Open Access

Draft genome sequence of Bacillus amyloliquefaciens HB-26

  • Xiao-Yan Liu1,
  • Yong Min1,
  • Kai-Mei Wang1,
  • Zhong-Yi Wan1,
  • Zhi-Gang Zhang1,
  • Chun-Xia Cao1,
  • Rong-Hua Zhou1,
  • Ai-Bing Jiang1,
  • Cui-Jun Liu1,
  • Guang-Yang Zhang1,
  • Xian-Liang Cheng1,
  • Wei Zhang2 and
  • Zi-Wen Yang1Email author
Standards in Genomic Sciences20149:9030775

DOI: 10.4056/sigs.4978673

Published: 15 June 2014

Abstract

Bacillus amyloliquefaciens HB-26, a Gram-positive bacterium was isolated from soil in China. SDS-PAGE analysis showed this strain secreted six major protein bands of 65, 60, 55, 34, 25 and 20 kDa. A bioassay of this strain reveals that it shows specific activity against P. brassicae and nematode. Here we describe the features of this organism, together with the draft genome sequence and annotation. The 3,989,358 bp long genome (39 contigs) contains 4,001 protein-coding genes and 80 RNA genes.

Keywords

Bacillus amyloliquefaciens HB-26 next-generation sequencing Plasmodiophora brassicae

Introduction

Bacillus amyloliquefaciens (B. amyloliquefaciens) is a species of bacterium in the genus Bacillus with high affinity of Bacillus subtilis. In the growth process, B. amyloliquefaciens can produce numerous antimicrobial or, more generally, bioactive metabolites with well-established activity in vitro such as surfactin, iturin and fengycin [1,2]. The production of all of these antibiotic compounds highlights B. amyloliquefaciens as a good candidate for the development of biocontrol agents [3,4].

Strain HB-26 belongs to the species B. amyloliquefaciens. The type strain of the species produces much bioactive metabolites showing specific activity against Plasmodiophora brassicae which could cause Clubroot, one of the most serious diseases of brassica crops worldwide [57]. Heavy infection by this pathogen of Chinese cabbage, cabbage, broccoli, turnip, oilseed rape, and other crucifers can lead to severe economic losses [811]. The root systems of infected plants show gall formation, which inhibits nutrient and water transport, stunts plant growth, and increases susceptibility to wilting [12,13]. Otherwise, bioassay results showed strain HB-26 also had some root-knot nematicidal activity.

Here, we present a summary classification and a set of features for B. amyloliquefaciens HB-26, together with the description of the genomic sequencing and annotation in order to improve the understanding of the molecular basis for its ability to inhibit Plasmodiophora brassicae and nematode.

Classification and features

Strain HB-26 colonies were milky white and matte with a wrinkled surface. Microscopy observations indicated that it was a Bacillus species (Figure 1A, Figure 1B and Table 1). SDS-PAGE analysis showed this strain secreted six major protein bands of 65, 60, 55, 34, 25 and 20 kDa (Figure 1C).
Figure 1.

General characteristics of B. amyloliquefaciens HB-26. (A) The colonial morphology pictures of strain HB-26. (B) Phase contrast micrograph of HB-26. (C) SDS-PAGE analysis of proteins of HB-26. Lane M, protein molecular weight marker; Lane 1, proteins of strain HB-26.

Table 1.

Classification and general features of B. amyloliquefaciens HB-26

MIGS ID

Property

Term

Evidence codea

 

Current classification

Domain Bacteria

TAS [14]

 

Phylum Firmicutes

TAS [1517]

 

Class Bacilli

TAS [18,19]

 

Order Bacillales

TAS [20,21]

 

Family Bacillaceae

TAS [20,22]

 

Genus Bacillus

TAS [20,23,24]

 

Species Bacillus amyloliquefaciens

TAS [2527]

 

Gram stain

Gram-positive

NAS

 

Cell shape

rod-shaped

IDA

 

Motility

mobile

NAS

 

Sporulation

Spore-forming

IDA

 

Temperature range

Room temperature

NAS

 

Optimum temperature

pH7.0

IDS

 

Carbon source

organic carbon source

NAS

 

Energy source

organic carbon source

NAS

MIGS-6

Habitat

Soil

IDA

MIGS-6.3

Salinity

salt tolerant

NAS

MIGS-22

Oxygen

Aerobic

NAS

MIGS-14

Pathogenicity

Avirulent

NAS

MIGS-4

Geographic location

Hubei, China

IDA

MIGS-4.1

Latitude

30.07N

 

MIGS-4.2

Longitude

112.23E

 

MIGS-4.3

Depth

5-10cm

 

MIGS-4.4

Altitude

about 35m

 

MIGS-5

Sample collection time

2009

IDA

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 [28].

A representative genomic 16S rDNA sequence of strain HB-26 was searched against GenBank database using BLAST [29]. Sequences showing more than 99% sequence identity to 16S rDNA of HB-26 were selected for phylogentic analysis, and 15 sequences were aligned with ClustalW algorithm. The tree was reconstructed by neighbor-Joining by using Kimura 2-parameter for distance calculation. The phylogenetic tree was assessed by bootstrapped for 1,000 times, and the consensus tree was shown in Figure 2.
Figure 2.

Neighbor-Joining Phylogenetic tree was generated using MEGA 4 based on 16S rRNA sequences. The strains and their corresponding GenBank accession numbers for 16S rDNA sequences are: A: B. amyloliquefaciens ML581 (KC692179.1); B: B. amyloliquefaciens JM-21 (KC752450.1); C: Bacillus strain HB-26 (HM138476); D: B. vallismortis WA3-7 (JF496475.1); E: B. sp.BYK1448 (HF549161.1); F: B. subtilis 2B (KF112078.1); G: B. methylotrophicus GZGL8 (JN999861.1); H: B. vallismortis D20 (KC441761.1); I: B. tequilensis L10 (JN700126.1); J: B. sp. C4(2013) (KC310834.1); K: B. subtilis WBZ (KC460988.1); L: B. Amyloliquefaciens CA81 (KF040978.1); M: B. sp. SWB30 (JX861886.1); N: B. methylotrophicus Ns7-22 (HQ831412.1); O: B. subtilis 26A (KC295415.1). The phylogenetic tree was constructed by using the neighbor-joining method within the MEGA software [30].

Genome sequencing information

Genome project history

This Bacillus strain was selected for sequencing due to its specific activity against Plasmodiophora brassicae and nematode. The complete high quality draft genome sequence is deposited in GenBank. The Beijing Genomics Institute (BGI) performed the sequencing and the NCBI staffs used the Prokaryotic Genome Annotation Pipeline (PGAAP) to complete the annotation. A summary of the project is given in Table 2.
Table 2.

Genome sequencing project information

MIGS ID

Property

Term

MIGS-31

Finishing quality

Draft

MIGD-28

Libraries used

One genomic libraries, one Illumina paired-end library (700 bp inserted size)

MIGS-29

Sequencing platform

Illumina Hiseq 2000

MIGS-31.2

Sequencing coverage

192 ×

MIGS-30

Assemblers

SOAPdenovo 1.05 version

MIGS-32

Gene calling method

Glimmer and GeneMark

 

GenBank Data of Release

August 31, 2016

 

NCBI project ID

AUWK00000000

 

Project relevance

Agricultural

Growth conditions and DNA isolation

B. amyloliquefaciens HB-26 was grown in 50 mL Luria-Broth for 6 h at 28°C. DNA was isolated by incubating the cells with lysozyme (10 mg/mL) in 2 mL TE (50 mM Tris base, 10 mM EDTA, 20% sucrose, pH8.0) at 4°C for 6 h. 4 mL of 2% SDS were added and the mixture was incubated at 55°C for 30 min; 2 mL 5M NaCl were added, and the mixture was incubated at 4°C for 10 min. DNA was purified by organic extraction and ethanol precipitation.

Genome sequencing and assembly

The genome of B. amyloliquefaciens HB-26 was sequenced using Illumina Hiseq 2000 platform (with a combination of a 251-bp paired-end reads sequencing from a 700-bp genomic library). Reads with average quality scores below Q30 or more than 3 unidentified nucleotides were eliminated. 2,605,589 paired-end reads (achieving 192 fold coverage [0.94 Gb]) was de novo assembled using SOAPdenovo 1.05 version [9]. The assembly consists of 39 contigs arranged in 39 scaffolds with a total size of 3,989,358 bp (including chromosome and plasmids).

Genome annotation

Genome annotation was completed using the Prokaryotic Genomes Automatic Annotation Pipeline (PGAAP). Briefly, Protein-coding genes were predicted using a combination of GeneMark and Glimmer [3133]. Ribosomal RNAs were predicted by sequence similarity searching using BLAST against an RNA sequence database and/or using Infernal and Rfam models [34,35]. Transfer RNAs were predicted using tRNAscan-SE [36]. In order to detect missing genes, a complete six-frame translation of the nucleotide sequence was done and predicted proteins (generated above) were masked. All predictions were then searched using BLAST against all proteins from complete microbial genomes. Annotation was based on comparison to protein clusters and on the BLAST results. Conserved domain Database and Cluster of Orthologous Group information is then added to the annotation.

Genome properties

The draft assembly of the genome consists of 39 contigs in 39 scaffolds, with an overall 47.37% G+C content. Of the 4,114 genes predicted, 4,001 were protein-coding genes, and 80 RNAs were also identified. The majority of the protein-coding genes (54.06%) were assigned a putative function while the remaining ones were annotated as hypothetical proteins. The distribution of genes into COGs functional categories is presented in Table 3, Table 4 and Figure 3.
Figure 3.

Graphical circular map of the Bacillus amyloliquefaciens HB-26 genome. From the outside to the center: genes on forward strand (color by COG categories), genes on reverse strand (color by COG categories), GC content, GC skew. The map was generated with the CGviewer server (Stothard Rearch Group: http://stothard.afns.ualberta.ca/cgview_server/).

Table 3.

Genome Statistics

Attribute

Value

% of total

Genome size (bp)

3,989,358

100.00

DNA coding region (bp)

3,486,615

87.39

DNA G+C content (bp)

1,889,758

47.37

Number of scaffolds

39

-

Extrachromosomal elements

unknown

-

Total genes

4,114

100.00

tRNA genes

76

1.85

rRNA genes

4

0.1

rRNA operons

0b

-

Protein-coding genes

4,001

97.25

Pseudo gene (Partial genes)

0 (36)

0 (0.87%)

Genes with function prediction (proteins)

2224

54.06%

Genes assigned to COGs

2,336

56.78%

Genes with signal peptides

328

7.97

CRISPR repeats

0

0

aThe 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.

bNone of the rRNA operons appears to be complete due to unresolved assembly problems.

Table 4.

Number of genes associated with the general COG functional categories

Code

Value

% age

Description

J

130

3.160

Translation, ribosomal structure and biogenesis

A

0

0.0

RNA processing and modification

K

262

6.368

Transcription

L

122

2.965

Replication, recombination and repair

B

1

0.024

Chromatin structure and dynamics

D

34

0.826

Cell cycle control, cell division, chromosome partitioning

Y

0

0

Nuclear structure

V

52

1.264

Defense mechanisms

T

153

3.719

Signal transduction mechanisms

M

182

4.424

Cell wall/membrane/envelope biogenesis

N

53

1.288

Cell motility

Z

0

0.000

Cytoskeleton

W

1

0.024

Extracellular structures

U

43

1.045

Intracellular trafficking, secretion, and vesicular transport

O

97

2.358

Posttranslational modification, protein turnover, chaperones

C

177

4.302

Energy production and conversion

G

249

6.053

Carbohydrate transport and metabolism

E

340

8.264

Amino acid transport and metabolism

F

79

1.920

Nucleotide transport and metabolism

H

123

2.990

Coenzyme transport and metabolism

I

117

2.844

Lipid transport and metabolism

P

205

4.983

Inorganic ion transport and metabolism

Q

116

2.820

Secondary metabolites biosynthesis, transport and catabolism

R

435

10.574

General function prediction only

S

287

6.976

Function unknown

 

856

20.81

Not in COGs

aThe total is based on the total number of protein coding genes in the annotated genome.

Declarations

Acknowledgements

This work was financially supported by the National Science and Technology Support Program (2008BADA5B03), the National 863 High Technology Research Program of China (2011AA10A201, 2011AA10A203), China 948 Program of Ministry of Agriculture (2011-G25), the National Science and Technology Support Program (2011BAB06B004-02), Hubei Province Development Plan (YJN0077) and the Science and Technology Support Program of Academy of Agricultural Sciences of Hubei Province (2012NKYJJ21).

Authors’ Affiliations

(1)
National Biopesticide Engineering Technology Research Center, Hubei Biopesticide Engineering Research Center, Hubei Academy of Agricultural Sciences
(2)
Department of Horticulture, Hubei Vocational College of Bio-technology

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Copyright

© The Author(s) 2014