Draft Genome Sequences of Sporulation-Impaired Bacillus pumilus Strain NRS576 and Its Native Plasmid p576

Bacillus pumilus spores can cause foodborne poisonings. B. pumilus strain NRS576 forms spores with a very reduced efficiency due to the presence of a plasmid, named p576.

O rganisms in the Bacillus genus are Gram-positive bacteria that can form spores resistant to radiation, heat, and chemicals. Bacillus pumilus is present in soil samples, but some strains are associated with food poisoning, and spore formation is relevant to its pathogenicity (1)(2)(3). B. pumilus NRS576 forms spores with low efficiency due to a plasmid (4), p576, which we have sequenced previously (5). Here, we report the chromosomal and plasmid sequences of strain NRS576 obtained from the Bacillus Genetic Stock Center. A total DNA sample (6), obtained from cells growing in LB medium at 37°C, was used for sequence determination with the Illumina MiSeq platform. DNA libraries were prepared with the NEBNext DNA library prep kit for Illumina (New England Biolabs). Briefly, 1 g DNA was sonicated, and fragments of ϳ675 or 1,075 bp were selected. DNA ends were repaired, A-tailed, and ligated to adapters. Next, fragments were PCR amplified (8 cycles) and quality checked on a DNA 7500 chip on a 2100 Bioanalyzer (Agilent). Library sizes of ϳ800 and 1,200 bp were isolated, validated (Bioanalyzer), and titrated with quantitative PCR (qPCR). After denaturation, the libraries were seeded on a flow cell (MiSeq v2, 2 ϫ 150 bp) at a density of 16 pM. The sequencing rendered 3,963,212 (800-bp library) and 2,015,074 (1,200-bp library) paired-end reads. Data processing was done with default parameters. Adapters (Cutadapt [7]) and low-quality sequences (Sickle 1.33 [8]) were removed. After verifying the quality of the processed data (FastQC [9]), de novo assembly was performed (SPAdes v3.9.1 [10][11][12][13]). Three apparent extrachromosomal elements were detected with plasmidSPAdes (13), (i) plasmid p576, (ii) bacteriophage phiX174 (added as a control for amplification and sequencing [14]), and (iii) sequences similar to part of the Brevibacillus laterosporus DSM25 bogC gene cluster. PhiX174 sequences and contigs smaller than 250 bp were removed, and sequences similar to bogC were considered genomic DNA. General statistics obtained with the bioinformatic tool Quast (15) gave an N 50 value of 313,965 bp and an L 50 value of 5.
One 43,328-bp contig corresponded to p576, which is 106 bp smaller than previously determined (5). The plasmid p576 contains direct-repeat sequences. The apparent deletions correspond to these repeats. Otherwise, the p576 sequences differ in one single base pair (gene 64 codon 492 [GAA462GGA]). We sequenced p576 and genomic DNA with mean coverages of about 70 and 30, respectively, and this implies that p576 has a copy number of two. The previously published p576 nomenclature (5) was respected. Sanger (5) and next-generation sequencing (NGS)-determined p576 sequences are publicly available.
Data availability. This whole-genome shotgun project has been deposited at DDBJ/ENA/GenBank under accession number UWJF00000000. The version described here is the first version. Raw sequence reads have been submitted to the Sequence Read Archive (SRA) (accession numbers ERR2811649 and ERR2811650, corresponding to 2 ϫ 150-bp paired-end sequences of 800-and 1,200-bp fragments, respectively). The p576 sequences are available under DDBJ/ENA/GenBank accession numbers LR026976 (Sanger) and LR026977 (NGS).

ACKNOWLEDGMENTS
We gratefully acknowledge David Abia and Ramón Peiró-Pastor for their help with the bioinformatics analyses.
This work was supported by the Ministry of Economy and Competitiveness of the Spanish Government (grant number BIO2016-77883-C2-1-P to W.J.J.M., which also funded J.V.-C., A.M.-A., and C.G.-C.). Part of the economic support of the aforementioned grants was provided by the Agencia Estatal de Investigación (AEI), Fondo Europeo de Desarrollo Regional (FEDER), and European Union (EU). This research was also supported by institutional grants from the Fundación Ramón Areces and Banco de Santander to the Centro de Biología Molecular Severo Ochoa. The funders had no role in the study design, data collection and interpretation, or decision to submit the work for publication.
We declare no conflict of interest.