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Genome sequences of the Psychrobacter sp. M13 isolated from intestine of Dokdo shrimp Lebbeus groenlandicus
Korean J. Microbiol. 2023;59(3):223-226
Published online September 30, 2023
© 2023 The Microbiological Society of Korea.

Hye-Jin Park1,2, Sang-Eon Kim1,2, and Kyoung-Ho Kim1,2*

1Department of Microbiology, Pukyong National University, Busan 48513, Republic of Korea
2School of Marine and Fisheries Life Science, Pukyong National University, Busan 48513, Republic of Korea
Correspondence to: *E-mail: kimkh@pknu.ac.kr; Tel.: +82-51-629-5611; Fax: +82-51-629-5619
Received August 8, 2023; Revised August 18, 2023; Accepted August 18, 2023.
Abstract
The complete whole genome sequences were obtained from a bacterial strain M13 isolated from the intestine of Dokdo shrimp, Lebbeus groenlandicus caught in the East Sea, near Ulleung island, South Korea. The strain was isolated at a low temperature (4°C) similar to those of the sea environment, where its host lived. Phylogenetic analysis showed that the strain contained 16S rRNA gene similarities lower than 98.55% of the species of genus Psychrobacter in the family Moraxellaceae. Whole genome sequencing using the PacBio RS II system yielded two circular contigs (a chromosome and a plasmid of 3,177,391 and 29,784 bp with G + C contents of 42.6 and 40.7 mol %, respectively). The genome has a total of 2,712 genes coding 2,654 CDSs, 46 tRNAs, 8 rRNAs, and 4 ncRNAs as well as an incomplete prophage region and a potential gene cluster for production of secondary metabolites. The genome also contains four genes coding cold-shock proteins that make bacteria adapt to cold temperatures.
Keywords : Lebbeus groenlandicus, Psychrobacter, Dokdo shrimp, whole genome sequencing
Body

Psychrobacter species are distributed in different animal hosts such as whale skin (Apprill et al., 2014), the throat and intestines of birds (Kämpfer et al., 2015), and fish (Svanevik and Lunestad, 2011) as well as environments such as seawater (Yoon et al., 2005), sea ice (Bowman et al., 1997), marine sediment (Matsuyama et al., 2015), glacial ice (Zeng et al., 2016), and permafrost soil (Bakermans et al., 2006). Based on the abundance of Psychrobacter in cold seawater and ice, they seem to have the characteristic of inhabiting cold environments. The genus Psychrobacter belongs to the family Moraxellaceae, and at the time of writing, a total of 44 species of Psychrobacter were validly published and had correct names (https://lpsn.dsmz.de/genus/psychrobacter).

Many strains of Psychrobacter are psychrophilic and psychrotolerant (grow between 4°C and 25°C ) but also some can grow at high temperatures (grow up to 37°C) (Welter et al., 2021). Their broad range of growth temperatures partially explains the reason why they were isolated in diverse habitats. A comparative genomic study hypothesized that the free-living and psychrophilic members of the genus Psychrobacter descended from their pathogenic ancestors related to the genus Moraxella (Welter et al., 2021).

Lebbeus groenlandicus is a shrimp species in the family Hippolytidae, order Decapoda, and phylum Arthropoda. This species inhabits mainly at depths of 200–400 m and a temperature of 4–6°C (Bae and Oh, 2014). The shrimps are usually caught with traps in the sea near Ulleung island, East Sea, South Korea and consumed domestically (Kim et al., 2013). In this study, the whole genome of strain M13, a psychrophilic strain isolated from the intestine of the shrimp L. groenlandicus, was analyzed.

Strain M13 was isolated from the intestine of L. groenlandicus. The content of the intestine was serially diluted using phosphate-buffered saline solution and incubated on marine agar 2216 (Difco) at 4°C for 5 days. The grown colonies were isolated in pure culture through transfer to fresh media. Phylogenetic analysis showed low 16S rRNA gene sequence similarities to close species Psychrobacter luti NF11T (98.55%), P. arcticus 273-4T (98.41%), P. fozii NF23T (98.34%), and P. cryohalolentis K5T (98.26%) of the genus Psychrobacter in the family Moraxellaceae.

The genomic DNA was then subjected to library construction and single-molecule real-time sequencing (SMRT) (Ardui et al., 2018) using PacBio RS II system (Pacific Biosciences). SMRT sequencing generated a total of 152,071 subreads (1,4 Gb; N50, 12,284 bp; mean length, 8,931 bp) and subreads were de novo-assembled by Hierarchical Genome Assembly Process (HGAP, Version 3.0, Pacific Biosciences). Strain M13 has one circular chromosome and one circular plasmid with sizes of 3,177,391 and 29,784 bp and G + C contents of 42.6 and 40.7 mol%, respectively (approximately 213 X folds, Table 1). The plasmid contig showed similarities with the plasmids of Psychrobacter species against the NCBI GenBank database. The genomes were annotated by NCBI Prokaryotic Genome Annotation Pipeline (PGAP) revision 6.5 (Tatusova et al., 2016) and a total of 2,712 genes including 2,654 CDSs, 46 tRNAs, 8 rRNAs, and 4 ncRNAs were annotated from the chromosome and plasmid.

Genomic features of strain M13
Characteristics of contigs Annotation results of whole genome
Contig Topology Length (bp) G + C content (mol%) Type No
Contig 1 Circular 3,177,391 42.6 Genes 2,712
Contig 2 Circular 29,784 40.7 CDS (total) 2,654
rRNA (5S, 16S, 23S) 8 (3, 3, 2)
tRNA 46
ncRNA 4
CDS (with protein) 2,634
Total 3,207,175 42.6 Pseudogenes 20


Analysis of prophage presence using PHASTER (Arndt et al., 2016) revealed that the contig 1 has an incomplete prophage region in a length of 6.8 kb with an attachment site and ten proteins including integrase, transposase, and tail genes sequences. The genes involved in the biosynthesis of secondary metabolites in strain M13 were identified and predicted by antiSMASH 6.1.1 (Blin et al., 2021). Only one cluster was predicted and associated with a beta-lactone containing protease inhibitor type (Fig. 1). Among the twenty genes of the cluster, only two genes showed significant BLAST hits with those of each of three different clusters called ‘plipastatin, NRP’, ‘mycosubtilin, NRP + Polyketide’, and ‘fengycin, NRP’ of the Minimum information about a biosynthetic gene cluster (MIBiG) database (Kautsar et al., 2020). The low similarity, that is, the small number of the common genes implies the gene cluster can produce an unknown secondary metabolite in the intestine of shrimp. The genes of Q9G97_RS02260, Q9G97_RS03105, Q9G97_RS05700, and Q9G97_RS11215 were related to cold-shock proteins that made bacteria adapt to cold temperatures (Jung et al., 2010). This genome analysis will provide a basis for understanding the characteristics of this novel Psychrobacter species and the ways in which the L. groenlandicus can adapt in cold environments.

Fig. 1. A cluster for the production of secondary metabolites in strain M13, predicted by antiSMASH.
(A) Genome region and gene organization of the cluster, (B) Known clusters showing similarities with the cluster predicted by antiSMASH. Three known clusters of the MiBIG database have similar genes to the cluster (e.g. query sequence). Number (e.g. BGC0000407) is the identifier of the compounds of the MiBIG database. Genes for (a) an AMP-binding protein (red), (b) a hydroxymethylglutaryl-CoA lyase (light blue), and (c) an acetyl/propionyl/methylcrotonyl-CoA carboxylase subunit alpha.

Nucleotide sequence and strain accession numbers

The strain is available at the Korean Collection for Type Cultures (KCTC 92916). The16S rRNA gene partial sequences and genome sequences were deposited at GenBank with accession numbers of OQ552727 and PRJNA1002627, respectively.

적 요

대한민국 울릉도 근처 동해에서 잡힌 독도새우, Lebbeus groenlandicus의 장에서 분리된 박테리아 균주 M13에서 전체 유전체 염기 서열을 얻었다. 균주는 숙주가 서식하는 바다 환경과 유사한 저온(4°C)에서 분리되었다. 이 균주는 계통학적 분석 결과 Moraxellaceae과의 Psychrobacter 속의 종들과 98.55% 이하의 낮은 16S rRNA 유전자 유사성을 보여주었다. PacBio RS II 시스템을 사용하여 전체 유전체 분석을 수행한 결과 2개의 원형 콘티그(각각 42.6 및 40.7 mol%의 G + C 함량을 갖는 3,177,391 및 29,784 bp의 염색체 및 플라스미드)를 얻었다. 2,654개의 CDS, 46개의 tRNA, 8개의 rRNA 및 4개의 ncRNA를 암호화하는 총 2,712개의 유전자와 불완전한 프로파지 영역 및 2차 대사산물 생산을 위한 잠재적인 유전자 클러스터가 유전체에 존재한다. 유전체에서 세균을 낮은 온도에 적응하는데 도움을 주는 cold-shock 단백질을 암호화하는 네 개의 유전자를 발견하였다.

Acknowledgments

This research was supported by a Research Grant of Pukyong National University (2021).

Conflict of Interest

Kyoung-Ho Kim is Associate Editor of KJM. He was not involved in the review process of this article. Also, Authors have no conflicts of interest to report.

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