Open Access

Complete genome sequence of Halorhodospira halophila SL1

  • Jean F. Challacombe
  • , Sophia Majid
  • , Ratnakar Deole,
  • , Thomas S. Brettin,
  • , David Bruce
  • , Susana F. Delano,
  • , John C. Detter
  • , Cheryl D. Gleasner
  • , Cliff S. Han
  • , Monica Misra
  • , Krista G. Reitenga
  • , Natalia Mikhailova,
  • , Tanja Woyke
  • , Sam Pitluck
  • , Matt Nolan
  • , Miriam L. Land
  • , Elizabeth Saunders
  • , Roxanne Tapia
  • , Alla Lapidus,
  • , Natalia Ivanova
  • and Wouter D. Hoff

DOI: 10.4056/sigs.3677284

Received: 15 April 2013

Accepted: 15 April 2013

Published: 15 June 2013

Abstract

Halorhodospira halophila is among the most halophilic organisms known. It is an obligately photosynthetic and anaerobic purple sulfur bacterium that exhibits autotrophic growth up to saturated NaCl concentrations. The type strain H. halophila SL1 was isolated from a hypersaline lake in Oregon. Here we report the determination of its entire genome in a single contig. This is the first genome of a phototrophic extreme halophile. The genome consists of 2,678,452 bp, encoding 2,493 predicted genes as determined by automated genome annotation. Of the 2,407 predicted proteins, 1,905 were assigned to a putative function. Future detailed analysis of this genome promises to yield insights into the halophilic adaptations of this organism, its ability for photoautotrophic growth under extreme conditions, and its characteristic sulfur metabolism.

Keywords:

halophilesaturated saltsulfur metabolismpurple sulfur bacteriumphototrophic

Introduction

Halorhodospira halophila is an anoxygenic photosynthetic halophile that was isolated from salt-encrusted mud along the shore of Summer Lake in Oregon [1], and from the hypersaline Wadi Natrun lakes in Egypt [2]. The original name of this organism, Ectothiorhodospira halophila, was modified to Halorhodospira halophila when the genus Ectothiorhodospira was divided into two genera (Ectothiorhodospira and Halorhodospira), and E. halophila was reclassified as a member of the genus Halorhodospira, serving as the type species of the new genus [3]. Over the last decade, the genomes of a number of extremely halophilic Archaea have been sequenced and analyzed, including Halobacterium salinarum [4,5], Haloarcula marismortui [6], Natronomonas pharaonis [7], and Haloquadratum walsbyi [8]. In addition, the genomes of three halophilic Bacteria have become available: Salinibacter ruber [9], Halothermothrix orenii [10], and ‘Halanaerobium hydrogenoformans’ [11]. All of these organisms are obligate chemotrophs. Thus, H. halophila is the first phototrophic extreme halophile to have its genome sequence determined and analyzed. In contrast to other extreme halophiles that grow well in saturated salt concentrations, H. halophila has a high flexibility with respect to the salt concentrations that it tolerates, and grows optimally at all NaCl concentrations from 15% to 35%, with growth down to 3.5% NaCl [12]. In contrast, the above extremely halophilic archaea and S. ruber require 15% NaCl for growth.

H. halophila is of significant interest because it is an obligately anaerobic purple sulfur bacterium, and among the most halophilic organisms known [13]. To date, genome sequences are available for two phototrophic purple sulfur bacteria, Allochromatium vinosum DSM 180 and the H. halophila SL1 genome reported here. H. halophila has very few growth requirements. However, it does need reduced sulfur compounds for growth, as does A. vinosum [14]. Its pathways for both photosynthetic electron transfer [15-17] and nitrogen fixation [18] have attracted attention. In addition, H. halophila contains photoactive yellow protein [19,20]. This is the first member of a novel class of blue light receptors, and triggers a negative phototaxis response in H. halophila [21]. The photoactive yellow protein (PYP) from H. halophila has been studied extensively for its biophysical characteristics [22-24].

The sulfur metabolism of H. halophila is unusual, resulting in the transient accumulation of extracellular sulfur globules via metabolic pathways that are not yet fully resolved [14]. While purple non-sulfur phototrophs such as Rhodobacter sphaeroides and Rhodospirillum rubrum use organic compounds like malate as electron donors, H. halophila obtains electrons from reduced sulfur compounds. The genome sequence of H. halophila promises to reveal insights into its adaptations to hypersaline environments, and to allow a better understanding of its unique combination of metabolic capabilities, combining properties from extreme halophiles, anoxygenic phototrophs, and purple sulfur bacteria.

Classification and features

H. halophila belongs to the Gammaproteobacteria [3] (Table 1). The 16S rRNA gene sequence of H. halophila SL1 reveals closer relationships with H. halochloris and Alkalilimnicola ehrlichii, the other representatives of the Ectothiorhodospiraceae (Figure 1), than with A. vinosum, a purple sulfur bacterium in the Chromatiaceae family, and the haloalkaliphilic chemolithoautotrophic Thioalkalivibrio strains.

Table 1

Classification and general features of H. halophila SL1 according to the MIGS recommendations [25].

MIGS ID

    Property

     Term

   Evidence codea

    Current classification

     Domain Bacteria

   TAS [26]

     Phylum Proteobacteria

   TAS [27]

     Class Gammaproteobacteria

   TAS [28,29]

     Order Chromatiales

   TAS [28,30]

     Family Ectothiorhodospiraceae

   TAS [31]

     Genus Halorhodospira

   TAS [32-34]

     Species Halorhodospira halophila

   TAS [32,33]

MIGS-7

    Subspecific genetic lineage

     DSM 244T

    Gram stain

     negative

   NAS

    Cell shape

     spiral

   TAS [1]

    Motility

     motile

   TAS [1]

    Sporulation

     non-sporulating

   NAS

    Temperature range

     mesophilic

   NAS

    Optimum temperature

     47°C

   TAS [1]

    Carbon source

     CO2, succinate, acetate

   TAS [35]

    Energy source

     photosynthesis

   TAS [1]

MIGS-6

    Habitat

     salt lake mud

   TAS [1]

MIGS-6.3

    Salinity

     Extreme halophile

   TAS [1]

MIGS-22

    Oxygen

     anaerobe

   TAS [1]

MIGS-15

    Biotic relationship

     free living

   NAS

MIGS-14

    Pathogenicity

     none

   NAS

MIGS-4

    Geographic location

     Summer Lake, Lake County, OR

   TAS [1]

MIGS-5

    Sample collection time

     about 1967

   TAS [1]

MIGS-4.1

    Latitude

     not reported

MIGS-4.2

    Longitude

     not reported

MIGS-4.3

    Depth

     not reported

MIGS-4.4

    Altitude

     not reported

aEvidence 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 [36]. 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.

Figure 1

Phylogram representation of a phylogenetic tree highlighting the position of Halorhodospira halophila strain SL1 relative to other organisms of interest, including members of the Ectothiorhodospiraceae, as well as additional strains that were included for comparison purposes, based on environmental and functional considerations. The strains (type=T) and their corresponding GenBank accession numbers (and coordinates) for 16S rRNA genes are: H. halophila strain SL1T, CP00544:380025-381562; Alkalilimnicola ehrlichii strain MLHE-1, CP00453:369818-369894; Thioalkalivibrio sp. HL-EbGR7, CP001339:2548250-2549775; Thioalkalivibrio sp. K90mix, CP001905:423231-424758; Allochromatium vinosum DSM 180T, CP001896:112452-113967; Ectothiorhodospira halochloris M59152; Burkholderia phytofirmans PsJN, CP001052:1541578-1543101; Desulfovibrio vulgaris subsp. vulgaris strain Hildenborough, AE017285:105921-107426; Rhodobacter sphaeroides 2.4.1, CP000143:1-1464; Rhodospirillum rubrum ATCC 11170, CP000230: 192528-194004; Escherichia coli B strain REL606, CP000819: 226609-228150. The 16S rRNA sequences were aligned by MUSCLE [37]. The tree was determined by the maximum likelihood model of PhyML [38] and rendered with TreeDyn [39], using the “one click” pipeline of the Phylogeny.fr web resource [40].

Genome sequencing and annotation

Genome project history

This organism was selected for sequencing to better understand its halophilic adaptations, its unusual sulfur metabolism, its photosynthetic pathways, and to provide a framework for better understanding signaling pathways for photoactive yellow protein. The complete genome sequence has been deposited in GenBank. Sequencing, finishing and annotation were performed by the DOE Joint Genome Institute (JGI). Table 2 presents the project information and its association with MIGS version 2.0 compliance [25].

Table 2

Project information

MIGS ID

     Property

    Term

MIGS-31

     Finishing quality

    Finished

MIGS-28

     Libraries used

    40kb, 8kb, 3kb

MIGS-29

     Sequencing platforms

    Sanger

MIGS-31.2

     Fold coverage

    12×

MIGS-30

     Assemblers

    phrap

MIGS-32

     Gene calling method

    Critica

     Genbank ID

    CP000544

     Genbank Date of Release

    January 12, 2012

     GOLD ID

    Gc00492

     Project relevance

    extremophile

Growth conditions and DNA isolation

H. halophila SL1 strain DSM 44T was obtained from Deutsche Sammlung vor Mikroorganismen und Zellkulturen (DSMZ), Braunschweig, Germany, and were grown in DSMZ 253 medium. The cells were grown anaerobically and photosynthetically by placing them in 20 ml glass culture tubes completely filled with growth medium and sealed with screw caps. The tubes were kept at 42ºC in a water bath and illuminated with 70 W tungsten light bulbs. Chromosomal DNA was purified from the resulting cell cultures using the CTAB procedure.

Genome sequencing and assembly

The random shotgun method was used in sequencing the genome of H. halophila SL1. Large (40 kb), median (8 kb) and small (3 kb) insert random sequencing libraries were sequenced for this genome project with an average success rate of 88% and average high-quality read lengths of 750 nucleotides. After the shotgun stage, reads were assembled with parallel phrap (High Performance Software, LLC). Possible mis-assemblies were corrected with Dupfinisher (unpublished, C. Han) or by transposon bombing of bridging clones (EZ-Tn5 <P6Kyori/KAN-2> Tnp Transposome kit, Epicentre Biotechnologies). Gaps between contigs were closed by editing, custom primer walks or PCR amplification. The completed genome sequence of H. halophila SL1 contains 36,035 reads, achieving an average of 12-fold sequence coverage per base with error rate less than 1 in 100,000.

Genome annotation

Identification of putative protein-encoding genes and initial automated annotation of the genome was performed by the Oak Ridge National Laboratory genome annotation pipeline. Additional gene prediction analysis and functional annotation was performed within the IMG platform [41].

Genome properties

The genome is 2,678,452 bp long and comprises one circular chromosome with 67% GC content (Figure 2). For the main chromosome, 2,493 genes were predicted, 2,407 of which are protein-coding genes. A total of 1,905 of protein coding genes were assigned to a putative function, with the remaining annotated as hypothetical proteins. In addition, 31 pseudo genes were identified. The properties and the statistics of the genome are summarized in Tables 3-4.

Figure 2

Graphical circular map of the genome. From outside to the center: Circle 1, genes on forward strand (colored by COG categories); Circle 2, genes on reverse strand (colored by COG categories); Circle 3, RNA genes (tRNAs green, rRNAs red, other RNAs black); Circle 4, mobile element genes; Circle 5, CRISPR-associated protein genes; Circle 6, GC content; Circle 7, GC skew.

Table 3

Nucleotide content and gene count levels of the genome

Attribute

     Value

    % of total

Genome size (bp)

     2,678,452

    100.00%

DNA coding region (bp)

     2,437,391

    91%

DNA G+C content (bp)

     1,794,562

    67%

Total genes

     2493

RNA genes

     63

    2.65%

rRNA operons

     2

Protein-coding genes

     2,407

    96.55%

Pseudo genes

     31

    1.24%

Genes in paralog clusters

     204

    8.19%

Genes assigned to COGs

     1,457

    58.44%

Genes with signal peptides

     499

    20.02%

Genes with transmembrane helices

     554

    22.22%

Table 4

Number of genes associated with the 25 general COG functional categories

Code

    Value

    %agea

     Description

J

    147

    5.9

     Translation

A

    1

    0.0

     RNA processing and modification

K

    86

    3.5

     Transcription

L

    125

    5.0

     Replication, recombination and repair

B

    1

    0.0

     Chromatin structure and dynamics

D

    36

    1.4

     Cell cycle control, mitosis and meiosis

Y

    0

    0.0

     Nuclear structure

V

    29

    1.2

     Defense mechanisms

T

    156

    6.3

     Signal transduction mechanisms

M

    144

    5.8

     Cell wall/membrane biogenesis

N

    93

    3.7

     Cell motility

Z

    0

    0.0

     Cytoskeleton

W

    0

    0.0

     Extracellular structures

U

    80

    3.2

     Intracellular trafficking and secretion

O

    103

    4.1

     Posttranslational modification, protein turnover, chaperones

C

    168

    6.7

     Energy production and conversion

G

    76

    3.1

     Carbohydrate transport and metabolism

E

    158

    6.3

     Amino acid transport and metabolism

F

    46

    1.9

     Nucleotide transport and metabolism

H

    152

    6.1

     Coenzyme transport and metabolism

I

    72

    2.9

     Lipid transport and metabolism

P

    122

    4.9

     Inorganic ion transport and metabolism

Q

    37

    1.5

     Secondary metabolites biosynthesis, transport and catabolism

R

    222

    8.9

     General function prediction only

S

    167

    6.7

     Function unknown

-

    493

    19.8

     Not in COGs

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

Conclusion

H. halophila is among the most halophilic eubacteria known. Further analysis and characterization of its genome will provide insights into the mechanisms it uses to adapt to hypersaline environments.

Declarations

Acknowledgements

This work was performed under the auspices of the US Department of Energy 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, UT-Battelle, and Oak Ridge National Laboratory under contract DE-AC05-00OR22725.


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. Raymond JC and Sistrom WR. The isolation and preliminary characterization of a halophilic photosynthetic bacterium. Arch Mikrobiol. 1967; 59:255-268View ArticlePubMed
  2. Imhoff JF, Hashwa F and Trüper HG. Isolation of extremely halophilic phototrophic bacteria from the alkaline Wadi Natrun, Egypt. Arch Hydrobiol. 1978; 84:381-388
  3. Imhoff JF and Suling J. The phylogenetic relationship among Ectothiorhodospiraceae: A reevaluation of their taxonomy on the basis of 16S rDNA analyses. Arch Microbiol. 1996; 165:106-113View ArticlePubMed
  4. Ng WV, Kennedy SP, Mahairas GG, Berquist B, Pan M, Shukla HD, Lasky SR, Baliga NS, Thorsson V and Sbrogna J. Genome sequence of Halobacterium species NRC-1. Proc Natl Acad Sci USA. 2000; 97:12176-12181View ArticlePubMed
  5. Pfeiffer F, Schuster SC, Broicher A, Falb M, Palm P, Rodewald K, Ruepp A, Soppa J, Tittor J and Oesterhelt D. Evolution in the laboratory: the genome of Halobacterium salinarum strain R1 compared to that of strain NRC-1. Genomics. 2008; 91:335-346View ArticlePubMed
  6. Baliga NS, Bonneau R, Facciotti MT, Pan M, Glusman G, Deutsch EW, Shannon P, Chiu Y, Weng RS and Gan RR. Genome sequence of Haloarcula marismortui: a halophilic archaeon from the Dead Sea. Genome Res. 2004; 14:2221-2234View ArticlePubMed
  7. Falb M, Pfeiffer F, Palm P, Rodewald K, Hickmann V, Tittor J and Oesterhelt D. Living with two extremes: conclusions from the genome sequence of Natronomonas pharaonis. Genome Res. 2005; 15:1336-1343View ArticlePubMed
  8. Bolhuis H, Palm P, Wende A, Falb M, Rampp M, Rodriguez-Valera F, Pfeiffer F and Oesterhelt D. The genome of the square archaeon Haloquadratum walsbyi: life at the limits of water activity. BMC Genomics. 2006; 7:169View ArticlePubMed
  9. Mongodin EF, Nelson KE, Daugherty S, Deboy RT, Wister J, Khouri H, Weidman J, Walsh DA, Papke RT and Sanchez Perez G. The genome of Salinibacter ruber: convergence and gene exchange among hyperhalophilic bacteria and archaea. Proc Natl Acad Sci USA. 2005; 102:18147-18152 View ArticlePubMed
  10. Mavromatis K, Ivanova N, Anderson I, Lykidis A, Hooper SD, Sun H, Kunin V, Lapidus A, Hugenholtz P and Patel B. Genome analysis of the anaerobic thermohalophilic bacterium Halothermothrix orenii. PLoS ONE. 2009; 4:e4192 View ArticlePubMed
  11. Brown SD, Begemann MB, Mormile MR, Wall JD, Han CS, Goodwin LA, Pitluck S, Land ML, Hauser LJ and Elias DA. Complete genome sequence of the haloalkaliphilic, hydrogen-producing bacterium Halanaerobium hydrogeniformans. J Bacteriol. 2011; 193:3682-3683View ArticlePubMed
  12. Deole R, Challacombe J, Raiford DW, Hoff WD. An Extremely Halophilic Proteobacterium Combines a Highly Acidic Proteome with a Low Cytoplasmic Potassium Content. J Biol Chem 2012.
  13. Ollivier B, Caumette P, Garcia JL and Mah RA. Anaerobic bacteria from hypersaline environments. Microbiol Rev. 1994; 58:27-38PubMed
  14. Frigaard NU and Dahl C. Sulfur metabolism in phototrophic sulfur bacteria. Adv Microb Physiol. 2009; 54:103-200 View ArticlePubMed
  15. Leguijt T, Engels PW, Crielaard W, Albracht SPJ and Hellingwerf KJ. Abundance, subunit composition, redox properties, and catalytic activity of the cytochrome bc1 complex from alkaliphilic and halophilic, photosynthetic members of the family Ectothiorhodospiraceae. J Bacteriol. 1993; 175:1629-1636PubMed
  16. Leguijt T and Hellingwerf KJ. Characterization of reaction center antenna complexes from bacteriochlorophyll a containing Ectothiorhodospiraceae. Biochim Biophys Acta. 1991; 1057:353-360View Article
  17. Lieutaud C, Alric J, Bauzan M, Nitschke W and Schoepp-Cothenet B. Study of the high-potential iron sulfur protein in Halorhodospira halophila confirms that it is distinct from cytochrome c as electron carrier. Proc Natl Acad Sci USA. 2005; 102:3260-3265View ArticlePubMed
  18. Tsuihiji H, Yamazaki Y, Kamikubo H, Imamoto Y and Kataoka M. Cloning and characterization of nif structural and regulatory genes in the purple sulfur bacterium, Halorhodospira halophila. J Biosci Bioeng. 2006; 101:263-270View ArticlePubMed
  19. Meyer TE. Isolation and characterization of soluble cytochromes, ferredoxins and other chromophoric proteins from the halophilic phototrophic bacterium Ectothiorhodospira halophila. Biochim Biophys Acta. 1985; 806:175-183View ArticlePubMed
  20. Meyer TE, Yakali E, Cusanovich MA and Tollin G. Properties of a water-soluble, yellow protein isolated from a halophilic phototrophic bacterium that has photochemical activity analogous to sensory rhodopsin. Biochemistry. 1987; 26:418-423View ArticlePubMed
  21. Sprenger WW, Hoff WD, Armitage JP and Hellingwerf KJ. The Eubacterium Ectothiorhodospira halophila is negatively phototactic, with a wavelength dependence that fits the absorption spectrum of the photoactive yellow protein. J Bacteriol. 1993; 175:3096-3104PubMed
  22. Cusanovich MA and Meyer TE. Photoactive yellow protein: A prototypic PAS domain sensory protein and development of a common signaling mechanism. Biochemistry. 2003; 42:4759-4770View ArticlePubMed
  23. Hellingwerf KJ, Hendriks J and Gensch T. Photoactive Yellow Protein, a new type of photoreceptor protein: Will this "yellow lab" bring us where we want to go? J Phys Chem A. 2003; 107:1082-1094View Article
  24. Kumauchi M, Hara M, Stalcup P, Xie A and Hoff WD. Identification of six new photoactive yellow proteins: diversity and structure-function relationships in a bacterial blue light photoreceptor. Photochem Photobiol. 2008; 84:956-969View ArticlePubMed
  25. 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; 26:541-547View ArticlePubMed
  26. 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; 87:4576-4579View ArticlePubMed
  27. 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.
  28. Validation of publication of new names and new combinations previously effectively published outside the IJSEM. List no. 106. Int J Syst Evol Microbiol. 2005; 55:2235-2238View Article
  29. 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.
  30. Imhoff J. Order I. Chromatiales ord. 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. 1-3.
  31. Imhoff JF. Reassignment of the Genus Ectothiorhodospira Pelsh 1936 to a New Family, Ectothiorhodospiraceae fam. nov., and Emended Description of the Chromatiaceae Bavendamm 1924. Int J Syst Bacteriol. 1984; 34:338-339View Article
  32. Validation of the publication of new names and new combinations previously effectively published outside the IJSB. List No. 62. Int J Syst Bacteriol. 1997; 47:915-916View Article
  33. Imhoff JF and Süling J. The phylogenetic relationship among Ectothiorhodospiraceae: a reevaluation of their taxonomy on the basis of 16S rDNA analyses. Arch Microbiol. 1996; 165:106-113View ArticlePubMed
  34. Hirschler-Réa A, Matheron R, Riffaud C, Mouné S, Eatock C, Herbert RA, Willison JC and Caumette P. Isolation and characterization of spirilloid purple phototrophic bacteria forming red layers in microbial mats of Mediterranean salterns: description of Halorhodospira neutriphila sp. nov. and emendation of the genus Halorhodospira. Int J Syst Evol Microbiol. 2003; 53:153-163View ArticlePubMed
  35. Raymond JC and Sistrom WR. Ectothiorhodospira halophila: a new species of the genus Ectothiorhodospira. Arch Mikrobiol. 1969; 69:121-126View ArticlePubMed
  36. 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; 25:25-29View ArticlePubMed
  37. Edgar RC. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004; 32:1792-1797View ArticlePubMed
  38. Guindon S and Gascuel O. A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol. 2003; 52:696-704View ArticlePubMed
  39. Chevenet F, Brun C, Bañuls AL, Jacq B and Christen R. TreeDyn: towards dynamic graphics and annotations for analyses of trees. BMC Bioinformatics. 2006; 7:439View ArticlePubMed
  40. Dereeper A, Guignon V, Blanc G, Audic S, Buffet S, Chevenet F, Dufayard JF, Guindon S, Lefort V, Lescot M and others. Phylogeny.fr: robust phylogenetic analysis for the non-specialist. Nucleic Acids Res. 2008;36(Web Server issue):W465-9. PMID:18424797
  41. Markowitz VM, Chen IM, Palaniappan K, Chu K, Szeto E, Grechkin Y, Ratner A, Jacob B, Huang J and Williams P. IMG: the Integrated Microbial Genomes database and comparative analysis system. Nucleic Acids Res. 2012; 40(Database issue):D115-D122View ArticlePubMed