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Complete genome sequence of Staphylococcus equorum KS1030 exhibiting acquired lincomycin resistance
Korean J. Microbiol. 2021;57(3):210-212
Published online September 30, 2021
© 2021 The Microbiological Society of Korea.

Tao Kim1†, Sojeong Heo1†, Jong-Hoon Lee2, and Do-Won Jeong1*

1Department of Food and Nutrition, Dongduk Women’s University, Seoul 02748, Republic of Korea
2Department of Food Science and Biotechnology, Kyonggi University, Suwon 16227, Republic of Korea
Correspondence to: E-mail: jeongdw@dongduk.ac.kr; Tel.: +82-2-940-4463; Fax: +82-2-940-4610
These authors contributed equally to this work.
Received March 17, 2021; Revised April 12, 2021; Accepted April 12, 2021.
Abstract
The complete genome sequence of Staphylococcus equorum KS1030 showing lincomycin resistance was determined. The genome is 2,952,351-bp long with a G + C content of 33.16%, and consists of a single circular 2,862,845-bp chromosome and four circular plasmids. The lincosamide O-nucleotidyltransferase gene, lnuA, conferring lincomycin resistance and the relaxase gene, rlx, for horizontal gene transfer were detected in plasmids pSELNU1 and pKS1030-3, respectively. This genetic information contributes to our understanding of the horizontal transfer of acquired antibiotic resistance genes.
Keywords : Staphylococcus equorum, horizontal gene transfer, lincomycin resistance, relaxase, saeu-jeot
Body

Staphylococcus equorum was previously isolated as a dominant bacterial species from the traditional Korean food saeu-jeot, which is made from salt-fermented shrimp (Guan et al., 2011). Staphylococcus equorum is one of several bacterial starter candidates used to enhance the sensory properties in high-salt fermented foods such as saeu-jeot, which are selected through safety and functional assessments (Jeong et al., 2014). However, four S. equorum strains among 126 examined exhibited lincomycin resistance and contained small plasmids carrying the lincosamide O-nucleotidyltransferase gene lnuA (Lee and Jeong, 2015). Of these four strains, KS1030 carries lnuA on plasmid pSELNU1 which contains the gene as an acquired resistance trait and was shown to exhibit horizontal transfer of pSELNU1 into other Staphylococcus species during soybean fermentation and mouse intestine passage, as well as in vitro (Lee and Jeong, 2015; Heo et al., 2019). The horizontal transfer mechanism of lnuA itself was unclear. Therefore, in the current study, the complete genome sequence of S. equorum strain KS1030 was determined to further understand the transfer mechanism of pSELNU1 into other bacteria during fermentation.

Whole-genome sequencing of S. equorum strain KS1030 was performed using a combination of the Illumina MiSeq system (Illumina) and the PacBio (Pacific Bioscience) single molecule real-time (SMRT) sequencing system by ChunLab, Inc. The generated PacBio sequencing reads (85,375 reads and 285.5-fold coverage) were assembled using the MaSurca algorithm (version 3.3.9) and plasmidSPAdes (version 3.13.0) software. Five contigs were generated from reads. The large sequence of contig 1 was assumed to be a chromosome and others to be plasmids. All contigs were confirmed to be of a circular form by gene walking. Genome annotation was performed using NCBI Prokaryotic Genome Annotation Pipeline (version 4.6) software. Open reading frames (ORFs) were predicted using Glimmer3 software, followed by annotation through a search against the Clusters of Orthologous Groups (COG) database (Tatusov et al., 1997).

The complete genome of S. equorum KS1030 consists of a single circular 2,862,845-bp chromosome and four circular plasmids (Table 1). The G + C content of the genome is 33.16%. The genome was predicted to contain 2,903 ORFs, 61 tRNA genes, and 22 rRNA genes. Genome analysis predicted 2,608 coding sequences (CDSs), of which 2,528 were in the chromosome, and 41, 27, 10, and two were in four different plasmids, respectively. The 2,608 CDSs were functionally assigned to categories based on the COG database, with the most abundant category related to amino acid transport and metabolism (232 genes, 8.9%), followed by carbohydrate transport and metabolism (227 genes, 8.7%). The high proportion of genes associated with protein and carbohydrate utilization indicates that strain KS1030 could degrade a wide range of proteins and carbohydrates in seafood.

Genome features of Staphylococcus equorum strain KS1030

Feature Value
Genome size (bp) 2,952,351
Chromosome 2,862,845
Plasmid pKS1030_1 42,637
Plasmid pKS1030_2 30,648
Plasmid pKS1030_3 13,583
Plasmid pSELNU1 2,638
G + C content (%) 33.16
Open reading frames 2,903
CDS assigned by COG 2,608
rRNA genes 22
tRNA genes 61

Abbreviations: CDS, coding DNA sequence; COG, Cluster of orthologous groups of proteins; tRNA, transfer RNA; rRNA, ribosomal RNA.



Although we previously reported the horizontal transfer of pSELNU1 from S. equorum KS1030 into other species (Lee and Jeong, 2015; Heo et al., 2019), we did not detect the mobile gene-containing relaxase gene in pSELNU1. The current complete genome of S. equorum KS1030 revealed that one of the four plasmids, a 13 kb plasmid designated pKS1030-3, possessed transferrable elements including the relaxase gene rlx (GenBank accession no. JL104_RS14645), which nicks an origin of transfer to initiate the process of DNA mobilization and transfer known as bacterial conjugation (Soler et al., 2019). Therefore, we assumed that rlx might assist the transfer of pSELNU1 carrying lnuA via a trans-acting mechanism.

Comparative genomic analysis was carried out of the genomes of strains KS1030 encoding rlx and lnuA, S. equorum KS1039 (lincomycin-sensitive; GenBank accession no. CP013114), S. equorum C2014 (lincomycin-sensitive; GenBank accession no. CP013714-CP013719), and S. equorum KM1031 (lincomycin-resistant; GenBank accession no. CP13980-CP13983) (Jeong et al., 2017). This identified a lincomycin resistance gene in strains KS1030 and KM1031, and mobile elements including the relaxase gene in KS1030 and C2014. Neither rlx nor lnuA were detected in the KS1039 strain genome, suggesting that they are strain-specific genes. Strain KM1031 possessing not rlx but lnuA did not transferred the plasmid into other strains by conjugation, unlike strain KS1030. These results suggested that rlx might be contributed the horizontal plasmid transfer by trans-acting origin. These genes may contribute to our understanding of the horizontal transfer of plasmids encoding antibiotic resistance genes and should help prevent such transfer during fermentation.

Nucleotide sequence accession numbers

Staphylococcus equorum KS1030 was deposited in the Korean Collection for Type Cultures (accession number: KCTC 43044) and its complete genome sequence was deposited in DDBJ/ENA/GenBank (accession number: CP068576–CP068580).

적 요

Lincomycin에 저항성을 보이는 Staphylococcus equorum KS1030 균주의 유전체를 분석하였다. GC 함량은 33.16%인 KS1030 균주의 유전체는 2,952,351 bp 크기로 단일 chromosome (2,862,845 bp)과 4개의 plasmid로 구성되어 있다. Lincomycin에 대한 단 저항성을 보이는 lincosamide O-nucleotydyltransferase, lnuA, 유전자와 수평적 전이에 관여하는 relaxase 유전자가 플라스미드 pSELNU1과 pKS1030-3에서 확인되었다. 이는 획득형 항생제 내성 유전자의 수평적 전이에 대한 기작 연구에 기여할 것이다.

Acknowledgments

This work was supported by the National Research Foundation of Korea (NRF) [NRF-2019R1A2C1003639 for Jeong and NRF-2020R1A6A3A13077341 for Heo].

We thank Sarah Williams, PhD, from Edanz Group (https://en-author-services.edanz.com/ac) for editing a draft of this manuscript.

Conflict of Interest

The authors declare that they have no conflicts of interest.

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