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Complete genome sequence of Treponema denticola KCOM 3500 isolated from the subgingival plaque of a human chronic periodontitis lesion
Korean J. Microbiol. 2022;58(4):338-341
Published online December 31, 2022
© 2022 The Microbiological Society of Korea.

Soon-Nang Park, Yun Kyong Lim, and Joong-Ki Kook*

Korean Collection for Oral Microbiology and Department of Oral Biochemistry, School of Dentistry, Chosun University, Gwangju 61452, Republic of Korea
Correspondence to: *E-mail: jkkook@chosun.ac.kr; Tel.: +82-62-230-6877; Fax: +82-62-236-2734
Received November 3, 2022; Revised December 20, 2022; Accepted December 24, 2022.
Abstract
Treponema denticola is a strictly anaerobic, Gram-negative, non-spore forming and motile, spiral-shaped cells. It is a major causative agent of periodontitis and is related to systemic diseases such as Alzheimer’s disease and cardiovascular diseases. T. denticola KCOM 3500 (= ChDC P019-Td1) was isolated from human chronic periodontitis lesion. In this study, we present the whole genome sequence of T. denticola KCOM 3500. The genome of strain KCOM 3500 was 2,778,422 bp in length and contained 2,507 protein-coding sequences, 6 rRNAs, and 44 tRNAs. The G + C content of it was 38.1%. It contained a dentilisin complex serine proteinase, a strong protease.
Keywords : Treponema denticola, chronic periodontitis, human
Body

Treponema denticola is a Gram-stain-negative, strictly anaerobic, motile, and non-spore forming spiral-shaped cells (Chan et al., 1993). T. denticola is frequently isolated from the subgingival dental plaque of chronic periodontitis lesions (Takeuchi et al., 2001) and may also be found in periapical endodontic lesions (Gomes et al., 2008). Recent epidemiological studies show that T. denticola is closely associated with systemic diseases such as Alzheimer’s disease (Su et al., 2021) and cardiovascular diseases (Beck et al., 2005). It has been reported that T. denticola has potential virulence genes, examples include: a major outer sheath membrane (Msp), an oligopeptide transporter unit (OppA), dentilisin (PrtP; a chymotrypsin-like protease), cystalysin (HlyA/HlyB: hemolyzes red blood cells), a hemin binding protein (HbpA/HbpB/troA-D/TroR), chemotaxis proteins (CheA/CheX/CheY/DmcA/DmcB), and FH-like binding protein (FhbB) (Ishihara, 2010).

Strain ChDC P019-Td1 was grown on agar plate using modified NOS medium (ATCC medium 1494; https://www.atcc.org/products/35405) from a subgingival dental plaque obtained from a male human suffering from chronic periodontitis (52 years old) in 2018 at the Chosun Dental Hospital (IRB No., CUDHIRB 1703001). The 16S rRNA gene (16S rDNA) was sequenced and deposited to GenBank (accession no. MW474827). To identify the strain at the species level, 16S rDNA sequence identification analysis was performed using a 16S-based ID program and the EzBioCloud database (https://www.ezbiocloud.net). The 16S rDNA sequence of the strain exhibited a 99.9% and 98.4% sequence identity with those of T. denticola ATCC 35405T (GenBank accession no. AE017226) and Treponema putidum OMZ 758T (GenBank accession no. CP009228), respectively. The phylogenetic analysis based on 16S rDNA was performed using the Mega program (version 11, https://megasoftware.net). The phylogenetic tree data showed that strain KCOM 3500 was included in same cluster of T. denticola ATCC 35405T (Fig. 1A). The strain was identified as T. denticola and deposited in the Korean Collection for Oral Microbiology (KCOM) (deposit no.: KCOM 3500). In this report, we present the whole genome sequence of the strain, T. denticola KCOM 3500.

Fig. 1. Phylogenetic tree based on the 16S ribosomal RNA gene (16S rDNA) sequences showing the relationships between strain KCOM 3500 and type strains of closely-related Treponema species. It was constructed using neighbor-joining evolution methods in MEGA software (version 11, https://megasoftware.net). Stability of the trees was assessed using bootstrap analysis of 1000 replicates. The GenBank accession number and/or sequence region of the 16S rDNA of each type strain is written in parenthesis. Bar indicates 0.02 changes per nucleotide position. (B) Heatmap constructed based on whole genome sequences of strain KCOM 3500 and each type strain of the three Treponema sp. closest to T. denticola generated by OrthoANI caculator (Lee et al., 2016). The GenBank accession numbers of Treponema denticola ATCC 35405T, Treponema putidum ATCC 700334T, and Treponema pedis T3552BT are NC_002967, NZ_VLLD01000001, and CP045670, respectively.

Strain KCOM 3500 was grown in a modified NOS medium in an anaerobic chamber (Model Bactron I) with a gaseous atmosphere of 85% N2, 10% CO2, and 5% H2. The bacterial genomic DNA was prepared as previously described (Cho et al., 2015). The genomic DNA of strain KCOM 3500 was sequenced using a PacBio Sequal1 sequencing platform using a 20 kb SMRTbell template library by Macrogen Inc. Approximately 720,805,033 bases (259.4×) with 72,199 subreads (mean subreads length: 9,983 bases, N50 was 12,579 bases) were generated and assembled into a single contig by HGAP (version: 3.0, default setting) in PacBiós SMRT portal (http://www.pacb.com/products-and-services/analytical-software/smrt-analysis). Genome annotation was conducted using the NCBI Prokaryotic Genome Annotation Pipeline (Tatusova et al., 2016).

The genome of strain KCOM 3500 was composed of one contig, 2,778,422 bp in length. The G + C content of the genome was 38.1% (Table 1). A total of 2,507 protein-coding sequences, 6 rRNAs, and 44 tRNAs were annotated (Table 1). To confirm the classification of strain KCOM 3500 at the species level, homologous analysis of the genome sequence was performed using the programs of average nucleotide identity (ANI) (OrthoANI, Lee et al., 2016) and genome-to-genome distance calculation (Meier-Kolthoff et al., 2013). The OrthoANI and GGD values between the genome sequence of strain KCOM 3500 and that of T. denticola ATCC 35405T were 97.26% (Fig. 1B) and 74.3% (71.3–77.1%), respectively. However, the OrthoANI and GGD values between strain KCOM 3500 and each type strain of the other two Treponema sp. closest to T. denticola were below 95% and 70%, respectively. These result show that strain KCOM 3500 belonged to T. denticola.

Genome features of the genome of <italic>Treponema denticola</italic> KCOM 3500
Attribute Value
Genome size (bp) 2,778,422
GC content (%) 38.1
No. of contig 1
Total genes 2584
Protein-coding genes 2,507
tRNA 44
Complete rRNA (5S, 16S, 23S) 6 (2, 2, 2)
ncRNA 3
Pseudogene 24
CRISPR arrays 1


The genome sequence of strain KCOM 3500 contained virulence-related genes: a dentilisin complex serine proteinase (subunit PrtP [OE909_RS09950], subunit PrcA [OE909_RS09955], subunit PrcB [OE909_RS09960]), four hemolysin family proteins (OE909_RS07900, OE909_RS00115, OE909_RS00120, OE909_RS03035), twenty two chemotaxis proteins (CheA [OE909_RS06755], CheX [OE909_RS06745], CheW [OE909_RS06750, OE909_RS06285], OE909_RS07260, OE 909_RS07710, OE909_RS08655, OE909_RS08865, OE909_RS09550, OE909_RS10435, OE909_RS11165, OE909_RS 11765, OE909_RS12215, OE909_RS12280, OE909_RS00205, OE909_RS00240, OE909_RS00245, OE909_RS00635, OE 909_RS01595, OE909_RS01990, OE909_RS02195, OE909_RS03280), tetracycline resistance ribosomal protection protein Tet(32) (OE909_RS01370). It also contained a type II and III secretion system protein (OE909_RS04395), forty toxin-antitoxin system proteins (OE909_RS06925, OE909_RS06930, OE 909_RS07290, OE909_RS07295, OE909_RS08330, OE909_RS09410, OE909_RS10270, OE909_RS10320, OE909_RS 10325, OE909_RS11025, OE909_RS11030, OE909_RS11105, OE909_RS11175, OE909_RS11180, OE909_RS11260, OE 909_RS11355, OE909_RS11360, OE909_RS12345, OE909_RS12520, OE909_RS12530, OE909_RS00025, OE909_RS 00030, OE909_RS00790, OE909_RS00795, OE909_RS00895, OE909_RS00900, OE909_RS01925, OE909_RS03330, OE 909_RS03335, OE909_RS03360, OE909_RS03910, OE909_RS04650, OE909_RS04735, OE909_RS04740, OE909_RS 04745, OE909_RS04750, OE909_RS05325, OE909_RS05970, OE909_RS06030, OE909_RS06035), two paired two-component systems (OE909_RS10350/OE909_RS10355, OE909_RS12115/OE909_RS12120), and an unmatched sensor histidine kinase (OE909_RS02780), five unmatched regulatory proteins (OE909_RS04540, OE909_RS11960, OE909_RS12040, OE 909_RS01735, OE909_RS04005), and phage-related proteins: phage protease (OE909_RS01745), phage virion morphogenesis protein (OE909_RS01765), phage tail sheath protein (OE909_RS01765), phage tail tube protein (OE909_RS01780), phage tail tape measure protein (OE909_RS01790), and phage GP 46 family protein (OE909_RS01810). The results of genes analysis in whole genome sequence of strain KCOM 3500 could offer some clues in understanding the mechanism of the pathogenicity of T. denticola in periodontal diseases.

Nucleotide sequence accession number

This whole genome sequence of strain KCOM 3500 was deposited in GenBank under the accession number CP109619.

적 요

Treponema denticola는 절대 혐기성, 그람 음성, 아포를 생성하지 않고 및 비운동성 나선모양의 세균이다. 이 세균 종은 치주염의 주요한 원인균 종이며 알츠하이머 질환과 심혈관계 질환과 같은 전신질환과도 연관이 있다. Treponema denticola KCOM 3500 (= ChDC P019-Td1) 균주는 사람 만성치주염 병소 부위의 치은연하 치면세균막에서 분리되었다. KCOM 3500 균주 전장 유전체는 2,778,422 bp로 구성되어 있고, 2,507개의 단백질 코딩 시퀀스, 6개 rRNAs 및 44개 tRNAs를 함유하고 있었다. 이 균주의 G+C 함량은 38.1%였다. 전장 유전체에는 강력한 단백분해효소인 dentilisin 복합 세린 단백분해효소를 함유하고 있었다.

Acknowledgments

This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. 2021R1A2C2009051).

Conflict of Interest

The authors have no conflict of interest to report.

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