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Complete genome sequence of a highly effective diazotroph Ensifer sesbaniae SDT23 isolated from soybean root nodules in Vietnam
Korean J. Microbiol. 2022;58(3):179-182
Published online September 30, 2022
© 2022 The Microbiological Society of Korea.

Thi Hieu Thu Nguyen, Thuy Hang Dinh, and Cao Son Trinh*

Institute of Microbiology and Biotechnology, Vietnam National University, Hanoi 122863, Vietnam
Correspondence to: *E-mail: caoson@vnu.edu.vn; Tel.: +84-2437547407; Fax: +84-2437547407
Received May 12, 2022; Revised June 16, 2022; Accepted June 21, 2022.
Abstract
The diazotroph Ensifer sesbaniae SDT23 was isolated from root nodules of healthy soybean plants in the Hanoi countryside. The strain possesses several agricultural beneficial characteristics, i.e. fixing nitrogen, producing Indole-3-acetic acid (IAA) and Extracellular polysaccharides (EPS), and thus has good potential in the application for organic agriculture and soil amendment. Here we show the draft genome sequence of strain SDT23 which is composed of 6,870,960 bp. with a GC content of 62.11% and approximately 6427 protein-coding sequences. The genome annotation indicated nitrogen fixation- and nodulation-related genes, along with genes involved in IAA, siderophore, and EPS biosynthesis, confirming the physiological properties of the strain.
Keywords : Ensifer sesbaniae, Diazotroph, draft genome, organic agriculture
Body

The diazotrophic microorganisms perform either symbiotic (endophytic) or free-living lifestyles, converting atmospheric N2 into usable form ammonium, supporting up to 60% of plant needs globally (Menéndez et al., 2017). The diazotrophs have been exploited in microbial formulas for use in agriculture, such as Gluconacetobacter diazotrophicus, Azorhizobium caulinodans, and Bradyrhizobium sp. (Azotic Technologies, Pivot Bio, etc.). In special cases, several diazotrophs also possess useful properties promoting plant growth such as enhancing nutrient uptake (via solubilizing K, P, and Fe species in soil) and producing phytohormones or compounds against phytopathogens (Choudhary et al., 2011; García-Faile et al., 2015).

The genus Ensifer (syn. Sinorhizobium) consists of both symbiotic and nonsymbiotic species, has been attracting attention in studying the evolution of symbiotic potential (Fagorzi et al., 2020). Ensifer species make up more than 1% of bacterial endophytes isolated, showing their potential in promoting organic agriculture. Heading to this line, the strain E. sesbaniae SDT23 (deposited at VTCC under accession number VTCC60024) owing several traits beneficial for organic agriculture was subjected to analysis of its whole genome.

The genomic DNA of Ensifer sesbaniae SDT2.3 was extracted using the E.Z.N.A Bacterial DNA kit (OMEGA D3350-01). The whole-genome sequencing of strain SDT23 was conducted at Macrogen Inc. using the Illumina Hiseq 2500-150PE system. Raw reads were processed as described previously (Chaitankar et al., 2016). Quality control was done by FastQC, and short and low-quality reads were trimmed by Trimmomatic (Bolger et al., 2014). After being quality-filtered, the de novo assembly was operated by SPAdes (Bankevich et al., 2012). Whole-genome annotation was accomplished by Prokka, and additional analyses of hypothesizing gene function were performed by Rapid Annotation using Subsystem Technology (RAST) with the SEED database (Aziz et al., 2008). The genome was compared against another E. sesbaniae genome, strain CCBAU 65729 (GCA_013283665.1), and several other Ensifer species, including E. glycinis (GCA_001651865.1), E. aridi (GCA_ 000510685.1), and E. psoraleae (GCA_013283645.1).

The coverage of the draft genome sequencing for strain SDT23 was 101.6×. The whole genome consists of 89 contigs with a total size of 6,870,960 bp, the G + C content of 62.11%, and approximately 6275 protein-coding sequences (Table 1). The 16S rRNA sequence of strain SDT23 is 1,345 bp long and has 100% similarity with Ensifer sesbaniae (OM570606). The whole genome sequences of Ensifer sesbaniae SDT23 showed high similarity to that of other Ensifer species, i.e. E. aridi, E. psoraleae, E. glycinis, and E. sesbaniae CCBAU 65729, with the average nucleotide identity (ANI) calculated as 80.27%, 80.21%, 81.98%, and 98.86% respectively (ANI calculator, Ezbiocloud). In particular, the high G + C content of around 62% seems to be the genus-specific characteristic of the Ensifer species (Table 1).

Genomic features of <italic>E. sesbaniae</italic> SDT23 in comparison with other <italic>Ensifer</italic> species
Genomic features E. sesbaniae SDT23 E. sesbaniae CCBAU 65729 E. glycinis CCBAU 23380 E. aridiTW10 E. psoraleae CCBAU 65732
Genome size (bp) 6,870,960 6,897,201 6,039,294 6,802,256 7,427,611
Contigs 89 12 68 57 67
G + C content (%) 62.11 62.1 62.4 61.7 61.3
Total gene number 6,560 6,573 5,536 6,232 7,028
CDS 6,275 6,192 5,327 5,946 6,531
rRNA genes 3 3 3 3 3
tRNA genes 49 53 48 50 53
Other RNA 4 4 1 4 4
Pseudo-genes 229 321 157 229 438


RAST analyses predicated 1692/6427 (25%) genes with functions categorized into groups in SEED viewer format. We found 11 nitrogen fixation-related genes in the nitrogen metabolism category, 5 siderophore antharachelin biosynthesis genes in the ion acquisition and metabolism category, 5 auxin biosynthesis genes in the secondary metabolism category, 4 EPS biosynthesis genes and 10 nodulation protein-encoding genes (Table 2).

Gene loci related to the agriculture-beneficial traits of strain <italic>E. sesbaniae</italic> SDT23
No. Gene Start–Stop Size (bp) Strand Predicted function
Nitrogen fixing
1 nifW 1639–1187 453 - Nitrogenase stabilizing/protective protein
2 nifT 2752–2522 231 - NifT protein
3 nifZ 3072–2749 324 - NifZ protein
4 nifB 4863–3382 1482 - Nitrogenase FeMo-cofactor synthesis FeS core scaffold and assembly protein
5 nifA 6734–5088 1647 - Nitrogenase (molybdenum-iron)-specific transcriptional regulator NifA
6 nifX 14567–13593 975 - Nitrogenase FeMo-cofactor carrier protein
7 nifN 15973–14564 1410 - Nitrogenase FeMo-cofactor scaffold and assembly protein
8 nifE 17473–15983 1491 - Nitrogenase FeMo-cofactor scaffold and assembly protein
9 nifH 235–1125 891 + Nitrogenase (molybdenum-iron) reductase and maturation protein
10 nifX2 283–771 489 + NifX-associated protein
11 nifQ 13115–13834 720 + Nitrogenase FeMo-cofactor synthesis molybdenum delivery protein
Nodulation
1 nod 3495–5288 1793 + Nodulation protein
2 nodD 5833–6492 659 + Nodulation protein D (transcriptional regulator, LysR family
13653–13868 215 +
3 nodD2 4596–5534 939 + Nodulation protein D2
13527–12562 966 -
4 nodC 8885–9532 647 + Nodulation protein C
5 nodB 9529–10119 590 + Nodulation protein B
6 nodA 10527–11150 623 + Nodulation protein A
7 nodN 188.427–188.978 551 + Nodulation protein N
8 nolO 3448–3561 113 + Nodulation protein nolO
5399–5527 128 +
9 nolB 17626–18348 722 + Nodulation protein nolB
10 nolU 16106–15480 627 - Nodulation protein NolU
Growth phytohormone biosynthesis
1 APRT 282.790–283.803 1014 + Anthranilate phosphoribosyltransferase
2 AAD 379.204–380.622 1419 + Aromatic-L-amina-acid decarboxylase
3 TSa 49.200–48.361 840 - Tryptophan synthase alpha chain
4 TSb 50.425–49.205 1221 - Tryptophan synthase beta chain
5 PRAI 51.096–50.422 675 - Phosphoribosylanthranilate isomerase
Siderophore production
1 FhuF 297.463–298.218 756 + Ferric reductase
2 HumS 623.951–625.009 1059 + Hemin transport protein
3 HBP 625.031–625.981 951 + Periplasmic hemin-binding protein
4 PP_7 625.998–627.086 1089 + Hemin ABC transporter, permease protein
5 ATPb 627.095–627.886 792 + ABC-type hemin transport system, ATPase component
EPS production
1 GP 176.288–178.825 2538 + Glycogen phosphorylase
2 GBr 178.822–181.032 2211 + 1,4-alpha-glucan (glycogen) branching enzyme, GH-13-type
3 GAT 181.153–182.415 1263 + Glucose-1-phosphate adenylyl-transferase
4 GS 182.427–183.869 1443 + Glycogen synthase, APD-glucose trans-glucosylase


The findings on the genome of strain SDT23 were intimately correlated to its physiological properties beneficial to agriculture, i.e. fixing nitrogen by forming plant root nodules, and producing IAA and EPS. The bacterium is a plant endophyte, it is expected that its colonization in plant compartments would lead to the plant growth enhancement effects that strain SDT23 displayed upon in planta conditions.

Nucleotide sequence accession number

The draft genome sequence of Ensifer sesbanise SDT23 was registered on NCBI GenBank under the accession number GCF_023108975.1 (WGS: JALMMB010000001-JALMMB0 10000089).

Acknowledgments

The study was supported by Grant No NVQG-2021/DT.07 from the Vietnam Ministry of Science and Technology.

Conflict of Interest

The authors have no conflict of interest to report.

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