Open Access

Genome sequence of Microvirga lupini strain LUT6T, a novel Lupinus alphaproteobacterial microsymbiont from Texas

  • Wayne Reeve1Email author,
  • Matthew Parker2,
  • Rui Tian1,
  • Lynne Goodwin3,
  • Hazuki Teshima3,
  • Roxanne Tapia3,
  • Cliff Han3,
  • James Han4,
  • Konstantinos Liolios4,
  • Marcel Huntemann4,
  • Amrita Pati4,
  • Tanja Woyke4,
  • Konstantinos Mavromatis4,
  • Victor Markowitz5,
  • Natalia Ivanova4 and
  • Nikos Kyrpides4
Standards in Genomic Sciences20149:9031159

https://doi.org/10.4056/sigs.5249382

Published: 15 June 2014

Abstract

Microvirga lupini LUT6T is an aerobic, non-motile, Gram-negative, non-spore-forming rod that can exist as a soil saprophyte or as a legume microsymbiont of Lupinus texensis. LUT6T was isolated in 2006 from a nodule recovered from the roots of the annual L. texensis growing in Travis Co., Texas. LUT6T forms a highly specific nitrogen-fixing symbiosis with endemic L. texensis and no other Lupinus species can form an effective nitrogen-fixing symbiosis with this isolate. Here we describe the features of M. lupini LUT6T, together with genome sequence information and its annotation. The 9,633,614 bp improved high quality draft genome is arranged into 160 scaffolds of 1,366 contigs containing 10,864 protein-coding genes and 87 RNA-only encoding genes, and is one of 20 rhizobial genomes sequenced as part of a DOE Joint Genome Institute 2010 Community Sequencing Project.

Keywords

root-nodule bacterianitrogen fixationrhizobia Alphaproteobacteria

Introduction

Microvirga is one of the most recently discovered genera of Proteobacteria known to engage in symbiotic nitrogen fixation with legume plants, and joins a diverse set of at least twelve other lineages of Proteobacteria that share this ecological niche [14]. Several genera of legume root-nodule symbionts have a world-wide distribution and interact with many legume taxa. By contrast, symbiotic strains of Microvirga are currently known from two distant locations and only two legume host genera [5,6]. The limited geographic and host distribution of Microvirga symbionts, along with the fact that root-nodule symbiosis is not characteristic of the genus Microvirga as a whole [7], suggest a relatively recent evolutionary transition to legume symbiosis in this group.

M. lupini is a specialized nodule symbiont associated with the legume Lupinus texensis, an annual plant endemic to a relatively small geographic area in central Texas and northeastern Mexico [5]. The genus Lupinus has about 270 annual and perennial species concentrated in western North America and in Andean regions of South America, and a much smaller number of species in the Mediterranean region of Europe and northern Africa [8]. Basal lineages of Lupinus all occur in the Mediterranean and are associated with bacterial symbionts in the genus Bradyrhizobium [9,10]. Bradyrhizobium is also the main symbiont lineage for most Lupinus species in North and South America, although a few Lupinus species utilize nodule bacteria in the genus Mesorhizobium [1013]. Thus, the acquisition of symbionts in the genus Microvirga by plants of L. texensis appears to be an unusual, derived condition for this legume genus.

L. texensis occurs in grassland and open shrub communities with an annual precipitation of 50 – 100 cm, on diverse soil types [14]. L. texensis appears to have a specialized symbiotic relationship with M. lupini in that existing surveys have failed to detect nodule symbionts of any other bacterial genus associated with this plant [5]. Moreover, inoculation experiments with other North American species of Lupinus, as well as other legume genera, have so far failed to identify any plant besides L. texensis that is capable of forming an effective, nitrogen-fixing symbiosis with M. lupini [5]. M. lupini strain Lut6T was isolated from a nodule collected from a L. texensis plant in Travis Co., Texas in 2006. Here we provide an analysis of the complete genome sequence of M. lupini strain Lut6T; one of the three described symbiotic species of Microvirga [15].

Classification and general features

M. lupini LUT6T is a non-motile, Gram-negative rod in the order Rhizobiales of the class Alphaproteobacteria. The rod-shaped form varies in size with dimensions of 1.0 µm for width and 1.5–2.0 µm for length (Figure 1 Left and Center). It is fast growing, forming colonies within 3–4 days when grown on half strength Lupin Agar (½LA) [16], tryptone-yeast extract agar (TY) [17] or a modified yeast-mannitol agar (YMA) [18] at 28°C. Colonies on ½LA are white-opaque, slightly domed and moderately mucoid with smooth margins (Figure 1 Right).
Figure 1.

Images of M. lupini LUT6T using scanning (Left) and transmission (Center) electron microscopy and the appearance of colony morphology on solid medium (Right).

Minimum Information about the Genome Sequence (MIGS) is provided in Table 1. Figure 2 shows the phylogenetic neighbor-hood of M. lupini LUT6T in a 16S rRNA sequence based tree. This strain shares 100% (1,358/1,358 bases) and 98% (1,344/1,367 bases) sequence identity to the 16S rRNA of Microvirga sp. Lut5 and Microvirga lotononidis WSM3557T, respectively.
Figure 2.

Phylogenetic tree showing the relationship of M. lupini LUT6T (shown in bold print) to other root nodule bacteria in the order Rhizobiales based on aligned sequences of the 16S rRNA gene (1,320 bp internal region). All sites were informative and there were no gap-containing sites. Phylogenetic analyses were performed using MEGA, version 5 [31]. The tree was built using the Maximum-Likelihood method with the General Time Reversible model [32]. Bootstrap analysis [33] with 500 replicates was performed 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 [34]. Published genomes are indicated with an asterisk.

Table 1.

Classification and general features of M. lupini LUT6T according to the MIGS recommendations [19,20]

MIGS ID

Property

Term

Evidence code

 

Current classification

Domain Bacteria

TAS [20]

 

Phylum Proteobacteria

TAS [21]

 

Class Alphaproteobacteria

TAS [22,23]

 

Order Rhizobiales

TAS [23,24]

 

Family Methylobacteriaceae

TAS [23,25]

 

Genus Microvirga

TAS [15,2628]

 

Species Microvirga lupini

TAS [15]

 

Strain LUT6T

 
 

Gram stain

Negative

TAS [15]

 

Cell shape

Rod

TAS [15]

 

Motility

Non-Motile

IDA

 

Sporulation

Non-sporulating

TAS [15]

 

Temperature range

Mesophile

TAS [15]

 

Optimum temperature

39°C

TAS [15]

 

Salinity

Non-halophile

TAS [15]

MIGS-22

Oxygen requirement

Aerobic

TAS [15]

 

Carbon source

Varied

TAS [15]

 

Energy source

Chemoorganotroph

TAS [15]

MIGS-6

Habitat

Soil, root nodule, on host

TAS [15]

MIGS-15

Biotic relationship

Free living, symbiotic

TAS [15]

MIGS-14

Pathogenicity

Non-pathogenic

NAS

 

Biosafety level

1

TAS [29]

 

Isolation

Root nodule of Lupinus texensis

TAS [5]

MIGS-4

Geographic location

Travis Co., Texas

TAS [5]

MIGS-5

Soil collection date

03 Jan 2006

IDA

MIGS-4.1

Latitude

−97.838

IDA

MIGS-4.2

Longitude

30.459

IDA

MIGS-4.3

Depth

0–10 cm

IDA

MIGS-4.4

Altitude

270 m

IDA

Evidence codes — IDA: Inferred from Direct Assay; TAS: Traceable Author Statement (i.e., a direct report exists in the literature); NAS: Non-traceable Author Statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project [30].

Symbiotaxonomy

M. lupini strain Lut6T was isolated in from a nodule collected from Lupinus texensis growing near Travis Co., Texas. The symbiotic characteristics of this isolate on a range of selected hosts are provided in Table 2.
Table 2.

Nodulation and N2 fixation properties of M. lupini Lut6T on selected legumes.

Legume Species

Nodulation

N2 fixation

Comment

Lupinus texensis

Nod+

Fix+

Highly effective

Lupinus perennis

Nod

Fix

No nodulation

Lupinus succulentus

Nod

Fix

No nodulation

Lupinus microcarpus

Nod

Fix

No nodulation

Phaseolus vulgaris

Nod

Fix

No nodulation

Macroptilium atropurpureum

Nod+

Fix

No fixation

Desmodium canadense

Nod

Fix

No nodulation

Cytisus scoparius

Nod+

Fix

No fixation

Mimosa pudica

Nod

Fix

No nodulation

Data compiled [5]. Note that ‘+’ and ‘-’ denote presence or absence, respectively, of nodulation (Nod) or N2 fixation (Fix).

Genome sequencing and annotation

Genome project history

This organism was selected for sequencing on the basis of its environmental and agricultural relevance to issues in global carbon cycling, alternative energy production, and biogeochemical importance, and is part of the Community Sequencing Program at the U.S. Department of Energy, Joint Genome Institute (JGI) for projects of relevance to agency missions. The genome project is deposited in the Genomes OnLine Database [34] and an improved-high-quality-draft genome sequence in IMG. Sequencing, finishing and annotation were performed by the JGI. A summary of the project information is shown in Table 3.
Table 3.

Genome sequencing project information for M. lupini LUT6T.

MIGS ID

Property

Term

MIGS-31

Finishing quality

Improved high-quality draft

MIGS-28

Libraries used

Illumina GAii shotgun and a paired end 454 libraries

MIGS-29

Sequencing platforms

Illumina GAii and 454 GS FLX Titanium technologies

MIGS-31.2

Sequencing coverage

3.5× 454 paired end, 300× Illumina

MIGS-30

Assemblers

Velvet version 1.0.13; Newbler 2.3, phrap SPS - 4.24

MIGS-32

Gene calling methods

Prodigal 1.4

 

GOLD ID

Gi06478

 

NCBI project ID

66529

 

Database: IMG

2508501050

 

Project relevance

Symbiotic N2 fixation, agriculture

Genome sequencing and assembly

The genome of M. lupini LUT6T was sequenced at the Joint Genome Institute (JGI) using a combination of Illumina [37] and 454 technologies [38]. An Illumina GAii shotgun library which generated 77,090,752 reads totaling 5,858.9 Mbp, and a paired end 454 library with an average insert size of 8 Kbp which generated 238,026 reads totaling 81.4 Mb of 454 data were generated for this genome [36].

All general aspects of library construction and sequencing performed at the JGI can be found at [36]. The initial draft assembly contained 1,719 contigs in 6 scaffolds. The 454 paired end data were assembled together with Newbler, version 2.3-PreRelease-6/30/2009. The Newbler consensus sequences were computationally shredded into 2 Kbp overlapping fake reads (shreds). Illumina sequencing data was assembled with VELVET, version 1.0.13 [39], and the consensus sequence computationally shredded into 1.5 Kbp overlapping fake reads (shreds). The 454 Newbler consensus shreds, the Illumina VELVET consensus shreds and the read pairs in the 454 paired end library were integrated using parallel phrap, version SPS - 4.24 (High Performance Software, LLC). The software Consed [4042] 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 [43]. Possible mis-assemblies were corrected using gapResolution (Cliff Han, unpublished) or Dupfinisher [44]. Some gaps between contigs were closed by editing in Consed. The estimated genome size is 10.3 Mb and the final assembly is based on 36.2 Mb of 454 draft data which provides an average 3.5x coverage of the genome and 3,090 Mbp of Illumina draft data which provides an average 300x coverage of the genome.

Genome annotation

Genes were identified using Prodigal [45] as part of the DOE-JGI annotation pipeline [46]. 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. The tRNAScanSE tool [47] was used to find tRNA genes, whereas ribosomal RNA genes were found by searches against models of the ribosomal RNA genes built from SILVA [48]. Other non-coding RNAs such as the RNA components of the protein secretion complex and the RNase P were identified by searching the genome for the corresponding Rfam profiles using INFERNAL [49]. Additional gene prediction analysis and manual functional annotation was performed within the Integrated Microbial Genomes (IMG-ER) platform [50].

Genome properties

The genome is 9,633,614 nucleotides long with 60.26% GC content (Table 4) and comprised of 160 scaffolds (Figure 3) of 1,366 contigs. From a total of 10,951 genes, 10,864 were protein encoding and 87 RNA only encoding genes. The majority of genes (63.25%) were assigned a putative function whilst the remaining genes were annotated as hypothetical. The distribution of genes into COGs functional categories is presented in Table 5.
Figure 3.

Graphical map of the genome of Microvirga lupini LUT6T showing the four largest scaffolds. From bottom to the top of each scaffold: 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.

Table 4.

Genome statistics for Microvirga lupini LUT6T

Attribute

Value

% of Total

Genome size (bp)

9,633,614

100.00

DNA coding region (bp)

7,880,506

81.80

DNA G+C content (bp)

5,805,078

60.26

Number of scaffolds

160

 

Number of contigs

1,366

 

Total genes

10,951

100.00

RNA genes

87

0.79

rRNA operons

1

0.01

Protein-coding genes

10,864

99.21

Genes with function prediction

6,927

63.25

Genes assigned to COGs

6,990

63.83

Genes assigned Pfam domains

7,343

67.05

Genes with signal peptides

768

7.01

Genes with transmembrane helices

2,006

18.32

CRISPR repeats

0

 
Table 5.

Number of protein coding genes of Microvirga lupini LUT6T associated with the general COG functional categories.

Code

Value

%age

COG Category

J

209

2.72

Translation, ribosomal structure and biogenesis

A

1

0.01

RNA processing and modification

K

571

7.43

Transcription

L

667

8.68

Replication, recombination and repair

B

10

0.13

Chromatin structure and dynamics

D

53

0.69

Cell cycle control, mitosis and meiosis

Y

  

Nuclear structure

V

104

1.35

Defense mechanisms

T

463

6.02

Signal transduction mechanisms

M

316

4.11

Cell wall/membrane biogenesis

N

69

0.9

Cell motility

Z

0

0

Cytoskeleton

W

1

0.01

Extracellular structures

U

95

1.24

Intracellular trafficking and secretion

O

249

3.24

Posttranslational modification, protein turnover, chaperones

C

401

5.22

Energy production conversion

G

602

7.83

Carbohydrate transport and metabolism

E

828

10.77

Amino acid transport metabolism

F

100

1.3

Nucleotide transport and metabolism

H

263

3.42

Coenzyme transport and metabolism

I

266

3.46

Lipid transport and metabolism

P

388

5.05

Inorganic ion transport and metabolism

Q

263

3.42

Secondary metabolite biosynthesis, transport and catabolism

R

976

12.70

General function prediction only

S

790

10.28

Function unknown

-

3,961

36.17

Not in COGS

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 research funding received from Murdoch University.

Authors’ Affiliations

(1)
Centre for Rhizobium Studies, Murdoch University
(2)
Biological Sciences Department, Binghampton University
(3)
Bioscience Division, Los Alamos National Laboratory
(4)
DOE Joint Genome Institute
(5)
Biological Data Management and Technology Center, Lawrence Berkeley National Laboratory

References

  1. Ardley JK. Symbiotic specificity and nodulation in the southern African legume clade Lotononis s. l. and description of novel rhizobial species within the Alphaproteobacterial genus Microvirga: Murdoch University, Murdoch, WA, Australia; 2012.Google Scholar
  2. Gyaneshwar P, Hirsch AM, Moulin L, Chen WM, Elliott GN, Bontemps C, Estrada-de Los Santos P, Gross E, Dos Reis FB, Sprent JI, et al. Legume-nodulating betaproteobacteria: diversity, host range, and future prospects. Mol Plant Microbe Interact 2011; 24:1276–1288. PubMed http://dx.doi.org/10.1094/MPMI-06-11-0172View ArticlePubMedGoogle Scholar
  3. Maynaud G, Willems A, Soussou S, Vidal C, Maure L, Moulin L, Cleyet-Marel JC, Brunel B. Molecular and phenotypic characterization of strains nodulating Anthyllis vulneraria in mine tailings, and proposal of Aminobacter anthyllidis sp. nov., the first definition of Aminobacter as legume-nodulating bacteria. Syst Appl Microbiol 2012; 35:65–72. PubMed http://dx.doi.org/10.1016/j.syapm.2011.11.002View ArticlePubMedGoogle Scholar
  4. Willems A. The taxonomy of rhizobia; an overview. Plant Soil 2006; 287:3–14. http://dx.doi.org/10.1007/s11104-006-9058-7View ArticleGoogle Scholar
  5. Andam CP, Parker MA. Novel alphaproteobacterial root nodule symbiont associated with Lupinus texensis. Appl Environ Microbiol 2007; 73:5687–5691. PubMed http://dx.doi.org/10.1128/AEM.01413-07PubMed CentralView ArticlePubMedGoogle Scholar
  6. Yates RJ, Howieson JG, Reeve WG, Nandasena KG, Law IJ, Bräu L, Ardley JK, Nistelberger HM, Real D, O’Hara GW. Lotononis angolensis forms nitrogen fixing, lupinoid nodules with phylogenetically unique, fast-growing, pink-pigmented bacteria, which do not nodulate L. bainesii or L. listii. Soil Biol Biochem 2007; 39:1680–1688. http://dx.doi.org/10.1016/j.soilbio.2007.01.025View ArticleGoogle Scholar
  7. Weon HY, Kwon SW, Son JA, Jo EH, Kim SJ, Kim YS, Kim BY, Ka JO. Description of Microvirga aerophila sp. nov. and Microvirga aerilata sp. nov., isolated from air, reclassification of Balneimonas flocculansTakeda et al. 2004 as Microvirga flocculans comb. nov. and emended description of the genus Microvirga. Int J Syst Evol Microbiol 2010; 60:2596–2600 PubMed http://dx.doi.org/10.1099/ijs.0.018770-0View ArticlePubMedGoogle Scholar
  8. Drummond CS, Eastwood RJ, Miotto ST, Hughes CE. Multiple continental radiations and correlates of diversification in Lupinus (Leguminosae): testing for key innovation with incomplete taxon sampling. Syst Biol 2012; 61:443–460. PubMed http://dx.doi.org/10.1093/sysbio/syr126PubMed CentralView ArticlePubMedGoogle Scholar
  9. Jarabo-Lorenzo A, Velazquez E, Perez-Galdona R, Vega-Hernandez MC, Martinez-Molina E, Mateos PF, Vinuesa P, Martinez-Romero E, Leon-Barrios M. Restriction fragment length polymorphism analysis of 16S rDNA and low molecular weight RNA profiling of rhizobial isolates from shrubby legumes endemic to the Canary islands. Syst Appl Microbiol 2000; 23:418–425. PubMed http://dx.doi.org/10.1016/S0723-2020(00)80073-9View ArticlePubMedGoogle Scholar
  10. Stepkowski T, Hughes CE, Law IJ, Markiewicz L, Gurda D, Chlebicka A, Moulin L. Diversification of lupine Bradyrhizobium strains: evidence from nodulation gene trees. Appl Environ Microbiol 2007; 73:3254–3264. PubMed http://dx.doi.org/10.1128/AEM.02125-06PubMed CentralView ArticlePubMedGoogle Scholar
  11. Barrera LL, Trujillo ME, Goodfellow M, Garcia FJ, Hernandez-Lucas I, Davila G, van Berkum P, Martinez-Romero E. Biodiversity of bradyrhizobia nodulating Lupinus spp. Int J Syst Evol Microbiol 1997; 47:1086–1091. PubMedGoogle Scholar
  12. Koppell JH, Parker MA. Phylogenetic clustering of Bradyrhizobium symbionts on legumes indigenous to North America. Microbiology 2012; 158:2050–2059. PubMed http://dx.doi.org/10.1099/mic0.059238-0View ArticlePubMedGoogle Scholar
  13. Simms EL, Taylor DL, Povich J, Shefferson RP, Sachs JL, Urbina M, Tausczik Y. An empirical test of partner choice mechanisms in a wild legume-Rhizobium interaction. Proceedings of Biological Sciences 2006;273(1582):77–81.View ArticleGoogle Scholar
  14. Nixon ES. Edaphic responses of Lupinus texensis and Lupinus subcarnosus. Ecology 1964; 45:459–469. http://dx.doi.org/10.2307/1936099View ArticleGoogle Scholar
  15. Ardley JK, Parker MA, De Meyer SE, Trengove RD, O’Hara GW, Reeve WG, Yates RJ, Dilworth MJ, Willems A, Howieson JG. Microvirga lupini sp. nov., Microvirga lotononidis sp. nov. and Microvirga zambiensis sp. nov. are alphaproteobacterial root-nodule bacteria that specifically nodulate and fix nitrogen with geographically and taxonomically separate legume hosts. Int J Syst Evol Microbiol 2012; 62:2579–2588. PubMed http://dx.doi.org/10.1099/ijs.0.035097-0View ArticlePubMedGoogle Scholar
  16. Howieson JG, Ewing MA, D’antuono MF. Selection for acid tolerance in Rhizobium meliloti. Plant Soil 1988; 105:179–188. http://dx.doi.org/10.1007/BF02376781View ArticleGoogle Scholar
  17. Beringer JE. R factor transfer in Rhizobium leguminosarum. J Gen Microbiol 1974; 84:188–198. PubMed http://dx.doi.org/10.1099/00221287-84-1-188PubMedGoogle Scholar
  18. Terpolilli JJ. Why are the symbioses between some genotypes of Sinorhizobium and Medicago suboptimal for N2 fixation? Perth: Murdoch University; 2009. 223 p.Google Scholar
  19. Field D, Garrity G, Gray T, Morrison N, Selengut J, Sterk P, Tatusova T, Thomson N, Allen M, Angiuoli SV, et al. 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. PubMed http://dx.doi.org/10.1038/nbt1360PubMed CentralView ArticlePubMedGoogle Scholar
  20. Woese CR, Kandler O, 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. PubMed http://dx.doi.org/10.1073/pnas.87.12.4576PubMed CentralView ArticlePubMedGoogle Scholar
  21. 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. 1View ArticleGoogle Scholar
  22. 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.View ArticleGoogle Scholar
  23. Validation List No. 107. List of new names and new combinations previously effectively, but not validly, published. Int J Syst Evol Microbiol 2006; 56:1–6. PubMed http://dx.doi.org/10.1099/ijs.0.64188-0
  24. 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.Google Scholar
  25. Garrity GM, Bell JA, Lilburn T. Family IX. Methylobacteriaceae fam. 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. 567.Google Scholar
  26. Kanso S, Patel BKC. Microvirga subterranea gen. nov., sp. nov., a moderate thermophile from a deep subsurface Australian thermal aquifer. Int J Syst Evol Microbiol 2003; 53:401–406. PubMed http://dx.doi.org/10.1099/ijs.0.02348-0View ArticlePubMedGoogle Scholar
  27. Zhang J, Song F, Xin YH, Zhang J, Fang C. Microvirga guangxiensis sp. nov., a novel alphaproteobacterium from soil, and emended description of the genus Microvirga. Int J Syst Evol Microbiol 2009; 59:1997–2001. PubMed http://dx.doi.org/10.1099/ijs.0.007997-0View ArticlePubMedGoogle Scholar
  28. Weon H-Y, Kwon S-W, Son J-A, Jo E-H, Kim S-J, Kim Y-S, Kim B-Y, Ka J-O. Description of Microvirga aerophila sp. nov. and Microvirga aerilata sp. nov., isolated from air, reclassification of Balneimonas flocculans Takeda et al. 2004 as Microvirga flocculans comb. nov. and emended description of the genus Microvirga. Int J Syst Evol Microbiol 2010; 60:2596–2600. PubMed http://dx.doi.org/10.1099/ijs.0.018770-0View ArticlePubMedGoogle Scholar
  29. Gubler M, Hennecke H, Fix A. B and C genes are essential for symbiotic and free-living, microaerobic nitrogen fixation. FEBS Lett 1986; 200:186–192. http://dx.doi.org/10.1016/0014-5793(86)80536-1View ArticleGoogle Scholar
  30. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, et al. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet 2000; 25:25–29. PubMed http://dx.doi.org/10.1038/75556PubMed CentralView ArticlePubMedGoogle Scholar
  31. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: Molecular Evolutionary Genetics Analysis using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Mol Biol Evol 2011; 28:2731–2739. PubMed http://dx.doi.org/10.1093/molbev/msr121PubMed CentralView ArticlePubMedGoogle Scholar
  32. Nei M, Kumar S. Molecular Evolution and Phylogenetics. New York: Oxford University Press; 2000.Google Scholar
  33. Felsenstein J. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 1985; 39:783–791. http://dx.doi.org/10.2307/2408678View ArticleGoogle Scholar
  34. Liolios K, Mavromatis K, Tavernarakis N, 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. PubMed http://dx.doi.org/10.1093/nar/gkm884PubMed CentralView ArticlePubMedGoogle Scholar
  35. Reeve WG, Tiwari RP, Worsley PS, Dilworth MJ, Glenn AR, Howieson JG. Constructs for insertional mutagenesis, transcriptional signal localization and gene regulation studies in root nodule and other bacteria. Microbiology 1999; 145:1307–1316. PubMed http://dx.doi.org/10.1099/13500872-145-6-1307View ArticlePubMedGoogle Scholar
  36. DOE Joint Genome Institute user homepage. http://my.jgi.doe.gov/general/index.html
  37. Bennett S. Solexa Ltd. Pharmacogenomics 2004; 5:433–438. PubMed http://dx.doi.org/10.1517/14622416.5.4.433View ArticlePubMedGoogle Scholar
  38. Margulies M, Egholm M, Altman WE, Attiya S, Bader JS, Bemben LA, Berka J, Braverman MS, Chen YJ, Chen Z, et al. Genome sequencing in microfabricated high-density picolitre reactors. Nature 2005; 437:376–380. PubMedPubMed CentralPubMedGoogle Scholar
  39. Zerbino DR. Using the Velvet de novo assembler for short-read sequencing technologies. Current Protocols in Bioinformatics 2010;Chapter 11:Unit 11 5.Google Scholar
  40. Ewing B, Green P. Base-calling of automated sequencer traces using phred. II. Error probabilities. Genome Res 1998; 8:186–194. PubMed http://dx.doi.org/10.1101/gr.8.3.175View ArticlePubMedGoogle Scholar
  41. Ewing B, Hillier L, Wendl MC, Green P. Base-calling of automated sequencer traces using phred. I. Accuracy assessment. Genome Res 1998; 8:175–185. PubMed http://dx.doi.org/10.1101/gr.8.3.175View ArticlePubMedGoogle Scholar
  42. Gordon D, Abajian C, Green P. Consed: a graphical tool for sequence finishing. Genome Res 1998; 8:195–202. PubMed http://dx.doi.org/10.1101/gr.8.3.195View ArticlePubMedGoogle Scholar
  43. LaButti K, Foster B, Lowry S, Trong S, Goltsman E, Lapidus A. POLISHER: a Tool for Using Ultra Short Read in Microbial Genome Finishing http://publications.lbl.gov/fedora/repository/ir%253A150163. Berkeley Lab Publications 2008.
  44. 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.Google Scholar
  45. Hyatt D, Chen GL, Locascio PF, Land ML, Larimer FW, Hauser LJ. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics 2010; 11:119. PubMed http://dx.doi.org/10.1186/1471-2105-11-119PubMed CentralView ArticlePubMedGoogle Scholar
  46. Mavromatis K, Ivanova NN, Chen IM, Szeto E, Markowitz VM, Kyrpides NC. The DOE-JGI Standard operating procedure for the annotations of microbial genomes. Stand Genomic Sci 2009; 1:63–67. PubMed http://dx.doi.org/10.4056/sigs.632PubMed CentralView ArticlePubMedGoogle Scholar
  47. Lowe TM, Eddy SR. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res 1997; 25:955–964. PubMed http://dx.doi.org/10.1093/nar/25.5.0955PubMed CentralView ArticlePubMedGoogle Scholar
  48. Pruesse E, Quast C, Knittel K. Ludwig W, Peplies J, Glöckner FO. SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB. Nucleic Acids Res 2007; 35:7188–7196. PubMed http://dx.doi.org/10.1093/nar/gkm864PubMed CentralView ArticlePubMedGoogle Scholar
  49. INFERNAL. http://infernal.janelia.org
  50. Markowitz VM, Mavromatis K, Ivanova NN, Chen IM, Chu K, Kyrpides NC. IMG ER: a system for microbial genome annotation expert review and curation. Bioinformatics 2009; 25:2271–2278. PubMed http://dx.doi.org/10.1093/bioinformatics/btp393View ArticlePubMedGoogle Scholar

Copyright

© The Author(s) 2014