Complete genome sequence of the molybdenum-resistant bacterium Bacillus subtilis strain LM 4–2
© You et al. 2015
Received: 27 April 2015
Accepted: 3 December 2015
Published: 10 December 2015
Bacillus subtilis LM 4–2, a Gram-positive bacterium was isolated from a molybdenum mine in Luoyang city. Due to its strong resistance to molybdate and potential utilization in bioremediation of molybdate-polluted area, we describe the features of this organism, as well as its complete genome sequence and annotation. The genome was composed of a circular 4,069,266 bp chromosome with average GC content of 43.83 %, which included 4149 predicted ORFs and 116 RNA genes. Additionally, 687 transporter-coding and 116 redox protein-coding genes were identified in the strain LM 4–2 genome.
KeywordsGram-positive Molybdate Bioremediation Molybdenum-resistance Bacillus subtilis LM 4–2
Bacillus subtilis LM 4–2 was a molybdenum-resistant strain isolated from a molybdenum mine. It has been reported that many microbes can resist the toxicity of molybdate ion though reduction of molybdate (Mo6+) to Mo-blue. Molybdenum-reducing microorganisms came from a variety of genera and included the following species, Klebsiella spp. [1, 2], Acidithiobacillus ferrooxidans , Enterobacter cloacae , Serratia marcescens [5, 6], Acinetobacter calcoaceticus , Pseudomonas spp. , and Escherichia coli K12 . The capability of molybdate-reduction presents potential possibility of molybdenum bioremediationin many polluted areas . Strain LM 4–2 showed stronger resistance to molybdate (up to 850 mM Na2MoO4) than many other reported molybdenum-resistant bacteria [11, 12]. However, no information related to the molecular mechanism of molybdenum-resistance has been identified, also in genus Bacillus . Thus, strain LM 4–2 might be a perfect subject for us to unveil the mechanism and evaluate its possibility utilization in bioremediation. Here we present the complete genome sequence and detailed genomic features of B. subtilis LM 4–2.
Classification and features
Classification and general features of Bacillus subtilis LM 4–2 according to the MIGS recommendations 
Species Bacillus subtilis
pH range; Optimum
organic carbon source
Genome sequencing information
Genome project history
Genome sequencing project information
Two libraries, 20 Kb PacBio library, 2 × 150 bpllumina library
PacBio RS II, Illumina Hi-Seq
Gene calling method
Glimmer 3.02 and GeneMark
GenBank Date of Release
April 23, 2015
Source Material Identifier
Growth conditions and genomic DNA preparation
Bacillus subtilis LM 4–2 was inoculated in 200 mL R2A medium and cultivated for 8 h at 30 °C in a shaker with speed of 200 rpm. 1.2 g of harvested cells was suspended in 5 mL TE (pH8.0) with 10 mg/mL lysozymeat 30 °C for 4 h. After centrifugation (12,000 rpm) for 10 min, genomic DNA was extracted by phenol-chloroform methods as described previously . DNA was dissolved in 2 mL sterilized deionized water with a final concentration of 12.67 μg/μL and 2.04 of OD260/OD280 ratio determined by NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). The genomic DNA was stored in −20 °C freezer.
Genome sequencing and assembly
The genome of Bacillus subtilis LM 4–2 was sequenced by a dual sequencing approach that using a combination of PacBio RS II and Genome Analyzer IIx sequence platforms. Approximately 121,583 PacBio and 1637 million Illumina reads were generated from PacBio platform and the Illumina platform (2 × 150 bp paired-end sequencing) with average sequence coverage of 213-and 409-fold.Sequence reads from the PacBio RS II were assembled by using hierarchical genome-assembly process assembler and finally only one self-cycled supper contig was generated. The Illumina reads were quality trimmed with the CLC Genomics Workbench and then utilized for error correction of the PacBio reads by using bowtie2 (version 2.1.0) software .
The Glimmer 3.02 and GeneMark programs were used to predict the positions of open reading frames [45, 46]. Protein function was predicted by the following methods: 1) homology searches in the GenBank and UniProt protein database ; 2) function assignment searches in CDD database ; and 3) domain or motif searches in the Pfam databases . The KEGG database was used to reconstruct metabolic pathways . Ribosomal RNAs and Transfer RNAs were predicted by using RNAmmer and tRNAscan-SE programs [51, 52]. Transporters were predicted by searching the TCDB database using BLASTP program [27, 53] with expectation value lower than 1e-05.
% of Total
Genome size (bp)
DNA coding (bp)
DNA G + C (bp)
Protein coding genes
Genes with function prediction
Genes assigned to COGs
Genes with Pfam domains
Genes with signal peptides
Genes with transmembrane helices
Number of genes associated with general COG functional categoriesa
Translation, ribosomal structure and biogenesis
RNA processing and modification
Replication, recombination and repair
Chromatin structure and dynamics
Cell cycle control, Cell division, chromosome partitioning
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
Molybdenum pollution has been reported in water and soils all around the world . Some Mo-resistance bacteria can be used to immobilize soluble molybdenum toinsoluble formsalong with reducing the toxicity. In this study we presented the complete genome sequence of Bacillus subtilis LM 4–2, which was isolated from a molybdenum mine in Luoyang city. Due to its strong resistance to molybdate and potential utilization in bioremediation of molybdate-polluted area, we sequence the genome and try to identify the possible molecular mechanism of molybdenum-resistance. Genomic analysis of strain LM 4–2 revealed 687 transporter-coding and 116 redox protein-coding genes were separated in the genome. Three genome islands were identified in the strain LM 4–2 genome, covering 2.71 % of the whole genome. Three gene clusters were involved in the non-ribosomal synthesis of lipopeptides, such as surfactin, fengycin, and dipeptide bacilysin. Additionally, one gene clusters for subtilosin A synthesis and one gene clusters for polyketide synthesis. No CRISPRs were identified in the strain LM 4–2 genome. The complete genome sequence of strain LM 4–2 will facilitate functional genomics to elucidate the molecular mechanisms that underlie molybdenum-resistance and it may facilitate the bioremediation of molybdenum-contaminated areas.
This work was financially supported by the National Natural Science Foundation of China (31200035, 41201224) and by the Research Fund for the Doctoral Program of Henan University of Science and Technology under Grant (09001608). Transmission electron microscopy was provided by instrument center of Institute of Microbiology, Chinese Academy of Sciences.
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