Open Access

Complete genome sequence of a plant associated bacterium Bacillus amyloliquefaciens subsp. plantarum UCMB5033

  • Adnan Niazi1Email author,
  • Shahid Manzoor1, 3,
  • Sarosh Bejai2,
  • Johan Meijer2 and
  • Erik Bongcam-Rudloff1
Standards in Genomic Sciences20149:9030718

https://doi.org/10.4056/sigs.4758653

Published: 15 June 2014

Abstract

Bacillus amyloliquefaciens subsp. plantarum UCMB5033 is of special interest for its ability to promote host plant growth through production of stimulating compounds and suppression of soil borne pathogens by synthesizing antibacterial and antifungal metabolites or priming plant defense as induced systemic resistance. The genome of B. amyloliquefaciens UCMB5033 comprises a 4,071,167 bp long circular chromosome that consists of 3,912 protein-coding genes, 86 tRNA genes and 10 rRNA operons.

Keywords

Bacillus amyloliquefaciens biocontrol rhizobacteria priming stress

Introduction

Bacillus amyloliquefaciens is a plant-associated species belonging to the family Bacillaceae. The members of the genus Bacillus are ubiquitous in nature and include biologically and ecologically diverse species, ranging from those beneficial for economically important plants, to pathogenic species that are harmful to humans. B. amyloliquefaciens UCMB5033 is a plant growth promoting bacterium (PGPB) that was isolated from a cotton plant [1]. Studies have shown that B. amyloliquefaciens UCMB5033 is an important tool for studies of plant-bacteria associations, has potential to confer protection against soil borne pathogens, and to stimulate growth of oilseed rape (Brassica napus) [2]. Such traits make UCMB5033 an important tool for studies of plant-bacteria associations and production of compounds that directly or indirectly promote plant growth or stress tolerance. Here we present a description of the complete genome sequencing of B. amyloliquefaciens UCMB5033 and its annotation.

Classification and features

Strain UCMB5033 was identified as a member of the B. amyloliquefaciens group based on phenotypic analysis [1]. The comparison of 16S rRNA gene sequences with the most recent databases from GenBank using NCBI BLAST [3] under default settings showed that B. amyloliquefaciens UCMB5033 shares 99% identity with many Bacillus species including Bacillus atrophaeus (CP002207.1) and Bacillus subtilis subsp. spizizenii str. W23 (CP002183.1). Figure 1 shows the phylogenetic relationship of B. amyloliquefaciens UCMB5033 with other species within the genus Bacillus. The tree highlights the close relationship of UCMB5033 with the B. amyloliquefaciens subsp. plantarum type strain FZB42. The other B. amyloliquefaciens type strain DSM 7T representing subsp. amyloliquefaciens, displayed less taxonomic relatedness and strain UCMB5033 can thus be regarded as belonging to the subsp. plantarum also in line with its plant associated characteristics [7].
Figure 1.

Phylogenetic tree showing the position of B. amyloliquefaciens UCMB5033 in relation to other species within the genus Bacillus. The tree is based on 16S rRNA gene sequences aligned with MUSCLE [4] was inferred under maximum likelihood criterion using MEGA5 [5] and rooted with Geobacillus thermoglucosidasius (a member of the family Bacillaceae). The numbers above the branches are support values from 1,000 bootstrap replicates if larger than 50% [6].

Morphology and physiology

B. amyloliquefaciens UCMB5033 is a Gram-positive, rod shaped, motile, spore forming, aerobic, and mesophilic microorganism (Table 1). Strain UCMB5033 is approximately 0.8 µm wide and 2 µm long that can grow on Luria Broth (LB) and potato dextrose agar (PDA) between 20 °C and 37 °C within the pH range 4–8. B. amyloliquefaciens UCMB5033 has properties as a plant growth promoting rhizobacterium (PGPR) [2]. The ability to catabolize plant derived compounds, resistance to metals and drugs; root colonization and biosynthesis of metabolites presumably give B. amyloliquefaciens UCMB5033 an advantage in developing a symbiotic relationship with plants in competition with other microorganims in the soil microbiota.
Table 1.

Classification and general features of B. amyloliquefaciens subsp. plantarum UCMB5033 according to the MIGS recommendation [8].

MIGS ID

Property

Term

Evidence codea

 

Classification

Domain Bacteria

TAS [9]

 

Phylum Firmicutes

TAS [1012]

 

Class Bacilli

TAS [13,14]

 

Order Bacillales

TAS [15,16]

 

Family Bacillaceae

TAS [15,17]

 

Genus Bacillus

TAS [15,18,19]

 

Species Bacillus amyloliquefaciens

TAS [2022]

 

Strain UCMB5033

 
 

Gram stain

Positive

IDA

 

Cell shape

Rod-shaped

IDA

 

Motility

Motile

IDA

 

Sporulation

Sporulating

IDA

 

Temperature range

Mesophilic

IDA

 

Optimum temperature

28°C

IDA

 

Carbon source

Glucose, fructose, trehalose, mannitol, sucrose, arabinose, raffinose

IDA

 

Energy source

--

 
 

Terminal electron receptor

--

 

MIGS-6

Habitat

Soil, Host (Plant)

IDA

MIGS-6.3

Salinity

up to 12% w/v

TAS [20,21]

MIGS-22

Oxygen

Aerobic

IDA

MIGS-15

Biotic relationship

Symbiotic (beneficial)

TAS [2]

MIGS-14

Pathogenicity

None

NAS

MIGS-4

Geographic location

Tajikistan

 

MIGS-5

Sample collection time

--

 

MIGS-4.1

Latitude

--

 

MIGS-4.2

Longitude

--

 

MIGS-4.3

Depth

--

 

MIGS-4.4

Altitude

--

 

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 [23].

Genome assembly and annotation

Growth conditions and DNA isolation

B. amyloliquefaciens UCMB5033 was grown in LB medium at 28°C for 12 hours (cells were in the early stationary phase). The genomic DNA was isolated using a QIAmp DNA mini kit (Qiagen).

Genome sequencing

B. amyloliquefaciens UCMB5033, originally isolated from cotton plant, was selected for sequencing on the basis of its ability to promote rapeseed growth and inhibit soil borne pathogens. Genome sequencing of B. amyloliquefaciens UCMB5033 using Illumina multiplex technology and Ion Torrent PGM systems was performed by Science for Life Laboratory (SciLifeLab) at Uppsala University. The genome project is deposited in the Genomes On Line Databases [24] and the complete genome sequence is deposited in the ENA database under accession number HG328253. A summary of the project information is shown in Table 2 and its association with MIGS identifiers.
Table 2.

Genome sequencing Project information

MIGS ID

Property

Term

MIGS-31

Finishing quality

Finished

MIGS-28

Libraries used

Illumina PE (75bp reads, insert size of 230bp), IonTorrent single end reads

MIGS-29

Sequencing platforms

Illumina GAii, IonTorrent PGM Systems

MIGS-31.2

Fold coverage

140× Illumina; 35× IonTorrent

MIGS-30

Assemblers

MIRA 3.4 and Newbler 2.8

MIGS-32

Gene calling method

PRODIGAL, AMIGene

 

ENA Project ID

PRJEB3961

 

Date of Release

September 8, 2013

 

INSDC ID

HG328253

 

GOLD ID

Gc0053646

 

Project relevance

Biocontrol, Agriculture

Genome assembly

The genome of B. amyloliquefaciens UCMB5033 was assembled using 21,919,534 Illumina paired-end reads (75bp) and 1,922,725 single-end reads (Ion Torrent). The chromosome of size 4,071,167 bp was assembled by providing paired-end reads to MIRA v.3.4 [25] for reference-guided assembly using the available genome sequence of B. amyloliquefaciens UCMB5036 (accession no. HF563562) [26]. Whereas, single-end reads were assembled with Newbler v.2.8 by a de novo assembly method. Both forms of assemblies were compared after alignment to identify indels and cover gap regions using Mauve genome alignment software [27].

Genome annotation

The genome sequence was annotated using a combination of several annotation tools via the Magnifying Genome (MaGe) Annotation Platform [28]. Genes were identified using Prodigal [29] and AMIGene [30] as part of the MaGe genome annotation pipeline followed by manual curation. Putative functional annotation of the predicted protein coding genes was done automatically by MaGe after BlastP similarity searches against the Uniprot and Trembl, TIGR-Fam, Pfam, PRIAM, COG and InterPro databases. The tRNAScanSE tool [31] was used to find tRNA genes. Ribosomal RNA genes were identified using RNAmmer tool [32].

Genome properties

The B. amyloliquefaciens UCMB5033 genome consists of a circular chromosome of size 4,071,168 bp. The genome having G+C content of 46.19% were predicted to contain 4,095 predicted ORFs including 10 copies each of 16S, 23S, and 5S rRNA; 86 tRNA genes, and 3,912 protein-coding sequences with the coding density of 87.51% (Figure 2). The majority of protein coding genes (81%) was assigned putative functions while those remaining were annotated as hypothetical or conserved hypothetical proteins (Table 3). The distribution into COG functional categories is presented in Table 4.
Figure 2.

Graphical circular map of the B. amyloliquefaciens UCMB5033 genome. From outer to inner circle: (1) GC percent deviation (GC window − mean GC) in a 1000-bp window. (2) Predicted CDSs transcribed in the clockwise direction. (3) Predicted CDSs transcribed in the counter-clockwise direction. Red and blue genes displayed in (2) and (3) are MaGe validated annotations and automatic annotations, respectively. (4) GC skew (G+C/G-C) in a 1,000-bp window. (5) rRNA (blue), tRNA (green), non-coding_RNA (orange), Transposable elements (pink) and pseudogenes (grey).

Table 3.

Nucleotide content and gene count levels of the genome

Attribute

Value

% of totala

Genome size (bp)

4,071,168

100

DNA cding region (bp)

3,565,936

87.5

DNA G+C content (bp)

1,880,879

46.1

Total number of genesb

4095

n/a

RNA genes

116

n/a

rRNA operons

10

n/a

Protein-coding genes

3912

100

CDSs with predicted functions

3170

81

Uncharacterized/Hypothetical genes

742

18.1

CDSs assigned to COGs

3506

89.6

CDSs with signal peptides

302

7.7

CDSs with transmembrane helices

1012

25.8

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 36 pseudogenes and 66 non-coding RNA.

Table 4.

Number of genes associated with the 25 general COG functional categories

Code

Value

%agea

Description

J

159

4.06

Translation

A

1

0.025

RNA processing and modification

K

287

7.33

Transcription

L

141

10.58

Replication, recombination and repair

B

1

0.025

Chromatin structure and dynamics

D

38

0.97

Cell cycle control, mitosis and meiosis

Y

0

0.00

Nuclear structure

V

50

1.27

Defense mechanisms

T

167

4.26

Signal transduction mechanisms

M

196

5.01

Cell wall/membrane biogenesis

N

63

1.61

Cell motility

Z

0

0

Cytoskeleton

W

0

0

Extracellular structures

U

54

1.38

Intracellular trafficking and secretion

O

98

2.5

Posttranslational modification, protein turnover, chaperones

C

181

4.62

Energy production and conversion

G

270

6.9

Carbohydrate transport and metabolism

E

313

8

Amino acid transport and metabolism

F

98

2.5

Nucleotide transport and metabolism

H

145

3.7

Coenzyme transport and metabolism

I

169

4.32

Lipid transport and metabolism

P

167

4.26

Inorganic ion transport and metabolism

Q

163

4.16

Secondary metabolites biosynthesis, transport and catabolism

R

426

10.88

General function prediction only

S

319

8.15

Function unknown

-

406

10.37

Not in COGs

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

Conclusion

Comparative genome analysis might reveal mechanisms by which UCMB5033 mediates plant protection and growth promotion, will further enable the investigations of the biochemical and regulatory mechanisms behind the symbiotic relationship, and will shed light on the activity of PGPR in different environments.

Declarations

Acknowledgements

This work was supported by the grants from Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (FORMAS) and the Higher Education Commission (HEC), Pakistan. The SNP&SEQ Technology Platform and Uppsala Genome Center performed sequencing supported by Science for Life Laboratory (Uppsala), a national infrastructure supported by the Swedish Research Council (VR-RFI) and the Knut and Alice Wallenberg Foundation. The Bioinformatics Infrastructure for the Life Sciences (BILS) supported the SGBC bioinformatics platform at SLU.

Authors’ Affiliations

(1)
Department of Animal Breeding and Genetics, SLU Global Bioinformatics Centre, Swedish University of Agricultural Sciences
(2)
Department of Plant Biology and Forest Genetics, Uppsala Biocenter, Swedish University of Agricultural Sciences and Linnéan Center for Plant Biology
(3)
University of the Punjab

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Copyright

© The Author(s) 2014