Draft Genome Sequence of Biocontrol Agent Bacillus cereus UW85.

Bacillus cereus UW85 was isolated from a root of a field-grown alfalfa plant from Arlington, WI, and identified for its ability to suppress damping off, a disease caused by Phytophthora megasperma f. sp. medicaginis on alfalfa. Here, we report the draft genome sequence of B. cereus UW85, obtained by a combination of Sanger and Illumina sequencing.

GENOME ANNOUNCEMENT

Bacillus cereus is a Gram-positive, ubiquitous spore-forming bacterium present in the soil, rhizosphere, and guts of several invertebrates (1). B. cereus UW85 was identified from 700 bacterial isolates from the roots of field-grown alfalfa plants. The collection was screened for the suppression of damping off caused by the oomycete Phytophthora megasperma f. sp. medicaginis on alfalfa seedlings (2). Damping off is characterized by browning of stem and root tissues, followed by girdling and seedling death. B. cereus UW85 produces two antibiotics, zwittermicin A (3) and kanosamine (4), which each exhibit broad-spectrum antimicrobial activity that contributes to the suppression of alfalfa seedling damping off, as demonstrated by analysis of mutants deficient in antibiotic production (4, 5). B. cereus UW85 can also protect tobacco seeds from infection by Phytophthora parasitica var. nicotianae (6) and cucumber fruit cotton leak, a disease caused by Phytophthora aphanidermatum (7). In the field, B. cereus UW85 enhances soybean nodulation (8) and can significantly increase the yield of several soybean cultivars (9). In addition, the presence of B. cereus UW85 on soybean seeds shapes the microbial community that develops subsequently in the rhizosphere (10).

The B. cereus UW85 genome was first sequenced using Sanger sequencing of a small-insert library (4 to 5 kb) and a large-insert library (10 to 12 kb), generating a total of ~51,000 reads, which were assembled using the Celera Assembler software (https://sourceforge.net/projects/wgs-assembler/) (11) into 271 contigs. Contigs were designated as originating from the chromosome or a plasmid with a BLAST comparison to the reference B. cereus isolates ATCC 10987 and ATCC 14579. Chromosomal contigs were ordered by Mauve (12) using the B. cereus ATCC 14579 genome (13) as a reference. A similar approach was used to assemble and order the plasmid contigs using several B. cereus group plasmids as references (14). Contigs were assembled manually by joining neighboring sequences with a linker sequence of unknown nucleotide characters labeled N. Gaps were filled using GapFiller (15) with 9,489,450 paired-end reads of 300 bp from an ~1-kb library generated on an Illumina MiSeq instrument, creating a merged assembly with both Sanger and Illumina data. The resulting assembled chromosome was 5,522,108 bp, consisting of 23 contigs, with an N50 contig size of 240,092 bp. Thirty-one contigs accounting for 881,969 bp, with an N50 contig size of 28,451 bp, showed greater similarity with B. cereus plasmid sequences. B. cereus is known to have an extremely varied plasmid profile, with strains carrying mixtures of plasmids ranging in size from 15 to 600 kb (16, 17). The zwittermicin A gene cluster (18, 19) is on one of the larger plasmid contigs, PC_11, which is ~150 kb. A second large plasmid, PC_30, is ~225 kb, has a region of similarity to the Bacillus anthracis pXO1 plasmid, and carries an uncharacterized gene cluster with similarity to a cluster encoding biosynthetic pathways for nonribosomal peptide synthesis machinery.

We anticipate that the genome sequence of B. cereus UW85, one of the best-characterized biocontrol agents, will facilitate discoveries about its plant growth-promoting activity, disease-suppressive properties, and potential for producing new antibiotics.

Accession number(s).

This whole-genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession no. LYVD00000000. The version described in this paper is version LYVD01000000.