Open Access

Complete genome sequence of Acetohalobium arabaticum type strain (Z-7288T)

  • Johannes Sikorski
  • , Alla Lapidus
  • , Olga Chertkov
  • , Susan Lucas
  • , Alex Copeland
  • , Tijana Glavina Del Rio
  • , Matt Nolan
  • , Hope Tice
  • , Jan-Fang Cheng
  • , Cliff Han,
  • , Evelyne Brambilla
  • , Sam Pitluck
  • , Konstantinos Liolios
  • , Natalia Ivanova
  • , Konstantinos Mavromatis
  • , Natalia Mikhailova
  • , Amrita Pati
  • , David Bruce,
  • , Chris Detter
  • , Roxanne Tapia
  • , Lynne Goodwin,
  • , Amy Chen
  • , Krishna Palaniappan
  • , Miriam Land,
  • , Loren Hauser,
  • , Yun-Juan Chang,
  • , Cynthia D. Jeffries,
  • , Manfred Rohde
  • , Markus Göker
  • , Stefan Spring
  • , Tanja Woyke
  • , James Bristow
  • , Jonathan A. Eisen,
  • , Victor Markowitz
  • , Philip Hugenholtz
  • , Nikos C. Kyrpides
  • and Hans-Peter Klenk
Corresponding author

DOI: 10.4056/sigs.1062906

Received: 20 August 2010

Published: 30 August 2010

Abstract

Acetohalobium arabaticum Zhilina and Zavarzin 1990 is of special interest because of its physiology and its participation in the anaerobic C1-trophic chain in hypersaline environments. This is the first completed genome sequence of the family Halobacteroidaceae and only the second genome sequence in the order Halanaerobiales. The 2,469,596 bp long genome with its 2,353 protein-coding and 90 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.

Keywords:

anaerobemesophilehalophilechemolithotrophmethylotrophorganotrophdegradation of betaineconsumption of trimethylaminehomoacetogenClostridiaHalanaerobialesGEBA

Introduction

Strain Z-7288T (= DSM 5501 = ATCC 49924) is the type strain of the species Acetohalobium arabaticum, which is the type species of the genus Acetohalobium [1,2]. The genus name derives from the Latin word ‘acetum’, meaning vinegar, and the Greek words ‘halos’ and ‘bios’, meaning salt and life, respectively, in order to indicate an acetate-producing organism living in salt [3]. The species name derives from Arabat, a peninsula between the Sea of Azov and Sivash [3], since the strain was isolated from lagoons of the Arabat spit (East Crimea) which separates Sivash lake from the Sea of Azov [2]. Currently, this is the only known strain in the genus Acetohalobium. A. arabaticum participates together with other halophilic bacteria and the genera Methanohalophilus and Methanohalobium in the C1-trophic chain in hypersaline environments [2]. Here we present a summary classification and a set of features for A. arabaticum Z-7288T, together with the description of the complete genomic sequencing and annotation.

Classification and features

The cells of A. arabaticum are bent rods, motile by one to two subterminal flagella (Table 1) [2]. The flagella are stated in the original description [2], though they are not visible in our study (Figure 1). The cells are single, in pairs or form short chains, being 0.7-1 µm in diameter and 1-5 µm in length [2]. Other typical cell aggregates are palisades and ribbons, which are formed by adhesion of cells having intimate contact (Figure 1) [2]. The multiplication is by binary fission. The outer membrane is typical of a Gram-negative organism [2]. Growth is completely inhibited by 100 µM/ml streptomycin, benzylpenicillin, bacitracin, erythromycin, gentamycin, kanamycin, vancomycin or tetracyclin [2]. Strain Z-7288T is obligately anaerobic, tolerating up to 12 mM H2S. Neither O2, S2O32-, SO42-, nor S0 can serve as electron acceptors. Strain Z-7288T requires a salt concentration of 10-25% NaCl, the optimum is 15-18% NaCl [2]. The optimal pH is between 7.6 and 8.0 [2].

Table 1

Classification and general features of A. arabaticum Z-7288T according to the MIGS recommendations [4]

MIGS ID

    Property

    Term

   Evidence code

    Current classification

    Domain Bacteria

   TAS [5]

    Phylum Firmicutes

   TAS [6,7]

    Class Clostridia

   TAS [8,9]

    Order Halanaerobiales

   TAS [10-12]

    Family Halobacteroidaceae

   TAS [11,12]

    Genus Acetohalobium

   TAS [1,13]

    Species Acetohalobium arabaticum

   TAS [1,13]

    Type strain Z-7288

   TAS [2]

    Gram stain

    negative

   TAS [2]

    Cell shape

    bent rod

   TAS [2]

    Motility

    motile, subterminal flagella

   TAS [2]

    Sporulation

    unknown; not observed

   NAS

    Temperature range

    max. 47°C

   TAS [2]

    Optimum temperature

    28-40°C

   TAS [2]

    Salinity

    10-25% (optimal 15-18%) NaCl

   TAS [2]

MIGS-22

    Oxygen requirement

    anaerobic

   TAS [2]

    Carbon source

    CO, CO2, TMA, betaine, lactate, pyruvate

   TAS [2]

    Energy source

    chemolithoautotroph, methylotroph,    organotroph

   TAS [2]

MIGS-6

    Habitat

    lagoon

   TAS [2]

MIGS-15

    Biotic relationship

    free-living

   TAS [2]

MIGS-14

    Pathogenicity

    not reported

    Biosafety level

    1

   TAS [14]

    Isolation

    lagoon

   TAS [2]

MIGS-4

    Geographic location

    Arabat Spit, Ukraine

   TAS [2]

MIGS-5

    Sample collection time

    1990 or before

   TAS [2]

MIGS-4.1MIGS-4.2

    Latitude    Longitude

    46.26     34.86

   NAS

MIGS-4.3

    Depth

    unknown

MIGS-4.4

    Altitude

    about 15 m

   NAS

Evidence codes - IDA: Inferred from Direct Assay (first time in publication); 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 of the Gene Ontology project [15]. If the evidence code is IDA, then the property was directly observed by one of the authors or an expert mentioned in the acknowledgements.

Figure 1

Scanning electron micrograph of A. arabaticum Z-7288T

A. arabaticum exhibits three modes of nutrition [2]: It is chemolithoautotrophic using H2 together with CO2 or CO; it is methylotrophic using trimethylamine (TMA); and it is organotrophic using betaine, lactate, pyruvate or histidine. Carbohydrates are not utilized. No growth occurs on methanol, monomethylamine (MMA), dimethylamine (DMA), dimethylglycine, choline or sarcosine [2]. When grown on TMA, an equimolar amount of acetate is formed along with lesser amounts of DMA and MMA [2]. Betaine is degraded mainly to acetate and minor amounts of methylamines [2].

Carbonic anhydrase (CA; carbonate hydrolyase, EC 4.2.1.1) has been studied in strain Z-7288T and in other acetogenic bacteria [16]. This zinc-containing enzyme is found in animals, plants, bacteria and archaea and catalyzes the following reaction: CO2 + H2O ↔ HCO3- and H+ [16]. Further biochemical details of CA are described elsewhere [16]. Strain Z-7288T displayed CA activities similar to those of other CA-containing bacteria [16-18]. With lactate as cultivation substrate the specific activity of CA in strain Z-7288T has been determined to be 2.1± 0.4 units per mg or protein [16]. It has been suggested that one physiological function for CA in acetogens is to increase intracellular CO2 levels [16].

The 16S rRNA genes of the other type strains in the family Halobacteroidaceae share between 85.9% (Orenia sivashensis [19]) and 95.1% (Sporohalobacter lortetii [20]) sequence identity with strain Z-7288T [21]. Uncultured clone sequences from environmental samples and metagenomic surveys do not surpass 84-86% sequence similarity to the 16S rRNA gene sequence of strain Z-7288T, indicating a lack of further members of the genus Acetohalobium in the habitats screened thus far (status June 2010).

Figure 2 shows the phylogenetic neighborhood of A. arabaticum Z-7288T in a 16S rRNA based tree. The sequences of the five 16S rRNA gene copies in the genome of Acetohalobium arabaticum Z-7288T differ from each other by up to one nucleotide, and differ by up to three nucleotides from the previously published 16S rRNA sequence generated from DSM 5501 (X89077).

Figure 2

Phylogenetic tree highlighting the position of A. arabaticum Z-7288T relative to the type strains of the other genera within the order Halanaerobiales. The trees were inferred from 1,308 aligned characters [22,23] of the 16S rRNA gene sequence under the maximum likelihood criterion [24] and rooted in accordance with the current taxonomy [25]. The branches are scaled in terms of the expected number of substitutions per site. Numbers above branches are support values from 300 bootstrap replicates [26] if larger than 60%. Lineages with type strain genome sequencing projects registered in GOLD [27] are shown in blue, published genomes [28] in bold.

Chemotaxonomy

No chemotaxonomic data are currently available for the genus Acetohalobium.

Genome sequencing and annotation

Genome project history

This organism was selected for sequencing on the basis of its phylogenetic position [29], and is part of the Genomic Encyclopedia of Bacteria and Archaea project [30]. The genome project is deposited in the Genome OnLine Database [27] and the complete genome sequence is deposited in GenBank. Sequencing, finishing and annotation were performed by the DOE Joint Genome Institute (JGI). A summary of the project information is shown in Table 2.

Table 2

Genome sequencing project information

MIGS ID

   Property

  Term

MIGS-31

   Finishing quality

  Finished

MIGS-28

   Libraries used

  Two genomic libraries: 454 pyrosequence standard  and paired ended 10kb library

MIGS-29

   Sequencing platforms

  454 GS Titanium, Illumina GAii

MIGS-31.2

   Sequencing coverage

  98.4× pyrosequence

MIGS-30

   Assemblers

  Newbler version 2.0.00.20-  PostRelease-11-05-2008-gcc-3.4.6,  phrap, Velvet

MIGS-32

   Gene calling method

  Prodigal 1.4, GenePRIMP

   INSDC ID

  CP002105

   Genbank Date of Release

  August 9, 2010

   GOLD ID

  Gc01329

   NCBI project ID

  32769

   Database: IMG-GEBA

  2502171194

MIGS-13

   Source material identifier

  DSM 5501

   Project relevance

  Tree of Life, GEBA

Growth conditions and DNA isolation

A. arabaticum Z-7288T, DSM 5501, was grown anaerobically in DSMZ medium 494 (Acetohalobium medium) [31] at 37°C. DNA was isolated from 0.5-1 g of cell paste using MasterPure Gram Positive DNA Purification Kit (Epicentre MGP04100). Two µl lysozyme and five µl mutanolysin were added to the standard lysis solution for 40min at 37°C followed by 1 hour incubation on ice after the MPC-step.

Genome sequencing and assembly

The genome of A. arabaticum Z-7288T was sequenced at using a combination of Illumina and 454 technologies. An Illumina GAii shotgun library with reads of 483 Mb a 454 Titanium draft library with average read length of 341 bases, and a paired end 454 library with average insert size of 10 kb were generated for this genome. All general aspects of library construction and sequencing can be found at Web Site. Illumina sequencing data was assembled with VELVET and the consensus sequences were shredded into 1.5 kb overlapped fake reads and assembled together with the 454 data. Draft assemblies were based on 241 Mb 454 draft data, and 454 paired end data. Newbler parameters are -consed -a 50 -l 350 -g -m -ml 20. The initial assembly contained 72 contigs in one scaffold. The initial 454 assembly was converted into a phrap assembly by making fake reads from the consensus, collecting the read pairs in the 454 paired end library. The Phred/Phrap/Consed software package (Web Site) was used for sequence assembly and quality assessment in the following finishing process [32]. After the shotgun stage, reads were assembled with parallel phrap (High Performance Software, LLC). Possible mis-assemblies were corrected with gapResolution (Web Site), Dupfinisher [32], or sequencing cloned bridging PCR fragments with subcloning or transposon bombing (Epicentre Biotechnologies, Madison, WI). Gaps between contigs were closed by editing in Consed, by PCR and by Bubble PCR primer walks (J.-F. Cheng, unpublished). A total of 292 additional reactions were necessary to close gaps and to raise the quality of the finished sequence. Illumina reads were used to improve the final consensus quality using an in-house developed tool (the Polisher [33], ). The completed genome sequences have an error rate of less than 1 in 100,000 bp.

Genome annotation

Genes were identified using Prodigal [34] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI Web Site [35]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) nonredundant database, UniProt, TIGRFam, Pfam, PRIAM, KEGG, COG, and InterPro databases. Additional gene prediction analysis and functional annotation was performed within the Web Site (IMG-ER) platform [36].

Genome properties

The genome consists of a 2,469,596 bp long chromosome with a 36.6% GC content (Table 3 and Figure 3). Of the 2,443 genes predicted, 2,353 were protein-coding genes, and 90 RNAs; Seventy-one pseudogenes were also identified. The majority of the protein-coding genes (76.4%) were assigned a putative function while the remaining ones were annotated as hypothetical proteins. The distribution of genes into COGs functional categories is presented in Table 4.

Table 3

Genome Statistics

Attribute

Value

  % of Total

Genome size (bp)

2,469,596

  100.00%

DNA coding region (bp)

2,147,537

  86.96%

DNA G+C content (bp)

904,645

  36.63%

Number of replicons

1

Extrachromosomal elements

0

Total genes

2,443

  100.00%

RNA genes

90

  3.67%

rRNA operons

5

Protein-coding genes

2,353

  96.33%

Pseudo genes

71

  3.30%

Genes with function prediction

1,873

  76.36%

Genes in paralog clusters

505

  20.58%

Genes assigned to COGs

1,861

  75.87%

Genes assigned Pfam domains

2,022

  82.43%

Genes with signal peptides

378

  15.41%

Genes with transmembrane helices

303

  12.35%

CRISPR repeats

1

Figure 3

Graphical circular map of the genome. 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 4

Number of genes associated with the general COG functional categories

Code

   value

  %age

  Description

J

   133

  6.5

  Translation, ribosomal structure and biogenesis

A

   0

  0.0

  RNA processing and modification

K

   119

  5.8

  Transcription

L

   139

  6.8

  Replication, recombination and repair

B

   2

  0.1

  Chromatin structure and dynamics

D

   26

  1.3

  Cell cycle control, cell division, chromosome partitioning

Y

   0

  0.0

  Nuclear structure

V

   21

  1.0

  Defense mechanisms

T

   88

  4.3

  Signal transduction mechanisms

M

   123

  6.0

  Cell wall/membrane/envelope biogenesis

N

   66

  3.2

  Cell motility

Z

   0

  0.0

  Cytoskeleton

W

   0

  0.0

  Extracellular structures

U

   56

  2.8

  Intracellular trafficking, secretion, and vesicular transport

O

   85

  4.2

  Posttranslational modification, protein turnover, chaperones

C

   150

  7.4

  Energy production and conversion

G

   62

  3.0

  Carbohydrate transport and metabolism

E

   187

  9.2

  Amino acid transport and metabolism

F

   61

  30

  Nucleotide transport and metabolism

H

   144

  7.1

  Coenzyme transport and metabolism

I

   43

  2.1

  Lipid transport and metabolism

P

   106

  5.2

  Inorganic ion transport and metabolism

Q

   19

  0.9

  Secondary metabolites biosynthesis, transport and catabolism

R

   224

  11.0

  General function prediction only

S

   183

  9.0

  Function unknown

-

   582

  24.1

  Not in COGs

Declarations

Acknowledgements

We would like to gratefully acknowledge the help of Maren Schröder (DSMZ) for growing cultures of A. arabaticum. 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, as well as German Research Foundation (DFG) INST 599/1-2 and SI 1352/1-2.


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. Zhilina TN and Zavarzin GA. A new extremely halophilic homoacetogen bacteria Acetohalobium arabaticum, gen. nov. sp. nov. Dokl Akad Nauk SSSR. 1990; 311:745-747
  2. Zhilina TN and Zavarzin GA. Extremely halophilic, methylotrophic, anaerobic bacteria. FEMS Microbiol Lett. 1990; 87:315-322 View Article
  3. 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 ArticlePubMed
  4. 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
  5. 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
  6. 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.
  7. Gibbons NE and Murray RGE. Proposals concerning the higher taxa of bacteria. Int J Syst Bacteriol. 1978; 28:1-6 View Article
  8. List editor. List of new names and new combinations previously effectively, but not validly, published. Int J Syst Evol Microbiol. 2010; 60:469-472 View Article
  9. Rainey FA. 2009. Class II. Clostridia class nov., p. 736. In P. De Vos, G. Garrity, D. Jones, N. R. Krieg, W. Ludwig, F. A. Rainey, K. H. Schleifer, and W. B. Whitman (ed.), Bergey’s Manual of Systematic Bacteriology, 3 ed, vol. 3. Springer, New York.
  10. . The nomenclatural types of the orders Acholeplasmatales, Halanaerobiales, Halobacteriales, Methanobacteriales, Methanococcales, Methanomicrobiales, Planctomycetales, Prochlorales, Sulfolobales, Thermococcales, Thermoproteales and Verrucomicrobiales are the genera Acholeplasma, Halanaerobium, Halobacterium, Methanobacterium, Methanococcus, Methanomicrobium, Planctomyces, Prochloron, Sulfolobus, Thermococcus, Thermoproteus and Verrucomicrobium, respectively. Opinion 79. Int J Syst Evol Microbiol. 2005; 55:517-518 View ArticlePubMed
  11. Rainey FA, Zhilina TN, Boulygina ES, Stackebrandt E, Tourova TP and Zavarzin GA. The taxonomic status of the fermentative halophilic anaerobic bacteria: description of Haloanaerobiales ord. nov., Halobacteroidaceae fam. nov., Orenia gen. nov. and further taxonomic rearrangements at the genus and species level. Anaerobe. 1995; 1:185-199 View ArticlePubMed
  12. . Validation of the publication of new names and new combinations previously effectively published outside the IJSB: List No. 55. Int J Syst Bacteriol. 1995; 45:879-880 View Article
  13. . List No. 35. Int J Syst Bacteriol. 1990; 40:470-471 View Article
  14. Classification of bacteria and archaea in risk groups. TRBA 466.Web Site
  15. 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
  16. Braus-Stromeyer SA, Schnappauf G, Braus GH, Gossner AS and Drake HL. Carbonic anhydrase in Acetobacterium woodii and other acetogenic bacteria. J Bacteriol. 1997; 179:7197-7200PubMed
  17. Alber BE and Ferry JG. A carbonic anhydrase from the archaeon Methanosarcina thermophila. Proc Natl Acad Sci USA. 1994; 91:6909-6913 View ArticlePubMed
  18. Karrasch M, Bott M and Thauer RK. Carbonic anhydrase activity in acetate grown Methanosarcina barkeri. Arch Microbiol. 1989; 151:137-142 View Article
  19. Zhilina TN, Turova TP, Kuznetsov BB, Kostrikina NA and Lysenko AM. Orenia sivashensis sp. nov., a new moderately halophilic anaerobic bacterium from Lake Sivash lagoons. Mikrobiologiya. 1999; 68:519-527
  20. Oren A, Pohla H and Stackebrandt E. Transfer of Clostridium lortetii to a new genus Sporohalobacter gen. nov. as Sporohalobacter lortetii comb. nov. and description of Sporohalobacter marismortui sp. nov. Syst Appl Microbiol. 1987; 9:239-246
  21. Chun J, Lee JH, Jung Y, Kim M, Kim S, Kim BK and Lim YW. EzTaxon: a web-based tool for the identification of prokaryotes based on 16S ribosomal RNA gene sequences. Int J Syst Evol Microbiol. 2007; 57:2259-2261 View ArticlePubMed
  22. Castresana J. Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol Biol Evol. 2000; 17:540-552PubMed
  23. Lee C, Grasso C and Sharlow MF. Multiple sequence alignment using partial order graphs. Bioinformatics. 2002; 18:452-464 View ArticlePubMed
  24. Stamatakis A, Hoover P and Rougemont J. A Rapid Bootstrap Algorithm for the RAxML Web Servers. Syst Biol. 2008; 57:758-771 View ArticlePubMed
  25. Yarza P, Richter M, Peplies J, Euzeby J, Amann R, Schleifer KH, Ludwig W, Glöckner FO and Rosselló-Móra R. The All-Species Living Tree project: A 16S rRNA-based phylogenetic tree of all sequenced type strains. Syst Appl Microbiol. 2008; 31:241-250 View ArticlePubMed
  26. 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
  27. Liolios K, Chen IM, Mavromatis K, Tavernarakis N, Hugenholtz P, Markovitzz 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
  28. Mavromatis K, Ivanova N, Anderson I, Lykidis A, Hooper SD, Sun H, Kunin V, Lapidus A, Hugenholtz P, Patel B and Kyrpides NC. Genome analysis of the anaerobic thermopholophilic bacterium Halothermothrix orenii. PLoS ONE. 2009; 4:e4192 View ArticlePubMed
  29. Klenk HP and Göker M. En route to a genome-based classification of Archaea and Bacteria? Syst Appl Microbiol. 2010; 33:175-182 View ArticlePubMed
  30. Wu D, Hugenholtz P, Mavromatis K, Pukall R, Dalin E, Ivanova NN, Kunin V, Goodwin L, Wu M and Tindall BJ. A phylogeny-driven genomic encyclopaedia of Bacteria and Archaea. Nature. 2009; 462:1056-1060 View ArticlePubMed
  31. List of growth media used at DSMZ: Web Site
  32. Sims D, Brettin T, Detter J, Han C, Lapidus A, Copeland A, Glavina Del Rio T, Nolan M, Chen F and Lucas S. Complete genome sequence of Kytococcus sedentarius type strain (541T). Stand Genomic Sci. 2009; 1:12-20 View Article
  33. 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
  34. Hyatt D, Chen GL, Locascio PF, Land ML, Larimer FW and Hauser LJ. Prodigal Prokaryotic Dynamic Programming Genefinding Algorithm. BMC Bioinformatics. 2010; 11:119 View ArticlePubMed
  35. Pati A, Ivanova N, Mikhailova N, Ovchinikova G, Hooper SD, Lykidis A and Kyrpides NC. GenePRIMP: A gene prediction improvement pipeline for microbial genomes. Nat Methods. 2010; 7:455-457 View ArticlePubMed
  36. Markowitz VM, 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