Whole-Genome Sequencing and Annotation of Pediococcus ethanolidurans and Pediococcus pentosaceus Isolates from Commercial Cucumber Fermentation

ABSTRACT

We report the whole-genome sequences, along with annotations, of five Pediococcus ethanolidurans and three Pediococcus pentosaceus isolates from commercial cucumber fermentations performed in North Carolina (n = 3) and Minnesota (n = 5), USA.

ANNOUNCEMENT

Pediococcus ethanolidurans and Pediococcus pentosaceus are lactic acid bacteria species relevant in the production of fermented foods. P. ethanolidurans is associated with spoilage in both olive fermentation (1) and in the production of cider (2) but is also important for the development of flavors in pickled radishes (3). P. pentosaceus is a significant organism for sauerkraut production (4). Both organisms were detected in unspoiled cucumber fermentations conducted in 2009 and 2010 in Mount Olive, NC, and Chaska, MN, respectively.

Pediococcus isolates were collected on days 1, 3, 7, 14, and 30 of commercial cucumber fermentations at depths of 2 and 8 ft from the brine surface in the collection tank (5). The isolation day and depth are incorporated into the genome sequence identification numbers. For example, genome sequence 14.2.45 was generated from a single colonial isolate (number 45) plated from fermentation brine collected on day 14 at a depth of 2 ft.

The isolates were derived from cucumber fermentation brine by spiral plating on Lactobacillus de Man Rogosa and Sharpe agar (MRS) supplemented with 0.0001% cycloheximide solution and incubated at 30°C (5). Anaerobic growth conditions were maintained via the BD GasPak EZ system (Franklin Lakes, NJ), which maintained an anaerobic atmosphere with ≥10% carbon dioxide. The isolated colonies were streaked onto MRS agar prior to preparing frozen stocks in MRS broth supplemented with 1.5% glycerol. Pure cultures were generated on MRS broth from the frozen stocks for DNA extraction. The MRS broth was incubated statically at 30°C for 24 h under aerobic conditions. DNA extraction was conducted using the Promega Wizard genomic DNA extraction kit (Madison, WI). The bacterial isolates had been previously identified using partial 16S rRNA gene sequences, as described by Pérez-Díaz and others (5). CosmosID (Rockville, MD) prepared libraries for Illumina reads using the Illumina Nextera XT kit and assessed the libraries for quality using Qubit (Thermo Fisher, Waltham, MA), prior to whole-genome sequencing. The samples were sequenced on an Illumina NextSeq 550 platform (San Diego, CA), producing paired-end reads at a maximum length of 150 bases. The estimated average raw read lengths are reported in Table 1, based on back calculation from the genome coverage (6). CosmosID also performed raw sequence data trimming for adapters and low-quality bases using BBDuk (https://sourceforge.net/projects/bbmap) in the spring of 2022, applying standard parameters (phred quality trimq = 22, and minimum length minlen = 36).

TABLE 1.

Accession numbers and genome statistics from P. ethanolidurans and P. pentosaceus isolates

Species	Isolate ID	GenBank accession no.	SRA accession no.	Assembly size (bp)	No. of contigs	Estimated coverage (×)	No. of read pairs	Avg raw read length (bp)	GC content (%)	N50 (bp)	Collection site (U.S. state)
Pediococcus ethanolidurans	14.2.5	JANJYK000000000	SRR20829250	2,148,770	175	442.575	3,525,283	134.8	37.45	33,153	NC
	3.8.1	JANQAL000000000	SRR20829249	2,208,096	164	360.416	2,906,518	136.9	37.34	34,310	NC
	1.2.28	JANQAM000000000	SRR20829248	2,170,406	166	433.77	3,601,207	130.7	37.46	31,828	NC
	14.2.45	JANJPN000000000	SRR20829251	2,163,868	167	277.284	2,164,405	138.6	37.39	33,670	MN
	30.2.11	JANQCL010000000	SRR21954066	2,129,602	162	409.327	3,261,290	133.6	37.42	37,022	MN
Pediococcus pentosaceus	7.2.43	JANKYZ000000000	SRR20964832	1,853,217	24	549.323	3,780,940	134.6	37.2	295,794	MN
	3.2.19	JANKZA000000000	SRR20964831	1,854,702	20	428.985	2,826,910	140.7	37.16	375,985	MN
	7.2.36	JANKZB000000000	SRR20964830	1,881,615	35	371.876	2,506,629	139.5	37.17	286,823	MN

The genome sequences were assembled and annotated using the software described below with default parameters unless otherwise specified. De novo assembly was performed using PATRIC v. p3-build-178 (7) via Unicycler v. 0.4.8 (8), with a minimum contig cutoff of 300. Quality assessment of the assemblies was performed using QUAST v. 5.0.2 (9), SAMtools v. 13 (10), and Pilon v. 1.23 (11). The closest reference genomes were identified using Mash/MinHash with the PATRIC database (12). Upon submission to GenBank, the assemblies were annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) v. 2022-04-14.build6021 (13).

Data availability.

The assemblies have been submitted to GenBank under BioProject accession number PRJNA674638. Strain identification and accession numbers for each genome annotation are listed in Table 1.