Genome sequence of Bacillus anthracis typing phage AP631
AP631, a virulent bacteriophage of Bacillus anthracis, is widely used in China to identify anthrax bacteria. In this study, we report the complete AP631 phage genome sequence as well as comparative genomic analysis with other bacteriophages of B. cereus and related species. The double-stranded circular DNA genome of phage AP631 was 39,549 bp in length with 35.01% G + C content. The phage genome contained 56 putative protein-coding genes but no rRNA or tRNA genes. Comparative phylogenetic analysis of the phage major capsid proteins and terminase large subunits showed that phage AP631 belongs to the B. cereus sensu lato phage clade II. Comparative genomic analysis revealed a high degree of sequence similarity between phage AP631 and B. anthracis phages Wbeta, Gamma, Cherry, and Fah, as well as three AP631-specific genes bearing no significant similarity to those of other phages.
Phages that infect prokaryotes were first reported by Frederick W. Twort in 1915 . Over time, bacteriophages have become useful tools for bacterial species and strain differentiation [2, 3]. In 1930, Cowles isolated the first Bacillus anthracis phage from crude sewage . Since then, several additional phages of B. anthracis have been isolated from sources worldwide [5, 6], including the well-known gamma, phage which is currently used as a diagnostic tool to distinguish B. anthracis from other B. cereus group members . Phage Fah was used widely in the former Soviet Union to identify anthrax bacteria . In 1963, Dong et al. isolated a B. anthracis phage from crude sewage and designated it AP631, indicating that this was the first B. anthracis phage isolated in China . Because AP631 can specifically infect non-encapsulated B. anthracis and form clear plaques, it is now widely used in China to identify anthrax bacteria. Under the electron microscope, AP631 has a hexagonal head around 50 nm in diameter and a long noncontractile tail around 180 nm in length . Thus, AP631 could be taxonomically assigned to the family Siphoviridae in the order Caudovirales. In 2016, Zhang et al. found that AP631 could nonspecifically lyse eight isolates of B. cereus preserved in their laboratory, forming turbid plaques . Molecular data on phage AP631 are not available. Here, we present the complete genome sequence of the AP631 bacteriophage and compare it with other phages of the B. cereus group. These data are useful for extending our understanding of the genomic characteristics of this group of phages and for enabling various applications related to the identification and control of B. anthracis.
Bacterial strains and phage DNA isolation
Phage AP631 was obtained from the Lanzhou Institute of Biological Products Co., Ltd., Gansu Province, China, and propagated on B. anthracis strain A16R by plating on BHI agar, followed by amplification in BHI nutrient broth. Bacterial cells were removed from culture supernatants by filtration through a 0.22-μm syringe filter prior to isolation of bacteriophage genomic DNA. Genomic DNA was purified using a QIAGEN Lambda DNA Extraction Kit (QIAGEN, Germany) following the manufacturer’s instructions.
Genome sequencing and annotation
Whole-genome sequencing was performed at the Beijing Novogene Bioinformatics Technology Co., Ltd. on the Illumina HiSeq PE150 platform with approximately 1340-fold coverage. High-quality paired-end reads were assembled de novo using SOAP [10, 11] (http://soap.genomics.org.cn/soapdenovo.html). Potential open reading frames (ORFs) were identified using PHASTER (http://phaster.ca) . Potential tRNAs were identified using tRNAscan-SE  (http://lowel.ab.ucsc.edu//tRNAscan-SE/). Comparative analysis of phage AP631 nucleotide and amino acid sequences with other known sequences was performed using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Comparative phylogenetic analysis was conducted using the neighbor-joining method in MEGA6  and ClustalW . Genomic comparisons between the AP631 and reference genome sequences (or among two or more genomes) were conducted using local BLAST+ tools, and the alignment results were visualized using Easyfig2.2.3  (http://easyfig.sourceforge.net/).
The general genomic features of AP631
Comparative genomic analysis
Nucleotide sequence accession number: The GenBank accession number for phage AP631 is MK085976.
We thank Liwen Bianji, Edanz Editing China (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript.
This study was funded by grants from the National Natural Science Foundation of China (No. 81571958 and No. 81601739).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
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