Draft Genome Assemblies of Phage AP50c-Resistant Derivatives of Bacillus anthracis Sterne Strain 7702 Lacking Plasmid pXO2

ABSTRACT

Mutants of the attenuated Bacillus anthracis (Sterne) strain 7702 that are resistant to phage AP50c have been previously described. Here, we report the draft genome assemblies of the parent strain, several phage-resistant derivatives, and mutants of genes in the pathways for synthesis and assembly of the S-layer.

ANNOUNCEMENT

Bacillus anthracis, a spore-forming, Gram-positive bacterium, is the causative agent of anthrax and a CDC category A select agent due to its potential to be used as a bioterrorism agent. Bacteriophages such as phages Wβ, γ, Cherry, and AP50c that specifically infect B. anthracis have been isolated and characterized for their potential diagnostic and surveillance utility (1). The concept of using these types of phages as therapeutics has also received recent interest due to the emergence of antimicrobial resistance in every realm of human interaction (2). When embarking on phage therapy, especially using a phage cocktail, it is critical to understand the phage resistance mechanisms of the targeted bacteria. In a previous study, we identified the bacterial receptor of phage AP50c as the S-layer protein, Sap, which is present on the B. anthracis vegetative cell surface. We constructed various mutants of sap and additional genes in the pathways for synthesis and assembly of the S-layer (3). To establish and strengthen the link between the genotypic changes, such as indels and single-nucleotide polymorphisms (SNPs), underlying these phage resistance phenotypes, we have sequenced the genomes of these mutants. Here, we report the whole-genome sequences of the parent and its derivatives.

For DNA preparation, all strains were recovered from glycerol stocks by plating them onto tryptic soy agar plates and incubating them overnight at 37°C. One colony was transferred to 50 mL of tryptic soy broth and grown overnight at 37°C with shaking at 180 rpm. The culture was centrifuged, and the pellet was used for DNA extraction using the Nanobind CBB Big DNA kit (PacBio; SKU 102-301-900), following the Gram-positive bacteria protocol for Nanobind high-molecular-weight (HMW) DNA extraction. DNA samples were quantified using the Qubit double-stranded DNA (dsDNA) broad-range (BR) assay. The integrity and purity of the DNA samples were determined using the Agilent D1000 ScreenTape assay.

Sequencing libraries were prepared using the Oxford Nanopore rapid barcoding sequencing kit (SQK-RBK004) and the Illumina Nextera XT kit and sequenced on the GridION vR9.4.1 and Illumina MiSeq v2 (midoutput 2 × 151-bp) instruments, respectively. Base calling, demultiplexing, quality control (QC; Phred score), and adapter trimming for Nanopore data were performed using MinKNOW v.4.3.28 software. MiSeq quality filters were used for QC, and adapter sequences were removed from the Illumina data using fastp v0.23.2 (4). All data were assembled using Unicycler v0.5.0 and Canu v2.2 and polished using Polypolish v0.5.0 and RagTag v2.1.0 (4, 5). The hybrid genome was annotated using Prokka v1.14.5, and reference mapping was performed using Minimap2-2.224 r1122 for the Nanopore data and BWA-0.7.17 r1188 for the Illumina data (5). Default parameters were used for all software except Minimap2 (-ax for long-read data). Analysis resulted in assemblies containing the complete chromosomal and plasmid pXO1 sequences for the parent and four mutant strains and draft assemblies containing multiple contigs for the remaining four mutant strains. Assessment using CheckM v1.1.6 estimated that all genomes were >99% complete and <3% contaminated (6).

Data availability.

The accession numbers and sequence statistics of the 9 genomes are listed in Table 1.

TABLE 1.

Genome sequence details of B. anthracis Sterne 7702 and its phage-sensitive/-resistant derivatives

Straina	Genotype (phage resistance)a	Illumina data		ONT datab			GenBank accession no.	Assembly size (bp)	No. of contigs	No. of CDSsc	Depth of coverage (×)d	Breadth of coverage (%)d
		SRA accession no.	Total no. of reads	SRA accession no.	Total no. of reads	N50 (bp)
7702	Wild type (N)	SRR21851321	1,245,458	SRR21851324	129,986	9,965	CP110279	5,415,265	2	5,661	182	100
BAP350	ΔcsaB (Y)	SRR21851319	1,291,907	SRR21851312	358,612	2,983	JAOZJJ000000000	5,369,429	9	5,615	68	99.97
BA749	ΔBAS0566 (N)	SRR21851318	1,380,162	SRR21851309	292,441	9,470	CP110281	5,480,422	2	5,713	325	99.98
BA750	Δsap (Y)	SRR21851317	1,797,176	SRR21851308	299,086	9,266	JAOZJK000000000	5,416,293	6	5,665	348	99.96
BA751	Δeag (N)	SRR21851316	1,370,002	SRR21851307	209,699	9,986	JAOZJL000000000	5,408,510	3	5,651	263	99.96
BA752	ΔBAS1792 (N)	SRR21851315	1,499,543	SRR21851306	87,351	10,217	JAOZJM000000000	5,400,779	5	5,638	142	99.98
BA754	Δspo0A (Y)	SRR21851313	1,615,048	SRR21851304	297,364	7,484	CP110283	5,406,662	2	5,647	291	99.99
BA755	Δspo0F (Y)	SRR21851311	1,237,687	SRR21851303	318,963	9,341	CP110285	5,408,021	2	5,646	361	99.99
DP-B-5747	Δspo0B (Y)	SRR21851310	1,611,136	SRR21851322	220,073	8,033	CP110287	5,482,710	2	5,735	248	99.99

^aGenotype/phenotype information on strains is from Plaut et al. (3). N, phage sensitive; Y, phage resistant.

^bONT, Oxford Nanopore Technology.

^cCDSs, coding sequences.

^dAgainst the reference sequences under GenBank accession numbers NZ_CP009541.1 (chromosome) and NZ_CP009540.1 (pXO1).