Genome sequence and description of the heavy metal tolerant bacterium Lysinibacillus sphaericus strain OT4b.31
© The Author(s) 2013
Published: 16 October 2013
Lysinibacillus sphaericus strain OT4b.31 is a native Colombian strain having no larvicidal activity against Culex quinquefasciatus and is widely applied in the bioremediation of heavy-metal polluted environments. Strain OT4b.31 was placed between DNA homology groups III and IV. By gap-filling and alignment steps, we propose a 4,096,672 bp chromosomal scaffold. The whole genome (consisting of 4,856,302 bp long, 94 contigs and 4,846 predicted protein-coding sequences) revealed differences in comparison to the L. sphaericus C3-41 genome, such as syntenial relationships, prophages and putative mosquitocidal toxins. Sphaericolysin B354, the coleopteran toxin Sip1A and heavy metal resistance clusters from nik, ars, czc, cop, chr, czr and cad operons were identified. Lysinibacillus sphaericus OT4b.31 has applications not only in bioremediation efforts, but also in the biological control of agricultural pests.
Biological control of vector-borne diseases, such as dengue and malaria, and agricultural pests have been an issue of special concern in the recent years. Since Kellen et al.  first described Lysinibacillus sphaericus as an insect pathogen, studies have shown mosquitoes to be the major target of this bacterium [2–4], but toxic activity against other species has also been reported [5,6]. L. sphaericus larvicidal toxicity has been reported due to vegetative mosquitocidal toxins (Mtx) , the binary toxin (BinA/BinB) , Cry48/Cry49 toxin  and recently the S-layer protein . To date, no larvicidal activity has been identified in Lysinibacillus sphaericus OT4b.31 against Culex quinquefasciatus .
On the other hand, Lysinibacillus species are potential candidates for heavy metal bioremediation. Some Bacillaceae strains have been successfully isolated from nickel contaminated soil , industrial landfills , naturally metalliferous soils  and a uranium-mining waste pile . In addition, native Colombian Lysinibacillus strains have been reported as potential metal bioremediators. Strain CBAM5 is resistant to arsenic, up to 200 mM, and contains the arsenate reductase gene . L. sphaericus OT4b.31 showed heavy metal biosorption in living and dead biomass. The S-layer protein was also shown to be present . We observed 19 mosquito-pathogenic L. sphaericus strains and 6 non-pathogenic strains (including OT4b.31) that were able to grow in arsenate, hexavalent chromium and/or lead . The moderate heavy metal tolerance in a Lysinibacillus strain isolated from a non-polluted environment generates interest in characterizing the genomic properties of L. sphaericus OT4b.31, in addition to its biotechnological potential in biological control.
Here we present a summary classification and a set of features for Lysinibacillus sphaericus OT4b.31 including previously unreported aspects of its phenotype, together with the description of the complete genomic sequencing and annotation.
Classification and features
Formerly known as Bacillus sphaericus, the species was defined as having a spherical terminal spore and by its inability to ferment sugars . According to physiological and phylogenetic analysis, it was reassigned to the genus Lysinibacillus . Strains of L. sphaericus can be divided into five DNA homology groups (I–V). Some mosquito pathogenic strains are allocated in subgroup II-A, while Lysinibacillus fusiformis species is in subgroup II-B . Later, according to 16S rDNA and lipid profile comparisons, Lysinibacillus sphaericus sensu lato was classified into seven similarity subgroups, of which only four retained the previous description by Krych et al. . Recently, by using 16S rDNA phylogenetic analysis some mosquito pathogenic native strains were found in group II with heterogeneous heavy metal tolerance levels. .
Classification and general features of Lysinibacillus sphaericus OT4b.31 according to the MIGS recommendations 
Species Lysinibacillus sphaericus
Type strain OT4b.31
Positive in vegetative cells, variable in sporulating stages
Mesophile, grows > 14°, < 37°C
Coleopteran (beetle) larvae
Growth in Luria-Bertani broth (5% NaCl)
Tenjo, Cundinamarca, Colombia
Sample collection time
2,685 m above sea level
Genome sequencing information
Genome project history
Genome sequencing project information
Improved high-quality draft
One paired end tags 90:90 bp with 500 bp insert
Illumina Hi-Seq 2000
CLC Assembly Cell version 4.0.10
Gene calling method
Genbank Date of Release
May 10, 2013
Biotechnology, metabolic pathway
Growth conditions and DNA isolation
Lysinibacillus sphaericus strain OT4b.31 was grown in nutrient broth for 16 hours at 30̱C and 150 rev/min. High molecular weight DNA was isolated using the EasyDNA® Kit (Carlsbad, CA, USA. Invitrogen) as indicated by the manufacturer. DNA purity and concentration were determined in a NanoDrop spectrophotometer (Wilmington, DE, USA. Thermo Scientific).
Genome sequencing and assembly
After DNA extraction, samples were sent to the Beijing Genome Institute (BGI) Americas Laboratory (Tai Po, Hong Kong). Purified DNA sample was first sheared into smaller fragments with a desired size by a Covaris E210 ultrasonicator. Then the overhangs resulting from fragmentation were converted into blunt ends by using T4 DNA polymerase, Klenow Fragment and T4 polynucleotide kinase. After adding an “A” base to the 3′ end of the blunt phosphorylated DNA fragments, adapters were ligated to the ends of the DNA fragments. The desired fragments were purified though gel-electrophoresis, then selectively enriched and amplified by PCR. The index tag was introduced into the adapter at the PCR stage as appropriate, and a library quality test was performed. Lastly, qualified, short, paired-ends of 90:90 bp length with 500 bp insert libraries were used to cluster preparation and to conduct whole-shotgun sequencing in Illumina Hi-Seq 2000 sequencers.
Using the FASTX-Toolkit version 0.6.1  and clean_reads version 0.2.3 from the ngs_backbone pipeline  reads were trimmed and quality filtered. Then, with the CLC Assembly Cell version 4.0.10 , assembly and scaffolding steps were conducted via a de novo assembly pipeline. The assembly included automatic scaffolding and k-mer/overlapping optimization steps. Some gaps were successfully filled by using GapFiller  within 30 iterations. No more gaps reached convergence by running more iterations. To obtain structural insight of a chromosomal scaffold, we used CONTIGuator.2 , using the Lysinibacillus sphaericus strain C3-41 chromosome (accession number: CP000817.1) as reference. Gap-filling steps and mapping to reference sequences were performed again to confirm convergence. Quality assessment of the assembly was performed with iCORN . The error rate of the final assembly is less than 1 in 1,000,000. Lastly, by using PROmer from the MUMmer  and Mauve  packages, we compared the chromosomal assembly and the chromosome of L. sphaericus C3-41.
The Glimmer 3 gene finder was used to identify and extract sequences for potential coding regions. To achieve the functional annotation steps, the RAST server  and Blast2GO pipelines  were used. Blast2GO performed the blasting, GO-mapping and annotation steps; which included a description according to the ProDom, FingerPRINTScan, PIR-PSD, Pfam, TIRGfam, PROSITE, ProDom, SMART, SuperFamily, Pattern, Gene3D, PANTHER, SignalIP and TM-HMM databases. The results were summarized with InterPro . Additionally, a GO-EnzymeCode mapping step was used to retrieve KEGG pathway-maps. tRNA genes were identified by using tRNAscan-SE  and rRNA genes by using RNAmmer . The possible orthologs of the genome were identified based on the COG database and classified accordingly . Prophage region prediction was also conducted by using the PHAST tool .
Summary of genome
Nucleotide content and gene count levels of the genome
% of totala
Genome size (bp)
DNA GC content (bp)
DNA coding region (bp)
Number of replicons
Genes in paralog clusters
Genes assigned to COGs
1 or more conserved domains
2 or more conserved domains
3 or more conserved domains
Genes with function prediction
Genes assigned Pfam domains
Genes with signal peptides
Genes with transmembrane helices
Number of genes associated with the 25 general COG functional categories
RNA processing and modification
Replication, recombination and repair
Chromatin structure and dynamics
Cell cycle control, mitosis and meiosis
Signal transduction mechanisms
Cell wall/membrane biogenesis
Intracellular trafficking and secretion
Posttranslational modification, protein turnover, chaperones
Energy production and conversion
Carbohydrate transport and metabolism
Amino acid transport and metabolism
Nucleotide transport and metabolism
Coenzyme transport and metabolism
Lipid transport and metabolism
Inorganic ion transport and metabolism
Secondary metabolites biosynthesis, transport and catabolism
General function prediction only
Not in COGs
Insights into the genome
The origin of replication of the chromosome of L. sphaericus OT4b.31 was estimated by similarities to several features of the corresponding regions in L. sphaericus C3-41, Bacillus sp. B-14905 and other close related bacteria, including colocalization of the genes: dnaX, recR, holB, dnaA, recG and recA; and GC nucleotide skew [(G−C)/(G+C)] analysis. In the first 40 Kbp of contig 1, we found dnaX, recR, and holB, while dnaA, recG and recA were found at the end (after 290 Kbp) of contig 13. This may suggest that contig 13 should be allocated immediately before contig 1. Besides, there was no evidence of multiple dnaA boxes around the potential origin. The replication termination site of the chromosomal scaffold is believed to be localized near 2.5 Mbp in the contig 18, according to GC skew analysis, and the coding bias for the two strands of the chromosome is for the majority of CDSs to be on the outer strand from 0 to ∼2.5 Mbp and on the inner strand from ∼2.5 Mbp to the end of the chromosomal scaffold (contig 26, Figure 4). This was also confirmed by the presence of parC (H131_12178) and parE (H131_12183), which encode the subunits of the chromosome-partitioning enzyme topoisomerase IV . Similar to the L. sphaericus C3-41 genome , we did not find the homolog of rtp (replication terminator protein-encoding gene) in the chromosomal assembly of OT4b.31.
A total of 42 hypothetical protein coding sequences were assigned as putative transposable elements, with the most frequent families being IS66, IS110, IS1272 and IS3. In addition, five prophage regions were identified, of which one region is intact and 4 regions are incomplete. Lactobacillus phage C5 (intact), Bacillus phage φ105, Clostridium phage c-st, Bacillus Phage SPP1 and Bacillus phage Wβ predicted regions were allocated at contigs 34, 8, 15, 18 and 37, respectively. Only lysis proteins were predicted in phages C5 and c-st regions. The only genes remaining in the phage φ105 region are those for coat proteins, integrase, and hypothetical and phage-like coding sequences. This is probably the remnant of phage invasion and genome deterioration during evolution. In addition, any previously reported phages in the genome of L. sphaericus C3-41 are in the genome of OT4b.31.
Two elements contain conserved domains from the Listeria pathogenicity island LIPI-1, functionally assigned as a thiol-activated cytolysin and a phosphatidylinositol phospholipase C. The first was confirmed to correspond to the L. sphaericus B354 sphaericolysin coding gene in contig 18 (H131_12483). Sphaericolysin B354 has been reported to be widespread across L. sphaericus DNA homology groups not only including IIA, IIB, IV and V  but also non-grouped species such as OT4b.31. Upstream, in the same contig, a Bacillus toxin from the family Mtx2 (PFam PF03318) was found and described as a hypothetical Sip1A toxin coding sequence (H131_12498). Purified from Bacillus thuringiensis strain EG2158, Sip1A is a secreted insecticidal protein of 38 KDa having activity against Colorado Potato beetle (Leptinotarsa decemlineata) . Considering that L. sphaericus OT4b.31 was isolated from beetle larvae, we suggest potential coleopteran larvicidal activity. To our knowledge, strain OT4b.31 is the first report of a predicted Sip1A-like toxin in a native Lysinibacillus sphaericus. Unexpectedly, mtx or bin mosquito pathogenic genes were not found in the OT4b.31 genome, despite a previous report showing positive evidence of BinA/B toxins with no larvicidal activity .
A total of 32 CDSs were described as surface (S) layer proteins or S-layer homologs (SLH). The putative S-layer gene sllB (H131_05299) previously reported in L. sphaericus JG-A12  was found in a 3,696 bp sequence allocated in contig 8. Three sequences with conserved domains similar to Slp5 and Slp6 were identified in contigs 8 (H131_05339, H131_05344) and 22 (H131_16838). Bacillus sp. B-14905 was the most similar sequence for the majority of S-layer protein domains. In addition, a putative glycoprotein (H131_22117), a bifunctional periplasmic precursor (H131_05993) and an S-layer fusion (H131_05409) coding sequence associated with S-layer proteins were recognized. On the other hand, a cluster of spore germination genes were determined near the termination of the replication site (including genes from the ger and ype operons) among other genes widespread in the genome. Three clusters of sporulation genes were allocated at contigs 1, 10 and 13 (including genes from spoII, spoV, yaa and sig operons).
Responses against toxic metal(oid)s in L. sphaericus OT4b.31 could be controlled by efflux pumps related genes in clusters found in contigs. Putative coding sequence order is as follows: yozA→czcD→csoR→copZA (contig 1, H131_00045: H131_00065); nikABC→oppD→nikD (contig 17, H131_11103:H131_11123); cadC-like→cadA (contig 24, H131_17086:H131_17081); arsRBC – putative extracellular secreted protein CDS – arsR-like→arsR-like→ putative excinuclease CDS (contig 18, H131_11998:H131_12028). The function of YozA is still unknown , but is similar to CzrA and CadC belonging to the ArsR transcriptional family regulators. YozA, CsoR (from the copper-sensitive operon), CadC-like and ArsR proteins seem to be the direct regulators of each cluster. At least one additional copy of ChrA, CzrB and CzcD CDSs were found. Upstream the nik cluster, we could not find transcriptional regulators. In summary, L. sphaericus OT4b.31 has protein encoding sequences probably involved in the resistance against Cd, Zn, Co, Cu, Ni, Cr, and As. In fact, prior reports of resistance to toxic metals [16,17] in L. sphaericus OT4b.31 may be explained due to participation of heavy-metal resistance proteins.
Strain OT4b.31 probably has a diverse defense repertoire according to the following responses and predicted genes: bacitracin stress responses, genes bceBASR and yvcPQRS; multidrug resistance, MATE (multidrug and toxin extrusion) family efflux pump genes ydhE/norM and acrB; antibiotics resistance, genes vanRSW, tetP-like group II, fusA (elongation factor G), fosB, blaZ and ampC-like. Based in the KEGG analysis, some predicted proteins might participate in peripheral pathways for the degradation of benzoate, aminobenzoate, quinate, toluene, naphthalene, geraniol, limonene, pinene, chloroalkane, chloroalkene, styrene, ethilbenzene, caprolactam and atrazine compounds, and biosynthesis of streptomycin, novobiocin, zeatin, ansamycins, penicillin and cephalosporins.
The native Colombian strain Lysinibacillus sphaericus OT4b.31, isolated from beetle larvae, is classified between DNA similarity groups III and IV. A comparison of the chromosomal sequences of strain OT4b.31 and its closest complete genome sequence, L. sphaericus C3-41, demonstrates the presence of only a few similar regions with syntenial rearrangements, and no prophage or putative mosquitocidal toxins are shared. Sphaericolysin B354 and the coleopteran toxin Sip1A were predicted in the strain OT4b.31, a finding which may be useful not only in bioremediation of polluted environments, but also for biological control of agricultural pests. Finally, Cd, Zn, Co, Cu, Ni, Cr and As resistances probably are supported by efflux pumps genes.
We would like to gratefully acknowledge the help of Dr. rer. nat. Diego Riaño-Pachón at Centro Nacional de Pesquisa em Energia e Materiais for his instructions in data analysis and the Group of Computational and Evolutionary Biology at University of Los Andes for providing us access to the computing grid cluster. This work was performed under the auspices of the Grant (1204-452-21129) of the Instituto Colombiano para el fomento de la Investigación Francisco José de Caldas, Colciencias and by the Centro de Investigaciones Microbiológicas - CIMIC laboratory.
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