1887

Abstract

Three bacterial strains, 1AS14I, 1AS12I and 6AS6, isolated from root nodules of , were characterized using a polyphasic approach. Phylogenetic analysis based on sequences placed all three strains within the complex. Further phylogeny, based on 1 756 bp sequences of four concatenated housekeeping genes (, , and ), revealed their distinction from known rhizobia species of the complex (Rlc), forming a distinct clade. The closest related species, identified as , with a sequence identity of 96.4% based on concatenated --- sequences. The type strain, 1AS14I, showed average nucleotide identity (ANI) values of 94.9, 94.3 and 94.1% and DNA–DNA hybridization values of 56.1, 57.4 and 60.0% with the type strains of closest known species: , and respectively. Phylogenomic analyses using 81 up-to-date bacteria core genes and the Type (Strain) Genome Server pipeline further supported the uniqueness of strains 1AS14I, 1AS12I and 6AS6. The relatedness of the novel strains to NCBI unclassified sp. (396 genomes) and metagenome-derived genomes showed ANI values from 76.7 to 94.8% with a species-level cut-off of 96%, suggesting that strains 1AS14I, 1AS12I and 6AS6 are a distinct lineage. Additionally, differentiation of strains 1AS14I, 1AS12I and 6AS6 from their closest phylogenetic neighbours was achieved using phenotypic, physiological and fatty acid content analyses. Based on the genomic, phenotypic and biochemical data, we propose the establishment of a novel rhizobial species, sp. nov., with strain 1AS14I designated as the type strain (=DSM 113914=LMG 33206). This study contributes to the understanding of microbial diversity in nitrogen-fixing symbioses, specifically within ecosystems in Tunisia.

Funding
This study was supported by the:
  • Agriculture and Agri-Food Canada (Award J-002272 & J-002295)
    • Principle Award Recipient: JamesT. Tambong
  • Ministère de l'Education supérieure et Recherche Scientifique, Tunisia. (Award 34MAG21)
    • Principle Award Recipient: BacemMnasri
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution.
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2024-09-05
2024-11-05
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References

  1. Gritli T, Ellouze W, Chihaoui S-A, Barhoumi F, Mhamdi R et al. Genotypic and symbiotic diversity of native rhizobia nodulating red pea (Lathyrus cicera L.) in Tunisia. Syst Appl Microbiol 2020; 43:126049 [View Article] [PubMed]
    [Google Scholar]
  2. Ilahi H, Hsouna J, Ellouze W, Gritli T, Chihaoui S-A et al. Phylogenetic study of rhizobia nodulating pea (Pisum sativum) isolated from different geographic locations in Tunisia. Syst Appl Microbiol 2021; 44:126221 [View Article] [PubMed]
    [Google Scholar]
  3. Tian CF, Young JPW, Wang ET, Tamimi SM, Chen WX. Population mixing of Rhizobium leguminosarum bv. viciae nodulating Vicia faba: the role of recombination and lateral gene transfer. FEMS Microbiol Ecol 2010; 73:563–576 [View Article] [PubMed]
    [Google Scholar]
  4. Saïdi S, Ramírez-Bahena M-H, Santillana N, Zúñiga D, Álvarez-Martínez E et al. Rhizobium laguerreae sp. nov. nodulates Vicia faba on several continents. Int J Syst Evol Microbiol 2014; 64:242–247 [View Article] [PubMed]
    [Google Scholar]
  5. Young JPW, Moeskjær S, Afonin A, Rahi P, Maluk M et al. Defining the Rhizobium leguminosarum species complex. Genes 2021; 12:111 [View Article]
    [Google Scholar]
  6. Jorrin B, Palacios JM, Peix Á, Imperial J. Rhizobium ruizarguesonis sp. nov., isolated from nodules of Pisum sativum L. Syst Appl Microbiol 2020; 43:126090 [View Article] [PubMed]
    [Google Scholar]
  7. Rahi P, Giram P, Chaudhari D, diCenzo GC, Kiran S et al. Rhizobium indicum sp. nov., isolated from root nodules of pea (Pisum sativum) cultivated in the Indian trans-Himalayas. Syst Appl Microbiol 2020; 43:126127 [View Article] [PubMed]
    [Google Scholar]
  8. Zhang J, Peng S, Andrews M, Liu C, Shang Y et al. Rhizobium changzhiense sp. nov., isolated from effective nodules of Vicia sativa L. in North China. Int J Syst Evol Microbiol 2019; 71: [View Article]
    [Google Scholar]
  9. Young JPW, Jorrin B, Moeskjær S, James EK. Rhizobium brockwellii sp. nov., Rhizobium johnstonii sp. nov. and Rhizobium beringeri sp. nov., three genospecies within the Rhizobium leguminosarum species complex. Int J Syst Evol Microbiol 2023; 73: [View Article]
    [Google Scholar]
  10. Hsouna J, Ilahi H, Han J-C, Gritli T, Ellouze W et al. Rhizobium acaciae sp. nov., a new nitrogen-fixing symbiovar isolated from root nodules of Acacia saligna in Tunisia. Int J Syst Evol Microbiol 2023; 73: [View Article] [PubMed]
    [Google Scholar]
  11. Hsouna J, Gritli T, Ilahi H, Ellouze W, Mansouri M et al. Genotypic and symbiotic diversity studies of rhizobia nodulating Acacia saligna in Tunisia reveal two novel symbiovars within the Rhizobium leguminosarum complex and Bradyrhizobium. Syst Appl Microbiol 2022; 45:126343 [View Article] [PubMed]
    [Google Scholar]
  12. Rogel MA, Ormeño-Orrillo E, Martinez Romero E. Symbiovars in rhizobia reflect bacterial adaptation to legumes. Syst Appl Microbiol 2011; 34:96–104 [View Article] [PubMed]
    [Google Scholar]
  13. Vincent J. A Manual for the Practical Study of the Root-Nodule Bacteria, 15th edn Oxford and Edinburgh: Blackwell Scientific Publications; 1970
    [Google Scholar]
  14. Terefework Z, Nick G, Suomalainen S, Paulin L, Lindstrom K. Phylogeny of Rhizobium galegae with respect to other rhizobia and agrobacteria. Int J Syst Bacteriol 1998; 48:349–356 [View Article]
    [Google Scholar]
  15. Kumar S, Stecher G, Tamura K. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Mol Biol Evol 2016; 33:1870–1874 [View Article] [PubMed]
    [Google Scholar]
  16. Kimura M. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 1980; 16:111–120 [View Article] [PubMed]
    [Google Scholar]
  17. Andrews S. A quality control tool for high throughput sequence data; 2010 https://www.bioinformatics.babraham.ac.uk/projects/fastqc
  18. Wick RR, Judd LM, Gorrie CL, Holt KE. Unicycler: resolving bacterial genome assemblies from short and long sequencing reads. PLoS Comput Biol 2017; 13:e1005595 [View Article] [PubMed]
    [Google Scholar]
  19. Olson RD, Assaf R, Brettin T, Conrad N, Cucinell C et al. Introducing the Bacterial and Viral Bioinformatics Resource Center (BV-BRC): a resource combining PATRIC, IRD and ViPR. Nucleic Acids Res 2023; 51:D678–D689 [View Article]
    [Google Scholar]
  20. Parks DH, Imelfort M, Skennerton CT, Hugenholtz P, Tyson GW. CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes. Genome Res 2015; 25:1043–1055 [View Article] [PubMed]
    [Google Scholar]
  21. Seemann T. Prokka: rapid prokaryotic genome annotation. Bioinformatics 2014; 30:2068–2069 [View Article] [PubMed]
    [Google Scholar]
  22. Jain C, Rodriguez-R LM, Phillippy AM, Konstantinidis KT, Aluru S. High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries. Nat Commun 2018; 9:5114 [View Article] [PubMed]
    [Google Scholar]
  23. Meier-Kolthoff JP, Auch AF, Klenk H-P, Göker M. Genome sequence-based species delimitation with confidence intervals and improved distance functions. BMC Bioinform 2013; 14:60 [View Article] [PubMed]
    [Google Scholar]
  24. Meier-Kolthoff JP, Göker M. TYGS is an automated high-throughput platform for state-of-the-art genome-based taxonomy. Nat Commun 2019; 10:2182 [View Article] [PubMed]
    [Google Scholar]
  25. Kim J, Na SI, Kim D, Chun J. UBCG2: Up-to-date bacterial core genes and pipeline for phylogenomic analysis. J Microbiol 2021; 59:609–615 [View Article]
    [Google Scholar]
  26. Kozlov AM, Darriba D, Flouri T, Morel B, Stamatakis A. RAxML-NG: a fast, scalable and user-friendly tool for maximum likelihood phylogenetic inference. Bioinformatics 2019; 35:4453–4455 [View Article]
    [Google Scholar]
  27. Chaumeil P-A, Mussig AJ, Hugenholtz P, Parks DH. GTDB-Tk v2: memory friendly classification with the genome taxonomy database. Bioinformatics 2022; 38:5315–5316 [View Article]
    [Google Scholar]
  28. Parks DH, Chuvochina M, Rinke C, Mussig AJ, Chaumeil P-A et al. GTDB: an ongoing census of bacterial and archaeal diversity through a phylogenetically consistent, rank normalized and complete genome-based taxonomy. Nucleic Acids Res 2022; 50:D785–D794 [View Article] [PubMed]
    [Google Scholar]
  29. Shaw J, Yu YW. Fast and robust metagenomic sequence comparison through sparse chaining with skani. Nat Methods 2023; 20:1661–1665 [View Article]
    [Google Scholar]
  30. Hyatt D, Chen G-L, Locascio PF, Land ML, Larimer FW et al. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinform 2010; 11:119 [View Article] [PubMed]
    [Google Scholar]
  31. Eddy SR. Accelerated profile HMM searches. PLoS Comput Biol 2011; 7:e1002195 [View Article] [PubMed]
    [Google Scholar]
  32. Ondov BD, Treangen TJ, Melsted P, Mallonee AB, Bergman NH et al. Mash: fast genome and metagenome distance estimation using MinHash. Genome Biol 2016; 17:132 [View Article] [PubMed]
    [Google Scholar]
  33. Matsen FA, Kodner RB, Armbrust EV. pplacer: linear time maximum-likelihood and Bayesian phylogenetic placement of sequences onto a fixed reference tree. BMC Bioinform 2010; 11:538 [View Article] [PubMed]
    [Google Scholar]
  34. Price MN, Dehal PS, Arkin AP. FastTree 2--approximately maximum-likelihood trees for large alignments. PLoS One 2010; 5:e9490 [View Article] [PubMed]
    [Google Scholar]
  35. González AJ, Cleenwerck I, De Vos P, Fernández-Sanz AM. Pseudomonas asturiensis sp. nov., isolated from soybean and weeds. Syst Appl Microbiol 2013; 36:320–324 [View Article]
    [Google Scholar]
  36. Mulet M, Lalucat J, García-Valdés E. DNA sequence-based analysis of the Pseudomonas species. Environ Microbiol 2010; 12:1513–1530 [View Article]
    [Google Scholar]
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