Open Access

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

  • Wayne Reeve
  • , Matthew Parker
  • , Rui Tian
  • , Lynne Goodwin
  • , Hazuki Teshima
  • , Roxanne Tapia
  • , Cliff Han
  • , James Han
  • , Konstantinos Liolios
  • , Marcel Huntemann
  • , Amrita Pati
  • , Tanja Woyke
  • , Konstantinos Mavromatis
  • , Victor Markowitz
  • , Natalia Ivanova
  • and Nikos Kyrpides
Corresponding author

DOI: 10.4056/sigs.5249382

Received: 01 March 2014

Accepted: 01 March 2014

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 fixationrhizobiaAlphaproteobacteria

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 [1-4]. 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 [10-13]. 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.

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 [15,26-28]

   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-4.1MIGS-4.2

LatitudeLongitude

   -97.838   30.459

    IDA    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].

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.

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 information

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

Growth conditions and DNA isolation

M. lupini LUT6T was cultured to mid logarithmic phase in 60 ml of TY rich media [35] on a gyratory shaker at 28°C. DNA was isolated from the cells using a CTAB (Cetyl trimethyl ammonium bromide) bacterial genomic DNA isolation method [36].

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 [40-42] 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.

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

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 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.


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