Open Access

Complete Genome sequence of Burkholderia phymatum STM815T, a broad host range and efficient nitrogen-fixing symbiont of Mimosa species

  • Lionel Moulin
  • , Agnieszka Klonowska
  • , Bournaud Caroline
  • , Kristina Booth
  • , Jan A.C. Vriezen
  • , Rémy Melkonian
  • , Euan K. James
  • , J. Peter W. Young
  • , Gilles Bena
  • , Loren Hauser
  • , Miriam Land
  • , Nikos Kyrpides
  • , David Bruce
  • , Patrick Chain
  • , Alex Copeland
  • , Sam Pitluck
  • , Tanja Woyke
  • , Michelle Lizotte-Waniewski
  • , Jim Bristow
  • and Margaret Riley
Corresponding author

DOI: 10.4056/sigs.4861021

Received: 25 March 2014

Accepted: 25 March 2014

Published: 15 June 2014

Abstract

Burkholderia phymatum is a soil bacterium able to develop a nitrogen-fixing symbiosis with species of the legume genus Mimosa, and is frequently found associated specifically with Mimosa pudica. The type strain of the species, STM 815T, was isolated from a root nodule in French Guiana in 2000. The strain is an aerobic, motile, non-spore forming, Gram-negative rod, and is a highly competitive strain for nodulation compared to other Mimosa symbionts, as it also nodulates a broad range of other legume genera and species. The 8,676,562 bp genome is composed of two chromosomes (3,479,187 and 2,697,374 bp), a megaplasmid (1,904,893 bp) and a plasmid hosting the symbiotic functions (595,108 bp).

Keywords:

BurkholderiasymbiosisMimosarhizobianitrogen fixation

Introduction

Rhizobia are a functional class of bacteria able to enter into nitrogen-fixing symbioses with legumes. The bacterial symbiont induces the formation of nodules on the roots of the plant where they differentiate into nitrogen-fixing bacteroids. Bacteria then allocate combined nitrogen to the plant, which in return provides the bacteria with energy derived from photosynthesis. This symbiosis confers agricultural advantages to the legumes by reducing the need for fertilization and allows them to be pioneer plants on degraded or contaminated soils.

Rhizobia are polyphyletic and are placed within two classes of Proteobacteria, the Alphaproteobacteria and the Betaproteobacteria. They are closely related to non-symbiotic species, including important human, animal or plant pathogens or saprophytes. Most research has focused on the α-rhizobia, since the β-rhizobia were only recently discovered [1,2]. The α-rhizobia include 10 genera (Sinorhizobium, Mesorhizobium, Rhizobium, Methylobacterium, Devosia, Azorhizobium, Bradyrhizobium, Ochrobactrum, Bosea and Phyllobacterium) and have a worldwide distribution associated with a diversity of legume species (from herbs to trees). To date, the β-rhizobia include only two genera, Burkholderia and Cupriavidus (ex Ralstonia), and a dozen species (for review [3], updated in [4]). They are found preferentially associated with Mimosa species (at least 68 nodulated species, and especially M. pudica, M. pigra, and M. bimucronata) in Asia, Australia, and Central and South America [5,6]. Based on a comparison of house-keeping and nodulation gene phylogenies, Burkholderia species have been postulated to be ancestral symbionts of South American Mimosa and Piptadenia species [4,5]. Here we describe the genome sequence of one of the first described β-rhizobia, the type strain of Burkholderia phymatum, STM815T.

Classification and features

Burkholderia phymatum STM815T is a motile, Gram-negative rod (Figure 1) in the order Burkholderiales of the class Betaproteobacteria. It is fast growing, forming colonies within 3-4 days when grown on yeast-mannitol agar (YMA [7],) at 28°C. It is one of the first described members of the β-rhizobia. The strain STM815T, which is the type strain of the species, was isolated from nodules of Machaerium lunatum in French Guiana in 2000 [1], and the species, B. phymatum, was described based on this single isolate [8]. However, the species has subsequently been shown not to nodulate Machaerium species [9], but it can nodulate species in the large genus Mimosa [9,10]. Indeed, the symbiotic abilities of STM815T have been demonstrated on numerous Mimosa species, and this strain is now considered to be an efficient symbiont of a broad range of legumes, particularly in Mimosa and related genera in the sub-family Mimosoideae [9]. Strain STM815T is also able to fix nitrogen in free-living conditions [9]. Many isolates of B. phymatum have been sampled from Mimosa pudica in French Guiana [10], Papua New Guinea [9], China [11] and India [12]. Phylogenetic analyses of core and symbiotic genes have illustrated the ancestral status of Burkholderia species in symbioses with Mimosa [4,5]. Burkholderia phymatum STM815T is now considered to be a model system for studying the adaptive processes of Burkholderia in symbioses with legumes, in comparison with α-rhizobia. The B. phymatum species is phylogenetically related to symbiotic and non-pathogenic species, and is distant from the “cepacia” clade of Burkholderia (which contains many pathogenic species) (Figure 2, Table 1).

Figure 1

Transmission electron microscopy of B. phymatum STM815 (credit: Geoffrey Elliott).

Figure 2

Phylogenetic tree highlighting the position of Burkholderia phymatum strain STM815T relative to other type strains within the genus Burkholderia. The 16S rDNA sequences from type strains were obtained from the ribosomal database project [13], aligned with muscle 3.6, and a neighbor-joining tree was built from a Kimura-2P corrected distance matrix using BioNJ on the Web Site server [14]. Numbers at nodes are % bootstraps from 1000 replicates (shown only if >50%). Accession numbers of 16S rDNA are indicated between parentheses for each strain. C. taiwanensis LMG19424T was used as outgroup.

Table 1

Classification and general features of Burkholderia phymatum STM815 according to MIGS recommendations [15]

MIGS ID

    Property

    Term

    Evidence codea

    Domain Bacteria

    TAS [16]

    Phylum Proteobacteria

    TAS [17]

    Class Betaproteobacteria

    TAS [18,19]

    Current classification

    Order Burkholderiales

    TAS [18,20]

    Family Burkholderiaceae

    TAS [18,21]

    Genus Burkholderia

    TAS [22-24]

    Species Burkholderia phymatum

    TAS [8,25]

    Type strain STM815

    Gram stain

    negative

    TAS [8]

    Cell shape

    straight rods

    TAS [8]

    Motility

    motile

    Sporulation

    non-sporulating

    TAS [8]

    Temperature range

    mesophile, no growth at 42°C

    TAS [8]

    Optimum temperature

    28°C

    TAS [8]

    Carbon source

    D-glucose, L-arabinose, D-mannose, D-mannitol, N-acteyl-D-glucosamine,    D-gluconate, caprate, D-galactose, citric acid, D-galacturonate acid,    methyl-pyruvate, L-aspartic acid, L-glutamic acid, L-asparagine, D,L-lactic acid

    TAS [8]    TAS [8]    IDA    IDA    IDA    IDA

    Energy source

    chemoorganotroph

    TAS [8]

MIGS-6

    Habitat

    Soil, nodule, host

    TAS [1]

MIGS-6.3

    Salinity

    Not reported

MIGS-22

    Oxygen

    Aerobic

    TAS [8]

MIGS-15

    Biotic relationship

    Free living, Symbiotic

    TAS [1,9]

MIGS-14

    Pathogenicity

    None

MIGS-4

    Geographic location

    Root nodule of Machaerium lunatum in French Guiana (Paracou)

    TAS [1]

MIGS-5

    Sample collection time

    2000

    TAS [1]

MIGS-4.1

    Latitude

    5°15’N

    TAS [1]

MIGS-4.2

    Longitude

    52°55’W

    TAS [1]

MIGS-4.3

    Depth

    Not reported

MIGS-4.4

    Altitude

    32 m

    TAS [1]

a) 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 [26].

Symbiotaxonomy

Burkholderia phymatum STM815T forms nodules (Nod+) and fixes N2 (Fix+) with a broad range of Mimosa species [6,9] as well as with other genera in the tribe Mimoseae in the Mimosoideae legumes sub-family [9]. Nodulation data were compiled in Table 2.

Table 2

Mimosoid legumes tested for nodulation with Burkholderia phymatum STM815

Tribe / Genus

     Species

    Nodulation by STM815

Tribe Mimoseae*

Acacia

     farnesiana, karroo, nilotica var. kraussiana, nilotica var. leiocarpa, pennatula, schaffneri, seyal, tortilis

    F

Anadenanthera

     pavonina, colubrina

    F

Desmanthus

     bicornutus, fruticosus, virgatus

    O

Dichrostachys

     cinerea, microcephala

    O

Leucaena

     collinsii, cuspidata, pulverulenta, trichodes

    N

     confertiflora, esculenta, greggii, retusa, salvadorensis

    O

     leucocephala, multicapitula

    F

Microlobius

     foetidus

    O

Mimosa

     aculeaticarpa1, luisana1, setosissima4

    O

     acutistipula1, albida1, albolanata4, artemisiana1, bimucronata1, caesalpiniifolia1, camporum1, cordistipula4, debilis4, diplotricha1, foliolosa4, flocculosa1, hexandra1, himalayana1, invisa1, latispinosa1, ophtalmocentra1, pigra1, polydactyla1, pudica1, somnians1, tenuiflora, setosa4, ursina4, velloziana4, xanthocentra4

    F

     adenocarpa1, affinis1, bahamensis1, blanchetii1, borealis1, callithrix4, claussenii4, decorticans4, delicatula1, densa4, dysocarpa1, melanocarpa4, menabeensis1, polyantha1, scabrella1, uruguensis1

    I

Neptunia

     dimorphantha, gracilis, majore, monosperma, plena

    O

     oleracea

    N

Parapiptadenia

     rigida

    N

Piptadenia

     gonoacantha, stipulacea, viridiflora2

    F

Pityrocarpa3

     moniliformis, obliqua

    F

Prosopis

     africana, farcta, glandulosa, velutina

    O

     chilensis, pubescens

    N

     juliflora

    F

Schleinitzia

     novo-guineensis

    O

Stryphnodendron

     coriaceum, guianensis, pulcherrimum

    O

Tribe Ingeae

Acacia (Ac)

     senegal

    N

Acacia (P)

     dealbata

    O

     mangium

    N

Albizia

     adenocephala, kalkora, niopoides

    O

     julibrissin

    N

Calliandra

     houstiana var. acapulcens, houstiana var. anomala, houstiana var. calothyrsus, juzepczukii, trinervia

    F

     physocalyx, rubescens

    N

Chloroleucon

     tortum

    O

Enterolobium

     cyclocarpum

    O

Faidherbia

     albida

    N

Pithecellobium

     dulce

    F

Samanea

     saman

    O

Zapoteca

     tetragona

    O

Legend: O = no nodules formed; N = outgrowths on roots, superficially similar to nodules but ineffective; I = nodules formed are inefficient; F = nitrogen fixing nodules formed (these may not all be fully effective, but plants gave acetylene reduction values at least twice that of non-nodulated control plants).

*This is taken to include Acacia subgenus Acacia, now thought to be closely related to tribe Mimoseae and given the generic name Vachellia by some.

This is taken to include Acacia, subgenera Aculeiferum (Ac) and Phyllodineae (P). The species listed below are now also included in genera Senegalia and Acacia respectively. Species from other genera in former Acacia have not been studied here.

1 Nodulation data from [9]; 2 This species has been transferred to an as yet unnamed genus by [27]; 3 This genus was formerly in Piptadenia [27]; 4 Nodulation data from [6]. Nodulation data for other legumes are from unpublished data from E.K. James and L. Moulin.

Genome sequencing information

Genome project history

The genome was selected by a consortium of researchers led by M. Riley, to be sequenced by the DOE Joint Genome Institute as part of the “Recommendations for Sequencing Targets in Support of the Science Missions of the Office of Biological and Environmental Research”. Initially, the strain was chosen to enrich genome data in the Burkholderia genus for comparative genomics. The genome was selected for genome determination because strain STM815T is a legume symbiont, as compared to the large number of genome sequences available for opportunistic and human-pathogens. The genome sequence was completed in 2007 and presented for public access on April 2008. Automatic annotation was performed using the JGI-Oak Ridge National Laboratory annotation pipeline [28]. Additional automatic and manual sequence annotation, as well as comparative genome analysis, were performed using the MicroScope platform at Genoscope [29]. Table 3 presents the project information and its association with MIGS version 2.0 compliance [30].

Table 3

Project information

MIGS ID

    Property

    Term

MIGS-31

    Finishing quality

    Complete

MIGS-28

    Libraries used

    3 kb, 8 kb and 40 kb (fosmid)

MIGS-29

    Sequencing platforms

    Sanger

MIGS-31.2

    Fold coverage

    11.2

MIGS-30

    Assemblers

    Phred/Phrap/Consed

MIGS-32

    Gene calling method

    DOE-JGI tools

    Genome Database release

    December 12, 2008

    Genbank ID

    CP001043 - CP001046

    Genbank Date of Release

    April 22, 2008

    NCBI BioProject ID

    PRJNA17409

    GOLD ID

    Gc00775

    Project relevance

    biotechnological

Growth conditions and DNA isolation

The strain was grown in 50 ml of broth Yeast-mannitol medium (YM [7],) and DNA isolation was performed using a CTAB (Cetyl trimethyl ammonium bromide) bacterial genomic DNA isolation method [31].

Genome sequencing and assembly

The genome of Burkholderia phymatum STM815T was sequenced by Sanger technology at the Joint Genome Institute (JGI) using a combination of 3 kb, 8 kb and 40 kb (fosmid) DNA libraries. All general aspects of library construction and sequencing performed at the JGI can be found at the DOE JGI website [32].

Draft assemblies were based on 115,329 total reads and resulted in approximately 11.2× coverage of the genome. The Phred/Phrap/Consed software package was used for sequence assembly and quality assessment [33-35]. Gaps between contigs were closed by custom primer walks on gap spanning clones or PCR products. A total of 1,282 additional reactions were necessary to close gaps and to raise the quality of the finished sequence. The completed genome sequences of B. phymatum STM815T contain 115,487 reads, achieving an average of 11.2-fold sequence coverage per base with an error rate less than 1 in 100,000.

Genome annotation

Automatic annotation was performed using the Integrated Microbial Genomes (IMG) platform [36] developed by the Joint Genome Institute, Walnut Creek, CA, USA [28]. Additional automatic and manual sequence annotation, as well as comparative genome analysis, were performed using the MicroScope platform at Genoscope [29]. Gene calling in Microscope resulted in the prediction of 940 additional protein coding sequences compared to the 7,496 detected at IMG. These additional genes were mostly short coding sequences considered as gene remnants or fragmented CDS, so that genome statistics presented here are from the IMG platform.

Genome properties

The genome includes two chromosomes and two plasmids, for a total size of 8,676,562 bp (62.3% GC content). Chromosome 1 is 3.48 Mb in size (63.0% GC), chromosome 2 is 2.69 Mb (62.3% GC), plasmid 1 is 1.90 Mb (62.0% GC) and plasmid 2 0.59 Mb (59.2% GC). For chromosomes 1 and 2, 3,140 and 2,358 genes were predicted, respectively. For plasmid 1 and 2, 1,627 and 449 genes were predicted, respectively. A total 7,496 of protein coding genes were predicted, of which 5,601 were assigned to a putative function with the remaining annotated as hypothetical proteins. 5,630 protein coding genes belong to COG families in this genome. The properties and the statistics of the genome are summarized in Tables 4-6, and circular maps of each replicon are shown in Figure 3 (chromosomes) and Figure 4 (plasmids). Plasmid 2 was identified as the symbiotic plasmid of STM815, as it carried nod, nif and fix genes directly involved in symbiosis as well as several other genes coding for proteins indirectly linked to symbiotic interactions with plants. Among them were found genes coding for the biosynthesis of phytohormones such as indol acetic acid (iaaHM), ACC deaminase (acdS), and genes involved in the biosynthesis of rhizobitoxine (rtxAC-like). A Type 4 secretion system was also identified on this plasmid, while no type 3 system could be detected in the whole genome.

Table 4

Summary of genome: two chromosomes and two plasmids

Label

    Size (Mb)

    Topology

    INSDC identifier

    Refseq identifier

Chromosome 1

    3.479189

    Circular

    NC_010622.1

    CP001043.1

Chromosome 2

    2.697376

    Circular

    NC_010627.1

    CP001044.1

Plasmid 1

    1.904895

    Circular

    NC_010623.1

    CP001045.1

Plasmid 2

    0.595110

    Circular

    NC_010625.1

    CP001046.1

Table 6

Number of genes associated with the 25 general COG functional categories

Code

    Value

    %agea

    Description

J

    195

    3.02

    Translation

A

    1

    0.02

    RNA processing and modification

K

    643

    10.00

    Transcription

L

    235

    3.65

    Replication, recombination and repair

B

    2

    0.03

    Chromatin structure and dynamics

D

    37

    0.58

    Cell cycle control, mitosis and meiosis

V

    68

    1.06

    Defense mechanisms

T

    397

    6.17

    Signal transduction mechanisms

M

    396

    6.16

    Cell wall/membrane biogenesis

N

    113

    1.76

    Cell motility

W

    1

    0.02

    Extracellular structures

U

    139

    2.16

    Intracellular trafficking and secretion

O

    213

    3.31

    Posttranslational modification, protein turnover, chaperones

C

    503

    7.82

    Energy production and conversion

G

    486

    7.55

    Carbohydrate transport and metabolism

E

    663

    10.31

    Amino acid transport and metabolism

F

    101

    1.57

    Nucleotide transport and metabolism

H

    223

    3.47

    Coenzyme transport and metabolism

I

    290

    4.51

    Lipid transport and metabolism

P

    287

    4.46

    Inorganic ion transport and metabolism

Q

    200

    3.11

    Secondary metabolites biosynthesis, transport and catabolism

R

    719

    11.18

    General function prediction only

S

    522

    8.11

    Function unknown

-

    1944

    25.67

    Not in COGs

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

Figure 3

Circular maps of Chromosome 1 (left) and Chromosome 2 (right) of B. phymatum STM815T. From outside to center: Genes on forward strand (color by COG categories as denoted by the IMG platform), Genes on reverse strand (color by COG categories), RNA genes (tRNAs green, sRNAs red, other RNAs black), GC content, GC skew. Replicons are not drawn to scale.

Figure 4

Circular maps of Plasmid 1 (left) and Plasmid 2 (right) of B. phymatum STM815T. From outside to center: Genes on forward strand (color by COG categories as denoted by the IMG platform), Genes on reverse strand (color by COG categories), RNA genes (tRNAs green, sRNAs red, other RNAs black), GC content, GC skew. Replicons are not drawn to scale.

Table 5

Nucleotide content and gene count levels of the genome

Attribute

    Value

   % of totala

Genome size (bp)

    8676562

   100.00%

DNA coding region (bp)

    7328930

   84.47%

DNA G+C content (bp)

    5404839

   62.29%

Total genesb

    7574

   100.00%

RNA genes

    78

   1.03%

Protein-coding genes

    7496

   98.93%

Genes assigned to COGs

    5630

   74.33%

Genes with signal peptides

    701

   9.26%

Genes with transmembrane helices

    1709

   22.56%

a) The total is based on either the size of the genome in base pairs or the total number of protein coding genes in the annotated genome.

b) Also includes 39 pseudogenes.

Comparison of Burkholderia phymatum STM815T with other fully sequenced genomes of Burkholderia

Venn diagram (family number)

Gene families specific to, or shared by, Burkholderia phymatum STM815T and 3 other Burkholderia species, were determined using MICFAM [Figure 5]. This tool is based on MicroScope gene families [39] which are computed using an algorithm implemented in the SiLiX software [40]: a single linkage clustering algorithm of homologous genes sharing an amino-acid alignment coverage and identity above a defined threshold. This algorithm operates on the “The friends of my friends are my friends” principle of gene comparison. If two genes are homologous, they are clustered. Moreover, if one of the genes is already clustered with another one, the three genes are clustered into the same MICFAM.

Figure 5

B. phymatum STM815T was compared to 3 others Burkholderia strains from similar and different ecological niches: a legume symbiont (B. phenoliruptrix BR3459a, a Mimosa flocculosa nodule symbiont from Brazil [37,38]; a soil bacterium (B. xenovorans LB400) and a human opportunistic pathogen (B. cenocepacia AU1054). The core genomes of all four bacteria yielded 1,582 gene families. Each bacterium had more gene families specific to its species, (from 3,002 to 5,656 depending on strain) than shared ones (1,582 core gene families). There were 418 gene families specific to the two Mimosa symbionts (STM815 and BR3459a), including symbiosis-related genes (nod genes) and nitrogen fixation genes (nif, fix), glutamine transporters, biosynthesis genes of the phytohormone indol acetic acid (IAA), and hydrogenase genes (hup, hyp).

Conclusion

Burkholderia phymatum STM815T possesses a large genome composed of two chromosomes and two plasmids, one of which encodes the symbiotic functions. Further studies on the genome of this bacterium will help elucidate the high nodulation competitiveness [41], broad host range and symbiotic efficiency of this strain.

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 the University of California, Lawrence Livermore 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, and French National Agency of Research (ANR) (Project “BETASYM” ANR-09-JCJC-0046).


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.

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