1 Introduction

Representatives of the genus Bacillus are widespread in the environment and traditional fermented meals. Therefore, they easily get to human intestine with food and drinks. Now, species of the genus Bacillus are regarded as resident rather than transient microorganisms in human GIT [1]. By secretion of enzymes, production of antibiotics and other small compounds, Bacillus species influence on intestinal microbiota as well as on human GIT. Bacillus altitudinis was described as a new species in 2006 during analysis of air samples collected at 41 km in atmosphere [2]. Later, it was found to be abundant in marine and soil environments. Additionally to high UV resistance, B. altitudinis possesses antagonistic traits in respect to fungi [3] and produces extracellular glucanase, xylanase, protease which can contribute to meal digestion [4–6] as well as autoinducer-2, a quorum sensing molecule which mediates interspecies signalling and affects bacterial behaviour [7]. Ribonuclease secreted by B. altitudinis is a homologue of B. pumilus binase showing antiviral and antitumour activities [8–10]. Decoding of B. altitudinis genome will generate an accurate reference for understanding its probiotic potential. Here, mate-pair next-generation sequencing was used to improve whole genome contigs obtained for B. altitudinis B-388 earlier [11].

2 Material and Methods

For genomic DNA extraction, B. altitudinis B-388 was grown on LB medium for 16–18 h. DNA isolation was performed using PureLink Genomic DNA Kit (Invitrogen, USA). Genomic DNA was sheared acoustically with a HydroShear DNA Shearing Device (Gene Machines, USA). Fragments with sizes of 2–3 and 5–6 kb were selected on an agarose gel. Two mate-pair libraries with an insert size of 2 and 5 kb were generated using SOLiD Mate-Paired Library Kit (Thermo Fisher Scientific, USA). Ligation with adaptors and circularization were performed with the help of Ion Xpress Barcode Adapters 1–16 Kit (Thermo Fisher Scientific, USA). Sequencing and initial sequencing data analysis were perform on the platform of Ion Personal Genome Machine System (Thermo Fisher Scientific, USA) and Torrent Suite Software (Thermo Fisher Scientific, USA), respectively. GS De Novo Assembler (Newbler software suite), v3.0 was used for genome assembly. The genome was annotated using the NCBI Prokaryotic Genome Annotation Pipeline version 3.1. Identification of prophage sequences within bacterial genome was performed using PHAST web server [12].

3 Results and Discussion

Genome of B. altitudinis B-388 was sequenced using whole-genome shotgun sequencing approach in 2014 [11]. By that time, it was a second B. altitudinis sequencing project (GenBank Acc. No JOVS00000000.1) after its type strain 41KF2b which was released early in 2014 under GenBank acc. No ASJC00000000.1. The sequencing with 20-fold overall B. altitudinis B-388 genome coverage resulted in obtaining of 59 contigs with an N50 size of 127,734 bp [11]. Contig 45 was assumed to represent a plasmid of 4,528 bp. The size of the genome was estimated to be 3,706,590 bp.

Accurate microbial genome sequence information is important for discovering genetic changes upon comparative analysis of microbial strains, understanding of genes regulation and prognostics of functional activities of the strains. Mate-pair sequencing allows decreasing gap regions and number of contigs with extending scaffold lengths compared to initial de novo whole genome sequencing. In this work mate-pair sequencing of B. altitudinis B-388 was performed with 262× overall genome coverage. For 2 kb and 5 kb insert libraries 72,295 and 75,268 reads were obtained, correspondingly. 19 contigs with an N50 size of 466,626 bp forming eight scaffolds with an N50 size of 865,860 bp were generated. The genome size was calculated as 3,743,699 bp. Gapped regions represent 0.08 % of total genome length. During annotation, 3645 protein coding sequences and 73 RNAs were detected. The number of frameshifted genes decreased twice compared to the results of de novo sequencing (Table 1). It was found that the scaffold 8 represents a cryptic plasmid of 4,529 bp encoding two hypothetical proteins, Rep protein and DNA-binding protein SoxR, involved in oxidative stress response. In addition to two prophages found in genome of B. altitudinis B-388 upon initial annotation, another incomplete phage sequence was revealed after mate-pairing (Table 1). Comparative analysis of B. altitudinis genome characteristics obtained after application of different sequencing techniques to four different strains demonstrated their high similarity with variation in prophage number and plasmid content (Table 1).

Table 1 Comparative analysis of characteristics of Bacillus altitudinis sequenced genomes
Full size table

4 Conclusions

Thus, by implication of mate-pair sequencing approach, we have improved the quality of B. altitudinis genome sequence, which will be used for comparative and functional studies involving this species. To date, the reported sequence is the most thorough presentation of B. altitudinis genomes deposited in GenBank.