Complete genome sequence of Corynebacterium sp. SCR221107, encoding biosynthesis of vitamin B₁₂ isolated from the rumen fluid of Holstein dairy cows

Members of the genus Corynebacterium are Gram-positive, non-acid-fast, non-motile, straight to curved rod-shaped bacteria and are classified as members of the order Mycobacteriales, class Actinomycetia, and phylum Actinobacteria [1]. To date, the genus comprises 140 species and four subspecies with validly published names. Corynebacterium has been isolated from soil, food, and animals, including humans. Some strains of the genus of which are recognized as pathogens related to human and animal diseases [2,3]. However, Corynebacterium vitaeruminis has been studied for its beneficial functions and has been known to be non-pathogenic and non-virulent [4,5]. C. vitaeruminis as a bacterium that is capable of synthesizing vitamin B within the rumen of cows [6].

In this study, Corynebacterium sp. SCR221107 was isolated from the rumen fluid of a 1-year-old healthy male Holstein dairy cow in Suncheon, Jeollanam-do, Republic of Korea. The sample was incubated in an anaerobic atmosphere with 5% carbon dioxide, 5% hydrogen, and 90% nitrogen at 37°C for 48 h on De Man, Rogosa and Sharpe (MRS) media. Genomic DNA was extracted from Corynebacterium sp. SCR221107 cell pellets using a Maxwell^® Prokaryote SEV DNA Purification Kit (Promega, Madison, WI, USA), in line with the manufacturer’s instructions. The genomic DNA obtained was sequenced commercially at Macrogen (Seoul, Korea) using the PacBio Sequel II system (Pacific Biosciences, Menlo Park, CA, USA) and the Illumina HiSeq platform. De novo assembly was performed using the Hierarchical Genome Assembly Process v3.0 (HGAP3) with default options within the SMRT Link v11.1 software. Read quality was confirmed by aligning shorter reads with longer reads using Basic Local Alignment with Successive Refinement v1 (BLASR) [7] and correcting errors using Pilon version 1.21 [8]. Genome annotation was performed using rapid prokaryotic genome annotation (Prokka) v1.14.6 [9] and the Basic Local Alignment Search Tool (BLAST+) v2.7.1+. Clustered regularly interspaced short palindromic repeats (CRISPR) were assessed using the CRISPR web server (http://crispr.i2bc.paris-saclay.fr) [10]. Resistance-related genes were analyzed using ResFinder 4.1 with a 90% threshold for gene identification [11].

A total of 159,928 reads with a mean subread length of 8,975 bases (N50) were obtained using PacBio sequencing, and 37,599,664 paired-end reads, totaling 5,677,549,264 base pair [bp], were obtained using Illumina sequencing. The genome statistics are presented in Table 1. The complete genome sequence of Corynebacterium sp. SCR221107 is composed of a single circular chromosome and does not contain plasmid DNA. The 3,043,024 bp genome with a G + C content of 60.1% contained 2,639 protein-coding sequences (CDS), 63 pseudogenes, and 72 RNA genes (15 rRNA genes, 57 tRNA genes, and three non-coding RNA genes), based on the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) (Fig. 1). Furthermore, 2,639 CDSs were clustered into 20 Clusters of Orthologous Groups (COGs) of protein-based functional categories (Fig. 1B). Many genes were classified into functional categories for amino acid transport and metabolism (n = 249); translation, ribosomal structure, and biogenesis (n = 190); inorganic ion transport and metabolism (n = 189); general function prediction only (n = 219); transcription (n = 185); and coenzyme transport and metabolism (n = 163). One confirmed CRISPR region and two questionable CRISPR 9 regions (1 and 2) were also detected. This pattern was identified as the CRISPR-CAS II type. A search using ResFinder returned no hits for antibiotic resistance genes in Corynebacterium sp. SCR221107.

Fig. 1. Genome map of Corynebacterium sp. SCR221107 (A) and the functional categorization of predicted coding sequences (B). Marked characteristics are shown from the outside to the center: coding sequence (CDS) on the forward strand, CDS on the reverse strand, tRNA, rRNA, guanine + cytosine (GC) content, and GC skew. COG, Clusters of Orthologous Group.

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Based on 16S rRNA gene sequence similarity data, it was found that the closest relatives of strain SCR221107 were C. vitaeruminis DSM 20294^T (98.5%) and C. felinum CCUG 39943^T (96.9%). As Corynebacterium sp. SCR221107 revealed close similarity with C. vitaeruminis DSM 20294^T, a known producer of B vitamin complex, the genomic analysis and annotation of coding regions unveiled a significant abundance of genes associated with vitamin biosynthesis. We identified cobalamin biosynthetic (vitamin B₁₂) and transport genes in Corynebacterium sp. SCR221107. In particular, Corynebacterium sp. SCR221107 possessed genes involved in the biosynthesis pathways of vitamin B₁₂ such as cobB, cobD, cobH, cobJ, cobK, cobL, cobM, cobN, cobQ, cobS, cobT, cobU, hemA, hemB, hemC, hemE, hemH, hemL, hemW, and hemY, and transport genes such as cbiM, cbiN, and cbiQ [12,13]. The vitamin B12 gene clusters, which contain hem-cob operons, consisted of 20 genes responsible for various enzymatic transformations along the cobalamin (vitamin B₁₂) pathway. In addition, the genes/enzymes are involved in the oxygen-dependent pathway.

These results suggest that Corynebacterium sp. SCR221107 is a potential probiotic candidate capable of synthesizing vitamin B₁₂. The genomic data obtained from this study provides valuable insights into the biosynthetic pathways of vitamin B₁₂ which might contribute for the development of vitamin B₁₂-enriched probiotics.