Open Access

Complete genome sequence of Serratia plymuthica strain AS12

  • Saraswoti Neupane
  • , Roger D. Finlay
  • , Sadhna Alström
  • , Lynne Goodwin,
  • , Nikos C. Kyrpides
  • , Susan Lucas
  • , Alla Lapidus
  • , David Bruce,
  • , Sam Pitluck
  • , Lin Peters
  • , Galina Ovchinnikova
  • , Olga Chertkov,
  • , James Han
  • , Cliff Han,
  • , Roxanne Tapia,
  • , John C. Detter,
  • , Miriam Land,
  • , Loren Hauser,
  • , Jan-Fang Cheng
  • , Natalia Ivanova
  • , Ioanna Pagani
  • , Hans-Peter Klenk
  • , Tanja Woyke
  • and Nils Högberg

DOI: 10.4056/sigs.2705996

Received: 01 May 2012

Published: 25 May 2012

Abstract

A plant-associated member of the family Enterobacteriaceae, Serratia plymuthica strain AS12 was isolated from rapeseed roots. It is of scientific interest because it promotes plant growth and inhibits plant pathogens. The genome of S. plymuthica AS12 comprises a 5,443,009 bp long circular chromosome, which consists of 4,952 protein-coding genes, 87 tRNA genes and 7 rRNA operons. This genome was sequenced within the 2010 DOE-JGI Community Sequencing Program (CSP2010) as part of the project entitled “Genomics of four rapeseed plant growth promoting bacteria with antagonistic effect on plant pathogens”.

Keywords:

Facultative anaerobegram-negativemotilenon-sporulatingmesophilicchemoorganotrophicagricultureEnterobacteriaceaeCSP 2010

Introduction

Plant associated Serratia species are commonly found as free-living bacteria in rhizosphere soil and as endophytes within plant roots. They include strains with the ability to stimulate plant growth and to inhibit the growth of soil borne pathogens of economically important agricultural plants [1-3]. One Serratia strain, S. plymuthica HRO-C48, is successfully used as an alternative to chemical agents for control of soil-borne fungal diseases in different crops such as strawberry and rapeseed [3,4]. Its ability to degrade chitin, a fungal cell wall component, may be responsible for antifungal activity, whereas the production of the plant hormone indole-3-acetic acid (IAA) could be involved in plant growth promotion [3]. S. plymuthica AS12 has chitinolytic activity and was isolated from rapeseed roots from Uppsala, Sweden in 1998 [5]. The reason for our interest in S. plymuthica AS12 is its ability to inhibit Verticillium longisporum (earlier V. dahliae), a soil borne fungal pathogen of rapeseed, thus promoting the rapeseed growth both directly and indirectly [5]. Here we present a description of the complete genome of S. plymuthica AS12 and its annotation.

Classification and features

A representative 16S rRNA gene sequence of the strain AS12 genome was used for comparison using NCBI BLAST [6] under default settings with the most recent databases. The relative frequencies of taxa and BLAST scores were determined. The most frequently occurring genus is Serratia where some of the ‘hits’ share a 100% identity. When considering high-scoring segment pairs (HSPs) from the best 250 hits, the most frequent matches were Serratia sp. (17.2%) with a maximum identity of 97-100%, while S. plymuthica (5.2%) had a maximum identity of 97-100%, S. proteomaculans (4.8%) with a maximum identity of 97-99%, S. marcescens (4.8%) with a maximum identity of 96-97% and different strains of Rahnella (7%) with a maximum identity of 97-98%.

A phylogenetic tree (Figure 1) was constructed using 16S rRNA sequences of S. plymuthica AS12 with other genera within the family Enterobacteriaceae including two species within the genus Serratia. The tree shows the position of S. plymuthica AS12 within the genus Serratia and its distinct clustering with S. plymuthica, which was confirmed by digital DNA-DNA hybridization values [11] above 70% with the (unpublished) draft genome sequence of the S. plymuthica type strain Breed K-7T from a DSM 4540 culture as well as with the complete genome sequence of S. plymuthica AS9 [12] using the GGDC web server [13].

Figure 1

Phylogenetic tree highlighting the position of S. plymuthica AS12 in relation to selected Serratia strains and other genera within the family Enterobacteriaceae. The tree was based on 1,535 characters of the 16S rRNA gene sequence aligned in ClustalW2 [7]. The tree was inferred under the maximum likelihood criterion using MEGA5 software [8] and rooted with Pseudomonas trivialis (a member of the Pseudomonadaceae family). The branches are mapped by the expected number of substitutions per site. The numbers above the branches are support values from 1,000 bootstrap replicates if larger than 60% [9]. Lineages with genome sequences registered in GOLD [10] are shown in blue.

The cells of strain AS12 stain Gram-negative and are rod shaped, 1-2 µm long, 0.5-0.7 µm wide (Figure 2 and Table 1) and motile. The culture forms red to pink colored colonies of 1-2 mm diameter on tryptic soy agar and potato dextrose agar, but the colony color depends on different factors such as the growth substrate, pH of the medium and growth temperature. The organism is a facultative anaerobe and grows at temperatures ranging from 4 °C - 40 °C and within a pH range of 4 - 10. It has the ability to utilize a wide range of carbon sources such as glucose, sucrose, succinate, mannitol and arabinose. It also has cellulolytic, phospholytic, chitinolytic and proteolytic activity [5]. The strain is deposited in the Culture Collection, University of Göteborg, Sweden (CCUG) as Serratia sp. AS12 (= CCUG 61397).

Figure 2

Scanning electron micrograph of S. plymuthica AS12

Table 1

Classification and general features of S. plymuthica AS12 according to MIGS recommendations [14]

MIGS ID

    Property

    Term

    Evidence codea

    Current classification

    Domain Bacteria

    TAS [15]

    Phylum Proteobacteria

    TAS [16]

    Class Gammaproteobacteria

    TAS [17,18]

    Order “Enterobacteriales

    TAS [19]

    Family Enterobacteriaceae

    TAS [20-22]

    Genus Serratia

    TAS [20,23,24]

    Species Serratia plymuthica

    TAS [20,25]

    Strain AS12

    IDA

    Gram stain

    Negative

    IDA

    Cell shape

    Rod-shaped

    IDA

    Motility

    Motile

    IDA

    Sporulation

    Non-sporulating

    IDA

    Temperature range

    Mesophilic, 4 – 40°C

    IDA

    Optimum temperature

    28°C

    IDA

    Carbon source

    Glucose, sucrose, fructose, succinate, trehalose, mannitol, inositol, arabinose

    IDA

    Energy metabolism

    Chemoorganotrophic

    IDA

MIGS-6

    Habitat

    Rapeseed roots

    IDA

MIGS-6.3

    Salinity

    Medium

    IDA

MIGS-22

    Oxygen

    Facultative

    IDA

MIGS-15

    Biotic relationship

    Endophyte

    TAS [5]

MIGS-14

    Pathogenicity

    None

    NAS

    Biosafety level

    1+

    TAS [26]

MIGS-4

    Geographic location

    Uppsala, Sweden

    NAS

MIGS-5

    Sample collection time

    Summer 1998

    NAS

MIGS-4.1

    Latitude

    59.8

    NAS

MIGS-4.2

    Longitude

    17.65

    NAS

MIGS-4.3

    Depth

    0.1 m

    NAS

MIGS-4.4

    Altitude

    24-25 m

    NAS

a) 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 [27]. If the evidence code is IDA, then the property should have been directly observed, for the purpose of this specific publication, for a live isolate by one of the authors, or an expert or reputable institution mentioned in the acknowledgements.

Chemotaxonomy

The cells of S. plymuthica AS12 contain a mixture of saturated and unsaturated fatty acids. The dominant fatty acids in strain AS12 are C16:0 (22.94%), C16:1ω7c (17.08%), C18:1ω7c (19.65%), C14:0 (5.11%), along with other minor fatty acid components. No information is available for other compounds. Previously it has been shown that Serratia spp. contain a mixture of C14:0, C16:0, C16:1 and C18:1+2 fatty acids in which 50-80% of the total fatty acid in the cell is C14:0 and others each less than 3% [28]. This is consistent with the observation that C14:0 is a characteristic fatty acid of the family Enterobacteriaceae.

Genome sequencing information

S. plymuthica AS12 was selected for sequencing on the basis of its ability to promote rapeseed plant growth as well as to inhibit fungal pathogens of rapeseed [5]. The genome sequence is deposited in the Genomes On Line Database [10] (GOLD ID = Gc01771) and in GenBank (INSDC ID = CP002774). Sequencing, finishing and annotation were performed by the DOE Joint Genome Institute (JGI). A summary of the project information and its association with MIGS identifiers is shown in Table 2.

Table 2

Genome sequencing project information

MIGS ID

    Property

      Term

MIGS-31

    Finishing quality

      Finished

MIGS-28

    Libraries used

      Three libraries: one 454 standard library,       one paired end 454 library (12 kb insert size) and one Illumina library

MIGS-29

    Sequencing platforms

      Illumina GAii, 454 GS FLX Titanium

MIGS-31.2

    Fold coverage

      59.0 × Illumina; 8.8 × pyrosequencing

MIGS-30

    Assemblers

      Velvet v. 1.0.13, Newbler v. 2.3, Phrap version SPS – 4.24

MIGS-32

    Gene calling method

      Prodigal 1.4, GenePRIMP

    NCBI project ID

      60453

    INSDC ID

      CP002774

    Genbank Date of Release

      October 12, 2011

    GOLD ID

      Gc01771

MIGS-13

    Source material identifier

      CCUG 61397

    Project relevance

      Biocontrol, Agricultural

Growth conditions and DNA isolation

The cells of S. plymuthica AS12 were grown in Luria Broth (LB) medium at 28°C with constant shaking at 200 rpm. The cells were harvested after 12 hours when the cells were in the early stationary phase. The cells were pelleted and resuspended in TE buffer (Sigma Aldrich). The DNA was extracted from the resuspended cells by following the standard CTAB protocol for bacterial genomic DNA isolation, which is available at JGI [29].

Genome sequencing and assembly

The genome of S. plymuthica AS12 was sequenced using a combination of Illumina [30] and 454 sequencing platforms [31]. The detailed information on library construction and sequencing can be found at the JGI website [29]. The sequence data from Illumina GAii (1,800 Mb) were assembled with Velvet [32] and the consensus sequence was computationally shredded into 1.5 kb overlapping fake reads. The sequencing data from 454 pyrosequencing (81.6 Mb) were assembled with Newbler. The initial draft assembly contained 61 contigs in one scaffold and consensus sequences were computationally shredded into 2 kb overlapping fake reads. The 454 Newbler consensus reads, the Illumina Velvet consensus reads and the read pairs in the 454 paired end library were integrated using a software parallel Phrap [33]. Possible mis-assemblies were corrected with gapResolution [29], Dupfinisher [34], or by sequencing cloned bridging PCR fragments with subcloning or transposon bombing (Epicentre Biotechnologies, Madison, WI). The gaps between contigs were closed by editing in the software Consed [35-37], by PCR and by Bubble PCR (J.-F. Chang, unpublished) primer walks. A total of 160 additional reactions was necessary to close gaps and to raise the quality of the finished sequence. The sequence reads from Illumina were used to correct potential base errors and increase consensus quality using the software Polisher developed at JGI [38]. The final assembly is based on 47.4 Mb of 454 draft data which provides an average 8.8 × coverage of the genome and 315 Mb of Illumina draft data which provides an average 59 × coverage of the genome.

Genome annotation

The S. plymuthica AS12 genes were identified using Prodigal [39] as part of the genome annotation pipeline at Oak Ridge National Laboratory (ORNL), Oak Ridge, TN, USA, followed by a round of manual curation using the JGI GenePRIMP pipeline [40]. The predicted CDS were translated and used to search the National Center for Biotechnology Information (NCBI) nonredundant database, Uniport, TIGR-Fam, Pfam, PRIAM, KEGG, COG and InterPro databases. The miscellaneous functions were predicted using tRNAScan-SE [41], RNAmmer [42], TMHMM [43], and signalP [44]. Additional gene prediction analysis and functional annotation was performed within the Integrated Microbial Genomes – Expert Review (IMG-ER) platform developed by the Joint Genome Institute, Walnut Creek, CA, USA [45].

Genome properties

The genome of S. plymuthica AS12 comprises a single circular chromosome of 5,443,009 bp with 55.96% GC content (Figure 3 and Table 3) and 5,140 predicted genes. Among those predicted genes, 4,952 were assigned as protein-coding genes and 88.71% of protein coding genes were assigned for putative function and the remaining ones were annotated as hypothetical proteins. There were 76 pseudogenes and 113 RNA genes with seven rRNA operons. The distribution of genes into the COG functional categories is presented in Table 4.

Figure 3

Graphical circular 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 3

Genome statistics

Attribute

    Value

    % of totala

Genome size (bp)

    5,443,009

    100.00%

DNA Coding region (bp)

    4,772,809

    87.69%

DNA G+C content (bp)

    3,045,986

    55.96%

Total genesb

    5,139

    100.00%

RNA genes

    112

    2.18%

rRNA operons

    7

    0.14%

Protein-coding genes

    4,952

    96.36%

Pseudo genes

    75

    1.46%

Genes in paralog clusters

    2721

    52.95%

Genes assigned to COGs

    3,808

    74.10%

Genes assigned in Pfam domains

    4,184

    81.41%

Genes with signal peptides

    675

    13.13%

Genes with transmembrane helices

    1,228

    23.89%

CRISPR repeats

    1

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.

Table 4

Number of genes associated with the 25 general COG functional categories

Code

    Value

     % age

     Description

J

    201

     4.27

     Translation, ribosomal structure and biogenesis

A

    1

     0.02

     RNA processing and modification

K

    481

     10.22

     Transcription

L

    160

     3.40

     Replication, recombination and repair

B

    1

     0.02

     Chromatin structure and dynamics

D

    37

     0.79

     Cell division and chromosome partitioning

Y

    0

     0.00

     Nuclear structure

V

    64

     1.36

     Defense mechanisms

T

    187

     3.97

     Signal transduction mechanisms

M

    265

     5.63

     Cell envelope biogenesis, Outer membrane

N

    94

     2.00

     Cell motility and secretion

Z

    0

     0.00

     Cytoskeleton

W

    0

     0.00

     Extracellular structure

U

    116

     2.47

     Intracellular trafficking and secretion

O

    153

     3.25

     Posttranslational modification, protein turnover, chaperones

C

    272

     5.78

     Energy production and conversion

G

    424

     9.01

     Carbohydrate transport and metabolism

E

    470

     9.99

     Amino acid transport and metabolism

F

    106

     2.25

     Nucleotide transport and metabolism

H

    185

     3.93

     Coenzyme metabolism

I

    135

     2.87

     Lipid metabolism

P

    285

     6.06

     Inorganic ion transport and metabolism

Q

    133

     2.83

     Secondary metabolite biosynthesis, transport and catabolism

R

    537

     11.41

     General function prediction only

S

    398

     8.46

     Function unknown

-

    918

     17.86

     Not in COGs

Declarations

Acknowledgements

We gratefully acknowledge the help of Elke Lang for providing a culture of reference bacterial strains, Evelyne-Marie Brambilla for extraction of DNA, and Anne Fiebig for assembly of the reference genomes required for digital DNA-DNA (all at DSMZ). The work was conducted by the U.S. Department of Energy Joint Genome Institute is supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231.

References

  1. Grimont PAD, Grimont F and Starr MP. Serratia species isolated from plants. Curr Microbiol. 1981; 5:317-322 View Article
  2. Kalbe C, Marten P and Berg G. Strains of genus Serratia as beneficial rhizobacteria of oilseed rape with antifungal properties. Microbiol Res. 1996; 151:433-439 View ArticlePubMed
  3. Kurze S, Bahl H, Dahl R and Berg G. Biological control of fungal strawberry diseases by Serratia plymuthica HRO–C48. Plant Dis. 2001; 85:529-534 View Article
  4. Müller H and Berg G. Impact of formulation procedures on the effect of the biocontrol agent Serratia plymuthica HRO-C48 on Verticillium wilt in oilseed rape. BioControl. 2008; 53:905-916 View Article
  5. Alström S. Characteristics of bacteria from oilseed rape in relation to their biocontrol activity against Verticillium dahliae. J Phytopathol. 2001; 149:57-64 View Article
  6. Altschul SF, Thomas LS, Alejandro AS, Jingui Z, Webb M and David JL. Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Res. 1997; 25:3389-3402 View ArticlePubMed
  7. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A and Lopez R. Clustal W and Clustal X version 2.0. Bioinformatics. 2007; 23:2947-2948 View ArticlePubMed
  8. 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 ArticlePubMed
  9. Pattengale ND, Alipour M, Bininda-Emonds ORP, Moret BME and Stamatakis A. How many bootstrap replicates are necessary? Lect Notes Comput Sci. 2009; 5541:184-200 View Article
  10. Liolios K, Chen IM, Mavromatis K, Tavernarakis N, Hugenholtz P, Markowitz VM and Kyrpides NC. The Genomes On Line Database (GOLD) in 2009: status of genomic and metagenomic projects and their associated metadata. Nucleic Acids Res. 2010; 38:D346-D354 View ArticlePubMed
  11. Auch AF, von Jan M, Klenk HP and Göker M. Digital DNA-DNA hybridization for microbial species delineation by means of genome-to-genome sequence comparison. Stand Genomic Sci. 2010; 2:117-134 View ArticlePubMed
  12. Neupane S, Högberg N, Alström S, Lucas S, Han J, Lapidus A, Cheng JF, Bruce D, Goodwin L and Pitluck S. , et al Complete genome sequence of the rapeseed plant-growth promoting Serratia plymuthica strain AS9. Stand Genomic Sci. 2012; 6:54-62PubMed
  13. Auch AF, Klenk HP and Göker M. Standard operating procedure for calculating genome-to-genome distances based on high-scoring segment pairs. Stand Genomic Sci. 2010; 2:142-148 View ArticlePubMed
  14. Field D, Garrity G, Gray T, Morrison N, Selengut J, Sterk P, Tatusova T, Thomson N, Allen MJ and Angiuoli SV. The minimum information about a genome sequence (MIGS) specification. Nat Biotechnol. 2008; 26:541-547 View ArticlePubMed
  15. 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 ArticlePubMed
  16. Garrity GM, Bell JA, Lilburn T. Phylum XIV. Proteobacteria phyl. nov. In: Garrity GM, Brenner DJ, Krieg NR, Staley JT (eds), Bergey's Manual of Systematic Bacteriology, Second Edition, Volume 2, Part B, Springer, New York, 2005, p. 1.
  17. . Validation of publication of new names and new combinations previously effectively published outside the IJSEM. List no. 106. Int J Syst Evol Microbiol. 2005; 55:2235-2238 View Article
  18. Garrity GM, Bell JA, Lilburn T. Class III. Gammaproteobacteria class. nov. In: Garrity GM, Brenner DJ, Krieg NR, Staley JT (eds), Bergey's Manual of Systematic Bacteriology, Second Edition, Volume 2, Part B, Springer, New York, 2005, p. 1.
  19. Garrity GM, Holt JG. Taxonomic Outline of the Archaea and Bacteria In: Garrity GM, Boone DR, Castenholz RW (eds), Bergey's Manual of Systematic Bacteriology, Second Edition, Volume 1, Springer, New York, 2001, p. 155-166.
  20. Skerman VBD, McGowan V and Sneath PHA. Approved Lists of Bacterial Names. Int J Syst Bacteriol. 1980; 30:225-420 View Article
  21. Rahn O. New principles for the classification of bacteria. Zentralbl Bakteriol Parasitenkd Infektionskr Hyg. 1937; 96:273-286
  22. . Conservation of the family name Enterobacteriaceae, of the name of the type genus, and designation of the type species OPINION NO. 15. Int Bull Bacteriol Nomencl Taxon. 1958; 8:73-74 View Article
  23. Sakazaki R. Genus IX. Serratia Bizio 1823, 288. In: Buchanan RE, Gibbons NE (eds), Bergey's Manual of Determinative Bacteriology, Eighth Edition, The Williams and Wilkins Co., Baltimore, 1974, p. 326-326.
  24. Bizio B. Lettera di Bartolomeo Bizio al chiarissimo canonico Angelo Bellani sopra il fenomeno della polenta porporina. Biblioteca Italiana o sia Giornale di Letteratura. [Anno VIII]. Scienze e Arti. 1823; 30:275-295
  25. Breed RS, Murray EGD, Hitchens AP. In: Breed RS, Murray EGD, Hitchens AP (eds), Bergey's Manual of Determinative Bacteriology, Sixth Edition, The Williams and Wilkins Co., Baltimore, 1948, p. 481-482.
  26. BAuA. 2010, Classification of bacteria and archaea in risk groups. TRBA 466, p. 200.Web Site
  27. 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. Nat Genet. 2000; 25:25-29 View ArticlePubMed
  28. Bergan T, Grimont AD and Grimont F. Fatty acids of Serratia determined by gas chromatography. Curr Microbiol. 1983; 8:7-11 View Article
  29. . Web Site
  30. Bennett S. Solexa Ltd. Pharmacogenomics. 2004; 5:433-438 View ArticlePubMed
  31. Margulies M, Egholm M, Altman WE, Attiya S, Bader JS, Bemben LA, Berka J, Braverman MS, Chen YJ and Chen Z. Genome sequencing in microfabricated high-density picolitre reactors. Nature. 2005; 437:326-327PubMed
  32. Zerbino DR and Birney E. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res. 2008; 18:821-829 View ArticlePubMed
  33. Phrap and Phred for Windows. MacOS, Linux, and Unix. Web Site
  34. Han C, Chain P. 2006. Finishing repeat regions automatically with Dupfinisher. In: Proceeding of the 2006 international conference on bioinformatics & computational biology. Arabina HR, Valafar H (eds), CSREA Press. June 26-29, 2006:141-146.
  35. Ewing B and Green P. Base-Calling of automated sequencer traces using Phred. II. error probabilities. Genome Res. 1998; 8:186-194PubMed
  36. Ewing B, Hillier L, Wendl MC and Green P. Base-Calling of automated sequencer traces using Phred. I. accuracy assessment. Genome Res. 1998; 8:175-185PubMed
  37. Gordon D, Abajian C and Green P. Consed: a graphical tool for sequence finishing. Genome Res. 1998; 8:195-202PubMed
  38. Lapidus A, LaButti K, Foster B, Lowry S, Trong S, Goltsman E. POLISHER: An effective tool for using ultra short reads in microbial genome assembly and finishing. AGBT, Marco Island, FL, 2008.
  39. Hyatt D, Chen GL, LoCascio PF, Land ML, Larimer FW and Hauser LJ. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics. 2010; 11:119 View ArticlePubMed
  40. Pati A, Ivanova NN, Mikhailova N, Ovchinnikova G, Hooper SD, Lykidis A and Kyrpides NC. GenePRIMP: a gene prediction improvement pipeline for prokaryotic genomes. Nat Methods. 2010; 7:455-457 View ArticlePubMed
  41. Schattner P, Brooks AN and Lowe TM. The tRNA Scan-SE, snoscan and snoGPS web servers for the detection of tRNAs and snoRNAs. Nucleic Acids Res. 2005; 33:W686-W689 View ArticlePubMed
  42. 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 ArticlePubMed
  43. Krogh A, Larsson B, von Heijne 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 ArticlePubMed
  44. 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 ArticlePubMed
  45. Markowitz VM, Mavromatis K, Ivanova NN, Chen IMA, Chu K and Kyrpides NC. IMG ER: a system for microbial genome annotation expert review and curation. Bioinformatics. 2009; 25:2271-2278 View ArticlePubMed