Open Access

Genome sequence of the acid-tolerant Burkholderia sp. strain WSM2230 from Karijini National Park, Australia

  • Robert Walker
  • , Elizabeth Watkin
  • , Rui Tian
  • , Lambert Bräu
  • , Graham O’Hara
  • , Lynne Goodwin
  • , James Han
  • , Elizabeth Lobos
  • , Marcel Huntemann
  • , Amrita Pati
  • , Tanja Woyke
  • , Konstantinos Mavromatis
  • , Victor Markowitz
  • , Natalia Ivanova
  • , Nikos Kyrpides
  • and Wayne Reeve
Corresponding author

DOI: 10.4056/sigs.5008793

Received: 31 December 2013

Accepted: 31 December 2013

Published: 15 June 2014

Abstract

Burkholderia sp. strain WSM2230 is an aerobic, motile, Gram-negative, non-spore-forming acid-tolerant rod isolated from acidic soil collected in 2001 from Karijini National Park, Western Australia, using Kennedia coccinea (Coral Vine) as a host. WSM2230 was initially effective in nitrogen-fixation with K. coccinea, but subsequently lost symbiotic competence. Here we describe the features of Burkholderia sp. strain WSM2230, together with genome sequence information and its annotation. The 6,309,801 bp high-quality-draft genome is arranged into 33 scaffolds of 33 contigs containing 5,590 protein-coding genes and 63 RNA-only encoding genes. The genome sequence of WSM2230 failed to identify nodulation genes and provides an explanation for the observed failure of the laboratory grown strain to nodulate. The genome of this strain is one of 100 sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project.

Keywords:

root-nodule bacterianitrogen fixationrhizobiaBetaproteobacteria

Introduction

Burkholderia spp. are ubiquitous in the environment and are found in nearly all terrestrial and some marine ecosystems. They have adapted to occupy numerous niches and may have saprophytic, parasitic, pathogenic or symbiotic lifestyles [1]. Emerging evidence suggests an ancient and stable symbiosis between Burkholderia and Mimosa genera within South America [2] and between Burkholderia and legumes from the Papilionoideae subfamily in South Africa [3,4]. Despite this, there is very little data regarding the symbiosis between Burkholderia and endemic legumes outside of South America and South Africa.

In Australia, legumes are predominately nodulated by species from the genera Bradyrhizobium, Ensifer, and Rhizobium [5,6]. There are no published genomes or species descriptions of symbiotic Burkholderia spp. isolated in Australia and there is a paucity of information on the interaction between Burkholderia and endemic Australia legumes. Burkholderia sp. WSM2230 was isolated from an effective nitrogen fixing nodule on Kennedia coccinea grown in an acidic soil (pH(CaCl2) 4.8) collected from Karijini National Park, Western Australia. Its symbiotic phenotype was authenticated in glasshouse experiments (Watkin, unpublished). Recently this isolate was revived from long-term storage from frozen glycerol stocks but failed to form nodules on K. coccinea in axenic glasshouse trials (Walker, unpublished). In this regard, it is interesting that the South African microsymbiont B. tuberum STM678T only infrequently forms effective nodules on Macroptilium atropurpureum (Siratro). A recent study [7] revealed that B. tuberum forms effective nodules on Siratro when water levels are reduced and temperature is increased. Unlike B. tuberum STM678T, the annotation of the genome sequence of the laboratory cultured strain of WSM2230 failed to identify nodulation genes and this offers an explanation for the lack of a nodulation phenotype.

Establishing the genomic sequences of Australian Burkholderia will be beneficial to understand the mutualistic interactions occurring between plant and rhizosphere organisms in low-pH soil. WSM2230 was only isolated from Karijini National Park acidic soil (pH(CaCl2) 4.8) and other sites where the soil pH was higher (pH(CaCl2) >7) did not contain any Burkholderia symbionts. In these more alkaline soils, numerous Bradyrhizobium and Rhizobium spp. were instead trapped (Watkin, unpublished). Soil pH is an edaphic variable that controls microbial biogeography [8] and the acid tolerance of Burkholderia has been shown to account for the biogeographical distribution of this genus [9].

The genome of WSM2230 is one of two Australian Burkholderia genomes (the other being that of WSM2232 (GOLD ID Gi08832)) that have now been sequenced through the Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) program. Here we present a preliminary description of the general features of the Burkholderia sp. WSM2230 together with its genome sequence and annotation. The genomes of WSM2232 and WSM2230 will be an important resource to identify the processes enabling such isolates to adapt to the infertile, highly acidic soils that dominate the Australian landscape.

Classification and features

Burkholderia sp. strain WSM2230 is a motile, non-sporulating, non-encapsulated, Gram-negative rod in the order Burkholderiales of the class Betaproteobacteria. The rod-shaped form varies in size with dimensions of 0.5 μm for width and 1.0-2.0 μm for length (Figure 1 Left and Center). It is fast growing, forming colonies within 1-2 days when grown on LB agar [10] devoid of NaCl and within 2-3 days when grown on half strength Lupin Agar (½LA) [11], tryptone-yeast extract agar (TY) [12] or a modified yeast-mannitol agar (YMA) [13] at 28°C. Colonies on ½LA are -opaque, slightly domed and moderately mucoid with smooth margins (Figure 1 Right).

Figure 1

Images of Burkholderia sp. strain WSM2230 using scanning (Left) and transmission (Center) electron microscopy and the appearance of colony morphology on a solid medium (Right).

Burkholderia sp. WSM2230 can solubilize inorganic phosphate, produces hydroxymate-like siderophores, and can tolerate a pH range of 4.5 - 9.0 (Walker, unpublished). Minimum Information about the Genome Sequence (MIGS) is provided in Table 1. Figure 2 shows the phylogenetic neighborhood of Burkholderia sp. strain WSM2230 in a 16S rRNA sequence based tree. This strain shares 99% (1352/1364 bp) sequence identity to the 16S rRNA gene of the sequenced strain Burkholderia sp. WSM2232 (Gi08831).

Table 1

Classification and general features of Burkholderia sp. strain WSM2230 according to the MIGS recommendations [14]

MIGS ID

    Property

    Term

    Evidence code

    Current classification

    Domain Bacteria

    TAS [15]

    Phylum Proteobacteria

    TAS [16]

    Class Betaproteobacteria

    TAS [17,18]

    Order Burkholderiales

    TAS [18,19]

    Family Burkholderiaceae

    TAS [18,20]

    Genus Burkholderia

    TAS [21-23]

    Species Burkholderia sp.

    IDA

    Strain WSM2230

    IDA

    Gram stain

    Negative

    IDA

    Cell shape

    Rod

    IDA

    Motility

    Motile

    IDA

    Sporulation

    Non-sporulating

    NAS

    Temperature range

    Mesophile

    IDA

    Optimum temperature

    30°C

    IDA

    Salinity

    Non-halophile

    IDA

MIGS-22

    Oxygen requirement

    Aerobic

    IDA

    Carbon source

    Varied

    IDA

    Energy source

    Chemoorganotroph

    NAS

MIGS-6

    Habitat

    Soil, root nodule, on host

    IDA

MIGS-15

    Biotic relationship

    Free living, symbiotic

    IDA

MIGS-14

    Pathogenicity

    Non-pathogenic

    IDA

    Biosafety level

    1

    IDA

    Isolation

    Root nodule of Kennedia coccinea

    IDA

MIGS-4

    Geographic location

    Karijini National Park, Australia

    IDA

MIGS-5

    Soil collection date

    September 2001

    IDA

MIGS-4.1MIGS-4.2

    Latitude    Longitude

    117.99    -22.5

    IDA    IDA

MIGS-4.3

    Depth

    0-10 cm

    IDA

MIGS-4.4

    Altitude

    Not reported

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 [24].

Figure 2

Phylogenetic tree showing the relationship of Burkholderia sp. strain WSM2230 (shown in bold print) to other members of the order Burkholderiales based on aligned sequences of the 16S rRNA gene (1,242 bp internal region). All sites were informative and there were no gap-containing sites. Phylogenetic analyses were performed using MEGA [25], version 5. The tree was built using the Maximum-Likelihood method with the General Time Reversible model [26]. Bootstrap analysis [27] 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 [28]. Published genomes are indicated with an asterisk.

Symbiotaxonomy

Burkholderia sp. WSM2230 formed nodules (Nod+) on, and fixed N2 (Fix+) with, K. coccinea when first isolated. However, after long term storage and its subsequent culture, it failed to nodulate Australian legume hosts (Table 2).

Table 2

Compatibility of WSM2230 with nine legume species for nodulation (Nod) and N2-Fixation (Fix)

Species name

    Common name

    Growth type

    Nod

    Fix

    Reference

K. coccinea

    Coral Vine

    Perennial

    +1

    +1

    IDA

Swainsona formosa

    Sturts Desert Pea

    Annual

    -

    -

    IDA

Indigofera trita

    -

    Annual

    -

    -

    IDA

Acacia acuminata

    Jam Wattle

    Perennial

    -

    -

    IDA

A. paraneura

    Weeping Mulga

    Perennial

    -

    -

    IDA

1result obtained from trapping experiment but the isolate failed to nodulate after long term storage.

IDA: Inferred from Direct Assay from the Gene Ontology project [24].

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 [28] 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 Burkholderia sp. WSM2230

MIGS ID

    Property

    Term

MIGS-31

    Finishing quality

    Improved high-quality draft

MIGS-28

    Libraries used

    1x Illumina library

MIGS-29

    Sequencing platforms

    Illumina HiSeq 2000

MIGS-31.2

    Sequencing coverage

    Illumina: 368×

MIGS-30

    Assemblers

    Velvet version 1.1.04; Allpaths-LG version r39750

MIGS-32

    Gene calling methods

    Prodigal 1.4

    GOLD ID

    Gi08831

    NCBI project ID

    165309

    Database: IMG

    2513237151

    Project relevance

    Symbiotic N2 fixation, agriculture

Growth conditions and DNA isolation

Burkholderia sp. strain WSM2230 was cultured to mid logarithmic phase in 60 ml of TY rich medium on a gyratory shaker at 28°C [29]. DNA was isolated from the cells using a CTAB (Cetyl trimethyl ammonium bromide) bacterial genomic DNA isolation method [30].

Genome sequencing and assembly

The genome of Burkholderia sp. strain WSM2230 was sequenced at the Joint Genome Institute (JGI) using Illumina technology [31]. An Illumina standard shotgun library was constructed and sequenced using the Illumina HiSeq 2000 platform which generated 15,498,652 reads totaling 2,324 Mbp.

All general aspects of library construction and sequencing performed at the JGI can be found at the JGI user home [30]. All raw Illumina sequence data was passed through DUK, a filtering program developed at JGI, which removes known Illumina sequencing and library preparation artifacts (Mingkun, L., Copeland, A. and Han, J., unpublished). The following steps were then performed for assembly: (1) filtered Illumina reads were assembled using Velvet [32] (version 1.1.04), (2) 1–3 Kbp simulated paired end reads were created from Velvet contigs using wgsim (Web Site), (3) Illumina reads were assembled with simulated read pairs using Allpaths–LG [33] (version r39750). Parameters for assembly steps were: 1) Velvet --v --s 51 --e 71 --i 2 --t 1 --f "-shortPaired -fastq $FASTQ" --o "-ins_length 250 -min_contig_lgth 500"), 2) wgsim (-e 0 -1 76 -2 76 -r 0 -R 0 -X 0), 3) Allpaths–LG (STD_1,project,assembly,fragment,1,200,35,,,inward,0,0 SIMREADS,project,assembly,jumping,1,,,3000,300,inward,0,0).

The final draft assembly contained 33 contigs in 33 scaffolds. The total size of the genome is 6.3 Mbp and the final assembly is based on 2,324 Mbp of Illumina data, which provides an average 368× coverage of the genome.

Genome annotation

Genes were identified using Prodigal [34] as part of the DOE-JGI annotation pipeline [35], followed by a round of manual curation using the JGI GenePrimp pipeline [36]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) non-redundant database, UniProt, TIGRFam, Pfam, PRIAM, KEGG, COG, and InterPro databases. The tRNAScanSE tool [37] was used to find tRNA genes, whereas ribosomal RNA genes were found by searches against models of the ribosomal RNA genes built from SILVA [38]. 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 [39]. Additional gene prediction analysis and manual functional annotation was performed within the Integrated Microbial Genomes (IMG-ER) platform [40,41].

Genome properties

The genome is 6,309,801 nucleotides 63.07% GC content (Table 4) and comprised of 33 scaffolds (Figures 3a,3b,3c and Figure 3d) of 33 contigs. From a total of 5,653 genes, 5,590 were protein encoding and 63 RNA only encoding genes. The majority of genes (83.42%) 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 Burkholderia sp. strain WSM2230

Attribute

    Value

    % of Total

Genome size (bp)

    6,309,801

    100.00

DNA coding region (bp)

    5,480,804

    86.86

DNA G+C content (bp)

    3,979,790

    63.07

Number of scaffolds

    33

Number of contigs

    33

Total gene

    5,653

    100.00

RNA genes

    63

    1.11

rRNA operons*

    1

    0.02

Protein-coding genes

    5,590

    98.89

Genes with function prediction

    4,716

    83.42

Genes assigned to COGs

    4,614

    81.62

Genes assigned Pfam domains

    4,843

    85.67

Genes with signal peptides

    571

    10.10

Genes with transmembrane helices

    1,343

    23.76

CRISPR repeats

    0

*4 copies of 5S, 2 copies of 16S and 1 copy of 23S rRNA.

Figure 3a

Graphical map of WSM2230_A3ACDRAFT_scaffold_0.1 of the genome of Burkholderia sp. strain WSM2230. 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.

Figure 3b

Graphical map of WSM2230_A3ACDRAFT_scaffold__3.4 of the genome of Burkholderia sp. strain WSM2230. 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.

Figure 3c

Graphical map of WSM2230_A3ACDRAFT_scaffold_1.2 of the genome of Burkholderia sp. strain WSM2230. 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.

Figure 3d

Graphical map of WSM2230_A3ACDRAFT_scaffold_2.3 of the genome of Burkholderia sp. strain WSM2230. 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 Burkholderia sp. strain WSM2230 associated with the general COG functional categories

Code

    Value

     %age

     Description

J

    179

     3.46

     Translation, ribosomal structure and biogenesis

A

    2

     0.04

     RNA processing and modification

K

    474

     9.17

     Transcription

L

    141

     2.73

     Replication, recombination and repair

B

    1

     0.02

     Chromatin structure and dynamics

D

    40

     0.77

     Cell cycle control, cell division, chromosome partitioning

Y

    0

     0.0

     Nuclear structure

V

    47

     0.91

     Defense mechanisms

T

    260

     5.03

     Signal transduction mechanisms

M

    357

     6.90

     Cell wall/membrane/envelope biogenesis

N

    103

     1.99

     Cell motility

Z

    0

     0.00

     Cytoskeleton

W

    2

     0.04

     Extracellular structures

U

    128

     2.48

     Intracellular trafficking, secretion, and vesicular transport

O

    169

     3.27

     Posttranslational modification, protein turnover, chaperones

C

    371

     7.17

     Energy production and conversion

G

    395

     7.64

     Carbohydrate transport and metabolism

E

    496

     9.59

     Amino acid transport and metabolism

F

    95

     1.84

     Nucleotide transport and metabolism

H

    197

     3.81

     Coenzyme transport and metabolism

I

    271

     5.24

     Lipid transport and metabolism

P

    233

     4.51

     Inorganic ion transport and metabolism

Q

    173

     3.35

     Secondary metabolite biosynthesis, transport and catabolism

R

    610

     11.80

     General function prediction only

S

    427

     8.26

     Function unknown

-

    1,039

     18.38

     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.


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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