Draft genome sequence of Bradyrhizobium manausense strain BR 3351T, an effective symbiont isolated from Amazon rainforest

Jean Luiz Simões-Araújo Norma Gouvêa Rumjanek Gustavo Ribeiro Xavier Jerri Édson Zilli About the authors

ABSTRACT

The strain BR 3351T (Bradyrhizobium manausense) was obtained from nodules of cowpea (Vigna unguiculata L. Walp) growing in soil collected from Amazon rainforest. Furthermore, it was observed that the strain has high capacity to fix nitrogen symbiotically in symbioses with cowpea. We report here the draft genome sequence of strain BR 3351T. The information presented will be important for comparative analysis of nodulation and nitrogen fixation for diazotrophic bacteria. A draft genome with 9,145,311 bp and 62.9% of GC content was assembled in 127 scaffolds using 100 bp pair-end Illumina MiSeq system. The RAST annotation identified 8603 coding sequences, 51 RNAs genes, classified in 504 subsystems.

Keywords:
Amazon forest; Biological nitrogen fixation; Next generation sequencing; Nodulation; Cowpea

Genome Announcement

Despite of great biodiversity present in the Amazon forest, ex. more than 3000 leguminous species, the knowledge about rhizobium diversity is scarce. Some studies have indicated Bradyrhizobium as an important nodulating-bacteria to different leguminous hosts.11 Guimarães AA, Jaramillo PMD, Nóbrega RSA, Florentino LA, Silva KB, Moreira FMS. Genetic and symbiotic diversity of nitrogen-fixing bacteria isolated from agricultural soils in the Western Amazon by using cowpea as the trap plant. Appl Environ Microbiol. 2012;78:6726-6733.

2 Silva FV, De Meyer SE, Simões de Araújo JL, et al. Bradyrhizobium manausense sp. nov., isolated from effective nodules of Vigna unguiculata grown in Brazilian Amazon rainforest soils. Int J Syst Evol Microbiol. 2014;64:2358-2363.

3 da Silva K, De Meyer SE, Rouws LF, et al. Bradyrhizobium ingae sp. nov., isolated from effective nodules of Inga laurina grown in Cerrado soil. Int J Syst Evol Microbiol. 2014;64:3395-3401.
-44 Zilli JE, Baraúna AC, da Silva K, et al. Bradyrhizobium neotropicale sp. nov., isolated from effective nodules of Centrolobium paraense. Int J Syst Evol Microbiol. 2014;64:3950-3957.B. manausense strain BR 3351T was isolated from a Vigna unguiculata root nodule growing as trap plant in a soil sample collected from the Brazilian Amazon rainforest.22 Silva FV, De Meyer SE, Simões de Araújo JL, et al. Bradyrhizobium manausense sp. nov., isolated from effective nodules of Vigna unguiculata grown in Brazilian Amazon rainforest soils. Int J Syst Evol Microbiol. 2014;64:2358-2363.,55 Silva FV, Simões-Araújo JL, Silva Júnior JP, Xavier GR, Rumjanek NG. Genetic diversity of Rhizobia isolates from Amazon soils using cowpea (Vigna unguiculata) as trap plant. Brazil Braz J Microbiol. 2012;43:682-691. This strain exhibited high capacity to fix nitrogen in symbiosis with V. unguiculata and the Phasoleae tribe seems to be the preferred host.22 Silva FV, De Meyer SE, Simões de Araújo JL, et al. Bradyrhizobium manausense sp. nov., isolated from effective nodules of Vigna unguiculata grown in Brazilian Amazon rainforest soils. Int J Syst Evol Microbiol. 2014;64:2358-2363. Here, we report the draft genome of strain BR 3351T, a type strain of B. manausense, a member of the Alphaproteobacteria isolated from Amazon rainforest.

B. manausense (BR 3351T) was grown on YMA medium66 Vincent JM. A Manual for the Practical Study of Root-nodule Bacteria. Oxford: Published for the International Biological Programme [by] Blackwell Scientific; 1970. for 5 days at 28 °C. A single colony was inoculated in 5 mL of YM medium and incubated at same condition to obtain cell biomass. After, 2 mL of the culture was centrifuged (16,000 × g; 4 min) and the pellet was submitted to DNA extraction with the kit Wizard miniprep. A sample of DNA, approximately 3 µg, was sent to Macrogen Inc. (Korea) for genome sequencing using the 100 bp pair-end Illumina MiSeq system. A total of 1,845,481,494 bp (aprox. 1.8 Gb) was generated, corresponding to 108 X genome coverage.

The FASTX-Toolkit (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) was used to trim the reads and only high quality bases (Q25) were used to assemble the genome. The ABySS software version 1.9.077 Simpson JT, Wong K, Jackman SD, Schein JE, Jones SJ, Birol I. ABySS: a parallel assembler for short read sequence data. Genome Res. 2009;19:1117-1123. was used to de novo assemble and contigs shorter than 200 bp were eliminated. The genome annotation and metabolic pathways identification was carried out by RAST version 2.0 server.88 Overbeek R, Olson R, Pusch GD, et al. The SEED and the Rapid Annotation of microbial genomes using Subsystems Technology (RAST). Nucleic Acids Res. 2013;42:D206-D214. In addition, the contigs were also submitted to GenBank and annotated by NCBI Prokaryotic Genome Annotation Pipeline (released 2013; http://www.ncbi.nlm.nih.gov/genomes/static/Pipeline.html).

The B. manausense (BR 3351T) draft genome consists of 127 contigs with 9,145,311 bp and 62.9% of GC content. This genome size and C + G content are compatible with other Bradyrhizobium. The RAST automatic annotation identified 51 copies of RNA genes and 8603 protein-coding gene sequences (CDSs), distributed in 504 subsystems. The nitrogen metabolism comprises 64 genes, including 8 genes for cyanate hydrolysis, 22 genes for nitrogen fixation, 14 genes for nitrate/nitrite/ammonification and 17 genes for ammonia assimilation. Furthermore, genes related to denitrification were not found, although the denitrification process has been described for some species on Bradyrhizobium genus.99 Sánchez C, Tortosa G, Granados A, et al. Involvement of Bradyrhizobium japonicum denitrification in symbiotic nitrogen. Soil Biol Biochem. 2011;43:212-217.,1010 Tortosa G, Hidalgo A, Salas A, Bedmar EJ, Mesa S, Delgado MJ. Nitrate and flooding induce N2O emissions from soybean nodule. Symbiosis. 2015;67:125-133. The carbohydrates metabolism seems to be quite complex, since a total of 698 genes related to this subsystem were annotated, including: 26 genes for CO2 fixation (photorespiration oxidative C2 cycle), 17 and 114 genes related to polysaccharides and monosaccharides metabolism, respectively. These genomic data add information to clarify the B. manausense metabolic strategies to nodulate and fix nitrogen in association with legumes, especially from the Phaseoleae tribe. Additional comparative genomic and transcriptomic studies on this bacterium will help to understand the symbiotic efficiency and host range capacity of the strain BR 3351T.

This whole genome sequence has been deposited in DDBJ/ENA/GenBank under the accession number LJYG00000000, the version described in this paper is the first version.

Acknowledgments

We would like to thank to Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for the financial support and research fellowship (PQ) given to the authors.

References

  • 1
    Guimarães AA, Jaramillo PMD, Nóbrega RSA, Florentino LA, Silva KB, Moreira FMS. Genetic and symbiotic diversity of nitrogen-fixing bacteria isolated from agricultural soils in the Western Amazon by using cowpea as the trap plant. Appl Environ Microbiol 2012;78:6726-6733.
  • 2
    Silva FV, De Meyer SE, Simões de Araújo JL, et al. Bradyrhizobium manausense sp. nov., isolated from effective nodules of Vigna unguiculata grown in Brazilian Amazon rainforest soils. Int J Syst Evol Microbiol 2014;64:2358-2363.
  • 3
    da Silva K, De Meyer SE, Rouws LF, et al. Bradyrhizobium ingae sp. nov., isolated from effective nodules of Inga laurina grown in Cerrado soil. Int J Syst Evol Microbiol 2014;64:3395-3401.
  • 4
    Zilli JE, Baraúna AC, da Silva K, et al. Bradyrhizobium neotropicale sp. nov., isolated from effective nodules of Centrolobium paraense Int J Syst Evol Microbiol 2014;64:3950-3957.
  • 5
    Silva FV, Simões-Araújo JL, Silva Júnior JP, Xavier GR, Rumjanek NG. Genetic diversity of Rhizobia isolates from Amazon soils using cowpea (Vigna unguiculata) as trap plant. Brazil Braz J Microbiol 2012;43:682-691.
  • 6
    Vincent JM. A Manual for the Practical Study of Root-nodule Bacteria Oxford: Published for the International Biological Programme [by] Blackwell Scientific; 1970.
  • 7
    Simpson JT, Wong K, Jackman SD, Schein JE, Jones SJ, Birol I. ABySS: a parallel assembler for short read sequence data. Genome Res 2009;19:1117-1123.
  • 8
    Overbeek R, Olson R, Pusch GD, et al. The SEED and the Rapid Annotation of microbial genomes using Subsystems Technology (RAST). Nucleic Acids Res 2013;42:D206-D214.
  • 9
    Sánchez C, Tortosa G, Granados A, et al. Involvement of Bradyrhizobium japonicum denitrification in symbiotic nitrogen. Soil Biol Biochem 2011;43:212-217.
  • 10
    Tortosa G, Hidalgo A, Salas A, Bedmar EJ, Mesa S, Delgado MJ. Nitrate and flooding induce N2O emissions from soybean nodule. Symbiosis 2015;67:125-133.

Publication Dates

  • Publication in this collection
    Oct-Dec 2017

History

  • Received
    29 July 2016
  • Accepted
    13 Oct 2016
Sociedade Brasileira de Microbiologia USP - ICB III - Dep. de Microbiologia, Sociedade Brasileira de Microbiologia, Av. Prof. Lineu Prestes, 2415, Cidade Universitária, 05508-900 São Paulo, SP - Brasil, Ramal USP 7979, Tel. / Fax: (55 11) 3813-9647 ou 3037-7095 - São Paulo - SP - Brazil
E-mail: bjm@sbmicrobiologia.org.br
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