Complete genome sequence of Streptococcus oralis subsp. tigurinus J22, a model strain for antagonistic interaction against Streptococcus mutans

TL;DR

This paper reports the complete genome sequence of a bacteria that can fight tooth decay-causing microbes.

Contribution

The study provides the complete genome sequence of Streptococcus oralis subsp. tigurinus J22, a model for antagonistic interactions.

Findings

The genome of S. oralis subsp. tigurinus J22 is 1,967,320 bp with 41.1% GC content.

The strain produces hydrogen peroxide and antagonizes Streptococcus mutans.

It serves as a model for studying microbial antagonism in the oral cavity.

Abstract

Streptococcus oralis subsp. tigurinus strain J22, a potent hydrogen peroxide-producing oral commensal. Given the antagonistic action against a cariogenic pathogen, this strain can be used as a model bacterium to characterize microbial antagonistic interactions. S. oralis J22 has a genome sequence of 1,967,320 bp along with 41.1% GC content.

Figures

Representative antagonistic activity of S. oralis J22 against S. mutans. (A) S. oralis J22 consistently inhibited S. mutans growth regardless of sugar concentration in the agar medium. In contrast, S. oralis ATCC 35037 exhibited sugar-dependent inhibition, with markedly reduced activity under high-glucose conditions (at 2% glucose compared to 0.2% glucose). (B) To test whether H2O2 is a sole chemical weapon generated by S. oralis J22, either peroxidase or protease was applied for 10 min prior to spotting of S. mutans in the proximity of S. oralis. Interestingly, the peroxidase completely diminished the inhibitory zone mediated by S. oralis, whereas the protease did not show any drastic changes in the growth inhibition when compared to the medium without treatments.

Tables

TABLE 1. Complete genome sequencing, de novo assembly, and annotation of S. oralis subsp. tigurinus strain J22

Category	Feature	S. oralis J22
HiFi reads	No. of reads	30,781
	Total bases (bp)	209,722,659
	N50 (bp)	7,641
Filtered Illumina reads	No. of reads	11,951,272
	Total bases (bp)	1,803,548,598
Assembly	No. of contig	1
Total	Genome size (bp)	1,967,320
	N50 (bp)	1,967,320
	GC content (%)	41.1
	Mean depth (×)	106.4
	No. of CDs	1,861
	Coding ratio (%)	98.5
	No. of tRNAs	61
	No. of rRNAs	12

Full text

ANNOUNCEMENT

Streptococcus oralis subsp. tigurinus strain J22 is a potent hydrogen peroxide (H_2_O_2_)-producing oral commensal. It has been used as a model bacterium to study bacterial antagonistic interactions against oral pathogens and to evaluate the inhibitory capacity of oral isolates in suppressing cariogenic biofilm formation (1–4). Its antagonistic activity is mediated by H_2_O_2_ production rather than proteinaceous compounds (e.g., bacteriocins) (Fig. 1).

Representative antagonistic activity of S. oralis J22 against S. mutans. (A) S. oralis J22 consistently inhibited S. mutans growth regardless of sugar concentration in the agar medium. In contrast, S. oralis ATCC 35037 exhibited sugar-dependent inhibition, with markedly reduced activity under high-glucose conditions (at 2% glucose compared to 0.2% glucose). (B) To test whether H2O2 is a sole chemical weapon generated by S. oralis J22, either peroxidase or protease was applied for 10 min prior to spotting of S. mutans in the proximity of S. oralis. Interestingly, the peroxidase completely diminished the inhibitory zone mediated by S. oralis, whereas the protease did not show any drastic changes in the growth inhibition when compared to the medium without treatments.

Compared to other oral commensal streptococci such as S. oralis ATCC 35037 (Fig. 1), strain J22 exhibits stronger inhibition of Streptococcus mutans under varying sugar conditions (3, 5). Despite its functional relevance, the whole genome sequence of strain J22 had not been available, limiting mechanistic studies, such as metatranscriptomics.

Strain J22, originally isolated from a human oral cavity (2), was kindly provided by Dr. Jens Kreth (Oregon Health & Science University). The glycerol stock was streaked onto brain heart infusion agar plates supplemented with 5% sheep blood and incubated at 37°C under 5% CO_2_ for 48 h. Genomic DNA was extracted using the MasterPure DNA Purification Kit (Epicenter, USA), beginning with enzymatic lysis using lysozyme and mutanolysin in TE buffer, followed by Proteinase K digestion and RNase A treatment (6). The purified gDNA was subjected to quality assessment using Agilent ScreenTape analysis (Agilent, USA).

The genome of S. oralis J22 was sequenced using both PacBio Sequel IIe (Pacific Biosciences, USA) and Illumina NovaSeq X (Illumina, USA) platforms operated at Microgen Inc. (Republic of Korea). For PacBio sequencing, 4 µg of genomic DNA was sheared using Megaruptor 3 (Diagenode), size-selected (7–12 kb fragments) with AMPure PB beads, and prepared with the PacBio SMRTbell prep kit 3.0. The libraries were annealed and sequenced using Sequel II Bind Kit 3.2 and SMRT Cell 8M with 15-h movie time on the PacBio Sequel IIe system. For Illumina sequencing, the TruSeq DNA Nano Kit (Illumina) was used to prepare the libraries, with 100 ng of fragmented DNA, and paired-end sequencing (2 × 150 bp) was performed on the NovaSeq X (Illumina). HiFi reads generated from PacBio were de novo assembled using the Microbial Genome Assembly pipeline in SMRT Link v13.0.0.207600, based on the Hierarchical Genome Assembly Process (7). Illumina reads were processed with Trimmomatic v0.38 (ILLUMINACLIP:Adapter.fasta:2:30:10:8:true LEADING:15 TRAILING:15 SLIDINGWINDOW:4:15MINLEN:36) (8). Reads with Phred score ≥30 were retained and used for three rounds of polishing with Pilon v1.22 (9). Circularization was confirmed by identifying overlapping terminal regions, and the genome was rotated to start at the origin of replication based on dnaA coordinates.

Genome annotation was performed using Prokka v1.14.6 (10) to predict coding sequences (CDSs), tRNAs, and rRNAs, while InterProScan v5.34-73.0 (11) and PSI-BLAST v2.6.0 (12) were used for further functional annotation, utilizing the EggNOG database v4.5 (13) for orthology.

The S. oralis J22 sequence was deposited at GenBank as one circular contig comprised of 1,967,320 bp along with 41.1% GC content, 1,861 CDSs, 61 tRNAs, and 12 rRNAs (Table 1).