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Complete genome sequence of Amycolatopsis sp. strain FBCC-B4732, an actinobacterium isolated from hyporheic freshwater§
Korean J. Microbiol. 2023;59(3):217-219
Published online September 30, 2023
© 2023 The Microbiological Society of Korea.

Ahyoung Choi*, Kiwoon Baek, Sumin Jang, Yoonjong Nam, Young Soo Ryu, Eu Jin Chung, and Shi Hyun Ryu

Bioresources Collection & Bioinformation Department, Nakdonggang National Institute of Biological Resources (NNIBR), Sangju 37242, Republic of Korea
Correspondence to: *E-mail: aychoi@nnibr.re.kr; Tel.: +82-54-530-0891; Fax: +82-54-530-0899
§Supplemental material for this article may be found at http://www.kjom.org/main.html
Received July 11, 2023; Revised July 25, 2023; Accepted August 4, 2023.
Abstract
An actinobacterial strain, designated Amycolatopsis sp. FBCC-B4732 (= KACC 23265) was isolated from hyporheic freshwater. The complete genome is of high quality and consists of a circular chromosome, measuring 10,190,959 bp with a 71.5% G + C content. Genome annotation revealed that the genome comprises 9,344 genes, including 9,120 protein-coding genes, 12 rRNAs, 51 tRNAs, 3 ncRNAs, and 158 pseudogenes. The analysis of the biosynthetic gene cluster for secondary metabolites revealed that FBCC-B4732 has 37 biosynthetic clusters. The analysis of the genome revealed the existence of pathways for synthesizing antibacterial compounds.
Keywords : Amycolatopsis sp., complete genome, hyporheic freshwater
Body

The genus Amycolatopsis, which was first proposed by Lechevalier et al. (1986), belongs to the family Pseudonocardiaceae, class Actinobacteria. Amycolatopsis spp. are Gram-positive, non-acid-fast, and non-motile actinomycetes bacteria. Various habitats induce Amycolatopsis to produce secondary metabolites abundantly (Ding et al., 2019; Kim et al., 2021; Song et al., 2021). The complete genome sequence of Amycolatopsis sp. FBCC-B4732 was isolated from a hyporheic freshwater sample collected in South Korea during the screening process for bacteria with antimicrobial activity. The original colony was obtained by isolating a freshwater suspension through serial dilution and plating on an R2A medium. The plate was then incubated for 1 week at 28°C.

A single colony of Amycolatopsis sp. FBCC-B4732 was grown for 3 days in R2A agar (BD Difco) at 28°C. Genomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen), following the manufacturer’s instructions. DNA quantity and quality were checked with a Qubit fluorometer (Thermo Fisher Scientific) and gel electrophoresis, respectively. The whole genome was sequenced using a combination of the PacBio Sequel I (Pacific Biosciences) and Illumina NovaSeq 6000 platforms (150-bp paired-end reads) (Illumina). For PacBio sequencing, DNA was fragmented to 20 kb using a g-TUBE (Covaris), and a DNA library was constructed using a SMRTbell template prep kit (Pacific Biosciences). For NovaSeq sequencing, DNA was fragmented to 350 bp using a Covaris LE220 ultrasonicator (Covaris), and a DNA library was constructed using a TruSeq DNA PCR-free library prep kit (Illumina). For NovaSeq sequencing, genomic DNA was randomly fragmented by an Ultrasonic breaker (Covaris) to an average size of 350 bp, and a DNA library was constructed using a TruSeq DNA PCR-free library prep kit (Illumina). The library quality was analyzed by Qubit and quantitative PCR (qPCR), and the average fragment size was estimated using an Agilent 2100 Bioanalyzer. De novo assembly was performed using CANU v.1.7 software (Koren et al., 2017). The genome was annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) after being submitted to GenBank (Tatusova et al., 2016). Bacterial identification utilizing whole genome sequences was conducted on the TrueBacTM ID system, a cloud-based service (ChunLab Inc.) (Ha et al., 2019) to reveal the genuine identification of bacterial isolates using a multitude of methods. Biosynthetic gene clusters (BGCs) were assessed using antiSMASH version 6.1.1 (Blin et al., 2021). Antimicrobial resistance gene sequences were identified with AMRFinder v3.9.8 (Feldgarden et al., 2019). All bioinformatics tools were used with default parameters.

PacBio sequencing yielded a total of 101,001 subreads (mean subread length, 8,695 bp) with an N50 value of 11,301 bp, totaling 878,240,383 bp. A total of 8,426,622 raw Illumina paired-end total reads were generated, and 4,274,142 filtered reads were used for error correction with Pilon v1.21 (Walker et al., 2014) for the final genome assembly. With the long and short reads combined, the total sequence data generated for 75.1× coverage. The complete genome sequence of Amycolatopsis sp. FBCC-B4732 consists of one circular chromosome with 10,190,959 bp and a GC content of 71.5%. The genome annotation predicted 9,120 protein coding DNA sequences (Table 1), which included 12 rRNAs, 51 tRNAs, and 3 ncRNAs. Here, using TrueBac ID system for bacterial identification based on the whole genome sequence of the FBCC-B4732 strain, it could be identified as new Amycolatopsis species (Supplementary data Table S1) (Richter and Rosselló-Móra, 2009; Meier-Kolthoff et al., 2013). However, further polyphasic research is required to confirm this taxonomic position. We identified various antibacterial genes in the genome of strain FBCC-B4732. AntiSMASH shell analysis revealed that the genome harbors 37 biosynthetic gene clusters (BGCs). The genome of strain FBCC-B4732 contained a variety of biosynthetic gene clusters for secondary metabolites (Supplementary data Table S2), particularly those for type I polyketide synthase (PKS) and non-ribosomal peptide synthases (NRPS). Other gene clusters included those for ectoin, beta-lactone, RiPP clusters, and multiple terpene clusters. AMRFinder identified genes and mutations that confer resistance to rifamycin monooxygenases (rox), D-Ala-D-Ala dipeptidase VanX (vanX), β-lactamase (bla), and Cmx/mrA family chloramphenicol efflux MFS transporter (cmx-cmrA). The complete genome sequence of Amycolatopsis sp. FBCC-B4732 can provide information for the synthesis of antimicrobial compounds.

General genomic features of the strain FBCC-B4732
Features Value
Genome size (bp) 10,190,959
GC content (mol%) 71.5
No. of contigs 1
Genes (total) 9,344
CDSs (total) 9,278
Genes (coding) 9,120
No. of rRNA genes (5S, 16S, 23S) 12 (4, 4, 4)
No. of tRNA genes 51
No. of ncNA genes 3
No. of pseudo genes 158


Strain and nucleotide sequence accession numbers

Strain FBCC-B4732 has been deposited in the Korean Agricultural Culture Collection (KACC) under the number KACC 23265. The GenBank/EMBL/DDBJ accession number for the complete genome sequence of strain FBCC-B4732 is CP095376. The associated BioProject and BioSample accession numbers are PRJNA823828 and SAMN27362481, respectively.

적 요

Amycolatopsis sp. FBCC-B4732 균주는 경상북도 예천의 혼합대에서 분리된 세균으로, 이 보고서에서는 FBCC-B4732 균주의 전장유전체에 대해서 서술한다. 해당 균주는 플라스미드가 없는 원형의 완성된 하나의 염색체를 갖고 있으며, 전체 10,190,959 bp 크기와 71.5%의 GC함량을 나타낸다. 총 9,344개 유전자를 갖고 있으며, 9,120 개의 protein-coding 유전자, 12개 rRNA, 51개 tRNA, 및 3개 ncRNA로 구성되어 있다. FBCC-B4732 균주는 Amycolatopsis 속의 신종 후보 균주로 판단되며, 유전체에서 2차 대사산물에 대한 생합성 유전자 클러스터가 발견되었다.

Acknowledgments

This work was supported by the Korea Environment Industry & Technology Institute (KEITI) through a project to make multi-ministerial national biological research resources a more advanced program, funded by the Korea Ministry of Environment (MOE) (2021003420001).

Conflict of Interest

The authors declare no conflict of interest.

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