ABSTRACT
We report 36 whole-genome sequences, along with annotations, of fermentative (n = 12) and spoilage associated (n = 6) lactic acid bacteria, Lysinibacillus (n = 3), Streptococcus (n = 1), and Proteobacteria (n = 14) isolated from commercial cucumber fermentations. Fifty-three percent of the genome sequence assemblies consist of 1–4 contigs, and the remainder have fewer than 16.
KEYWORDS: fermentation, food microbiology
ANNOUNCEMENT
We document genome sequences of multiple Bacilli and Proteobacteria isolated from commercial cucumber fermentation. Nine genome sequences are higher quality versions of previous submissions (1). Three genome sequences represent the nisin producer Lactococcus lactis (2) and putative bacteriocin producing Pediococcus pentosaceus and Leuconostoc mesenteroides strains (3). The remainder are Proteobacteria (n = 14) associated with fermented cucumber bloater defect (4) and Bacilli (n = 9) relevant to the instability of cucumber fermentation (5).
Table 1 describes the collection site of the isolates. Most fermentative lactic acid bacteria were collected, classified, and identified in surveys of commercial cucumber fermentations conducted between 2009 and 2010 as described by Pérez-Díaz et al. (6). LA0445 was derived from anaerobic fermentation of cucumbers (7). The isolations of Lactococcus lactis NCK401 (2) and Pediococcus pentosaceus LA0061 (previously identified as P. cerevisiae L-7230) (3) were conducted in Raleigh, NC, USA from cover brine samples derived from undefined sources. Leuconostoc mesenteroides FFL48 (previously identified as P. cerevisiae FBB-61) was collected and isolated in Marshall, MI, USA (8). The Proteobacteria and spoilage associated lactic acid bacteria included here were isolated as described by Pérez-Díaz et al. (6) and Pérez-Díaz et al. (4), respectively. Lysinibacillus strains were isolated from spoiled commercial cucumber fermentations in Mt. Olive, NC, USA as described by Medina et al. (5).
TABLE 1.
Accession numbers and genome statistics for isolates described in this announcementa
| Strain IDs | NCBI accession number | SRA accession number | Assembly size (bp) | Contigs | Estimated coverage (×) | Reads | Average raw read length | GC % | N50 (bp) | Collection site |
|---|---|---|---|---|---|---|---|---|---|---|
| Lactiplantibacillus pentosus | ||||||||||
| 7.2.15 | JAVLAU000000000 | SRR25916667 | 3,919,042 | 8 | 157.542 | 63,240 | 9,772 | 45.93 | 3,645,019 | Mount Olive, NC |
| LA0445 | JAHLEN000000000.2 | SRR25916659 | 3,945,202 | 11 | 71.017 | 42,596 | 6,588 | 45.84 | 3,580,249 | Mount Olive, NC |
| 1.8.18 | JAVLAN000000000 | SRR25916663 | 3,897,544 | 15 | 108.48 | 47,598 | 8,910 | 45.82 | 2,062,758 | Mount Olive, NC |
| 1.8.9 | JAVLAO000000000 | SRR25916661 | 3,734,812 | 12 | 90.879 | 50,246 | 6,761 | 45.92 | 3,419,212 | Mount Olive, NC |
| 14.8.42 | JAVGXB000000000 | SRR25916660 | 4,050,922 | 16 | 62.214 | 32,012 | 7,882 | 45.78 | 789,853 | Mount Olive, NC |
| 7.8.46 | JAVLAQ000000000 | SRR25916658 | 3,966,095 | 12 | 106.639 | 46,231 | 9,160 | 45.73 | 2,973,648 | Mount Olive, NC |
| 7.8.2 | CP134788-CP134793 | SRR26196798 | 3,919,042 | 8 | 157.542 | 63,240 | 9,772 | 45.93 | 3,645,019 | Chaska, MN |
| Lactiplantibacillus plantarum | ||||||||||
| 7.8.4 | JAVLAR000000000 | SRR25916657 | 3,490,038 | 15 | 44.239 | 23,524 | 6,609 | 44.23 | 2,712,281 | Mount Olive, NC |
| Lactococcus lactis | ||||||||||
| NCK401 | CP137623 | SRR26043614 | 2,551,975 | 1 | 75.178 | 25,782 | 7,450 | 34.86 | 2,551,975 | Raleigh, NC |
| LA0312 | CP134164 | SRR25916659 | 2,563,933 | 1 | 172.327 | 49,924 | 8,857 | 34.86 | 2,563,933 | Mount Olive, NC |
| Pediococcus pentosaceus | ||||||||||
| LA0061 | CP137627-CP137629 | SRR26043613 | 1,945,268 | 3 | 250.134 | 51,502 | 9,455 | 37.19 | 1,847,864 | Raleigh, NC |
| Leuconostoc mesenteroides | ||||||||||
| FFL48 | CP137625-CP137626 | SRR26043612 | 1,816,909 | 2 | 252.299 | 53,393 | 8,589 | 38.02 | 1,799,974 | Marshall, MI |
| Companilactobacillus alimentarius | ||||||||||
| 7.2.6 | JAVKYT000000000 | SRR25916668 | 2,665,037 | 10 | 192.679 | 56,034 | 9,171 | 35.5 | 1,641,583 | Mount Olive, NC |
| Levilactobacillus namurensis | ||||||||||
| 1.2.9 | JAVLAL000000000 | SRR25916666 | 2,932,609 | 10 | 173.356 | 64,197 | 7,925 | 50.77 | 2,656,307 | Mount Olive, NC |
| 3.8.38 | JAVLAM000000000 | SRR25916665 | 2,983,047 | 14 | 152.717 | 57,348 | 7,938 | 50.68 | 2,566,380 | Mount Olive, NC |
| 90.8.26 | CP134159-CP134163 | SRR25916664 | 2,776,426 | 5 | 196.758 | 59,866 | 9,290 | 51.4 | 2,674,724 | Mount Olive, NC |
| Latilactobacillus zymae | ||||||||||
| 90.8.36 | JAVLAS000000000 | SRR25916656 | 2,892,482 | 8 | 170.533 | 56,995 | 8,701 | 51.49 | 2,645,371 | Mount Olive, NC |
| Latilactobacillus curvatus | ||||||||||
| 3.8.43 | JAVLAK000000000 | SRR25916651 | 2,056,476 | 4 | 237.549 | 62,061 | 7,860 | 42.03 | 1,616,338 | Chaska, MN |
| Lysinibacillus capsici | ||||||||||
| Lys1 | CP137622 | SRR26060012 | 4,682,737 | 1 | 94.303 | 49,304 | 8,969 | 37.58 | 4,682,737 | Mount Olive, NC |
| Lys24 | CP134502 | SRR26060010 | 4,687,025 | 1 | 87.806 | 48,098 | 8,564 | 37.62 | 4,687,025 | Mount Olive, NC |
| Lysinibacillus louembei | ||||||||||
| Ll15 | CP137624S | SRR26043611 | 3,960,254 | 1 | 93.807 | 45,092 | 8,245 | 38.53 | 3,960,254 | Mount Olive, NC |
| Streptococcus parasanguinis | ||||||||||
| 30.8.10 | CP134147-CP134148 | SRR25916648 | 2,128,578 | 2 | 199.9238186 | 51,124 | 8,334 | 42.01 | 2,118,583 | Mount Olive, NC |
| Enterobacter cancerogenus | ||||||||||
| 3.2.13 | JAVLAJ000000000 | SRR25916674 | 4,902,923 | 4 | 72.304 | 53,067 | 6,685 | 55.64 | 4,466,362 | Chaska, MN |
| 3.2.17 | CP134403-CP134405 | SRR25916652 | 4,882,814 | 3 | 131.134 | 68,845 | 9,309 | 55.63 | 4,668,754 | Chaska, MN |
| Enterobacter hormaechei | ||||||||||
| 1.2.3 | CP134406-CP134411 | SRR25916671 | 3,862,785 | 6 | 121.153 | 51,423 | 9,112 | 46.03 | 3,635,834 | Mount Olive, NC |
| 3.2.8 | JAVLAI000000000 | SRR25916670 | 5,031,271 | 6 | 68.791 | 52,737 | 6,568 | 55.41 | 3,591,916 | Mount Olive, NC |
| Kluyvera cryocrescens | ||||||||||
| 1.8.5 |
CP134165 | SRR25916669 | 4,849,988 | 1 | 120.883 | 60,604 | 9,685 | 53.92 | 4,849,988 | Mount Olive, NC |
| Leclercia pneumoniae | ||||||||||
| 1.2.7 | CP134503-CP134506 | SRR26196799 | 4,932,015 | 4 | 129.539 | 68,633 | 9,317 | 54.49 | 4,521,154 | Mount Olive, NC |
| Pantoea agglomerans | ||||||||||
| 1.2.4 | CP134149-CP134151 | SRR25916650 | 4,870,469 | 3 | 125.758 | 69,211 | 8,857 | 55.14 | 4,084,927 | Mount Olive, NC |
| Pseudomonas putida | ||||||||||
| 1.8.4 | CP137621 | SRR26043609 | 5,946,927 | 1 | 124.581 | 80,233 | 9,246 | 61.9 | 5,946,927 | Chaska, MN |
| Pseudomonas inefficax | ||||||||||
| 7.8.24 | CP134401 | SRR25916649 | 5,927,592 | 1 | 113.28 | 73,885 | 9,099 | 62.78 | 5,927,592 | Chaska, MN |
| Erwinia toletana | ||||||||||
| 1.2.20 | CP134152-CP134153 | SRR25916662 | 5,014,558 | 2 | 117.097 | 67,679 | 8,696 | 52.22 | 4,923,789 | Chaska, MN |
| Siccibacter colletis | ||||||||||
| 3.2.4 | CP134402 | SRR25916654 | 4,158,854 | 1 | 135.985 | 67,089 | 8,438 | 57.41 | 4,158,854 | Chaska, MN |
| Hafnia alvei | ||||||||||
| 3.8.4 | CP134154-CP134158 | SRR25916653 | 4,855,803 | 5 | 109.27 | 56,367 | 9,421 | 48.81 | 4,631,589 | Chaska, MN |
| Stenotrophomonas indicatrix | ||||||||||
| 7.2.7 | JAVYIJ000000000 | SRR26060011 | 4,573,902 | 9 | 108.262 | 72,052 | 6,878 | 66.29 | 2,356,763 | Chaska, MN |
| Brucella pseudogrignonensis | ||||||||||
| 1.2.1 | JAVLAT000000000 | SRR25916655 | 4,453,607 | 7 | 130.227 | 63,658 | 9,118 | 51.87 | 2,323,104 | Chaska, MN |
sites of sample collection are abbreviated as NC, MI, and MN for North Carolina, Michigan, and Minnesota, USA, respectively.
Pure cultures of lactic acid bacteria and bacilli were transferred from frozen stocks to Lactobacilli deMan, Rogosa, and Sharpe (MRS) broth, while proteobacteria were transferred to Brain Heart Infusion (BHI) broth and incubated statically and aerobically at 30°C prior to DNA extraction. The Promega Wizard Genomic DNA Extraction Kit (Madison, WI) was used to lyse cells and precipitate proteins. To produce a higher DNA yield than possible with the standard kit protocol, cell lysates were treated with 25:24:1 phenol:chloroform:isoamyl alcohol followed by ethanol precipitation (9).
Genomic DNA was sheared using Covaris G-tubes (Woburn, MA) targeting 10 kb fragments. Sheared DNA was prepared for PacBio sequencing using the SMRTbell Prep Kit 3.0 (PacBio, Menlo Park, CA). Samples were barcoded and libraries were size selected with AMpure PB beads (Pacific Biosciences, Menlo Park, CA) to remove fragments less than 3 kb. Sequencing was performed on a Sequel IIe System (Pacific Biosciences, Menlo Park, CA) using Binding Kit 3.2, Sequel II Sequencing Kit 2.0 and SMRTCell 8M. To target HiFi reads, the library was sequenced using a 30-hour movie time using Instrument Control Software Version 11. Raw subreads were converted to HiFi data by processing with CCS to call a single high-quality consensus sequence for each molecule, using a 99.5% consensus accuracy cutoff. De novo assembly was performed in BV-BRC v. 330.19a (10) via Unicycler version 0.4.8 (11) with a minimum contig cut-off of 300. Quality assessment of assemblies was performed with QUAST version 5.0.2 (12), SamTools version 13 (13), and Pilon version 1.23 (14). Closest reference genomes were identified by Mash/MinHash employing the PATRIC database (15). Upon submission to GenBank (Bioproject PRJNA674638), assemblies were reannotated using the NCBI Prokaryotic Genome Annotation Pipeline v. 6.5 (16). Default parameters for the software were used.
ACKNOWLEDGMENTS
We thank Sandra Parker and Research Leaders Drs. Muquarrab Qureshi and Brian Scheffler with the United States Department of Agriculture (USDA) - Agricultural Research Service for their administrative efforts to facilitate this collaborative research. We also thank Linda Ballard, IT specialist, with the USDA-ARS Genomics and Bioinformatics Research Unit in Stoneville, MS, USA for her assistance with SCINet downloads. We thank Dr. Michael Taveirne, Associate Teaching Professor in the Department of Biological Sciences, and Dr. Sarah O’Flaherty, Research Scholar in the Department of Food, Bioprocessing and Nutrition Sciences, both at North Carolina State University for reviewing the manuscript.
This work was performed in part at the Analytical Instrumentation Facility (AIF) at North Carolina State University, which is supported by the State of North Carolina and the National Science Foundation (award number ECCS-2025064). This work made use of instrumentation at AIF acquired with support from the National Science Foundation (DMR-1726294). The AIF is a member of the North Carolina Research Triangle Nanotechnology Network (RTNN), a site in the National Nanotechnology Coordinated Infrastructure (NNCI).
Contributor Information
Ilenys M. Pérez-Díaz, Email: ilenys.perez-diaz@usda.gov.
J. Cameron Thrash, University of Southern California, USA.
DATA AVAILABILITY
Strain identification and accession numbers for each genome annotation and sequence read archive are included in Table 1.
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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
Strain identification and accession numbers for each genome annotation and sequence read archive are included in Table 1.