ABSTRACT
Limosilactobacillus reuteri is a lactic acid bacterium with several probiotic properties. Here, we present the draft genome sequence of L. reuteri isolated from human breast milk. The average genome size was estimated as 2,087,202 bp, with a guanine-cytosine (GC) content of 51.6%. GC content is the percentage of nitrogenous bases in a DNA or RNA molecule that are either guanine (G) or cytosine (C). De Man, Rogosa and Sharpe, often abbreviated to MRS, is a selective culture medium designed to favor the luxuriant growth of lactobacilli for lab study.
KEYWORDS: whole genome, gram-positive bacteria
ANNOUNCEMENT
The present work represents the draft genome sequence of Limosilactobacillus reuteri to understand its safety, probiotic characteristics, and technological genomic properties. The present strain was isolated from a 36-year-old woman’s breast milk in India. Fresh breast milk (1 mL) was serially diluted, and 109 dilutions were applied to the MRS media (1). The inoculum was incubated in anaerobic conditions at 37°C for 24 h. Later, 16S rRNA sequencing was performed, and the strain was confirmed as KT000042 Limosilactobacillus reuteri.
KT000042 was anaerobically and statically grown in MRS broth for 24 h at 37°C in preparation for long-read sequencing. Genomic DNA (gDNA) was extracted with the Maxwell RSC system from Promega (Promega, Madison, WI, USA), and sheared to 8–13 kb with the Megaruptor 3 system from Diagenode (Megaruptor 3; Diagenode, Denville, NJ, USA); finally, AMPure PB beads were used to remove small fragments (<3 kb) (Pacific Biosciences [PacBio], CA, USA]. The sequencing library was built with the SMRTbell Express template preparation kit v2.0 (PacBio) with 3 µg of gDNA. The Sequel system (PacBio) was used to sequence the library, yielding 72,860 subreads, N50 10,519 bp, with an average length of 9,035 bp. The data were assembled in SMRT Link v10.1.0.119588 (PacBio) using the Microbial Assembly protocol, comprising reads quality control, error correction, adapter filtering, circularity checking, and overlap trimming (2). For short-read sequencing, 100 ng of gDNA was sheared using Adaptive Focused Acoustics technology (Covaris, MA, USA), and a ~350 bp sequencing library was formed using the TruSeq Nano DNA high-throughput library preparation kit (Illumina, San diego, CA, USA). The HiSeq X Ten platform (Illumina) was used for the sequencing, which generated 33,384,528 raw reads and 19,646,034 filtered paired-end reads (90% of bases with a Phred score >30 or higher). The Illumina reads were then mapped to the PacBio assembled genome using BWA-MEMv0.7.17 (3) after adapter and quality trimmed with CANU v1.5 (4). The final error correction was conducted three times with Pilon v1.21 (5) with a default minDepth value of 0.015, resulting in a total of 14 contigs spanning 2,087,202 bp with an average contig length of 682,680 bp, N50 of 496,276 bp, 51.6% G = C, and 43.2 Χ total depth (Table 1). The genome annotation by the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) v6.1 (6) and Prokka (v1.13) (7) was used for gene prediction and basic annotation with following options: compliant, rnammer, and addgenes predicting 2,051 protein-coding genes, 59 tRNA genes, 15 rRNA genes, 9 rRNA (5S) genes, 6 16S rRNA genes, and 3 23S rRNA genes. For additional annotation, predicted protein sets were subjected to InterProScan v5.30.69.0 (8) and psi blast v2.4.0 with EggNOG database v4.5 (9). All tools were run with default parameters unless otherwise specified.
TABLE 1.
Summary of assembly and annotation of draft genome
| Names | Contig 1 | Contig 2 | Contig 3 | Contig 4 | Contig 5 | Contig 6 | Contig 7 | Contig 8 | Contig 9 | Contig 10 | Contig 11 | Contig 12 | Contig 13 | Contig 14 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Feature | Chromosomal | Chromosomal | Chromosomal | Chromosomal | Chromosomal | Chromosomal | Chromosomal | Chromosomal | Chromosomal | Chromosomal | Chromosomal | Chromosomal | Plasmid | Plasmid |
| Length (bases) | 682,680 | 496,276 | 240,718 | 150,217 | 105,335 | 93,424 | 71,775 | 63,696 | 57,359 | 57,148 | 26,488 | 19,602 | 12,854 | 9,630 |
| GC% | 52.3 | 52.0 | 51.6 | 47.5 | 52.3 | 53.7 | 49.7 | 51.6 | 52.1 | 49.2 | 48.5 | 53.4 | 44.0 | 53.2 |
| CDSs a | 638 | 501 | 249 | 146 | 106 | 105 | 69 | 60 | 59 | 49 | 21 | 17 | 18 | 13 |
| Depth | 40.7 | 40.5 | 63.3 | 46.0 | 33.8 | 41.7 | 51.9 | 41.3 | 32.2 | 49.9 | 39.5 | 12.8 | 6.5 | 16.1 |
| tRNA | 24 | 2 | 22 | 0 | 1 | 1 | 0 | 2 | 1 | 0 | 6 | 0 | 0 | 0 |
| rRNA | 9 | 0 | 3 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 3 | 0 | 0 | 0 |
| Total length (bases) | 2,087,202 | |||||||||||||
| Total GC% | 51.6% | |||||||||||||
| Total CDS | 2,051 | |||||||||||||
| Total depth | 43.2 | |||||||||||||
| Coverage % (>=1x) | 99.75% | |||||||||||||
| GenBank accession no. | JAMKCD000000000 | |||||||||||||
| BioProject accession no. | PRJNA834610 | |||||||||||||
| BioSample accession no. | SAMN28052912 | |||||||||||||
| SRA accession no. | SRR21943033 | |||||||||||||
CDS (coding sequence) is the coding region of a gene.
The present genome was found closer to L. fermentum NZ_BJLV00000000.1. In contrast, 16S rRNA and our previous research studies (10, 11) confirm it as L. reuteri strain. Additionally, literature has evidence that L. reuteri is an L. fermentum biotype II (12); therefore, the similarity is possible between the strains.
ACKNOWLEDGMENTS
The fourth Brain Korea financially supported this research (BK) 21 Plus Project (grant no. 4299990913942), financed by the Korean Government, Republic of Korea.
We are grateful to everyone who participated in this research; we especially would like to thank Macrogen, Inc., Seoul, Korea, for genome sequencing.
Contributor Information
Deog-Hwan Oh, Email: deoghwa@kangwon.ac.kr.
Julie C. Dunning Hotopp, University of Maryland School of Medicine, Baltimore, Maryland, USA
DATA AVAILABILITY
The draft genome sequence has been deposited in the GenBank database under the accession number JAMKCD000000000, BioProject number PRJNA834610, and BioSample number SAMN28052912 and in the Sequence Read Archive (SRA) database under accession number SRR21943033.
REFERENCES
- 1. Łubiech K, Twarużek M. 2020. Lactobacillus bacteria in breast milk. Nutrients 12:3783. doi: 10.3390/nu12123783 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2. Chin C-S, Alexander DH, Marks P, Klammer AA, Drake J, Heiner C, Clum A, Copeland A, Huddleston J, Eichler EE, Turner SW, Korlach J. 2013. Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data. Nat Methods 10:563–569. doi: 10.1038/nmeth.2474 [DOI] [PubMed] [Google Scholar]
- 3. Li H, Durbin R. 2009. Fast and accurate short read alignment with burrows-wheeler transform. Bioinformatics 25:1754–1760. doi: 10.1093/bioinformatics/btp324 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4. Koren S, Walenz BP, Berlin K, Miller JR, Bergman NH, Phillippy AM. 2017. Canu: scalable and accurate long-read assembly via adaptive K-MER weighting and repeat separation. Genome Res 27:722–736. doi: 10.1101/gr.215087.116 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5. Walker BJ, Abeel T, Shea T, Priest M, Abouelliel A, Sakthikumar S, Cuomo CA, Zeng Q, Wortman J, Young SK, Earl AM, Wang J. 2014. Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLoS ONE 9:e112963. doi: 10.1371/journal.pone.0112963 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6. Tatusova T, DiCuccio M, Badretdin A, Chetvernin V, Ciufo S, Li W. 2013. Prokaryotic genome annotation pipeline, the NCBI Handbook. In National center for biotechnology information (US) [Google Scholar]
- 7. Seemann T. 2014. Prokka: rapid prokaryotic genome annotation. Bioinformatics 30:2068–2069. doi: 10.1093/bioinformatics/btu153 [DOI] [PubMed] [Google Scholar]
- 8. Jones P, Binns D, Chang H-Y, Fraser M, Li W, McAnulla C, McWilliam H, Maslen J, Mitchell A, Nuka G, Pesseat S, Quinn AF, Sangrador-Vegas A, Scheremetjew M, Yong S-Y, Lopez R, Hunter S. 2014. Interproscan 5: genome-scale protein function classification. Bioinformatics 30:1236–1240. doi: 10.1093/bioinformatics/btu031 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9. Huerta-Cepas J, Szklarczyk D, Forslund K, Cook H, Heller D, Walter MC, Rattei T, Mende DR, Sunagawa S, Kuhn M, Jensen LJ, von Mering C, Bork P. 2016. eggNOG 4.5: a hierarchical orthology framework with improved functional annotations for eukaryotic, prokaryotic and viral sequences. Nucleic Acids Res 44:D286–93. doi: 10.1093/nar/gkv1248 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10. Tyagi A, Shabbir U, Chelliah R, Daliri E-M, Chen X, Oh D-H. 2021. Limosilactobacillus reuteri fermented brown rice: a product with enhanced bioactive compounds and antioxidant potential. Antioxidants (Basel) 10:1077. doi: 10.3390/antiox10071077 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11. Tyagi A, Yeon S-J, Daliri E-M, Chen X, Chelliah R, Oh D-H. 2021. Untargeted metabolomics of Korean fermented brown rice using UHPLC Q-TOF MS/MS reveal an abundance of potential dietary antioxidative and stress-reducing compounds. Antioxidants (Basel) 10:626. doi: 10.3390/antiox10040626 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12. Gangaiah D, Mane SP, Tawari NR, Lakshmanan N, Ryan V, Volland A, Susanti D, Patel M, Abouzeid A, Helmes EB, Kumar A. 2022. In Silico, in vitro and in vivo safety evaluation of Limosilactobacillus reuteri strains ATCC PTA-126787 & ATCC PTA-126788 for potential probiotic applications. PLoS One 17:e0262663. doi: 10.1371/journal.pone.0262663doi:35081129 [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
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Data Availability Statement
The draft genome sequence has been deposited in the GenBank database under the accession number JAMKCD000000000, BioProject number PRJNA834610, and BioSample number SAMN28052912 and in the Sequence Read Archive (SRA) database under accession number SRR21943033.