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Microbiology and Biotechnology Letters

Genome Report(Note)

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Genome Report  |  Genome Report

Microbiol. Biotechnol. Lett. 2024; 52(3): 331-334

https://doi.org/10.48022/mbl.2408.08001

Received: August 2, 2024; Revised: September 2, 2024; Accepted: September 6, 2024

Complete Genome Sequence of Priestia megaterium Strain 10 Isolated from Soybean Rhizosphere (Glycine max)

Amani Sliti1 and Jae-Ho Shin1,2,3*

1Department of Applied Biosciences, Kyungpook National University, Daegu 41566, Republic of Korea
2NGS Core Facility, Kyungpook National University, Daegu 41566, Republic of Korea
3Department of Integrative Biotechnology, Kyungpook National University, Daegu 41566, Republic of Korea

Correspondence to :
Jae-Ho Shin,      jhshin@knu.ac.kr

Abstract

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We present the complete genome sequence analysis of Priestia megaterium strain 10, isolated from the soybean rhizosphere. The genome consists of a single circular chromosome of 4,815,034 bp with a G+C content of 38.2% and 4 plasmids named P1 (198,305 bp), P2 (139,815 bp), P3 (79,328 bp), and P4 (61,901 bp).

Keywords: Complete genome, Priestia megaterium, rhizosphere, soybean

Body

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Plants are a major living organism form, essential for ecosystem sustainability. Due to their beneficial roles, plants provide nutrient sources, regulate the hydroclimate, improve soil structure and function, and clean the biosphere of contaminants (gas emissions, biochemical fertilizers, etc.) [1, 2]. However, various environmental stress conditions, including abiotic (drought, salinity, floods, cold/heat) and biotic factors (pathogen infection), can have detrimental impacts on plant growth and agricultural productivity. To mitigate the impact of harsh environmental conditions and enhance crop yield, the application of plant growth-promoting bacteria (PGPB) has shown promising results [3].

P. megaterium, previously known as Bacillus megaterium is a Gram-positive, rod-shaped bacterium belonging to the Bacillota. Several studies have demonstrated its plant growth-promoting ability in normal and stress conditions. Through nutrient uptake, phytohormone synthesis, and enhancement of plant defense mechanisms, P. megaterium has been recognized as a PGPB [4].

In the present study, P. megaterium strain 10 was isolated from the rhizosphere of soybean soil collected from Daegu, South Korea (35°52'43.1"N 128°47'37.3"E). Briefly, 1 g of soil was serially diluted, then dilutions from 10-1 to 10-5 were spread on tryptic soy agar (TSA) and incubated at 30℃ for 24 to 48 h. Additionally, and for purification purposes, a bacterial colony was selected and sub-cultured twice on TSA agar and subsequently incubated for 24 to 48 h.

The genomic DNA extraction was performed from an overnight cultured bacterial cell at 30℃ in tryptic soy broth (TSB) using the Wizard® Genomic DNA Purification Kit (Promega, USA) following the manufacturer's instructions. The concentration and quality of DNA were assessed using the Qubit fluorometer 2.0 (Thermo Fisher Scientific, USA) and the NanoDrop UV-Vis spectrophotometer (Thermo Fisher Scientific), respectively. The sequencing library was prepared without DNA size selection and was generated using the Oxford Nanopore technology (ONT). The V14 kit chemistry (SQK-LSK114, Oxford Nanopore Technologies, United Kingdom) and the ligation kit NEBNext® Companion Module (New England Biolabs, USA) were utilized for library preparation based on the manufacturer's protocol. The genomic DNA was sequenced on an R10.4.1 flow cell using the MinION device of the ONT. In addition, Guppy v4.4.1 software was used to produce FASTQ files of the sequenced DNA [5]. Low-quality reads (5% worst fastq reads) were removed using Filtlong v0.2.1 with default settings. The genomic DNA sequencing was performed at the KNU NGS Core Facility (Republic of Korea).

The sequencing data showed the generation of 144,000 reads equivalent to 862,137,845 bp with an approximate coverage of 164 x and a relative N50 of 11,632 bp. Flye 2.9.1-b1780 was used for de novo genome assembly using the default parameters except for genome size which was set to 5 m (--genome-size 5m) [6]. Assembly results showed the presence of 5 circular contigs, with the biggest corresponding to P. megaterium chromosome of 4,815,034 bp with a G+C content of 38.2%. The rest of the four contigs correspond to plasmids P1, P2, P3, and P4 with sizes of 198,305 bp, 139,815 bp, 79,328 bp, and 61,901 bp, and G+C contents of 39.8%, 34.2%, 35.1%respectively. The bacterial genome was annotated using the Prokaryotic Genome Annotation Pipeline (PGAP), and the Rapid Annotation using Subsystem Technology (RAST server) version 2.0 [7, 8].

The annotation indicated that the chromosome of P. megaterium 10 encodes 5317 coding genes, 38 ribosomal RNAs, 130 transfer RNAs, 8 non-coding RNAs, and 45 pseudogenes (Table 1). Bacterial chromosome and plasmids were visualized using Proskee tool [9] (Fig. 1).

Table 1 . Genomic features of P. megaterium 10.

FeatureNumber
Number of contigs5
Chromosome size (bp)4,815,034
Coding genes (CDSs)5317
Ribosomal RNAs (rRNAs)38
Transfer RNAs (tRNAs)130
Non-coding RNAs (ncRNAs)8
Pseudogenes45


Figure 1.Circular genome map of P. megaterium 10. The outer blue circles present the annotation, location, and direction of predicted genes, the middle black circles show the GC% content and the inner circle indicates the GC skew, positive (green) and negative (purple).

The average nucleotide identity (ANI) was performed between the genome of P. megaterium strain 10 and the NCBI deposited sequence of P. megaterium strain WSH-002 with the accession number CP003017.1. An ANI of 99.2% was shared between the two genomes.

The functional annotation of P. megaterium genome revealed the presence of plant growth-promoting (PGP) and stress-resistance genes, as highlighted in Table 2. Notably, genes involved in nitrogen metabolism, such as NorDQ, Amt, and GltD were identified. Additionally, genes associated with phosphate, sulfate, potassium, and indole-3-acetic acid (IAA) metabolism, including PhoHPUR, CysAC, KdpABC, and TrpBD, were present [10, 11]. The annotation further showed the presence of iron acquisition systems, specifically siderophores production, which play a role in pathogen inhibition and nutrient assimilation [12]. These systems are encoded by genes X-ABC3, Fe-ABC1, ABC2, and ABC3 [13]. Furthermore, genes associated with stress resistance, including BetB, OpuAA, OpuAB, GbsB, SodBC, and GLPFK, were detected [14]. Osmoprotectant molecules like betaines, amino acids, and sugars synthesized under stress conditions are crucial for reactive oxygen species (ROS) detoxification [15]. Similarly, other detoxification genes, such as Chb, and ProAB, involved in stress alleviation, were also identified.

Table 2 . PGP and stress resistance genes annotation of P. megaterium 10.

GeneProductChromosome location
PGP related genesNorDNitric oxide reductase activation1842547-1844463
NorQNitric oxide reductase activation1841645-1842535
AmtAmmonium transporter2288510-2287227
GltDGlutamate synthase small subunit1772515-1773996
PhoHPhosphate starvation-inducible protein1103104-1104432
PhoPAlkaline phosphatase synthesis transcriptional regulatory protein4271064-4270348
PhoUPhosphate transport system regulatory protein4024271-4023612
PhoRPhosphate regulon sensor protein PhoR4270355-4268583
CysASulfate and thiosulfate import ATP-binding protein CysA1798136-1799202
CysCAdenylyl-sulfate kinase4420749-4421348
KdpAPotassium-transporting ATPase A chain2715008-2713341
KdpBPotassium-transporting ATPase B chain2713319-2711244
KdpCPotassium-transporting ATPase C chain2711230-2710664
X-ABC3Uncharacterized iron compound ABC uptake transporter, ATP-binding protein2840979-2840221
Fe-ABC1Iron compound ABC uptake transporter substrate-binding protein2840197-2839241
TrpBAnthranilate phosphoribosyltransferase3848094-3844706
TrpDIndole-3-glycerol phosphate synthase3847079-3846312
Stress response related genesAhpCAlkyl hydroperoxide reductase subunit C-like protein1073806-1074354
BetBBetaine aldehyde dehydrogenase4592262-4593746
OpuAAGlycine betaine ABC transport system, ATP-binding protein OpuAA1272624-1273877
OpuABGlycine betaine ABC transport system, permease protein OpuAB1273880-1274743
GbsBAlcohol dehydrogenase GbsB (type III)4593768-4594970
SodBSuperoxide dismutase [Fe]2390241-2389357
SodCSuperoxide dismutase [Cu-Zn] precursor1813126-1813716
GlpFGlycerol uptake facilitator protein262037-262864
GlpKGlycerol kinase262932-264422
ChbCyanoglobin; Hemoglobin-like protein HbN2080787-2081149
ProAGamma-glutamyl phosphate reductase1969310-1970572
ProBGlutamate 5-kinase / RNA-binding C-terminal domain PUA1968089-1969195


P. megaterium 10 has been proven to harbor potential genes that enhance plant growth and stress resistance. Hence, the agricultural application of this strain may improve plant yield and stress resilience under both biotic and abiotic conditions. The investigation of the functional properties of P. megaterium 10 has shown its potential role as a beneficial PGPB for maintaining plant fertility and productivity.

Nucleotide Sequence Accession Number(s)

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The genome of P. megaterium strain 10 and its plasmids P1, P2, P3, and P4 have been submitted to DDKJ/ENA/GenBank under the accession numbers CP162497.1, CP162498.1, CP162499.1, CP162500.1, and CP162501.1 respectively. BioProject and BioSample numbers are PRJ-NA1133222 and SAMN42379998 respectively.

Acknowledgments

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This research was supported by the Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ015697)" Rural Development Administration, and the biological materials specialized graduate program through the Korea Environmental Industry & Technology Institute(KEITI), funded by the Ministry of Environment(MOE), and by a grant from the Korea Basic Science Institute (National Research Facilities and Equipment Center) funded by the Ministry of Education(2021R1A6C101A416), Republic of Korea.

Conflict of Interest

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The authors have no financial conflicts of interest to declare.

References

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