MEM INST OSWALDO CRUZ, RIO DE JANEIRO, 110(6) September 2015
PAGES: 822-823 DOI: 10.1590/0074-02760150273 Genome announcement and Highlights
Draft genome sequence of Bacillus thuringiensis 147, a Brazilian strain with high insecticidal activity

Luiz Carlos Bertucci Barbosa1,+, Débora Lopes Farias2, Isabella de Moraes Guimarães Silva2, Fernando Lucas Melo3, Bergmann Morais Ribeiro3, Raimundo Wagner de Souza Aguiar2

1Universidade Federal de Itajubá, Instituto de Recursos Naturais, Itajubá, MG, Brasil
2Universidade Federal do Tocantins, Departamento de Engenharia de Bioprocessos e Biotecnologia, Gurupi, TO, Brasil
3Universidade de Brasília, Departamento de Biologia Celular, Brasília, DF, Brasil

Abstract

Bacillus thuringiensis is a ubiquitous Gram-positive and sporulating bacterium. Its crystals and secreted toxins are useful tools against larvae of diverse insect orders and, as a consequence, an alternative to recalcitrant chemical insecticides. We report here the draft genome sequence of B. thuringiensis 147, a strain isolated from Brazil and with high insecticidal activity. The assembled genome contained 6,167,994 bp and was distributed in seven replicons (a chromosome and 6 plasmids). We identified 12 coding regions, located in two plasmids, which encode insecticidal proteins.

Bacillus thuringiensis is a Gram-positive bacterium that has been isolated from a range of ecosystems including soil, water and dead insects, among others. B. thuringiensis is a spore-forming bacterium that synthesises parasporal crystalline inclusions containing Cry and Cyt proteins (also known as δ-endotoxins) and some of these are toxic against a wide range of insect orders, nematodes and human cancer cells (Palma et al. 2014). B. thuringiensis isolates can also synthesise and secrete other insecticidal proteins during the vegetative growth phase, which are designated vegetative insecticidal proteins (Vip) and secreted insecticidal protein (Sip). Furthermore, other predicted toxins are also produced by B. thuringiensis strains, but their toxicity has yet to be proven (Palma et al. 2014).

The crystals and secreted toxins of B. thuringiensis are highly specific for their hosts and have therefore gained worldwide importance as an alternative to chemical insecticides, motivating the search for new B. thuringiensis isolates to identify and characterise new insecticidal proteins (Pardo-López et al. 2013, Palma et al. 2014). Accordingly, whole genome sequence of these isolates can be an important starting point. In this study, we determined the draft genome sequence of B. thurin-giensis 147, a strain isolated from soil samples in the state of Tocantins, Brazil. Toxicity assays of this strain have shown high insecticidal activity against larvae from Aedes aegypti (Diptera: Culicidae) and Spodoptera frugiperda (Lepidoptera: Noctuidae).

For genome sequencing, total DNA (chromosome and plasmids) was isolated using the Wizard Genomic DNA Purification kit (Promega) from fresh overnight cultures. Whole-genome sequencing was performed with the MiSeq platform (Illumina, USA), located at the High-Performance Genome Centre of Federal District (Brasília, Brazil) using the 600-cycle MiSeq reagent kit v.3 (Illumina). A total of 2,614,978 paired-end reads were generated at a read length of 150 bp. A quality control of these reads was performed with the FastQC tool (bioinformatics.babraham.ac.uk/projects/fastqc/). De novo genome assembly was carried out with SPAdes 3.5.0 (Bankevich et al. 2012). The final draft genome assembly consisted of 94 contigs (length > = 500 bp), with a total size of 6,167,994 bp, N50 value of 205,568 and a mean guanine-cytosine content of 34.90%. A BLAST analysis (blast.ncbi.nlm.nih.gov/blast/Blast.cgi) of each contig showed that the assembled genome was distributed in seven replicons: a circular chromosome and six plasmids. The genetic information about these replicons is summarised in Table I.

 

 

Automated annotation, carried out using the RAST annotation server (Aziz et al. 2008), showed that the draft genome of B. thuringiensis strain 147 contains 6,319 predicted protein-coding sequences and 138 predicted RNAs (rRNAs and tRNAs). These data are consistent with other published complete genomes from B. thuringiensis strains (Doggett et al. 2013, Liu et al. 2014, Johnson et al. 2015). In addition to the analysis performed by the RAST annotation server, the identification and annotation of insecticidal genes were performed with BLAST (Altschul et al. 1997), using a custom insecticidal toxin database from B. thuringiensis. The local custom database was constructed with amino acid sequences of δ-endotoxins (Cry and Cyt), secreted toxins (Vip and Sip), proteins called "mosquitocidal toxin" and haemagglutinin-related proteins, all retrieved from the curated UniProtKB database (uniprot.org/uniprot/). All insecticidal proteins identified using a local database were confirmed using remote BLAST (blast.ncbi.nlm.nih.gov/blast/Blast.cgi). Insecticidal genes were confined to two plasmids. Plasmid 1 and plasmid 4 were found to harbour one and 11 insecticidal genes, respectively. Table II summarises the regions of the assembled contigs that encode insecticidal proteins.

This Whole Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession LFXM00000000. The version described in this paper is version LFXM01000000.

 

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Financial support: CNPq, SECT-TO
+ Corresponding author: This e-mail address is being protected from spambots. You need JavaScript enabled to view it.
Received 21 July 2015
Accepted 21 August 2015

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