Open Access

Draft genome sequence of Bacillus amyloliquefaciens HB-26

  • Xiao-Yan Liu
  • , Yong Min
  • , Kai-Mei Wang
  • , Zhong-Yi Wan
  • , Zhi-Gang Zhang
  • , Chun-Xia Cao
  • , Rong-Hua Zhou
  • , Ai-Bing Jiang
  • , Cui-Jun Liu
  • , Guang-Yang Zhang
  • , Xian-Liang Cheng
  • , Wei Zhang
  • and Zi-Wen Yang
Corresponding author

DOI: 10.4056/sigs.4978673

Received: 15 March 2014

Accepted: 15 March 2014

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-26The Next-Generation sequencingPlasmodiophora 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 [5-7]. Heavy infection by this pathogen of Chinese cabbage, cabbage, broccoli, turnip, oilseed rape, and other crucifers can lead to severe economic losses [8-11]. 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

      Domain Bacteria

       TAS [14]

      Phylum Firmicutes

       TAS [15-17]

      Class Bacilli

       TAS [18,19]

       Current classification

      Order Bacillales

       TAS [20,21]

      Family Bacillaceae

       TAS [20,22]

      Genus Bacillus

       TAS [20,23,24]

      Species Bacillus amyloliquefaciens

       TAS [25-27]

       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 [31-33]. 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.

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

       0**

       -

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

**: none 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

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: Web Site).

Declarations

Acknowledgments

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).


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