ABSTRACT
Leuconostoc carnosum is a bacterial species commonly associated with meat spoilage. However, some strains exhibit preservative effects due to bacteriocin production. Here, we report the complete genome sequences for two strains, L. carnosum 4010 and AMS1. Bacteriocin-related gene clusters were found on the plasmids of both strains.
KEYWORDS: Leuconostoc carnosum, bacteriocins, genome sequences
ANNOUNCEMENT
Leuconostoc carnosum is a group of lactic acid bacteria often found in meat including chill-stored meats and poultry products (1–4), some strains of L. carnosum presented bacteriocin production properties (3, 5). Here, we report the complete genomes of two strains: L. carnosum 4010, isolated from vacuum-packed meat products (3); L. carnosum AMS1, isolated from lightly fried beef/pork minced meat in this study by soft-agar overlay method (6).
The strains were grown in calcium-citrate-sucrose medium (2% tryptone, 0.5% yeast extract, 0.4% NaCl, 0.2% sodium citrate, 0.8% calcium lactate, 0.005% MnSO4, and 1% sucrose) at 28°C. Genomic DNA (gDNA) was isolated from a 2 mL overnight culture using Qiagen MagAttract HMW DNA Kit (Qiagen, Hilden, Germany).
Isolated AMS1 gDNA was sequenced on a high-throughput sequencer Pacbio Sequel II System (Pacific Biosciences, San Francisco, CA, USA) at the DNA Sequencing and Genomics Laboratory (Helsinki Institute of Life Science). DNA template prep kit no. 2.0 and DNA/polymerase binding kit P6 (Pacific Biosciences) were used to generate 3–10 kb fragments and DNA polymerase/template library complex for sequencing. The reads were de novo assembled by SMRT Link 9 Analysis software (Pacific Biosciences). The PacBio pipeline generated three polished contigs with a total contig length of 1,851,151 bp (read N50, 16,133 bp).
The 4010 gDNA was sequenced using in-house Illumina and Nanopore technologies (KU Leuven, Belgium), and size selection was done before sequencing (7). Short reads of the genome were obtained from Illumina MiniSeq (Illumina, CA, USA) using a paired-end 2 × 150 bp approach with a DNA library prepared with Nextera Flex (Illumina). The Illumina sequencing generated 159,693 read pairs with an average length of 151 bp (approximately 48 Mbp). The Illumina reads were processed using Trimmomatic v0.39 to remove adapters and exclude sequences shorter than 50 bp (8) and the resulting data set was quality controlled using FastQC v0.11.8 (9). The same gDNA aliquot was also prepared for long reads sequencing using the Rapid barcoding kit SQK-RBK004 and sequenced on a MinION sequencer (Oxford Nanopore Technologies, United Kingdom) with R9.4.1 flow cell with Guppy (v3.1.5) as basecaller. The Nanopore reads were processed using the Nanopack software suite v1.27.0 (length >1,000 bp, phred score >10) (10), resulting in 147,216 raw reads with an average length of 9,103 bp (approximately 1,340 Mb). The 4010 genome was de novo assembled using the hybrid assembler Unicycler v0.4.8 (11). The obtained draft genome was polished by Pilon v1.22 (12). Default parameters were used except where otherwise indicated.
Complete genome sequences were circularized using a genome assembly program (Gap4, Staden package) (13), and overlapping ends were removed manually. Default parameters were used for all software unless otherwise noted. The annotation of the genomes was performed using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) version 4.12 (14). The detailed annotated information is listed in Table 1. The genomes were further annotated for the presence of bacteriocin-related gene clusters using BAGEL4 (15). Genes responsible for the leucocin production were found on pLC4010-2 of strain 4010 and pLCAMS1-2 of strain AMS1.
TABLE 1.
Genome information of L. carnosum 4010 and AMS1a
| Parameters | Data for strain: | |
|---|---|---|
| AMS1 | 4010 | |
| BioSample accession no. | SAMN36664906 | SAMN15099347 |
| BioProject accession no. | PRJNA997118 | PRJNA637255 |
| GenBank accession no. | CP130703.1 (chromosome) | CP054418.1 (chromosome) |
| CP130704.1 (plasmid pLCAMS1-1) | CP054419.1 (plasmid pLC4010-1) | |
| CP130705.1 (plasmid pLCAMS1-2) | CP054420.1 (plasmid pLC4010-2) | |
| CP054421.1 (plasmid pLC4010-3) | ||
| SRA accession no. | SRX21119575 | SRX21104482 |
| Assembly accession no. | GCA_030585795.1 | GCA_030585425.1 |
| Contig N50 (bp) | 1,809,605 | 1,748,147 |
| Chromosome size (bp) | 1,809,605 | 1,748,147 |
| Genome coverage | 4,600 × | 830 × |
| CDS | 1,799 | 1,723 |
| tRNA | 68 | 68 |
| rRNA | 12 | 12 |
| GC% | 37.6 | 37.5 |
| Plasmids | pLCAMS1-1: 26,558 bp, 32 CDS | pLC4010-1: 38,425 bp, 45 CDS |
| pLCAMS1-2: 14,988 bp, 20 CDS | pLC4010-2: 33,269 bp, 37 CDS | |
| (Bacteriocin-related genes: lcnI, lcnA, lebI, lebB, lecX, lecT, lecS) | (Bacteriocin-related genes: lcnI, lcnA, lebI, lebB, lecI, lecC, lecX, lecT, lecS) | |
| pLC4010-3: 29,604 bp, 33 CDS | ||
lcnI, leucocin A immunity gene; lcnA, leucocin A precursor gene; lebI, leucocin B immunity gene; lebB, leucocin B precursor gene; lecI, leucocin C immunity gene; lecC, leucocin C precursor gene; lecX, accessory gene; lecT, translocator gene; lecS, secretion gene.
ACKNOWLEDGMENTS
X.W. received a personal grant from the Jane and Aatos Erkko Foundation (grant No. 170120).
C.L. is supported by a doctoral fellowship from the Research Foundation—Flanders (FWO) (grant No. 1S64720N). R.L. is granted by the Natural Science Foundation of Sichuan Province (grant No. 2023NSFSC1160).
L. carnosum 4010 was kindly provided by Chr. Hansen (Hørsholm, Denmark).
Contributor Information
Ran Li, Email: liran@sicau.edu.cn.
Xing Wan, Email: xing.wan@helsinki.fi.
David Rasko, University of Maryland School of Medicine, Baltimore, USA.
DATA AVAILABILITY
The complete genomic sequences are available in GenBank under the accession numbers in Table 1.
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Associated Data
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Data Availability Statement
The complete genomic sequences are available in GenBank under the accession numbers in Table 1.