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Draft genome sequence of an Alteromonadaceae bacterium BrNp21-10 isolated from seawater
Korean J. Microbiol. 2023;59(4):346-349
Published online December 31, 2023
© 2023 The Microbiological Society of Korea.

Ji-Sung Oh and Dong-Hyun Roh*

Department of Biological Sciences and Biotechnology, Chungbuk National University, Cheongju 28644, Republic of Korea
Correspondence to: *E-mail: dhroh@chungbuk.ac.kr; Tel.: +82-43-261-3368; Fax: +82-43-264-9600
Received October 30, 2023; Revised November 9, 2023; Accepted November 9, 2023.
Abstract
The draft genome sequence of an Alteromonadaceae bacterium BrNp21-10 isolated from coastal seawater was determined using the Illumina Hiseq X-ten platform. The assembled genome consists of 33 contigs totaling 4,217,876 bp with N50 values of 212,777 bp. The genomic DNA G + C content was 42.5%. The draft genome comprised 3,674 protein-coding, 5 rRNA, 48 tRNA, 4 non-coding RNA genes, and 15 pseudogenes. There were a series of genes related to CRISPR, flagellar assembly, and assimilatory sulfate reduction in the genome. The isolated strain BrNp21-10 is considered to be a novel strain belonging to the family Alteromonadaceae by 16S rRNA gene sequence and digital DDH analysis.
Keywords : Alteromonadaceae, draft genome sequence, novel genus strain
Body

The family Alteromonadaceae belongs to the order Alteromonadales, the class Gammaproteobacteria, and the phylum Pseudomonadota (Ivanova and Mikhailov, 2001). This family was originally described by Ivanova and Mikhailov (2001), based on the phenotypic, genotypic, and phylogenetic characteristics comprising various genera of Gram-negative aerobic and facultative rod bacteria to form a phylogenetic cluster with a 16S rRNA gene sequence homology of 90% or higher. The type genus is Alteromonas (Ivanova et al., 2004; Parte et al., 2020). Most members in this family have been isolated from coastal, open, and deep-sea water, marine sediment, sea ice, and invertebrates from marine environments (Ivanova and Mikhailov, 2001). Here, the draft genome sequence and annotation of an Alteromonadaceae bacterium BrNp21-10 isolated from coastal seawater, in Boryeong, Republic of Korea, were described.

For the extraction of genomic DNA, Alteromonadaceae bacterium BrNp21-10 was incubated in Marine broth (MB, Difco) at 25°C for 3 days and then the genomic DNA was extracted using MagAttract® HMW DNA kit (Qiagen) according to the manufacturer’s instructions. The sequencing of strain BrNp21-10 genome was performed by Macrogen Inc. as the Illumina Hiseq X-ten platform with TruSeq Nano DNA (350 bp insert size) library. Raw reads were qualified by FastQC (version 0.11.5) and were assembled by SPAdes (version 3.13.0). Genome quality and CRISPR site were analyzed with the Bacterial and Viral Bioinformatics Resource Center (BV-BRC, https://www.bv-brc.org/). The genome annotation was conducted using NCBI Prokaryotic Genome Annotation Pipeline (PGAP) (Tatusova et al., 2016), and the additional functions of the predicted genes were conducted by BlastKOALA with the KEGG database (https://www.genome.jp/kegg/), Rapid Annotation of microbial genomes using Subsystems Technology (RAST) server with SEED database, and the BV-BRC web resources. The identification of an isolated strain using 16S rRNA gene sequences was performed with EZBioCloud (https://eztaxon-e.ezbiocloud.net/) and the whole genome-based taxonomic analysis was performed with the Type Strain Genome Server (TYGS) (Meier-Kolthoff et al., 2021). AntiSMASH 7.0 (https://antismash.secondarymetabolites.org/) was used to detect and characterize biosynthetic gene clusters for secondary metabolite.

The NCBI PGAP annotation for the genome of Alteromonadaceae bacterium BrNp21-10 revealed that the genome consists of 33 contigs with a total length of 4,217,876 bp and N50 value of 212,777 bp (Table 1). Genome coverage was 147.4 x and the G + C content was 42.5%. The draft genome comprised 3,746 genes consisting of 3,674 protein-coding genes, 15 pseudogenes, 2, 2, 1 rRNA genes (5S, 16S, 23S), 48 tRNA genes, and 4 non-coding RNA genes. The genome quality was good representing that coarse consistency, fine consistency, and CheckM completeness were 95.5%, 94.8%, and 98.7%, respectively.

Genomic features of Alteromonadaceae bacterium BrNp21-10
Feature type Value
Genome size (bp) 4,217,876
Number of contigs 33
Contig N50 212,777
Genome Coverage (X) 147.4
G+C content (%) 42.5
CDS (with protein) 3,674
rRNA genes (5S, 16S, 23S) 2, 2, 1
RNA genes 57
Pseudogenes 15
Accession number (GenBank) JAUHQD000000000


The isolated strain BrNp21-10 revealed potential new species and did not belong to any species found in the TYGS database. The partial 16S rRNA gene sequence (1,319 bp) of BrNp21-10 had the highest similarity of 94.3% and branched with Neptunicella marina S27-2 in phylogeny (Data not shown) (Liu et al., 2018). The whole-proteome-based Genome Blast Distance Phylogeny (GBDP) also showed the same branched pattern with a high bootstrap value of 100% (Fig. 1). The dDDH values (formula d0, d4, d6) between strain BrNp21-10 and strain Neptunicella marina S27-2 were 13.5, 19.3, and 13.7, respectively.

Fig. 1. Tree inferred with FastME 2.1.6.1 (Lefort et al., 2015) from whole-proteome-based GBDP distances.
The branch lengths are scaled via GBDP distance formula d5. Branch values are GBDP pseudo-bootstrap support values > 60% from 100 replications, with an average branch support of 98.0%. The tree was midpoint-rooted (Farris, 1972). GenBank assembly accession numbers are given in parentheses.

Interestingly, the genome contained a CRISPR array region with 46 repeats with sequence TTTCTAAGCTGCCTGTG CGGCAGTFAAC and 45 spacers from 88190 to 90917 on accession JAUHQD010000002. Nearby, type I-F CRISPR-associated endoribonuclease Cas6/Cys4 (cas6f, locus_tag= QX776_04050), type I-Fv CRISPR-associated protein Cas5fv (cas5fv, locus_tag=QX776_04055) and type I-Fv CRISPR-associated protein Cas7fv (cas7fv, locus_tag=QX776_04060) were located.

Two secondary metabolite coding regions were found in the genome. The one was 55% similarity aryl polyenes Vf biosynthetic gene cluster related producing protective pigments from Alliivbrio fischeri ES114 (Cimermancic et al., 2014) in contig JAUHQD010000004.1. The other was a 13% similarity capsular polysaccharide biosynthetic gene cluster from Staphylococcus aureus in contig JAUHQD010000003.1 (Luong et al., 2002).

The closest strain Neptunicella marina had a flagellum (Liu et al., 2018). Therefore, the basal body/hook and filament, stator, and regulator required flagellar assembly were checked in the genome of strain BrNp21-10. The genome contained flagella basal body/hook-related genes, which consisted of MS/C ring and type III secretion system (T3SS) coding genes (fliE, F, G, H, I, J, L, M, N, O, P, Q, and R), and T3SS coding genes (flhA and B), Rod, P/L ring and hook coding genes (flgA, B, C, D, E, F, G, H, I, and J), hook-filament junction coding genes (flgK and L), H ring coding genes (flgP and T), and T ring coding genes (motX and Y). A filament coding gene (fliC) and filament cap and chaperone coding genes (fliD and fliS) for filament were found. The stator coding genes (motA and B), and a regulator gene (flgN) were also found in the genome. Besides, it also contained global regulator coding genes (rpoD, flrA, rpoN, flrC, and fliA).

The strain BrNp21-10 also had assimilatory sulfate reduction pathway modules required from sulfate to hydrogen sulfide; adenylylsulfate kinase [EC 2.7.1.25], sulfate adenylyltransferase subunit 2 [EC 2.7.7.4], bifunctional enzyme CysN/CysC [EC 2.7.7.4 2.7.1.25], phosphoadenosine phosphosulfate reductase [EC 1.8.4.8 1.8.4.10], sulfite reductase (NADPH) flavoprotein α-component [EC 1.8.1.2] and sulfite reductase (NADPH) hemoprotein β component [EC 1.8.1.2].

Considering the results, the isolated strain BrNp21-10 is thought to be a novel genus strain of the family Alteromonadaceae, and the determined base sequence is expected to help in the classification of similar strains and the use of useful genes in the future.

Nucleotide sequence accession numbers

The draft genome sequence and strain Alteromonadaceae bacterium BrNp21-10 have been deposited to Genbank and the Korean Culture Center of Microorganisms under the accession numbers JAUHQD000000000, and KCCM 43491, respectively.

적 요

바닷물에서 분리된 Alteromonadaceae 세균 BrNp21-10의 초안 유전체 서열을 Illumina Hiseq X-ten platform을 사용하여 결정하였다. 조립된 유전체의 총 길이는 4,217,876 bp이고 N50 값은 212,777 bp이며, 33개의 콘티그로 구성되었다. 유전체의 G + C 함량은 42.5% 이었다. 초안 유전체는 3,674개의 단백질 코드 유전자와 5개의 rRNA 유전자, 48개의 tRNA 유전자, 4개의 non-coding RNA 유전자, 15개 위유전자(pseudo gene)를 암호화하였다. 유전체에는 CRISPR와 편모조립 유전자, 황산염 환원에 관련된 일련의 유전자들이 존재하였다. 분리된 균주 BrNp21-10은 16S rRNA 유전자 염기서열 및 디지털 DDH 분석에 의해 Alteromonadaceae과에 속하는 새로운 균주로 간주된다.

Acknowledgments

This work was supported by a funding for the academic research program of Chungbuk National University in 2023.

Conflict of Interest

The authors have no conflict of interest to report.

References
  1. Cimermancic P, Medema MH, Claesen J, Kurita K, Brown LCW, Mavrommatis K, Pati A, Godfrey PA, Koehrsen M, and Clardy J, et al. 2014. Insights into secondary metabolism from a global analysis of prokaryotic biosynthetic gene clusters. Cell 158, 412-421.
    Pubmed KoreaMed CrossRef
  2. Farris JS. 1972. Estimating phylogenetic trees from distance matrices. Am. Natur. 106, 645-668.
    CrossRef
  3. Ivanova EP, Flavier S, and Christen R. 2004. Phylogenetic relationships among marine Alteromonas-like proteobacteria: emended description of the family Alteromonadaceae and proposal of Pseudoalteromonadaceae fam. nov., Colwelliaceae fam. nov., Shewanellaceae fam. nov., Moritellaceae fam. nov., Ferrimonadaceae fam. nov., Idiomarinaceae fam. nov. and Psychromonadaceae fam. nov. Int. J. Syst. Evol. Microbiol. 54, 1773-1788.
    Pubmed CrossRef
  4. Ivanova E and Mikhailov V. 2001. A new family, Alteromonadaceae fam. nov., including marine proteobacteria of the genera Alteromonas, Pseudoalteromonas, Idiomarina, and Colwellia. Microbiology 70, 10-17.
  5. Lefort V, Desper R, and Gascuel O. 2015. FastME 2.0: a comprehensive, accurate, and fast distance-based phylogeny inference program. Mol. Biol. Evol. 32, 2798-2800.
    Pubmed KoreaMed CrossRef
  6. Liu X, Lai Q, Du Y, Zhang X, Liu Z, Sun F, and Shao Z. 2018. Neptunicella marina gen. nov., sp. nov., isolated from surface seawater. Int. J. Syst. Evol. Microbiol. 68, 1423-1428.
    Pubmed CrossRef
  7. Luong TT, Ouyang S, Bush K, and Lee CY. 2002. Type 1 capsule genes of Staphylococcus aureus are carried in a Staphylococcal cassette chromosome genetic element. J. Bacteriol. 184, 3623-3629.
    Pubmed KoreaMed CrossRef
  8. Meier-Kolthoff JP, Carbasse JS, Peinado-Olarte RL, and Göker M. 2021. TYGS and LPSN: a database tandem for fast and reliable genome-based classification and nomenclature of prokaryotes. Nucleic Acids Res. 50, D801-D807.
    Pubmed KoreaMed CrossRef
  9. Parte AC, Sardà Carbasse J, Meier-Kolthoff JP, Reimer LC, and Göker M. 2020. List of prokaryotic names with standing in nomenclature (LPSN) moves to the DSMZ. Int. J. Syst. Evol. Microbiol. 70, 5607-5612.
    Pubmed KoreaMed CrossRef
  10. Tatusova T, DiCuccio M, Badretdin A, Chetvernin V, Nawrocki EP, Zaslavsky L, Lomsadze A, Pruitt KD, Borodovsky M, and Ostell J. 2016. NCBI prokaryotic genome annotation pipeline. Nucleic Acids Res. 44, 6614-6624.
    Pubmed KoreaMed CrossRef


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