# Whole-Genome Sequence of Burkholderia ambifaria Strain Q53, a Potential Plant Growth Promoter Isolated from the Rhizosphere of Peanut

Fiorela Nievas, Santiago Revale, Emiliano Foresto, María Evangelina

Carezzano, Emiliano Primo, Pedro Alzari, Mariano Martinez, Mathilde

Ben-Assaya, Damien Mornico, Maricel Santoro, et al.

► To cite this version:

Fiorela Nievas, Santiago Revale, Emiliano Foresto, María Evangelina Carezzano, Emiliano Primo, et al. Whole-Genome Sequence of Burkholderia ambifaria Strain Q53, a Potential Plant Growth Promoter Isolated from the Rhizosphere of Peanut. Microbiology Resource Announcements, 2023, 12 (5), pp.e0002123. 10.1128/mra.00021-23. pasteur-04567337

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# Whole-Genome Sequence of *Burkholderia ambifaria* Strain Q53, a Potential Plant Growth Promoter Isolated from the Rhizosphere of Peanut

Fiorela Nievas,ª Santiago Revale,ᵇ Emiliano Foresto,ª María Evangelina Carezzano,ª Emiliano Primo,ª Pedro Alzari,ᶜ Mariano Martínez,ᶜ Mathilde Ben-Assaya,ᶜ Damien Mornico,ᵈ Maricel Santoro,ᵉ Walter Giordano,ª Pablo Boginoª

Fiorela Nievas and Santiago Revale contributed equally to this work. Author order was determined by seniority in working with peanut rhizospheric strains.

**ABSTRACT** We report the complete genome sequence of *Burkholderia ambifaria* strain Q53, an environmental rhizobacterium isolated from the rhizosphere of peanut plants. The genome consists of 7.4 Mbp distributed into three circular chromosomes and was determined using a hybrid long- and short-read assembly approach.

Members of the *Burkholderia* genus are versatile betaproteobacteria that inhabit diverse biotic and abiotic environments (1). They have a wide range of ecological roles (2, 3) which have attracted scientific interest due to their potential biotechnological applications (4).

Here, we announce the complete annotated genome of *Burkholderia ambifaria* Q53, a rhizospheric strain isolated from peanut roots. The plants were collected from an agricultural field in the south of the province of Córdoba, Argentina (33°31'38"S, 64°39'13"W). The collection of the rhizospheric soil and the isolation of the strain were carried out as described previously (5).

A pure culture of the strain was aerobically grown in LB medium (6) at 30°C until reaching the late exponential growth phase. This was the source for the total DNA, obtained with a DNeasy Blood & Tissue kit (Qiagen) for Illumina sequencing and a Promega Wizard HMW DNA extraction kit (Promega) for Oxford Nanopore Technologies sequencing. The genome was assembled through a hybrid approach including short Illumina reads and long Oxford Nanopore reads. The library was prepared with a Nextera XT DNA library preparation kit and sequenced on Illumina NextSeq 500, with a paired-end (PE) 150-bp read configuration. The sample was processed with an Oxford Nanopore Technologies rapid barcoding sequencing kit (SQK-RBK004) and a native barcoding genomic DNA sequencing kit (SQK-LSK109 with EXP-NBD104). The products of each were sequenced in two Flongle flow cells. Data were base called with Guppy v4.2.2, using the high-accuracy model and the -trim_barcodes option.

We obtained 5,397,708 Illumina PE reads and 43,449 Nanopore long reads (average of 4,981 bp, N₅₀ of 9,460 bp), predicting 109-fold coverage and 29-fold coverage, respectively. Hybrid genome assembly was performed on the raw reads using the nf-core/bacass pipeline (commit ceebac0) with default parameters (7). This resulted in three contigs that were closed by manually analyzing the overlapped ends on Geneious software version 2019 2.1 (8). The reported genome consists of three chromosomes, which agrees with reports of

**Editor** David A. Baltrus, University of Arizona

**Copyright** © 2023 Nievas et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

Address correspondence to Pablo Bogino, pbogino@exa.unrc.edu.ar, or Walter Giordano, wgiordano@exa.unrc.edu.ar.

The authors declare no conflict of interest.

**Received** 12 January 2023

**Accepted** 29 March 2023

**Published** 11 April 2023

<footnote>ªInstituto de Biotecnología Ambiental y Salud (INBIAS-CONICET), Departamento de Biología Molecular, Facultad de Ciencias Exactas, Físico-Químicas y Naturales, Universidad Nacional de Río Cuarto, Río Cuarto, Córdoba, Argentina</footnote>

<footnote>ᵇWellcome Centre for Human Genetics, University of Oxford, Oxford, United Kingdom</footnote>

<footnote>ᶜUnité de Microbiologie Structurale, Institut Pasteur, CNRS UMR 3528, Université de Paris, Paris, France</footnote>

<footnote>ᵈHub de Bioinformatique et Biostatistique - Département Biologie Computationnelle, CNRS USR 3756, Institut Pasteur, Paris, France</footnote>

<footnote>ᵉDepartment of Biochemistry, Max Planck for Chemical Ecology, Jena, Germany</footnote>
several large replicons for this genus (9). The first chromosome had 3,495,354 bp, the second 2,728,612 bp, and the third 1,179,612 bp. Their G+C content was 66.8%, 66.6%, and 66.3%, respectively.

The complete genome, which was annotated using the NCBI Prokaryotic Genomes Annotation Pipeline (PGAP) (10–12), consists of 6,453 protein-coding sequences, six complete ribosomal operons, and 66 tRNAs.

As reported earlier for *B. ambifaria* (13, 14), the genome of Q53 harbors several genes related to plant growth promotion. For instance, there are genes predicted to encode the biosynthesis, transport, export, and iron uptake of siderophores, others predicted to be responsible for auxin biosynthesis, and yet others predicted to encode secondary metabolite biosynthesis, such as those related to antifungal activity. In addition, the genome has predicted genes associated with motility, biofilm development, and quorum sensing, all of which are typical of an environmental rhizospheric lifestyle. This complete sequence could be crucial for more in-depth research on the biological features of plant-associated *Burkholderia*.

**Data availability.** The complete genome sequence of *Burkholderia* sp. strain Q53 is available at NCBI GenBank under accession numbers CP092843.1, CP092844.1, and CP092845.1 with BioProject identifier (ID) PRJNA782308 and BioSample accession number SAMN23371909. Raw data reads are available at NCBI's Sequence Read Archive under accession numbers SRR17038419 to SRR17038423.

## ACKNOWLEDGMENTS

This work was supported by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT). We thank OGC at the Wellcome Centre for Human Genetics for the sequencing data and BMRC for processing them (supported by Wellcome Trust Core Award grant 203141/Z/16/Z and the NIHR Oxford BRC).

We are also grateful to Vincent Enouf from Unité de Génétique Moléculaire des Virus à ARN-UMR3569 CNRS, Université de Paris, Centre National de Référence Virus des Infections Respiratoires (dont la grippe), to Plateforme de Microbiologie Mutualisée (P2M) and the Pasteur International Bioresources network (PIBnet), to Institut Pasteur Paris for providing the resources for Illumina sequencing, and finally, to F. Sgarlatta for proofreading the manuscript.

The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, or the Department of Health.

## REFERENCES

1. Coenye T, Vandamme P. 2003. Diversity and significance of Burkholderia species occupying diverse ecological niches. *Environ Microbiol* 5:719–729. https://doi.org/10.1046/j.1462-2920.2003.00471.x.

2. Vial L, Chapalain A, Groleau MC, Deziel E. 2011. The various lifestyles of the Burkholderia cepacia complex species: a tribute to adaptation. *Environ Microbiol* 13:1–12. https://doi.org/10.1111/j.1462-2920.2010.02343.x.

3. Tagele SB, Kim SW, Lee HG, Kim HS, Lee YS. 2018. Effectiveness of multi-trait Burkholderia contaminans KNU17B1 in growth promotion and man-agement of banded leaf and sheath blight in maize seedling. *Microbiol Res* 214:8–18. https://doi.org/10.1016/j.micres.2018.05.004.

4. Kunakom S, Eustáquio AS. 2019. Burkholderia as a source of natural products. *J Nat Prod* 82:2018–2037. https://doi.org/10.1021/acs.jnatprod.8b01068.

5. Bogino P, Abod A, Nievas F, Giordano W. 2013. Water-limiting conditions alter the structure and biofilm-forming ability of bacterial multispecies communities in the alfalfa rhizosphere. *PLoS One* 8:e79614. https://doi.org/10.1371/journal.pone.0079614.

6. Sambrook JF, Russell DW. 2001. Molecular cloning: a laboratory manual, 3rd ed, p A2.2. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

7. Ewels PA, Peltzer A, Fillinger S, Patel H, Alneberg J, Wilm A, Garcia MU, Di Tommaso P, Nahnsen S. 2020. The nf-core framework for community-curated bioinformatics pipelines. *Nat Biotechnol* 38:276–278. https://doi.org/10.1038/s41587-020-0439-x.

8. Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, Buxton S, Cooper A, Markowitz S, Duran C, Thierer T, Ashton B, Meintjes P, Drummond A. 2012. Geneious basic: an integrated and extensible desktop software platform for the organization and analysis of sequence data. *Bioinformatics* 28:1647–1649. https://doi.org/10.1093/bioinformatics/bts199.

9. Johnson SL, Bishop-Lilly KA, Ladner JT, Daligault HE, Davenport KW, Jaissle J, Frey KG, Koroleva GI, Bruce DC, Coyne SR, Broomall SM, Li P-E, Teshima H, Gibbons HS, Palacios GF, Rosenzweig CN, Redden CL, Xu Y, Minogue TD, Chain PS. 2015. Complete genome sequences for 59 Burkholderia isolates, both pathogenic and near neighbor. *Genome Announc* 3:e00159-15. https://doi.org/10.1128/genomeA.00159-15.

10. Tatusova T, DiCuccio M, Badretdin A, Chetvernin V, Nawrocki EP, Zaslavsky L, Lomsadze A, Pruitt KD, Borodovsky M, Ostell J. 2016. NCBI Prokaryotic Genome Annotation Pipeline. *Nucleic Acids Res* 44:6614–6624. https://doi.org/10.1093/nar/gkw569.

11. Haft DH, DiCuccio M, Badretdin A, Brover V, Chetvernin V, O'Neill K, Li W, Chitsaz F, Derbyshire MK, Gonzales NR, Gwadz M, Lu F, Marchler GH, Song JS, Thanki N, Yamashita RA, Zheng C, Thibaud-Nissen F, Geer LY, Marchler-Bauer A, Pruitt KD. 2018. RefSeq: an update on prokaryotic genome annotation and curation. *Nucleic Acids Res* 46:D851–D860. https://doi.org/10.1093/nar/gkx1068.

12. Li W, O'Neill KR, Haft DH, DiCuccio M, Chetvernin V, Badretdin A, Coulouris G, Chitsaz F, Derbyshire MK, Durkin AS, Gonzales NR, Gwadz M, Lanczycki CJ,
several large replicons for this genus (9). The first chromosome had 3,495,354 bp, the second 2,728,612 bp, and the third 1,179,612 bp. Their G+C content was 66.8%, 66.6%, and 66.3%, respectively.

The complete genome, which was annotated using the NCBI Prokaryotic Genomes Annotation Pipeline (PGAP) (10–12), consists of 6,453 protein-coding sequences, six complete ribosomal operons, and 66 tRNAs.

As reported earlier for *B. ambifaria* (13, 14), the genome of Q53 harbors several genes related to plant growth promotion. For instance, there are genes predicted to encode the biosynthesis, transport, export, and iron uptake of siderophores, others predicted to be responsible for auxin biosynthesis, and yet others predicted to encode secondary metabolite biosynthesis, such as those related to antifungal activity. In addition, the genome has predicted genes associated with motility, biofilm development, and quorum sensing, all of which are typical of an environmental rhizospheric lifestyle. This complete sequence could be crucial for more in-depth research on the biological features of plant-associated *Burkholderia*.

**Data availability.** The complete genome sequence of *Burkholderia* sp. strain Q53 is available at NCBI GenBank under accession numbers CP092843.1, CP092844.1, and CP092845.1 with BioProject identifier (ID) PRJNA782308 and BioSample accession number SAMN23371909. Raw data reads are available at NCBI's Sequence Read Archive under accession numbers SRR17038419 to SRR17038423.

## ACKNOWLEDGMENTS

This work was supported by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT). We thank OGC at the Wellcome Centre for Human Genetics for the sequencing data and BMRC for processing them (supported by Wellcome Trust Core Award grant 203141/Z/16/Z and the NIHR Oxford BRC).

We are also grateful to Vincent Enouf from Unité de Génétique Moléculaire des Virus à ARN-UMR3569 CNRS, Université de Paris, Centre National de Référence Virus des Infections Respiratoires (dont la grippe), to Plateforme de Microbiologie Mutualisée (P2M) and the Pasteur International Bioresources network (PIBnet), to Institut Pasteur Paris for providing the resources for Illumina sequencing, and finally, to F. Sgarlatta for proofreading the manuscript.

The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, or the Department of Health.

## REFERENCES

1. Coenye T, Vandamme P. 2003. Diversity and significance of Burkholderia species occupying diverse ecological niches. *Environ Microbiol* 5:719–729. https://doi.org/10.1046/j.1462-2920.2003.00471.x.

2. Vial L, Chapalain A, Groleau MC, Deziel E. 2011. The various lifestyles of the Burkholderia cepacia complex species: a tribute to adaptation. *Environ Microbiol* 13:1–12. https://doi.org/10.1111/j.1462-2920.2010.02343.x.

3. Tagele SB, Kim SW, Lee HG, Kim HS, Lee YS. 2018. Effectiveness of multi-trait Burkholderia contaminans KNU17B1 in growth promotion and man-agement of banded leaf and sheath blight in maize seedling. *Microbiol Res* 214:8–18. https://doi.org/10.1016/j.micres.2018.05.004.

4. Kunakom S, Eustáquio AS. 2019. Burkholderia as a source of natural products. *J Nat Prod* 82:2018–2037. https://doi.org/10.1021/acs.jnatprod.8b01068.

5. Bogino P, Abod A, Nievas F, Giordano W. 2013. Water-limiting conditions alter the structure and biofilm-forming ability of bacterial multispecies communities in the alfalfa rhizosphere. *PLoS One* 8:e79614. https://doi.org/10.1371/journal.pone.0079614.

6. Sambrook JF, Russell DW. 2001. Molecular cloning: a laboratory manual, 3rd ed, p A2.2. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

7. Ewels PA, Peltzer A, Fillinger S, Patel H, Alneberg J, Wilm A, Garcia MU, Di Tommaso P, Nahnsen S. 2020. The nf-core framework for community-curated bioinformatics pipelines. *Nat Biotechnol* 38:276–278. https://doi.org/10.1038/s41587-020-0439-x.

8. Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, Buxton S, Cooper A, Markowitz S, Duran C, Thierer T, Ashton B, Meintjes P, Drummond A. 2012. Geneious basic: an integrated and extensible desktop software platform for the organization and analysis of sequence data. *Bioinformatics* 28:1647–1649. https://doi.org/10.1093/bioinformatics/bts199.

9. Johnson SL, Bishop-Lilly KA, Ladner JT, Daligault HE, Davenport KW, Jaissle J, Frey KG, Koroleva GI, Bruce DC, Coyne SR, Broomall SM, Li P-E, Teshima H, Gibbons HS, Palacios GF, Rosenzweig CN, Redden CL, Xu Y, Minogue TD, Chain PS. 2015. Complete genome sequences for 59 Burkholderia isolates, both pathogenic and near neighbor. *Genome Announc* 3:e00159-15. https://doi.org/10.1128/genomeA.00159-15.

10. Tatusova T, DiCuccio M, Badretdin A, Chetvernin V, Nawrocki EP, Zaslavsky L, Lomsadze A, Pruitt KD, Borodovsky M, Ostell J. 2016. NCBI Prokaryotic Genome Annotation Pipeline. *Nucleic Acids Res* 44:6614–6624. https://doi.org/10.1093/nar/gkw569.

11. Haft DH, DiCuccio M, Badretdin A, Brover V, Chetvernin V, O'Neill K, Li W, Chitsaz F, Derbyshire MK, Gonzales NR, Gwadz M, Lu F, Marchler GH, Song JS, Thanki N, Yamashita RA, Zheng C, Thibaud-Nissen F, Geer LY, Marchler-Bauer A, Pruitt KD. 2018. RefSeq: an update on prokaryotic genome annotation and curation. *Nucleic Acids Res* 46:D851–D860. https://doi.org/10.1093/nar/gkx1068.

12. Li W, O'Neill KR, Haft DH, DiCuccio M, Chetvernin V, Badretdin A, Coulouris G, Chitsaz F, Derbyshire MK, Durkin AS, Gonzales NR, Gwadz M, Lanczycki CJ,
Song JS, Thanki N, Wang J, Yamashita RA, Yang M, Zheng C, Marchler-Bauer A, Thibaud-Nissen F. 2021. RefSeq: expanding the Prokaryotic Genome Annotation Pipeline reach with protein family model curation. Nucleic Acids Res 49: D1020–D1028. https://doi.org/10.1093/nar/gkaa1105.

13. Batista BD, Taniguti LM, Monteiro-Vitorello CB, Azevedo JL, Quecine MC. 2016. Draft genome sequence of *Burkholderia ambifaria* RZ2MS16, a plant growth-promoting rhizobacterium isolated from guarana, a tropical

plant. Genome Announc 4:e00125-16. https://doi.org/10.1128/genomeA.00125-16.

14. Alvarez F, Simonetti E, Draghi WO, Vinacour M, Palumbo MC, Fernández Do Porto D, Montecchia MS, Roberts IN, Ruiz JA. 2022. Genome mining of Burkholderia ambifaria strain T16, a rhizobacterium able to produce antimicrobial compounds and degrade the mycotoxin fusaric acid. World J Microbiol Bio-technol 38:114. https://doi.org/10.1007/s11274-022-03299-0.
Song JS, Thanki N, Wang J, Yamashita RA, Yang M, Zheng C, Marchler-Bauer A, Thibaud-Nissen F. 2021. RefSeq: expanding the Prokaryotic Genome Annotation Pipeline reach with protein family model curation. Nucleic Acids Res 49: D1020–D1028. https://doi.org/10.1093/nar/gkaa1105.

13. Batista BD, Taniguti LM, Monteiro-Vitorello CB, Azevedo JL, Quecine MC. 2016. Draft genome sequence of *Burkholderia ambifaria* RZ2MS16, a plant growth-promoting rhizobacterium isolated from guarana, a tropical

plant. Genome Announc 4:e00125-16. https://doi.org/10.1128/genomeA.00125-16.

14. Alvarez F, Simonetti E, Draghi WO, Vinacour M, Palumbo MC, Fernández Do Porto D, Montecchia MS, Roberts IN, Ruiz JA. 2022. Genome mining of Burkholderia ambifaria strain T16, a rhizobacterium able to produce antimicrobial compounds and degrade the mycotoxin fusaric acid. World J Microbiol Bio-technol 38:114. https://doi.org/10.1007/s11274-022-03299-0.