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Complete genome sequence of plant-pathogenic Pseudomonas sp. N3-W isolated from subarctic tundra soil
Korean J. Microbiol. 2022;58(4):302-304
Published online December 31, 2022
© 2022 The Microbiological Society of Korea.

Dockyu Kim*, Sungho Woo, Jungeun Lee, Sanghee Kim, and Yung Mi Lee

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 September 13, 2022; Revised October 7, 2022; Accepted November 4, 2022.
Abstract
Pseudomonas sp. N3-W, a bacterial plant pathogen isolated from a subarctic Alaskan tundra soil for its ability to secrete strong extracellular protease(s), was found to be capable of producing brown spots on hot pepper leaves. The complete genome sequence consisted of one circular chromosome with 6,674,931 bp (G + C content of 59.7%). A computer-aided analysis discovered 5,726 protein coding sequences (CDSs), of which at least nine CDSs were putatively assigned to code for various proteolytic enzymes. The production of these proteases would explain N3-W’s high degree of extracellular proteolytic activity, which could contribute to the evasion and deactivation of defense mechanisms of plant hosts.
Keywords : bacterial pathogen, cold-adapted, crop yield, global warming, permafrost soil
Body

Permafrost soils have been shown to be a reservoir of diverse uncharacterized microorganisms that can be harmful to plants (Wu et al., 2022). Plant pathogens include fungi, bacteria, protists, nematodes, and viruses that can infect almost all types of plants and significantly reduce crop yield. Thus, outbreak and spread of plant disease by novel pathogens will be of interest with regard to ecosystem sustainability and global food supply. During permafrost thawing in (sub)Arctic tundra, however, the potential for plant pathogens outbreaks remains largely unexplored. Recent rapid temperature rising has extended the period and soil depth of permafrost thawing, increasing the soil temperature and soil water content (main ecological factors). A data from a global field survey and a field experiment showed that warmer temperatures increased the relative abundances of some soil-borne potential fungal plant pathogens worldwide (Delgado-Baquerizo et al., 2020). Because the individual effects of the ecological factors on microbial activity and survival are still poorly understood, even less is known about their combined impact on the composition and activity of potential pathogens in the tundra permafrost soils during summer seasons. Understanding the current and future distribution and activity of potential plant pathogens from permafrost soils is critical to maintaining stable ecosystem and food supply.

To isolate cold-adapted plant pathogenic bacteria, a tundra melted soil containing decaying plant material was sampled from the north subarctic Council, Alaska, USA, in 2019 September. Among many bacterial isolates from the soil, one strain (N3-W) was selected for strong proteolytic activity on skim milk-containing R2A agar plate and its cell suspension was infiltrated into the leaves of hot pepper seedlings (Capsicum sp. cultivar Maepgona). When the pathogenicity was examined, a positive strain (Pseudomonas syringae pv. tomato KACC 15103) and N3-W produced brown spots on the inoculated pepper leaves. These experiments indicated that N3-W is a plant pathogenic bacterium that can infect and proliferate in pepper leaves. This strain was deposited in Korean Agricultural Culture Collection under accession number KACC 22935.

Whole genome sequencing of N3-W was performed using the PacBio Sequel I and Illumina NovaSeq 6000 platforms (Macrogen Inc.). The resulting filtered total subreads was 90,469 (total subread bases, 782,611,869 bp; mean subread length, 8,650 bp; N50, 11,216 bp; genome coverage, 117×). 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 6,674,931 bp, with a G + C content of 59.7% (Fig. 1 and Table 1). The TrueBac ID-Genome database (www.truebacid.com; Ha et al., 2019) search showed that the N3-W whole genome sequence (WGS) had the highest 16S rRNA sequence identity (99.86%) and average nucleotide identity (ANI) value of 88.09% with Pseudomonas phylotype LIRD_s strain In5 (GenBank accession number LIRD01000005) from Greenland agricultural soil. Consequently, this isolate could not be categorized at the species level, and thus assigned as a novel species in genus Pseudomonas.

Genomic features of <italic>Pseudomonas</italic> sp. N3-W
Feature type Value
Size (bp) 6,674,931
Contigs 1
Circular chromosome Yes
Plasmids 0
G + C content (%) 59.7
CDSs (total) 5,823
CDSs (with protein) 5,726
rRNAs (5S, 16S, 23S) 19 (7, 6, 6)
tRNAs 72
GenBank accession number CP103965


Fig. 1. The circular map of N3-W genome. Marked characteristics are shown from outside to the center: CDS on forward strand, CDS on reverse strand, tRNA, rRNA, GC content, and GC skew.

Following GenBank submission, the genes in complete genome sequence were annotated with NCBI Prokaryotic Genome Annotation Pipeline (PGAP) using the annotation method GeneMarkS-2+ v6.2. The genome annotation revealed 5,726 protein coding sequences (CDSs), 72 tRNA genes, and 19 rRNA genes containing six genes for complete 16S rRNA. Other genomic features are presented in Table 1. Interestingly, at least nine CDSs were assigned to code for various proteolytic enzymes including metalloprotease (locus_tag NYP20_04160, 04540, 04550, 07115, 09555, 11625, 15270, and 21270) and serine protease (NYP20_00790). Among them, one (469 amino acids, NYP20_11625) was identified to have higher similarities (a maximum of 92.5%) with members in the serralysin family of zinc metalloproteases from Pseudomonas spp. An opportunistic pathogen Pseudomonas aeruginosa pv. tomato DC3000 secretes alkaline protease AprA in the serralysin family. AprA was reported to be an important virulence factor, which helps to evade host immunity by degrading its own flagellin monomers, strong inducers of innate immune responses in plants (Pel et al., 2014). Overall, this observation suggests that N3-W produces brown spots on pepper leaves by producing extracellular proteases in order to evade the host immunity and/or and contribute to the deactivation of the defense response pathways of plant hosts (Figaj et al., 2019).

적 요

아북극권 미국 알래스카주 툰드라 토양으로부터 강력한 단백질분해 활성을 나타내는 세균 균주 Pseudomonas sp. N3-W를 분리하였으며, 작물 고추 잎에서 갈색 반점을 생성할 수 있는 식물병원균임을 검증하였다. N3-W은 하나의 원형 염색체(6,674,931 bp, 59.7% G + C content)를 보유하고 있는데, 유전체 서열 내 유전자들의 생물학적 기능예측(gene annotation)을 통해 총 5,726개의 단백질 코딩 서열(coding sequence)을 확인하였다. 이들 가운데 최소 9개 코딩 서열들이 다양한 종류의 단백질분해 효소를 암호화하리라 예측하였다. 위의 유전체 서열 분석 결과를 통해, N3-W는 고활성 단백질분해 효소들을 사용하여 기주식물체의 방어기작을 회피하거나 불활성화시키는 것으로 추정하였다.

Acknowledgments

This work was supported by Korea Polar Research Institute (KOPRI) grant funded by the Ministry of Oceans and Fisheries (KOPRI PE22140).

Conflict of Interest

The authors have no conflict of interest to report.

References
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December 2022, 58 (4)