [41] and PHAST [42] 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.

Declarations

Acknowledgements

The authors thank Mr. Julien Paganini at Xegen Company (Web Site) for automating the genomic annotation process and Laetitia Pizzo for her technical assistance.


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References

  1. Merhej V, Falsen E, Raoult D and 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; View ArticlePubMedWeb Site
  2. Euzéby JP. List of Bacterial Names with Standing in Nomenclature: a folder available on the Internet. Int J Syst Bacteriol. 1997; 47:590-592; View ArticlePubMedWeb Site
  3. Collins MD, Goodfellow M and Minnikin DE. A survey of the structures of mycolic acids in Corynebacterium and related taxa. J Gen Microbiol. 1982; 128:129-149; .PubMed
  4. von Graevenitz A, Punter V, Gruner E, Pfyffer GE and Funke G. Identification of coryneform and other gram-positive rods with several methods. APMIS. 1994; 102:381-389; View ArticlePubMedWeb Site
  5. Khamis A, Raoult D and La Scola B. rpoB gene sequencing for identification of Corynebacterium species. J Clin Microbiol. 2004; 42:3925-3931; View ArticlePubMedWeb Site
  6. Pascual C, Lawson PA, Farrow JA, Gimenez MN and Collins MD. Phylogenetic analysis of the genus Corynebacterium based on 16S rRNA gene sequences. Int J Syst Bacteriol. 1995; 45:724-728; View ArticlePubMedWeb Site
  7. Ruimy R, Riegel P, Boiron P, Monteil H and Christen R. Phylogeny of the genus Corynebacterium deduced from analyses of small-subunit ribosomal DNA sequences. Int J Syst Bacteriol. 1995; 45:740-746; View ArticlePubMedWeb Site
  8. Feurer C, Clermont D, Bimet F, Candrea A, Jackson M, Glaser P, Bizet C and 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; View ArticlePubMedWeb Site
  9. Funke G, Lawson PA and 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; View ArticlePubMedWeb Site
  10. Funke G, Hutson RA, Hilleringmann M, Heizmann WR and Collins MD. Corynebacterium lipophiloflavum sp. nov. isolated from a patient with bacterial vaginosis. FEMS Microbiol Lett. 1997; 150:219-224; View ArticlePubMedWeb Site
  11. Funke G, Lawson PA and Collins MD. Corynebacterium mucifaciens sp. nov., an unusual species from human clinical material. Int J Syst Bacteriol. 1997; 47:952-957; View ArticlePubMedWeb Site
  12. Funke G, Osorio CR, Frei R, Riegel P and Collins MD. Corynebacterium confusum sp. nov., isolated from human clinical specimens. Int J Syst Bacteriol. 1998; 48:1291-1296; View ArticlePubMedWeb Site
  13. Riegel P, Creti R, Mattei R, Nieri A and von Hunolstein C. Isolation of Corynebacterium tuscaniae sp. nov. from blood cultures of a patient with endocarditis. J Clin Microbiol. 2006; 44:307-312; View ArticlePubMedWeb Site
  14. Yassin AF. Corynebacterium ureicelerivorans sp. nov., a lipophilic bacterium islated from blood culture. Int J Syst Evol Microbiol. 2007; 57:1200-1203; View ArticlePubMedWeb Site
  15. Collins MD, Hoyles L, Foster G, Sjoden B and Falsen E. Corynebacterium capitovis sp. nov., from a sheep. Int J Syst Evol Microbiol. 2001; 51:857-860; View ArticlePubMedWeb Site
  16. Collins MD, Hoyles L, Foster G and Falsen E. Corynebacterium caspium sp. nov., from a Caspian seal (Phoca caspica). Int J Syst Evol Microbiol. 2004; 54:925-928; View ArticlePubMedWeb Site
  17. Fernández-Garayzábal M and Collins MD. Corynebacterium ciconiae sp. nov., isolated from the trachea of black storks (Ciconia nigra). Int J Syst Evol Microbiol. 2004; 54:2191-2195; View ArticlePubMedWeb Site
  18. Vela AI, Mateos A, Collins MD, Briones V, Hutson RA, Dominguez L and Fernandez-Garayzabal JF. Corynebacterium suicordis sp. nov., from pigs. Int J Syst Evol Microbiol. 2003; 53:2027-2031; View ArticlePubMedWeb Site
  19. Chen HH, Li WJ, Tang SK, Kroppenstedt RM, Stackebrandt E, Xu LH and Jiang CL. Corynebacterium halotolerans sp. nov., isolated from saline soil in the west of China. Int J Syst Evol Microbiol. 2004; 54:779-782; View ArticlePubMedWeb Site
  20. Fudou R, Jojima Y, Seto A, Yamada K, Kimura E, Nakamatsu T, Hiraishi A and Yamanaka S. Corynebacterium efficiens sp. nov., a glutamic-acid-producing species from soil and vegetables. Int J Syst Evol Microbiol. 2002; 52:1127-1131; View ArticlePubMedWeb Site
  21. Zhou Z, Yuan M, Tang R, Chen M, Lin M and Zhang W. Corynebacterium deserti sp. nov., isolated from desert sand. Int J Syst Evol Microbiol. 2012; 62:791-794; View ArticlePubMedWeb Site
  22. Tamura K, Peterson D, Peterson N, Stecher G, Nei M and Kumar S. MEGA5: Molecular Evolutionary Genetics Analysis using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Mol Biol Evol. 2011; 28:2731-2739; View ArticlePubMedWeb Site
  23. Woese CR, Kandler O and 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; View ArticlePubMedWeb Site
  24. 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.
  25. Stackebrandt E, Rainey FA and Ward-Rainey NL. Proposal for a New Hierarchic Classification System, Actinobacteria classis nov. Int J Syst Bacteriol. 1997; 47:479-491;. View Article
  26. Skerman VBD, McGowan V and Sneath PHA. Approved Lists of Bacterial Names. Int J Syst Bacteriol. 1980; 30:225-420;. View Article
  27. Buchanan RE. Studies in the nomenclature and classification of bacteria. II. The primary subdivisions of the Schizomycetes. J Bacteriol. 1917; 2:155-164; .PubMed
  28. Zhi XY, Li WJ and Stackebrandt E. An update of the structure and 16S rRNA gene sequence-based definition of higher ranks of the class Actinobacteria, with the proposal of two new suborders and four new families and emended descriptions of the existing higher taxa. Int J Syst Evol Microbiol. 2009; 59:589-608; View ArticlePubMedWeb Site
  29. Lehmann KB, Neumann R. Lehmann's Medizin, Handatlanten. X Atlas und Grundriss der Bakteriologie und Lehrbuch der speziellen bakteriologischen Diagnostik., Fourth Edition, Volume 2, J.F. Lehmann, München, 1907, p. 270.
  30. Lehmann KB, Neumann R. Atlas und Grundriss der Bakteriologie und Lehrbuch der speziellen bakteriologischen Diagnostik, First Edition, J.F. Lehmann, München, 1896, p. 1-448.
  31. Bernard KA, Wiebe D, Burdz T, Reimer A, Ng B, Singh C, Schindle S and Pacheco AL. Assignment of Brevibacterium stationis (ZoBell and Upham 1944) Breed 1953 to the genus Corynebacterium, as Corynebacterium stationis comb.nov., and emended description of the genus Corynebacterium to include isolates that can alkalinize citrate. Int J Syst Evol Microbiol. 2010; 60:874-879; View ArticlePubMedWeb Site
  32. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS and Eppig JT. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet. 2000; 25:25-29; View ArticlePubMedWeb Site
  33. Prodigal Web Site
  34. GenBank database. Web Site
  35. Tatusov RL, Galperin MY, Natale DA and Koonin EV. The COG database: a tool for genome-scale analysis of protein functions and evolution. Nucleic Acids Res. 2000; 28:33-36; View ArticlePubMedWeb Site
  36. Lowe TM and Eddy SR. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res. 1997; 25:955-964; . View ArticlePubMed
  37. Lagesen K, Hallin P, Rodland EA, Staerfeldt HH, Rognes T and Ussery DW. RNAmmer: consistent and rapid annotation of ribosomal RNA genes. Nucleic Acids Res. 2007; 35:3100-3108; View ArticlePubMedWeb Site
  38. Krogh A, Larsson B, von Heijni G and Sonnhammer EL. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol. 2001; 305:567-580; View ArticlePubMedWeb Site
  39. Bendtsen JD, Nielsen H, von Heijne G and Brunak S. Improved prediction of signal peptides: SignalP 3.0. J Mol Biol. 2004; 340:783-795; View ArticlePubMedWeb Site
  40. Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, Formsma K, Gerdes S, Glass EM and Kubal M. The RAST Server: Rapid Annotations using Subsystems Technology. BMC Genomics. 2008; 9:75-89; View ArticlePubMedWeb Site
  41. Bose M and Barber RD. Prophage Finder: a prophage loci prediction tool for prokaryotic genome sequences. In Silico Biol. 2006; 6:223-227; .PubMed
  42. Zhou Y, Liang Y, Lynch KH, Dennis JJ and Wishart DS. PHAST: a fast phage search tool. Nucleic Acids Res. 2011; 39:W347-W352; View ArticlePubMedWeb Site