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Genomic Analysis of Halophilic Bacterium, Lentibacillus Sp Ahn et al. Gut Pathog (2021) 13:41 https://doi.org/10.1186/s13099-021-00436-2 Gut Pathogens GENOME REPORT Open Access Genomic analysis of halophilic bacterium, Lentibacillus sp. CBA3610, derived from human feces Seung Woo Ahn1, Se Hee Lee1, Hong‑Seok Son2, Seong Woon Roh1* and Yoon‑E Choi3* Abstract Background: Lentibacillus species are gram variable aerobic bacteria that live primarily in halophilic environments. Previous reports have shown that bacteria belonging to this species are primarily isolated from salty environments or food. We isolated a bacterial strain CBA3610, identifed as a novel species of the genus Lentibacillus, from a human fecal sample. In this report, the whole genome sequence of Lentibacillus sp. CBA3610 is presented, and genomic analyses are performed. Results: Complete genome sequence of strain CBA3610 was obtained through PacBio RSII and Illumina HiSeq plat‑ forms. The size of genome is 4,035,571 bp and genes estimated to be 4714 coding DNA sequences and 64 tRNA and 17 rRNA were identifed. The phylogenetic analysis confrmed that it belongs to the genus Lentibacillus. In addition, there were genes related to antibiotic resistance and virulence, and genes predicted as CRISPR and prophage were also identifed. Genes related to osmotic stress were found according to the characteristics of halophilic bacterium. Genomic diferences from other Lentibacillus species were also confrmed through comparative genomic analysis. Conclusions: Strain CBA3610 is predicted to be a novel candidate species of Lentibacillus through phylogenetic analysis and comparative genomic analysis with other species in the same genus. This strain has antibiotic resistance gene and pathogenic genes. In future, the information derived from the results of several genomic analyses of this strain is thought to be helpful in identifying the relationship between halophilic bacteria and human gut microbiota. Keywords: Lentibacillus sp. CBA3610, Complete genome sequence, Gut microbiota, Halophile Background close phylogenetic relationship, based on 16S rRNA gene Lentibacillus is a gram-variable, aerobic or facultatively sequence analysis and phenotypic characteristics, such as anaerobic, and halophilic bacterial genus of the fam- unique lipid content and fatty acid profle [2]. Te pres- ily Bacillaceae in the phylum Firmicutes [1]. Tis genus ence of halophilic prokaryotes in the human gut has been has been classifed as a new genus and species, difer- confrmed by various molecular biological and next-gen- ent from the genus Virgibacillus, Salibacillus, Gracili- eration sequencing (NGS) techniques. However, little had bacillus, and Halobacillus, which was identifed to have been known about the information of halophilic micro- organisms inhabiting the human gut [3]. Recently, halo- philic microorganisms have been isolated and reported *Correspondence: [email protected]; [email protected] through development of culturomics [4, 5]. Te previous 1 Microbiology and Functionality Research Group, World Institute study suggested that the presence of halophilic microbi- of Kimchi, Gwangju 61755, Republic of Korea 3 Division of Environmental Science and Ecological Engineering, Korea ota in the gut is associated with high salinity in the gut. University, Seoul 02841, Republic of Korea High salinity of human gut changes the halophilic micro- Full list of author information is available at the end of the article biota which could be related to human diseases such as © The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Ahn et al. Gut Pathog (2021) 13:41 Page 2 of 6 obesity [5]. Terefore, further studies of halophilic bacte- parsimony (MP) [11], and maximum likelihood (ML) ria isolated from the human gut could be helpful in elu- [12] algorithms using 1000 bootstrap value [13]. Func- cidating the relationship between halophilic bacteria and tional genes were predicted and annotated using Rapid human health. We isolated a bacterium belonging to the Annotation using Subsystem Technology (RAST; https:// Lentibacillus species from human fecal sample, identifed rast. nmpdr. org/) [14]. PathogenFinder (https:// cge. cbs. its whole genome sequence through NGS, and analyzed dtu. dk/ servi ces/ Patho genFi nder/) was used for predict- information on the genes that could have a pathogenic ing pathogenicity towards humans [15]. Te presence efect on humans. In addition, we performed phyloge- of Clustered Regularly Interspaced Short Palindromic netic analysis based on 16S rRNA gene sequence and Repeats (CRISPRs) was detected using the CRISPRfnder comparative genomic analysis with other species of genus server (https:// crispr. i2bc. paris- saclay. fr/ Server/) [16]. Lentibacillus. Prophages were confrmed using the PHASTER database (https:// phast er. ca/), a phage search tool [17]. Methods Bacterial strain isolation Comparative genomics analysis Strain CBA3610 was isolated from a stool sample from a Comparative genome analysis was performed using 12 28-year-old healthy male in Gwangju, Republic of Korea. reference strains belonging to the genus Lentibacillus Te fecal sample was enriched in Deutsche Sammlung along with strain CBA3610. Te genome and amino acid von Mikroorganismen und Zellkulturen (DSMZ) sequences of 12 reference strains are available in the Gen- medium 372 broth under aerobic conditions at 37 °C for Bank of National Center for Biotechnology Information 7 days, after which 100 mL of the enriched broth was (NCBI, Accessed 22 September 2020). Te list of strains spread on DSMZ medium 372 agar plates to isolate bac- used in the analysis is summarized in Additional fle 1: terial strains under aerobic conditions at 37 °C for 24 h. Table S1. Pan-genome analysis was performed using Strain CBA3610 was isolated from several colonies, and Bacterial Pan Genome Analysis tool (BPGA). Te 50% subculturing was performed under the same conditions sequence identity cut-of was applied to obtain the core at least three times. genomes of a total of 13 strains using USEARCH (ver. 9.0) [18]. Te core genome tree was constructed with the Genome sequencing, assembly, and gene annotation aligned amino acid sequences of common genes of 13 Te genomic DNA of the isolated strain was extracted strains using MAFFT (ver. 7.471) [19] and the MEGA 7 and purifed using the MG genomic DNA purifcation kit with NJ algorithm [10, 13]. Te OrthoANI value was cal- (MGmed, Seoul, Korea). Te whole genome sequencing culated using the Orthologous Average Nucleotide Iden- was performed using Pacifc Biosciences RS II (Pacifc tity Tool (OAT) provided by EzBioCloud database [20]. Biosciences, Menlo Park, CA) and Illumina HiSeq X Ten (Illumina, San Diego, CA). Each library used for sequenc- Quality assurance ing was constructed using a 20-kb SMRTbell template Before genomic DNA extraction, the single colony of preparation kit and a TruSeq Nano DNA High Trough- strain CBA3610 was transferred three times in DSMZ put Library kit. Te genome was assembled using the medium 372 to obtain pure single colony. After obtain- protocol of Unicycler ver. 0.4.6 with PacBio SMRT analy- ing the whole genome sequence of strain CBA3610, the sis ver. 2.3 [6] and Pilon ver. 1.21 with Illumina HiSeq for sequence of the 16S rRNA gene, extracted using RNAm- error correction [7]. Te subread fltering of the PacBio mer 1.21 server, was confrmed through the EzBioCloud sequences was performed based on the following criteria: database. minimum subread length 50, minimum polymerase read quality 75, and minimum polymerase read length 50. Results and discussion Adapter/primer contamination of HiSeq raw sequences Genome characteristics and annotation data was confrmed using FastQC (v0.11.9). Te genome was After the PacBio subreads fltering process, the total annotated using the Pathosystems Resource Integra- number of bases was 1,186,149,844 and the number of tion Center (PATRIC; https:// www. patri cbrc. org/) ver. reads was 111,990. After the HiSeq raw data fltering pro- 3.6.7, the bacterial bioinformatics database and analysis cess, the total number of bases in the fltered dataset was resource [8]. We constructed a phylogenetic tree based 796,687,476 and number of reads was 5,276,076. In the de on 16S rRNA gene sequences. To construct the phylo- novo assembly process, long-reads of PacBio were assem- genetic tree, the sequences of 16S rRNA gene of strain bled using the default option. After de novo assembly CBA3610 and related species were aligned using Clustal with PacBio subreads and error correction using HiSeq W [9]. Phylogenetic trees were constructed using MEGA reads, the complete genome of Lentibacillus sp. CBA3610 7, based on the neighbor-joining (NJ) [10], maximum consists of one chromosome (Total length: 4,035,571 bp). Ahn et al. Gut Pathog (2021) 13:41 Page 3 of 6 No plasmid was identifed. Chromosome was circular Table 1 Complete genome features of Lentibacillus sp. CBA3610 with 42% G + C content. According to the PATRIC anno- Property Value tation results, the genome has 4714 predicted genes, 166 repeat regions, 64 tRNA genes, and 17 rRNA genes.
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