ABSTRACT
Priestia megaterium MWU16-30321 was isolated from a mixed bacterial culture in a cranberry stem gall in Massachusetts following a severe winter. The genome is 5,623,390 bp in size and putatively encodes indole-3-acetic acid acetyltransferase, a key enzyme in tryptophan-dependent indole-3-acetic acid production.
ANNOUNCEMENT
Tumorous formations in cranberry (Vaccinium macrocarpon Ait.) can result from mixed bacterial infections of stem tissue following mechanical or frost injury to the epidermis (1–3). These hypertrophic and hyperplastic galls form in response to bacterially produced indole-3-acetic acid (IAA); this results in stem girdling that causes the death of reproductive meristems, thus reducing fruit yield. Priestia megaterium MWU16-30321 (basonym Bacillus megaterium de Bary [4]) was isolated from a single stem gall in a commercial cranberry bog in Carver, Massachusetts, in 2015 by spreading surface-sterilized gall tissue on nonselective medium. The isolate was transferred to King’s medium B (KMB) agar supplemented with 50 μg mL−1 each of cycloheximide and ampicillin, incubated at 26°C for 24 h, colony purified three times on KMB agar, and stored at −80°C in 34% glycerol. A population of MWU16-30321 was inoculated into overnight KMB broth cultures for genomic DNA isolation with a DNeasy blood and tissue kit (Qiagen, USA). An Illumina-compatible genomic DNA library was generated with a KAPA HyperPlus library preparation kit (Kapa Biosystems KK8514; Roche, USA). DNA was enzymatically sheared to ~500-bp fragments, end repaired, and A-tailed, Illumina-compatible adapters with unique indexes (00989130v2; Integrated DNA Technologies, Coralville, IA) were individually ligated to each sample, and the DNA was cleaned using KAPA Pure beads (Kapa Biosystems KK8002) and amplified with HiFi enzyme (Kapa Biosystems KK2502). The library was analyzed for fragment size with an Agilent TapeStation and quantified by quantitative PCR (qPCR) (KAPA library quantification kit KK4835 with QuantStudio v5 [Thermo Fisher Scientific]) before multiplex pooling and sequencing on an Illumina MiSeq system with a 2 × 250-bp flow cell. All kits used in this work were used according to the manufacturer’s instructions. Raw reads were assembled with Unicycler v0.4.8 (5) and polished with Pilon v1.23 (6) within the PATRIC Comprehensive Genome Analysis pipeline v3.6.12, using default settings except for the trim setting, which was set to true (7). Adapter trimming and quality control were provided as part of the PATRIC pipeline by Trim Galore v0.4.0 (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore) and QUAST v5.0.2 (8). Genome sequences were annotated using RASTtk v1.073 (9) as part of the PATRIC pipeline.
The 5,623,390-bp genome was assembled into 104 contigs, with a G+C content of 37.65 mol%, an N50 value of 610,290 bp, and 130× coverage. The average read length was 235.51 bp, from 3,115,330 total reads. There were 4 predicted rRNA genes and 84 tRNA genes. Of the 6,086 predicted genes, 4,017 had functional assignments, including IAA acetyltransferase, a key enzyme in tryptophan-dependent IAA production that has been observed in several Bacillus species (10–13).
MWU16-30321 was placed with high confidence in the species P. megaterium by phylogenetic analysis using the Type (Strain) Genome Server (TYGS) v342 (14). The digital DNA-DNA hybridization (dDDH) value (formula d4) was 94.2% with respect to P. megaterium ATCC 14581T (GenBank accession number FOPA01000000).
Data availability.
The whole-genome shotgun project has been deposited in DDBJ/EMBL/GenBank under BioProject accession number PRJNA765055, with BioSample accession number SAMN30839142, genome accession number JAOTPB000000000, and SRA accession number SRR18445423. The version described in this paper is JAOTPB010000000. RASTtk annotation is available under open license at Zenodo (https://doi.org/10.5281/zenodo.6415975).
ACKNOWLEDGMENTS
This research was supported by the Office of Research and Sponsored Programs, College of Graduate Studies, and the Biomedical Sciences Program, Midwestern University.
MWU16-30321 was originally isolated by Erika Salaau-Rojas, Ocean Spray Cranberry Cooperative (Middleborough, MA), while at the University of Massachusetts Cranberry Station (East Wareham, MA). Library construction and Illumina sequencing were performed at the Arizona State University Genomics Core Facility. We gratefully acknowledge the generous cooperation of the University of Massachusetts Cranberry Station and members of the Massachusetts Cranberry Growers Association for access to plant materials.
The manuscript satisfies a course requirement for K.S.
Contributor Information
Scott Soby, Email: ssobyx@midwestern.edu.
Leighton Pritchard, SIPBS, University of Strathclyde.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The whole-genome shotgun project has been deposited in DDBJ/EMBL/GenBank under BioProject accession number PRJNA765055, with BioSample accession number SAMN30839142, genome accession number JAOTPB000000000, and SRA accession number SRR18445423. The version described in this paper is JAOTPB010000000. RASTtk annotation is available under open license at Zenodo (https://doi.org/10.5281/zenodo.6415975).