Complete Genome Sequence of Kitasatospora Sp. MMS16-CNU292, an Acidophilic Actinobacterium

Complete genome sequence of Kitasatospora sp. MMS16-CNU292, an acidophilic actinobacterium

Min Ji Kim and Seung Bum Kim* Department of Microbiology and Molecular Biology, College of Bioscience and Biotechnology, Chungnam National University, Daejeon 34134, Republic of Korea

호산성 방선균인 Kitasatospora sp. MMS16-CNU292 균주의 유전체 분석

김민지 ･ 김승범* 충남대학교 생명시스템과학대학 미생물 ․ 분자생명과학과

(Received September 11 2019; Revised September 23 2019; Accepted September 25 2019)

Kitasatospora sp. MMS16-CNU292 is an acidophilic actino- and galactose in their cell walls (Kämpfer, 2012). Kitasatospora bacterium showing antibacterial activity, and its complete is an aerobic, Gram-stain-positive, non-acid-alcohol-fast acti- genome was analyzed. The genome consisted of three contigs nobacterial group which forms intensively branched, non- and a total length of 8,587,212 bp. The G + C content was 71.5 fragmenting mycelia (Takahashi et al., 1999). Kitasatospora mol%, and the annotation results revealed presence of 6,934 species were found to produce a large number of secondary protein coding genes, 30 rRNAs, 79 tRNAs, and 4 ncRNA genes. Based on 16S rRNA gene sequence, the strain was metabolites with diverse structures and interesting activities, in T closest to Kitasatospora azatica KCTC 9699 , showing 98.75% particular those active against eukaryotes (Takahashi et al., similarity. The analysis of biosynthetic gene clusters for secon- 2017). dary metabolites indicated presence of 30 clusters including In this study, an acidophilic actinobacterial strain designated those for non-ribosomal peptide synthetases, polypeptide syn- Kitasatospora sp. MMS16-CNU292 was isolated from a pine thases, NRPS-PKS hybrids, lanthipeptides, terpenes, and lasso- grove soil in Daejeon, Korea (36°22'13.3''N, 127°21'02.6''E). peptide. The strain was isolated on acidified starch casein agar (SCA, Keywords: Kitasatospora , acidophilc actinobacterium, biosynthetic BD) supplemented with antifungal agents cycloheximidine gene cluster, complete genome and nystatin, and also gentamicin, an antibacterial agent active mainly against Gram-negative bacteria (each at 50 µg/ml). The strain was deposited in the Korean Collection for Type The genus Kitasatospora, first proposed by Omura et al. Cultures (KCTC) under the accession number KCTC 49011. (1982), belongs to the family Streptomycetaceae (Waksman Extraction of the genomic DNA for the whole genome and Henrici, 1943) together with the genera Streptacidiphilus sequence of strain MMS16-CNU292 was performed using and Streptomyces. Kitasatospora and Streptomyces are sister commercial genomic DNA extraction kit (Solgent). The taxa and share a number of phenotypic properties, but the analysis of whole genome shotgun sequencing for MMS16- former contains major amounts of meso-diaminopimelic acid CNU292 was carried out using single molecule realtime sequencing technology (SMRT) with the PacBio RS II system *For correspondence. E-mail: [email protected]; Tel.: +82-42-821-6412; Fax: +82-42-822-7367 (Pacific Biosciences). Generation of the long and highly accurate 406 ∙ Kim and Kim

sequences, contigs and final assembly were conducted by cluster for lasso peptide. SMRT analysis HGAP 3.0. The NCBI Prokaryotic Genome Annotation Pipeline (PGAP) 4.5 was used for the annotation of Nucleotide sequence accession number genes. The BioProject number for Kitasatospora sp. MMS16- The whole genome sequencing by the PacBio platform CNU292 is PRJNA532311, and the sequence accession number produced a total length of 8,587,212 bp composed 3 contigs for the genome is NZ_VIGB00000000. with the DNA G + C content of 71.5 mol%. The annotation results indicated the presence of the total 7,664 coding sequences (CDS) of which 6,934 were protein-coding genes 적 요 and 730 were pseudogenes, and 30 rRNAs, 79 tRNAs, and 4 ncRNAs were also identified (Table 1). 소나무 숲에서 분리한 호산성 방선균 Kitasatosproa sp. On the basis of 16S rRNA gene sequence similarity, the MMS16-CNU292 균주는 다수의 세균에 대한 항균 활성을 나 strain formed a novel evolutionary lineage within Kitasatospora 타냈다. 본 연구에서는 MMS16-CNU292 분리주의 유전체 분 and shared highest similarities with Kitasatospora azatica 석을 실시하였고, 그 결과 3개의 콘티그로 구성된 총 연장 T KCTC 9699 (Nakagaito et al., 1992) (98.75%), Kitasatospora 8,587,212 bp의 유전체 정보를 확보하였다. DNA G + C 함량 T kifunensis IFO 15206 (Groth et al., 2003) (98.74%), Kitasa- 은 71.5 mol% 였으며, 6,934개의 단백질 지정 유전자, 30개의 T tospora purpeofusca NRRL B-1817 (Labeda et al., 2017) rRNA, 79개의 tRNA 및 4개의 ncRNA 유전자를 확인하였다. T (98.61%), Kitasatospora nipponensis HKI 0315 Groth et al. 16S rRNA 유전자 염기서열 분석 결과 MMS16-CNU292는 T (2004) (98.42%), and Streptomyces novaecaesareae NRRL Kitasatospora azatica KCTC 9699 와 가장 유사했고, 98.75% T B-1267 (Waksman et al., 1948) (98.41%). 의 유사도를 보였다. 이차대사산물에 대한 생합성 유전자 클 The orthoANI values between strain MMS16-CNU292 and 러스터 분석 결과 non-ribosomal peptide synthetase (NRPS), the two mostly related type strains K. azatica and K. purpeofusca polyketide synthase (PKS), NRPS-PKS 하이브리드, lanthi- were calculated as 83.87 and 78.19% respectively, which are peptide, terpene 및 lassopeptide 생합성 유전자 클러스터 등 총 clearly below the suggested cutoff for species boundary of 95% 30개 유전자 클러스터를 보유하는 것으로 나타났다. (Chun et al., 2018). The analysis of biosynthetic gene clusters for secondary metabolites showed that the MMS16-CNU292 strain contains Acknowledgements 30 biosynthetic clusters in total. These include 5 clusters for non-ribosomal peptide synthetases (NRPS), 2 clusters for This work was supported by the 2019 CNU Research Grant polyketide synthase (PKS), 9 clusters for NRPS-PKS hybrids, of Chungnam National University (grant no. 2019-0853-01). 5 clusters for lanthipeptides, 3 clusters for terpenes, and 1

Table 1. The features of MMS16-CNU292 genome References

Feature Value Chun J, Oren A, Ventosa A, Christensen H, Arahal DR, da Costa MS, Genome size 8,587,212 bp Rooney AP, Yi H, Xu XW, De Meyer S, et al. 2018. Proposed G + C content 71.5 mol% minimal standards for the use of genome data for the taxonomy Contigs 3 of prokaryotes. Int. J. Syst. Evol. Microbiol. 68, 461–466. Total CDS 7,664 Groth I, Rodríguez C, Schütze B, Schmitz P, Leistner E, and Protein coding CDS 6,934 Goodfellow M. 2004. Five novel Kitasatospora species from Pseudogenes 730 soil: Kitasatospora arboriphila sp. nov., K. gansuensis sp. nov., rRNA genes (5S, 16S, 23S) 30 (10, 10, 10) K. nipponensis sp. nov., K. paranensis sp. nov. and K. terrestris sp. nov. Int. J. Syst. Evol. Microbiol. 54, 2121–2129. tRNA genes 79 Groth I, Schütze B, Boettcher T, Pullen CB, Rodriguez C, Leistner E, ncRNAs 4

미생물학회지 제55권 제4호 Genome sequence of Kitasatospora sp. MMS16-CNU292 ∙ 407

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Korean Journal of Microbiology, Vol. 55, No. 4