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Draft genome sequence of a malachite green-decolorizing Bacillus velezensis N1 isolated from Indonesian chicken crop
Korean J. Microbiol. 2022;58(1):47-48
Published online March 31, 2022
© 2022 The Microbiological Society of Korea.

Reinhard Pinontoan1*, Hans Victor1, Dikson1, Steven Ryan Susanto Tan1, Ellen Tanudjaja1, and Melanie Cornelia2

1Department of Biology, Universitas Pelita Harapan, Jl. MH Thamrin 1100 Blvd, Tangerang, Indonesia
2Department of Food Technology, Universitas Pelita Harapan, Jl. MH Thamrin 1100 Blvd, Tangerang, Indonesia
Correspondence to: E-mail: reinhard.pinontoan@uph.edu; Tel.: +62-21-546-0901; Fax: +62-21-547-0910
Received November 25, 2021; Revised December 22, 2021; Accepted January 19, 2022.
Abstract
A whole-genome sequencing was performed on Bacillus velezensis strain N1 isolated from an Indonesian chicken crop. The draft genome sequence bears the length of 3,878,255 bp and GC content of 46.51% with a high degree of similarity with several Bacilus velezensis genomes. Furthermore, genome annotation indicated the existence of laccase enzyme gene sequence reported as capable of degrading triphenylmethane dye groups, which in turn confirmed the ability of Bacillus velezensis N1 in degrading malachite green.
Keywords : Bacillus velezensis, decolorization, malachite green, whole genome sequencing
Body

Several species of bacteria produce enzymes capable of degrading malachite green (MG), a type of triphenylmethane dye used in dyeing textiles and treating aquatic animals for fungal infection, but found to be toxic towards organisms (Wang et al., 2012). One such species is the Bacillus velezensis strain N1 isolated from the Indonesian chicken crop, which exhibited strong MG-degrading capabilities based on a prior unpublished study.

Upon sub-culturing and extraction of genomic DNA (gDNA) of Bacillus velezensis N1, the gDNA sample was delivered to Theragen Bio, for whole-genome sequencing using Illumina HiSeq 2500. The sequencing reads were assembled and scaffolded using the A5-MiSeq genome assembly pipeline (Coil et al., 2015) with minimum contig size of 500 bp. The assembled scaffolds were reordered against B. velezensis BY6 (CP051011.1) using Mauve Contig Mover (Darling et al., 2004; Rissman et al., 2009). The genome sequence was submitted to DFAST (Tanizawa et al., 2018) and eggNOG-mapper (Huerta-Cepas et al., 2019; Cantalapiedra et al., 2021) for annotation and functional protein categorization into Clusters of Orthologous Groups (COGs) respectively. The genome was compared against B. velezensis BvL03 (CP041192.1), B. velezensis BY6 (CP051011.1), B. amyloliquefaciens X030 (CP040672.1), B. amyloliquefaciens subsp. plantarum FZB42 (CP000560.2), and B. subtilis 168 (NC_000964.3) genomes for their average nucleotide identity (ANI) percentages using Kostas Lab ANI Matrix Calculator (Rodriguez-R and Konstantinidis, 2016).

Bacillus velezensis N1 whole genome paired-end raw reads contain 21,778,380 reads with a total length of 2,199,616,380 bp which were assembled into a draft genome sequence containing 14 scaffolds with a total length of 3,878,255 bp with N50 value of 1,006,304 bp and GC content of 46.5%. DFAST annotation of the Bacillus velezensis N1 genome predicted 3,724 coding sequences (CDS), 79 tRNA genes, and 4 rRNA genes. The eggNOG-Mapper2 categorized 3,514 of the CDS into COGs.

Furthermore, Bacillus velezensis N1 genome bears high ANI percentage between B. velezensis BvL03 (99.14%) and B. velezensis BY6 (98.68%), followed by B. amyloliquefaciens X030 (99.13%) and B. amyloliquefaciens subsp. plantarum FZB42 (97.65%) as well as the outgroup B. subtilis 168 (79.65%). This finding further confirmed the genome’s identity at species level as B. velezensis.

Bacillus velezensis N1 capabilities of degrading MG was confirmed from laccase enzyme sequence identified from its whole-genome sequencing and annotation. The laccase enzyme sequence shared 99.22% identity with B. velezensis TCCC 111904 Laccase enzyme sequence characterized for its capabilities of degrading azo, anthraquinonic, and triphenylmethane dyes (Li et al., 2020). The Bacillus velezensis N1 laccase enzyme was categorized as COG category Q/COG2132 (Secondary metabolite biosynthesis, transport and catabolism/multicopper oxidase) with EC number 1.10.3.2. In conclusion, whole-genome sequencing of Bacillus velezensis N1 identified a gene encoding malachite green-degrading laccase enzyme and confirmed the isolate’s ability to degrade malachite green.

Draft genome sequence accession number

The whole-genome draft of B. velezensis N1 was submitted to International Nucleotide Sequence Database Collaboration via DNA Data Bank of Japan under the accession number BQFE00000000.

Acknowledgments

The authors acknowledge The Directorate of Higher Education, Indonesian Ministry of Education, Culture, Research and Technology [grant number: 1218/LL3/PG/2021].

Conflict of Interest

The authors declare no notable conflict of interest.

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March 2022, 58 (1)
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