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Microbiology and Biotechnology Letters

Genome Report(Note)

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Genome Report  |  Genome Report

Microbiol. Biotechnol. Lett. 2023; 51(3): 296-299

https://doi.org/10.48022/mbl.2307.07010

Received: July 24, 2023; Revised: September 13, 2023; Accepted: September 20, 2023

Whole-Genome Sequence of Priestia aryabhattai Strain S2 Isolated from the Rhizosphere of Soybean (Glycine max)

Amani Sliti1, Min-Ji Kim1, GyuDae Lee1, Yeong-Jun Park2, and Jae-Ho Shin1,2*

1Department of Applied Biosciences, 2NGS Core Facility, 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 of Priestia aryabhattai strain S2 isolated from the soybean rhizosphere. The genome consists of a single circular chromosome of 5,070,860 bp with a G+C content of 38.3% and 2 plasmids, P1(148,124 bp, GC content 33.3%) and P2 (76,418 bp, GC content 36.5%).

Keywords: Priestia aryabhattai, genome, soybean, rhizosphere

Body

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The increasing reliance on chemical fertilizers for enhancing agricultural productivity has raised grave environmental concerns. The uncontrolled usage of these chemicals has led to the pollution of the ecosystem, the contamination of food, and significant risks to human health. As an alternative approach, exploring the potential of plant growth-promoting bacteria (PGPB) offers a sustainable solution to improve crop production and stress resilience while reducing our dependence on harmful chemicals such as pesticides [1]. Consequently, biofertilizers based on PGPB inoculants are becoming more prominent due to their eco-friendly nature and ability to enhance soil fertility and plant growth sustainably [2, 3].

Priestia aryabhattai, formerly known as Bacillus aryabhattai is, a rod-shaped Gram-positive bacterium [4]. It is known for its remarkable ability to promote plant growth through various mechanisms such as the synthesis of phytohormones, the assimilation of nutrients, and the enhancement of plant defense mechanisms, earning its classification as PGPB [5, 6].

In this study, P. aryabhattai strain S2 was isolated from the soybean rhizosphere collected in Daegu, South Korea (35°52'43.1"N 128°47'37.3"E). In short, 1 g of soil was serially diluted; subsequently, dilutions from 10-1 to 10-6 were spread on tryptic soy agar (TSA). The plates were incubated at 30℃ for 48 to 72 h. A bacterial colony of the strain was selected and sub-cultured twice on TSA for purification and isolation of a single colony.

Genomic DNA was extracted from bacterial cells grown overnight at 30℃ in tryptic soy broth using the Wizard® Genomic DNA Purification Kit (Promega, USA) following the manufacturer’s instructions. Further, DNA concentration and quality were measured using the Qubit fluorometer 2.0 (Thermo Fisher Scientific, USA) and the NanoDrop UV-Vis spectrophotometer (Thermo Fisher Scientific), respectively. Prior to the construction of the sequencing library, genomic DNA was not subjected to size selection. Furthermore, the sequencing library was constructed with the Oxford Nanopore Technology using the V14 kit chemistry (SQK-LSK114, Oxford Nanopore Technologies, UK) according to the manufacturer’s instructions. This technique includes minimal fragmentation of the genomic input DNA in a sequence independent manner. The ligation kit was used to prepare the sequencing library using the NEBNext ® Module (New England Biolabs, USA). Additionally, Genomic DNA was sequenced on an R10.4.1 flow cell using the MinION device. FASTQ files were generated by Guppy v4.4.1 software with high accuracy mode. In addition, low-quality reads (5% of worst fastq reads) were removed using Filtlong v0.2.1 with default parameters [7]. The sequencing was performed at the KNU NGS Core Facility (Korea).

The sequencing produced a total of 116,000 reads (791,903,685 bp) with an estimated genome coverage of 149 x and a relative N50 (rN50) of 13,578 bp. A de novo genome assembly was conducted using Flye 2.9-b1768 with the default parameters expect for genome size (--genome-size 5.3m), number of iterations (--iterations 5), and assembly coverage (--asm-coverage 40) [8]. The genome assembly was evaluated using the Quality Assessment Tool for Genome Assemblies v5.0.2 software [9]. The assembly resulted in the generation of 3 circular contigs, with the largest one corresponding to P. aryabhattai chromosome of 5,070,860 bp, with a GC content of 38.3%. The second and the third contigs correspond to plasmids P1 and P2 with sizes of 148,124 bp and 76,418 bp, and GC content of 33.3% and 36.5%, respectively. The bacterial genome was annotated using the Prokaryotic Genome Annotation Pipeline (PGAP) build6494, and the Rapid Annotation using Subsystem Technology (RAST server) version 2.0, respectively [10, 11]. The annotation revealed that P. aryabhattai S2 chromosome encodes 5,324 coding genes, 42 ribosomal RNAs, 131 transfer RNAs, 6 non-coding RNAs and 42 pseudogenes (Table 1). Finally, bacterial genome and plasmids were visualized using Proskee online tool [12] (Fig. 1).

Table 1 . Genome features of P. aryabhattai S2 annotated by PGAP.

Genome featuresValue
Number of contigs3
Chromosome size (bp)5,070,860 bp
Coding genes (CDSs)5,324
Ribosomal RNAs (rRNAs)42
Transfer RNAs (tRNAs)131
Non-coding RNAs (ncRNAs)6
Pseudogenes42


Figure 1.Circular genome map of Priestia aryabhattai S2 with its plasmids P1 and P2. 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 was conducted between the genome of Priestia aryabhattai strain S2 and the NCBI deposited Priestia aryabhattai strain K13 with the accession numbers CP024035.1, CP024036.1, CP024037.1.The comparison tool showed a 99.46% similarity between the 2 genomes.

Genomic functional annotation of P. aryabhattai S2 revealed the presence of potent genes associated with plant growth promotion (PGP) and stress resistance, as outlined in Table 2. Notable examples include gltD, involved in nitrogen assimilation; trpABCDE responsible for indole-3-acetic acid (IAA) synthesis; ureC, which catalyzes the hydrolysis of urea to produce ammonia; ilvB, responsible for the synthesis of acetoin, a plant growth-promoting signalling molecule; pstAC, facilitating the uptake and utilization of phosphate; and nirBD responsible for nitrite reduction and assimilation. Additionally, we assessed the ability of P. aryabhattai S2 to thrive under high salinity stress conditions, with concentrations of up to 10%, using TSA medium supplemented with NaCl. Furthermore, genomic analysis revealed the presence of genes with antioxidant properties that can mitigate oxidative stress in plants during both biotic and abiotic stress conditions, including trxB, msrA, hmpA, and nhaC. trxB and msrA are involved in suppressing oxidative stress responses, similarly hmpA is a NO-inducible flavohemoprotein contributing to the detoxification of free radicals under stress conditions, and nhaC plays a role in maintaining ion homeostasis by regulating extracellular ion concentrations. Owing to its unique PGP features, P. aryabhattai S2 has the potential to revolutionize sustainable agriculture practices and reduce reliance on chemical fertilizers and pesticides. For instance, it can be applied as a biofertilizer to enrich soil fertility and enhance nutrient uptake in plants by promoting phosphate solubilization and nitrogen. Furthermore, P. aryabhattai S2 can be employed to mitigate the impact of abiotic stress factors, such as salinity and drought, on plants through its genomic features related to oxidative stress control and alleviation.

Table 2 . Priestia aryabhattai S2 PGP genes annotation.

GeneProteinChromosome Location
gltDGlutamate synthase [NADPH] small subunit3287188-3288669
trpATryptophan synthase subunit beta1195140-1195955
trpBTryptophan synthase beta chain1193939-1195153
trpCIndole-3-glycerol phosphate synthase1192558-1193325
trpDAnthranilate phosphoribosyltransferase1191543-1192568
trpEAnthranilate synthase component I1190036-1191550
ureCUrease subunit alpha2367895-2369604
ilvBAcetolactate synthase large subunit825575-827302
pstAPhosphate ABC transporter permease908697-909626
pstCPhosphate ABC transporter permease subunit907813..908700
nirBNitrite reductase large subunit4172757-4175171
nirDnitrite reductase small subunit4172412-4172738
trxBThioredoxin-disulfide reductase483345-484298
nhaCNa+/H+ antiporter483345-484298
hmpANO-inducible flavohemoprotein4963721-4964902
msrAPeptide-methionine (S)-S-oxide reductase4972793-4973308


The applications of P. aryabhattai S2 can be extended to other crops, including wheat, rice, and tomato plants, which are susceptible to many abiotic stressors such as salinity. Other studies showed the potential of distinctive strains of P. aryabhattai to enhance crop production by the alleviation of salinity stress impact on plants growth, yield, and quality [13, 14].

The genomic investigation of P. aryabhattai strain S2 has been pivotal to explore its unique genetic features. This valuable insight highlights the promising potential of this PGPB for future application as a sustainable biofertilizer to enhance plant growth, yield and stress tolerance.

Nucleotide Sequence Accession Number(s)

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The genome sequence of P. aryabhattai strain S2 and its plasmids P1 and P2 have been deposited in DDBJ/ENA/GenBank under the accession numbers CP129633, CP129634, and CP129635 respectively. The associated BioProject accession number is PRJNA991722, the Bio-Sample accession number is SAMN36315346 and the SRA accession number is SRR25269555.

Acknowledgments

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This work was carried out with the support of “The Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ015697)” Rural Development Administration, Republic of Korea. Supported by Korea Basic Science Institute (National Research Facilities and Equipment Center) grant funded by the Ministry of Education (2021R1A6C101A416) and by a project to train professional personnel in biological materials by the Ministry of Environment. We express our sincere appreciation to the Kyungpook National University NGS Core Facility for collaborative efforts in facilitating the Nanopore sequencing.

Conflict of Interest

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The authors have no financial conflicts of interest to declare.

References

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