Genomic Analysis of Bacillus licheniformis CBA7126 Isolated from a Human Fecal Sample

摘要

Introduction Bacillus licheniformis is a Gram-positive, endospore-forming, saprophytic organism that occurs in plant and soil (Veith et al., 2004 ). A taxonomical approach shows that it is closely related to Bacillus subtilis (Lapidus et al., 2002 ; Xu and C?te, 2003 ; Rey et al., 2004 ). Generally, most bacilli are predominantly aerobic; however, B. licheniformis is a facultative anaerobe compared to other bacilli in ecological niches (Alexander, 1977 ). The commercial utility of the extracellular products of B. licheniformis makes this microorganism an economically interesting species (Kovács et al., 2009 ). For example, B. licheniformis is used industrially for manufacturing biochemicals, enzymes, antibiotics, and aminopeptidase. Several proteases such as α-amylase, penicillinase, pentosanase, cycloglucosyltransferase, β-mannanase, and certain pectinolytic enzymes are synthesized industrially using B. licheniformis (Rodríguez-Absi and Prescott, 1978 ; Rey et al., 2004 ). The proteases are used in the detergent industry and the amylases are utilized for starch hydrolysis, desizing of textiles, and sizing of paper (Erickson, 1976 ). In addition, certain strains are utilized to produce peptide antibiotics, specialty chemicals, and poly-γ-glutamic acid (Nierman and Maglott, 1989 ; Rey et al., 2004 ).The annotated genome sequence of B. licheniformis has been previously analyzed to assess the biotechnological importance of the organism (Veith et al., 2004 ). Since the first sequencing, the genomes of specific B. licheniformis strains have been sequenced to completely realize its industrial potential. In this study, genome sequencing of B. licheniformis CBA7126 isolated from a human fecal sample was performed to understand bacterial specificity. The genome sequence of CBA7126 revealed features such as stress response genes, antibiotic-resistance genes, and genes for resistance to toxic compounds, which are of considerable biotechnological value. Materials and methods Bacterial isolation, culture conditions, and DNA extraction B. licheniformis CBA7126 was isolated from the feces of a 74-year-old man in Geochang-gun, South Korea and was cultured under anaerobic conditions in Gifu Anaerobic Medium (GAM) (containing per liter of deionized distilled water: 10 g peptone, 3 g soytone, 10 g proteose peptone, 13.5 g bovine serum albumin, 5 g yeast extract, 2.2 g beef extract, 2.5 g monopotassium phosphate, 1.2 g liver extract, 3 g sodium chloride, 0.3 g l -cystein, 0.3 g sodium thioglychollate, 3 g dextrose, 5 g soluble starch) at 37°C for 48 h. Genomic DNA of strain CBA7126 was extracted using the QIAamp DNA extraction kit (Qiagen, USA) and QuickGene DNA tissue kit S (Kurabo, Japan), and purified using the MG genomic DNA purification kit (Doctor Protein, Korea) according to the manufacturer's instructions. The purity and concentration of the extracted genomic DNA were measured using the Nanodrop spectrophotometer (NanoDrop Technologies, UK). Genome sequencing, assembly, and annotation The genome of B. licheniformis CBA7126 was sequenced using a 20-kb SMRTbell library and PacBio RS II system (Pacific Biosciences, USA), and de novo assembly was performed using the HGAP2 protocol in PacBio SMRT Analysis version 2.3.0. rRNAs and tRNAs were analyzed using RNAmmer 1.2 (Lagesen et al., 2007 ) and tRNAscan-SE 1.21 (Lowe and Eddy, 1997 ), respectively. The potential coding regions and functional genes were predicted via a combination of Glimmer 3.02 (Delcher et al., 1999 ), COG database (Tatusov et al., 2003 ), the Rapid Annotation Search Tool (RAST) (Aziz et al., 2008 ), and the National Center for Biotechnology Information (NCBI) prokaryotic genome annotation pipeline (PGAP) 4.1 (Tatusova et al., 2016 ). Prophages in the genome were identified using the PHAge Search Tool (PHAST) (Zhou et al., 2011 ). In addition, pathogenicity of strain CBA7126 was predicted using PathogenFinder 1.1 (Cosentino et al., 2013 ). Carbohydrate-active enzymes were annotated using dbCAN (Yin et al., 2012 ). Comparative genomic analysis To identify the unique features of strain CBA7126, the genomes of B. licheniformis and Bacillus sp. strains ( B. licheniformis B4164, B. licheniformis VTM3R78, B. licheniformis V30, B. licheniformis B4124, and Bacillus sp. H15-1) were selected for comparative genomic analysis using the NCBI genome database ( http://www.ncbi.nlm.nih.gov/genome/ ). For calculation of overall genome relatedness, average nucleotide identity (ANI), and orthologous average nucleotide identity (OrthoANI) analysis of B. licheniformis CBA7126 was performed on sequences of related species using the ANI calculator ( http://enve-omics.ce.gatech.edu/ani/ ) and orthologous average nucleotide identity tool (OAT) of ChunLab (Lee et al., 2016 ). The genome structure of strain CBA7126 was compared to those of B. licheniformis B4164 (LQYQ00000000.1), B. licheniformis VTM3R78 (FOFE00000000.1), B. licheniformis V30 (LQRR00000000.1), Bacillus sp. H15-1 (C