Open Access

Complete genome sequence of Mesorhizobium ciceri bv. biserrulae type strain (WSM1271T)

  • Kemanthi Nandasena
  • , Ron Yates,
  • , Ravi Tiwari
  • , Graham O’Hara
  • , John Howieson
  • , Mohamed Ninawi
  • , Olga Chertkov
  • , Chris Detter
  • , Roxanne Tapia
  • , Shunseng Han
  • , Tanja Woyke
  • , Sam Pitluck
  • , Matt Nolan
  • , Miriam Land
  • , Konstantinos Liolios
  • , Amrita Pati
  • , Alex Copeland
  • , Nikos Kyrpides
  • , Natalia Ivanova
  • , Lynne Goodwin
  • , Uma Meenakshi
  • and Wayne Reeve
Corresponding author

DOI: 10.4056/sigs.4458283

Received: 31 December 2013

Accepted: 31 December 2013

Published: 15 June 2014

Abstract

Mesorhizobium ciceri bv. biserrulae strain WSM1271T was isolated from root nodules of the pasture legume Biserrula pelecinus growing in the Mediterranean basin. Previous studies have shown this aerobic, motile, Gram negative, non-spore-forming rod preferably nodulates B. pelecinus – a legume with many beneficial agronomic attributes for sustainable agriculture in Australia. We describe the genome of Mesorhizobium ciceri bv. biserrulae strain WSM1271T consisting of a 6,264,489 bp chromosome and a 425,539 bp plasmid that together encode 6,470 protein-coding genes and 61 RNA-only encoding genes.

Keywords:

root-nodule bacterianitrogen fixationevolutionlateral gene transferintegrative and conjugative elementssymbiosisAlphaproteobacteria

Introduction

The productivity of sustainable agriculture around the world is heavily dependent on the provision of bioavailable nitrogen (N) [1]. The demand for N by non-leguminous and leguminous plants can be supplied by the application of chemically synthesized nitrogenous fertilizer onto crops and pastures. However, the production of fertilizer is costly and requires the burning of fossil fuels in the manufacturing process which increases greenhouse gas emissions. Furthermore, high application rates of fertilizer can contaminate ecosystems and waterways, and result in leaching into the environment.

In contrast, the demand for N by leguminous plants can be sustainably met through the biological process of N fixation that occurs following the successful formation of an effective symbiosis. This symbiotic nitrogen fixation (SNF) process can account for approximately 70% of the bioavailable nitrogen supplied to legumes [1].

One legume that has many beneficial agronomic attributes is Biserrula pelecinus L., which is an annual herbaceous legume native to the Mediterranean basin that was introduced into Australian soil in 1994 [2]. The beneficial agronomic attributes of this legume include drought tolerance, hard seed production, easy harvesting characteristics, insect tolerance and most importantly, a capacity to grow well in the acidic duplex soils of Australia [2,3]. This monospecific legume specifically forms an effective nitrogen fixing symbiosis with the root nodule bacterium Mesorhizobium ciceri bv. biserrulae type strain WSM1271T (= LMG23838 = HAMBI2942) [4,5]. Australian indigenous rhizobial populations were found to be incapable of nodulating B. pelecinus L [2]. However, within six years of the introduction of the inoculant into Australia, the in situ evolution of a diverse range of competitive strains capable of nodulating B. pelecinus L. compromised optimal N2-fixation with this host. This rapid emergence of less effective strains threatens the establishment of this legume species in the Australian agricultural setting. The sub-optimal strains appear to have evolved from indigenous mesorhizobia that acquired the island of genes associated with symbiosis from the original inoculant, WSM1271T, following a horizontal gene transfer event [6].

In this report, a summary classification and a set of general features for M. ciceri bv. biserrulae strain WSM1271T are presented together with the description of the complete genome sequence and its annotation.

Classification and features

M. ciceri strain WSM1271T is a motile, Gram-negative, non-spore-forming rod (Figure 1 and Figure 2) in the order Rhizobiales of the class Alphaproteobacteria. They are moderately fast growing, forming 2-4 mm diameter colonies within 3-4 days, and have a mean generation time of 4-6 h when grown in half Lupin Agar (½LA) broth [7] at 28 °C. Colonies on ½LA are white-opaque, slightly domed, moderately mucoid with smooth margins (Figure 3).

Figure 1

Image of Mesorhizobium ciceri bv. biserrulae strain WSM1271T using scanning electron microscopy.

Figure 2

Image of Mesorhizobium ciceri bv. biserrulae strain WSM1271T using transmission electron microscopy.

Figure 3

Image of Mesorhizobium ciceri bv. biserrulae strain WSM1271T using the appearance of colony morphology on solid media.

The organism tolerates a pH range between 5.5 and 9.0. Carbon source utilization and fatty acid profiles have been described before [6]. Minimum Information about the Genome Sequence (MIGS) is provided in Table 1. Figure 4 shows the phylogenetic neighborhood of M. ciceri bv. biserrulae strain WSM1271T in a 16S rRNA sequence based tree. This strain clustered in a tight group, which included M. australicum, M. ciceri, M. loti and M. shangrilense and had >99% sequence identity with all four type strains. Our polyphasic taxonomic study indicates that WSM1271T is a new biovar of nodulating bacteria [5].

Table 1

Classification and features of Mesorhizobium ciceri bv. biserrulae strain WSM1271T according to the MIGS recommendations [8,9].

MIGS ID

       Property

       Term

       Evidence code

       Current classification

       Domain Bacteria

       TAS [9]

       Phylum Proteobacteria

       TAS [10]

       Class Alphaproteobacteria

       TAS [11,12]

       Order Rhizobiales

       TAS [11,13]

       Family Phyllobacteriaceae

       TAS [11,14]

       Genus Mesorhizobium

       TAS [15]

       Species Mesorhizobium ciceri bv biserrulae

       TAS [15]

       Gram stain

       Negative

       TAS [6]

       Cell shape

       Rod

       TAS [6]

       Motility

       Motile

       TAS [6]

       Sporulation

       Non-sporulating

       TAS [16]

       Temperature range

       Mesophile

       TAS [16]

       Optimum temperature

       28°C

       TAS [6]

       Salinity

       Unknown

       NAS

MIGS-22

       Oxygen requirement

       Aerobic

       TAS [16]

       Carbon source

       Arabinose, β-gentibiose, glucose, mannitol & melibiose

       TAS [6]

       Energy source

       Chemoorganotroph

       TAS [16]

MIGS-6

       Habitat

       Soil, root nodule, host

       TAS [6]

MIGS-15

       Biotic relationship

       Free living, Symbiotic

       TAS [6]

MIGS-14

       Pathogenicity

       None

       NAS

       Biosafety level

       1

       TAS [17]

       Isolation

       Root nodule

       TAS [5,6]

MIGS-4

       Geographic location

       5 km before Bottida, Sardinia

       TAS [2,5]

MIGS-5

       Nodule collection date

       April 1993

       TAS [4]

MIGS-4.1

       Longitude

       9.012008

       NAS

MIGS-4.2

       Latitude

       40.382709

       NAS

MIGS-4.3

       Depth

       10 cm

       NAS

MIGS-4.4

       Altitude

       295 m

       TAS [5]

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). Evidence codes are from the Gene Ontology project [18].

Figure 4

Phylogenetic tree showing the relationships of Mesorhizobium ciceri bv. biserrulae WSM1271T (shown in bold print) with root nodule bacteria in the order Rhizobiales based on aligned sequences of the 16S rRNA gene (1,290 bp internal region). All sites were informative and there were no gap-containing sites. Phylogenetic analyses were performed using MEGA [19]. The tree was built using the Maximum-Likelihood method with the General Time Reversible model. Bootstrap analysis [20] was performed with 500 replicates to assess the support of the clusters. Type strains are indicated with a superscript T. Brackets after the strain name contain a DNA database accession number and/or a GOLD ID (beginning with the prefix G) for a sequencing project registered in GOLD [21]. Published genomes are indicated with an asterisk.

Symbiotaxonomy

M. ciceri bv. biserrulae strain WSM1271T has an extremely narrow legume host range for symbiosis only forming highly effective nitrogen-fixing root nodules on Biserrula pelecinus. L. This strain also nodulates the closely related species Astragalus membranaceus but does not nodulate 21 other legume species nodulated by Mesorhizobium spp [5]. The high degree of specificity in the symbiotic relationships of this strain is representative of root nodule bacteria isolated from B. pelecinus L. growing in undisturbed landscapes in the Mediterranean basin, and is an important example of a highly specific legume host-root nodule bacteria relationship in an annual herbaceous legume used as a forage species in agriculture.

Genome sequencing and annotation

Genome project history

The Joint Genome Institute (JGI) operated by US Department of Energy (DOE) sequenced, finished and annotated WSM1271 as part of the Community Sequencing Program (CSP). The genome project is deposited in the Genomes OnLine Database [21]. The finished genome sequence is in GenBank. The CSP selects projects on the basis of environmental and agricultural relevance to issues in global carbon cycling, alternative energy production, and biogeochemical importance. Table 2 summarizes the project information.

Table 2

Genome sequencing project information for Mesorhizobium ciceri bv. biserrulae strain WSM1271T

MIGS ID

       Property

       Term

MIGS-31

       Finishing quality

       Finished

MIGS-28

       Libraries used

       Illumina GAii shotgun library,       454 Titanium standard library and paired end 454 libraries

MIGS-29

       Sequencing platforms

       Illumina and 454 technologies

MIGS-31.2

       Sequencing coverage

       454 (26.8x) and Illumina (124x)

MIGS-30

       Assemblers

       Newbler, version 2.3 and Velvet version 0.7.63, PHRAP and CONSED

MIGS-32

       Gene calling method

       Prodigal, GenePrimp

       Genbank ID

       CP002447       CP002448

       Genbank Date of Release

       November 10, 2012

       GOLD ID

       Gc01578

       NCBI project ID

       48991

       Database: IMG

       649633066

       Project relevance

       Symbiotic nitrogen fixation, agriculture

Growth conditions and DNA isolation

M. ciceri bv. biserrulae strain WSM1271T was grown to mid logarithmic phase in TY rich medium [22] on a gyratory shaker at 28 °C. DNA was isolated from 60 mL of cells using a CTAB (Cetyl trimethyl ammonium bromide) bacterial genomic DNA isolation method [23].

Genome sequencing and assembly

The Joint Genome Institute (JGI) generated the draft genome of M. ciceri bv. biserrulae WSM1271T using a combination of Illumina [24] and 454 technologies [25]. The sequencing of an Illumina GAii shotgun library generated 23,461,369 reads totaling 844.6 Mb, a 454 Titanium standard library which generated 277,881 reads and a paired end 454 libraries with average insert size of 1.137 +/- 2.842 Kb and 4.378 +/- 1.094 kb which generated 40,653 and 130,843 reads totaling 244.0 Mb of 454 data. All general aspects of library construction and sequencing performed at the JGI can be found at the JGI website [23]. The initial draft assembly contained 32 contigs in 2 scaffolds. The 454 Titanium standard data and the 454 paired end data were assembled together with Newbler, version 2.3. The Newbler consensus sequences were computationally shredded into 2 Kb overlapping fake reads (shreds). Illumina sequencing data was assembled with VELVET, version 0.7.63 [26], and the consensus sequences were computationally shredded into 1.5 Kb overlapping fake reads (shreds). We integrated the 454 Newbler consensus shreds, the Illumina VELVET consensus shreds and the read pairs in the 454 paired end library using parallel phrap, version SPS - 4.24 (High Performance Software, LLC). The software Consed [27-29] was used in the following finishing process. Illumina data was used to correct potential base errors and increase consensus quality using the software Polisher developed at JGI (Alla Lapidus, unpublished). Possible mis-assemblies were corrected using gapResolution (Cliff Han, unpublished), Dupfinisher [30], or sequencing cloned bridging PCR fragments with subcloning. Gaps between contigs were closed by editing in Consed, by PCR and by Bubble PCR (J-F Cheng, unpublished) primer walks. A total of 49 additional reactions were necessary to close gaps and to raise the quality of the finished sequence. The total size of the genome is 6,890,027 bp and the final assembly is based on 112.0 Mb of 454 draft data which provides an average 26.8× coverage of the genome and 832.1 Mb of Illumina draft data which provides an average 124× coverage of the genome.

Genome annotation

Genes were identified using Prodigal [31] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePrimp pipeline [32]. 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 [33], RNAMMer [34], Rfam [35], TMHMM [36], and SignalP [37]. Additional gene prediction analyses and functional annotation were performed within the Integrated Microbial Genomes (IMG-ER) platform [38].

Genome properties

The genome is 6,690,028 bp long with a 62.56% GC content (Table 3) and comprises a single chromosome and a single plasmid. From a total of 6,531 genes, 6,470 were protein encoding and 61 RNA only encoding genes. Within the genome, 206 pseudogenes were also identified. The majority of genes (70.74%) were assigned a putative function while the remaining genes were annotated as hypothetical. The distribution of genes into COGs functional categories is presented in Table 4, and Figures 5,6 and 7.

Table 3

Genome Statistics for Mesorhizobium ciceri bv. biserrulae strain WSM1271T.

Attribute

       Value

       % of Total

Genome size (bp)

       6,690,028

       100.00

DNA coding region (bp)

       5,791,860

       86.57

DNA G+C content (bp)

       4,185,397

       62.56

Number of replicons

       2

Extrachromosomal elements

       1

Total genes

       6,531

       100.00

RNA genes

       61

       0.93

Protein-coding genes

       6,470

       99.07

Genes with function prediction

       4,620

       70.74

Genes assigned to COGs

       5174

       79.22

Genes assigned Pfam domains

       5398

       82.65

Genes with signal peptides

       597

       9.14

Genes with transmembrane helices

       1528

       23.40

Table 4

Number of protein coding genes of Mesorhizobium ciceri bv. biserrulae WSM1271T associated with the general COG functional categories.

Code

       Value

      %age

        COG Category

J

       193

      3.35

        Translation, ribosomal structure and biogenesis

A

       1

      0.02

        RNA processing and modification

K

       492

      8.53

        Transcription

L

       156

      2.71

        Replication, recombination and repair

B

       6

      0.10

        Chromatin structure and dynamics

D

       35

      0.61

        Cell cycle control, mitosis and meiosis

Y

       0

      0.00

        Nuclear structure

V

       63

      1.09

        Defense mechanisms

T

       238

      4.13

        Signal transduction mechanisms

M

       290

      5.03

        Cell wall/membrane biogenesis

N

       62

      1.08

        Cell motility

Z

       0

      0.00

        Cytoskeleton

W

       2

      0.03

        Extracellular structures

U

       124

      2.15

        Intracellular trafficking and secretion

O

       185

      3.21

        Posttranslational modification, protein turnover, chaperones

C

       356

      6.17

        Energy production conversion

G

       535

      9.28

        Carbohydrate transport and metabolism

E

       732

      12.70

        Amino acid transport metabolism

F

       92

      1.60

        Nucleotide transport and metabolism

H

       204

      3.54

        Coenzyme transport and metabolism

I

       235

      4.08

        Lipid transport and metabolism

P

       274

      4.75

        Inorganic ion transport and metabolism

Q

       175

      3.04

        Secondary metabolite biosynthesis, transport and catabolism

R

       731

      12.68

        General function prediction only

S

       585

      10.15

        Function unknown

-

       1,357

      20.78

        Not in COGS

Figure 5

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

Figure 6

Graphical circular map of the plasmid of Mesorhizobium ciceri bv. biserrulae WSM1271T. From outside to the center. Genes on forward strand (color by COG categories as denoted by the IMG platform), Genes on reverse strand (color by COG categories), RNA genes (tRNAs green, sRNAs red, other RNAs black), GC content, GC skew.

Figure 7

Color code for Figure 5 and 6.

Declarations

Acknowledgements

This work was performed under the auspices of the US Department of Energy’s Office of Science, Biological and Environmental Research Program, and by the University of California, Lawrence Berkeley National Laboratory under contract No. DE-AC02-05CH11231, Lawrence Livermore National Laboratory under Contract No. DE-AC52-07NA27344, and Los Alamos National Laboratory under contract No. DE-AC02-06NA25396. We gratefully acknowledge the funding received from Australian Research Council Discovery grant (DP0880896), Murdoch University Strategic Research Fund through the Crop and Plant Research Institute (CaPRI) and the Centre for Rhizobium Studies (CRS) at Murdoch University. The authors would like to thank the Australia-China Joint Research Centre for Wheat Improvement (ACCWI) and SuperSeed Technologies (SST) for financially supporting Mohamed Ninawi’s PhD project.


This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

References

  1. O'Hara GW. The role of nitrogen fixation in crop production. J Crop Prod. 1998; 1(2):115-138 View Article
  2. Howieson JG, Loi A and Carr SJ. Biserrula pelecinus L. - a legume pasture species with potential for acid, duplex soils which is nodulated by unique root-nodule bacteria. Aust J Agric Res. 1995; 46:997-1009 View Article
  3. Loi A, Howieson JG, Nutt BJ and Carr SJ. A second generation of annual pasture legumes and their potential for inclusion in Mediterranean-type farming systems. Aust J Exp Agric. 2005; 45:289-299 View Article
  4. Nandasena KG, O'Hara GW, Tiwari RP, Sezmis E and Howieson JG. In situ lateral transfer of symbiosis islands results in rapid evolution of diverse competitive strains of mesorhizobia suboptimal in symbiotic nitrogen fixation on the pasture legume Biserrula pelecinus L. Environ Microbiol. 2007; 9:2496-2511 View ArticlePubMed
  5. Nandasena KG, O'Hara GW, Tiwari RP, Willlems A and Howieson JG. Mesorhizobium ciceri biovar biserrulae, a novel biovar nodulating the pasture legume Biserrula pelecinus L. Int J Syst Evol Microbiol. 2007; 57:1041-1045 View ArticlePubMed
  6. Nandasena KG, O'Hara GW, Tiwari RP, Willems A and Howieson JG. Mesorhizobium australicum sp. nov. and Mesorhizobium opportunistum sp. nov., isolated from Biserrula pelecinus L. in Australia. Int J Syst Evol Microbiol. 2009; 59:2140-2147 View ArticlePubMed
  7. Howieson JG, Ewing MA and D'antuono MF. Selection for acid tolerance in Rhizobium meliloti. Plant Soil. 1988; 105:179-188 View Article
  8. Field D, Garrity G, Gray T, Morrison N, Selengut J, Sterk P, Tatusova T, Thomson N, Allen M and Angiuoli SV. Towards a richer description of our complete collection of genomes and metagenomes "Minimum Information about a Genome Sequence " (MIGS) specification. Nat Biotechnol. 2008; 26:541-547 View ArticlePubMed
  9. 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
  10. 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.
  11. . 107. List of new names and new combinations previously effectively, but not validly, published. Int J Syst Evol Microbiol. 2006; 56:1-6 View ArticlePubMed
  12. Garrity GM, Bell JA, Lilburn T. Class I. Alphaproteobacteria class. nov. In: Garrity GM, Brenner DJ, Krieg NR, Staley JT (eds), Bergey's Manual of Systematic Bacteriology, Second Edition, Volume 2, Part C, Springer, New York, 2005, p. 1.
  13. Kuykendall LD. Order VI. Rhizobiales ord. nov. In: Garrity GM, Brenner DJ, Kreig NR, Staley JT, editors. Bergy's Manual of Systematic Bacteriology. Second ed: New York: Springer - Verlag; 2005. p 324.
  14. Mergaert J, Swings J. Family IV. Phyllobacteriaceae In: Garrity GM, Brenner DJ, Kreig NR, Staley JT, editors. Bergy's Manual of Systematic Bacteriology. Second ed: New York: Springer - Verlag; 2005. p 393.
  15. Jarvis BDW, Van Berkum P, Chen WX, Nour SM, Fernandez MP, Cleyet-Marel JC and Gillis M. Transfer of Rhizobium loti, Rhizobium huakuii, Rhizobium ciceri, Rhizobium mediterraneum, Rhizobium tianshanense to Mesorhizobium gen.nov. Int J Syst Evol Microbiol. 1997; 47:895-898
  16. Chen WX, Wang ET, Kuykendall LD. The Proteobacteria New York: Springer - Verlag; 2005.
  17. Agents B. Technical rules for biological agents. TRBA ():466.Web Site
  18. 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 ArticlePubMed
  19. Kumar S, Tamura K and Nei M. MEGA3: Integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment. Brief Bioinform. 2004; 5:150-163 View ArticlePubMed
  20. Felsenstein J. Confidence limits on phylogenies: an approach using the bootstrap. Evolution. 1985; 39:783-791 View Article
  21. Liolios K, Mavromatis K, Tavernarakis N and Kyrpides NC. The Genomes On Line Database (GOLD) in 2007: status of genomic and metagenomic projects and their associated metadata. Nucleic Acids Res. 2008; 36:D475-D479 View ArticlePubMed
  22. Reeve WG, Tiwari RP, Worsley PS, Dilworth MJ, Glenn AR and Howieson JG. Constructs for insertional mutagenesis, transcriptional signal localization and gene regulation studies in root nodule and other bacteria. Microbiology. 1999; 145:1307-1316 View ArticlePubMed
  23. . Web Site
  24. Bennett S. Solexa Ltd. Pharmacogenomics. 2004; 5:433-438 View ArticlePubMed
  25. 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; 437:376-380PubMed
  26. Zerbino DR. Using the Velvet de novo assembler for short-read sequencing technologies. Current Protocols in Bioinformatics 2010;Chapter 11:Unit 11 5.
  27. Ewing B and Green P. Base-calling of automated sequencer traces using phred. II. Error probabilities. Genome Res. 1998; 8:175-185 View ArticlePubMed
  28. Ewing B, Hillier L, Wendl MC and Green P. Base-calling of automated sequencer traces using phred. I. Accuracy assessment. Genome Res. 1998; 8:175-185 View ArticlePubMed
  29. Gordon D, Abajian C and Green P. Consed: a graphical tool for sequence finishing. Genome Res. 1998; 8:195-202 View ArticlePubMed
  30. Han C, Chain P. Finishing repeat regions automatically with Dupfinisher. In: Valafar HRAH, editor. Proceeding of the 2006 international conference on bioinformatics & computational biology: CSREA Press; 2006. p 141-146.
  31. 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
  32. 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
  33. Lowe TM and Eddy SR. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res. 1997; 25:955-964PubMed
  34. 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
  35. Griffiths-Jones S, Bateman A, Marshall M, Khanna A and Eddy SR. Rfam: an RNA family database. Nucleic Acids Res. 2003; 31:439-441 View ArticlePubMed
  36. 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
  37. 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
  38. Markowitz VM, Mavromatis K, Ivanova NN, Chen IM, Chu K and Kyrpides NC. IMG ER: a system for microbial genome annotation expert review and curation. Bioinformatics. 2009; 25:2271-2278 View ArticlePubMed