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Complete genome sequence of plant-pathogenic Pseudomonas sp. G2-4 from rhizosphere soil in Northern Greenland
Korean J. Microbiol. 2023;59(2):154-157
Published online June 30, 2023
© 2023 The Microbiological Society of Korea.

Sungho Woo, Mincheol Kim, Yung Mi Lee, and Dockyu Kim*

Division of Life Sciences, Korea Polar Research Institute, Incheon 21990, Republic of Korea
Correspondence to: *E-mail: envimic@kopri.re.kr; Tel.: +82-32-760-5525; Fax: +82-32-760-5509
Received June 15, 2023; Accepted June 20, 2023.
Abstract
A novel bacterium, Pseudomonas sp. G2-4, was isolated from the rhizosphere soil of Oxyria digyna in Northern Greenland. This strain exhibited a notable ability to secrete strong extracellular proteases and was capable of developing brown spots on hot pepper leaves and soft rot on potatoes. The complete genome sequence of the phytopathogen G2-4 consisted of one circular chromosome with 6,651,895 bp (G + C content of 60.4%). Genomic analysis discovered 5,692 protein coding sequences (CDSs), among which one CDS was identified as a putative virulence factor responsible for encoding the extracellular protease. The extracellular proteolytic activity of G2-4 would at least partially contribute to the development of disease in the pepper and potato plants.
Keywords : bacterial pathogen, cold-adapted, crop yield, global warming, permafrost soil
Body

Permafrost soil continuously remains below 0°C for at least two consecutive years. The approximately 14.6% of the exposed land area in the Northern Hemisphere, including regions in Alaska, Greenland, Canada, and Siberia, consists of permafrost (Obu et al., 2019). Under global warming, the Arctic permafrost has experienced a temperature increase of 2–3°C since the 1970s, with some sites recording a rise of 0.3°C from 2007 to 2016 (Scheel et al., 2022). This recent rapid temperature rising has resulted in the thawing of permafrost soil, a reservoir of diverse uncharacterized microorganisms (Scheel et al., 2022; Wu et al., 2022). These microbes include soil-borne potential plant pathogens, such as fungi, bacteria, and viruses, that can infect plants and significantly reduce crop yields. Until now, the outbreaks of plant pathogens from melted permafrost have not yet reported, although ancient DNA fragments of fungal plant pathogens were detected in ancient Siberia permafrost, 16,000–32,000 years old (Bellemain et al., 2013).

Therefore, monitoring the emergence and distribution of these potential plant pathogens from thawed permafrost soil is critical for maintaining stable ecosystems and food supplies.

To isolate cold-adapted soil bacteria harmful to crop plant, rhizosphere soils (82°47.650' N, 42°25.728' W) of Oxyria digyna were collected from Sirius Passet in Northern Greenland, in July 2022. Among many bacterial isolates obtained from the soils, one strain (G2-4) was selected based on its ability to produce dark-brown pigment and display strong proteolytic activity on R2A agar plate containing skim milk (1% w/v). For pathogenicity tests of G2-4, its cell suspension was infiltrated into the leaves of hot pepper seedlings (Capsicum sp. cultivar Maepgona) and inoculated on potato tubers (Solanum tuberosum). G2-4 produced brown spots on the inoculated pepper leaves (hypersensitivity response) and potato tuber tissue maceration (soft rot), respectively. These results indicated that G2-4 is a plant pathogenic bacterium that can infect and proliferate in the two different crops. This strain was deposited in the Korean Agricultural Culture Collection under the accession number KACC 23303.

Whole genome sequencing of G2-4 was performed using the PacBio Sequel II HiFi and Illumina NovaSeq 6000 platforms (Macrogen Inc.). The resulting filtered total HiFi reads were 48,357 (total HiFi bases, 280,159,734 bp; mean length, 5,793 bp; HiFi N50, 7,097 bp; genome coverage, 42×). De novo assembly of subreads was carried out using Microbial Assembly Application within the PacBio SMRT analysis pipeline (v8.0). Error correction of the assembled genome was performed with Pilon (v1.21) using the Illumina reads. The complete genome sequence consisted of one circular chromosome with 6,651,895 bp, with a G + C content of 60.4% (Fig. 1 and Table 1). The TrueBac ID-Genome database (www.truebacid.com;Ha et al., 2019) search showed that whole genome sequence of G2-4 had the highest 16S rRNA sequence similarities (99.79% and 99.45%) and average nucleotide identity (ANI) values (93.69% and 86.20%) with Pseudomonas sp. DF41 and Pseudomonas migulae CIP 105470T (GenBank accession numbers CP007410 and AF074383) isolated from phyllosphere of canola plants and mineral waters, respectively. ANI is a widely accepted overall genome related index, with a typical species boundary set at ≥ 95% ANI (Richter and Rosselló-Móra, 2009). Based on ANI comparisons, G2-4 could not be classified at the species level, and was designated as a novel species within the genus Pseudomonas.

Genomic features of Pseudomonas sp. G2-4
Feature type Value
Size (bp) 6,651,895
Contigs 1
Circular chromosome Yes
Plasmids 0
G + C content (%) 60.4
CDSs (total) 5,780
CDSs (with protein) 5,692
rRNAs (5S, 16S, 23S) 16 (6, 5, 5)
tRNAs 67
GenBank accession number CP125957


Fig. 1. Circular map of Pseudomonas sp. G2-4 genome.
Marked characteristics are shown from outside to the center: coding sequences (CDS) on the forward strand, CDS on the reverse strand, tRNA, rRNA, GC content, and GC skew.

The genes were annotated with the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) using the annotation method GeneMarkS-2+ v6.5. The genome annotation revealed 5,692 protein coding sequences (CDSs), 65 tRNA genes, and five copies of ribosomal RNA operons. Additional genomic features are presented in Table 1. Remarkably, one CDS (485 amino acids, locus_tag = QNH97_13980) showed significant similarities (88.73–97.53%) to members in serralysin family metalloproteases found in diverse environmental, non-pathogenic Pseudomonas spp. The TrueBac ID system screened potential virulence factors (VFs) from the G2-4 genome sequence by comparing it to the virulence factor database (VFDB, www.mgc.ac/VFs/; Liu et al., 2019). QNH97_13980 was identified as one VF (alkaline protease AprA), which showed high similarities (52.54–52.97%) with the serralysin family metalloproteases from Dickeya chrysanthemi strains. These strains cause soft rot and wilting diseases in a wide range of host plants in tropical, subtropical, and temperate regions. The phytopathogen Pseudomonas syringae pv. tomato DC3000 secretes AprA, an important VF in tomato infections (Pel et al., 2014). The extracellular proteases from plant pathogenic bacteria are involved in diverse processes associated with plant infection (Figaj et al., 2019) and G2-4 can infect pepper and potato. Thus, it is likely that QNH97_13980, with a structural similarity to VF proteases, contributes at least partially to the development of leaf brown spot and soft rot diseases by G2-4.

적 요

북그린랜드 나도수영 근권 토양으로부터 강력한 단백질분해 활성을 나타내는 세균 균주 Pseudomonas sp. G2-4를 분리하였으며, 작물 고추 잎에서 갈색 반점과 감자 괴경에서 무름 증상을 유발할 수 있는 식물 병원균임을 검증하였다. G2-4은 하나의 원형 염색체(6,651,895 bp, 60.4% G + C content)를 보유하고 있는데, 유전체 서열 내 유전자들의 생물학적 기능 예측(gene annotation)을 통해 총 5,692개의 단백질 코딩 서열(coding sequence)을 확인하였다. 이들 가운데 1개 코딩 서열을 식물 병원성 인자로 추정되는 외분비성 단백질 분해효소를 암호화하리라 예측하였다. 위의 유전체 서열 분석 결과를 통해, G2-4의 고활성 단백질 분해효소가 고추잎 갈색 반점과 감자 괴경 무름 증상 유발에 최소한의 역할을 담당하리라 추정하였다.

Acknowledgments

This work was supported by Korea Polar Research Institute (KOPRI) grant funded by the Ministry of Oceans and Fisheries (KOPRI PE23140).

Conflict of Interest

The authors have no conflict of interest to report.

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