Tips and suggestions for the practising rhizobial taxonomist

General recommendations for the description of symbiotic rhizobia:

  • Consider the recommendations found in the Notes on the characterization of prokaryote strains for taxonomic purposes (Tindall et al. 2010). See below a selected list of key papers on the nature of prokaryotic species and their description, as viewed from different perspectives: polyphasic taxonomy, phylogenetics, population genetics, ecology and genomics.
  • How many strains should be used to describe a new rhizobial species? We recommend that new taxon descriptions are based on a minimum of at least three distinct strains. By distinct we mean three isolates with different genotypes, as revealed by proper molecular markers (like IGS-PCR-RFLPs, rep-PCR or AFLP genomic fingerprints, sequence data ...). Whenever possible, sample several populations, each with > 12 isolates. Population sampling should be made from different ecological settings (i.e. geographic regions, ecosystems and/or hosts). This is the only means to learn about the ecological and gentic diversity of any species. Phenotypic analyses based on a single or a few isolates are of very limited value given the high phenotypic and genotypic diversity found within rhizobia and other free-living bacteria with large genomes capable of inhabiting different ecological niches. In conclusion: New species descriptions based on a single isolate are strongly discouraged!

  • How many and which molecular markers should be used? Use a range of different molecular markers suitable to uncover and analyze genetic diversity at different phylogenetic/taxonomic depths. We recommend to use some kind of high-resolution molecular typing method apropriate to reveal diversity within species (i.e. rep-PCR or AFLP genomic fingerprints) combined with multilocus sequence analyses of at least 3 protein-coding loci. The former methods will provide insights on the genetic structure of the populations (levels of clonality and epidemicity), data that are very valuable to make an informed selection of strains for MLSA. Some loci like recA and rpoB have large sequence databases which are publicly available at the NCBI/DDBJ/EMBL DBs, and have proven very useful in molecular systematics of diverse prokaryotic groups, including alpha and beta rhizobia. It is always very interesting to gain sequence data also for accessory loci such as symbiotic or virulence genes. Large nifH, nodA and nodC sequence databases are available. A good compromise would be to generate full-length 16S rDNA sequences for a few carefully selected strains, along with the partial sequencing of two protein-coding core loci (e.g. recA and rpoB) and at least one sym locus.
  • What phenotypic tests should be performed? It is always of great ecological and evolutionary interest to perform extensive host range tests for new isolates, specially when combined with sequence analysis of sym loci. This is a very powerful strategy to uncover symbiotic ecotypes or biovarieties, as shown in a number of studies. Other potentially relevant attributes such as pH and temperature growth-range, salt tolerance, growth on different C and N sources, as well as antibiotic resistance profiling and fatty acid methyl-ester analysis (FAME) have been commonly included in rhizobial species descriptions. Of particular interest are phenotypes and chemotaxonomic markers that are relevant or expressed in the niches potentially occupied by the target organisms (i.e. ecological adaptive traits). When coupled with clear genetic determinants, such ecological traits are very informative and of extraordinary taxonomic value.
  • What about DNA-DNA hybridization or reassociation experiments? These experiments are of great potential value only if properly performed. We think that it is essential that at least three clearly distinct strains of a new potential taxon should be included in such experiments in order to get an estimate of the standard deviations of homology values (genome heterogeneity) within the new taxon, as compared to the homology values across closely related taxa. However, we favour the use a thorough multilocus sequence analysis combined with key phenotypic analyses for species demarcation. This evidence should be ideally further extended with 16S rDNA sequencing of the type strain and two other strains, combined with DNA-DNA hybridization data for these reference strains and the relevant phylogenetic relatives.
  • Suggested reading:

Cavalier-Smith, T. (2007). Concept of a bacterium still valid in prokaryote debate. Nature 446, 257.

Cohan, F. M. & Perry, E. B. (2007). A systematics for discovering the fundamental units of bacterial diversity. Curr Biol 17, R373-386.

Felis, G. E. & Dellaglio, F. (2007). On species descriptions based on a single strain: proposal to introduce the status species proponenda (sp. pr.). Int J Syst Evol Microbiol 57, 2185-2187.

Fenchel, T. & Finlay, B. J. (2006). The diversity of microbes: resurgence of the phenotype. Philos Trans R Soc Lond B Biol Sci 361, 1965-1973.

Figueras, M. J., Alperi, A., Guarro, J. & Martinez-Murcia, A. J. (2006). Genotyping of isolates included in the description of a novel species should be mandatory. Int J Syst Evol Microbiol 56, 1183-1184.

Gevers, D., Cohan, F. M., Lawrence, J. G. & other authors (2005). Opinion: Re-evaluating prokaryotic species. Nat Rev Microbiol 3, 733-739.

Gevers, D., Dawyndt, P., Vandamme, P., Willems, A., Vancanneyt, M., Swings, J. & De Vos, P. (2006). Stepping stones towards a new prokaryotic taxonomy. Philos Trans R Soc Lond B Biol Sci 361, 1911-1916.

Goris, J., Konstantinidis, K. T., Klappenbach, J. A., Coenye, T., Vandamme, P. & Tiedje, J. M. (2007). DNA-DNA hybridization values and their relationship to whole-genome sequence similarities. Int J Syst Evol Microbiol 57, 81-91.

Kämpfer P. Systematics of prokaryotes: the state of the art. Antonie Van Leeuwenhoek. 2012 Jan;101(1):3-11. Epub 2011 Nov 1. Review. PubMed PMID: 2041978.

Kämpfer P, Glaeser SP. Prokaryotic taxonomy in the sequencing era--the polyphasic approach revisited. Environ Microbiol. 2012 Feb;14(2):291-317. doi: 10.1111/j.1462-2920.2011.02615.x. Epub 2011 Oct 31. Review. PubMed PMID: 22040009.

Konstantinidis, K. T. & Tiedje, J. M. (2005). Towards a Genome-Based Taxonomy for Prokaryotes. J Bacteriol 187, 6258-6264.

Konstantinidis, K. T., Ramette, A. & Tiedje, J. M. (2006). Toward a More Robust Assessment of Intraspecies Diversity, Using Fewer Genetic Markers. Appl Environ Microbiol 72, 7286-7293.

Martens M, Dawyndt P, Coopman R, Gillis M, De Vos P, Willems A. Advantages of multilocus sequence analysis for taxonomic studies: a case study using 10 housekeeping genes in the genus Ensifer (including former Sinorhizobium). Int J Syst Evol Microbiol. 2008 Jan;58(Pt 1):200-14. PubMed PMID: 18175710.

Rivas R, Martens M, de Lajudie P, Willems A. Multilocus sequence analysis of the genus Bradyrhizobium. Syst Appl Microbiol. 2009 Apr;32(2):101-10. Epub 2009 Feb 6. PubMed PMID: 19201125.

Shapiro BJ, Friedman J, Cordero OX, Preheim SP, Timberlake SC, Szabó G, Polz MF, Alm EJ. Population genomics of early events in the ecological differentiation of bacteria. Science. 2012 Apr 6;336(6077):48-51. PubMed PMID: 22491847; PubMed Central PMCID: PMC3337212.

Stackebrandt, E., Frederiksen, W., Garrity, G. M. & other authors (2002). Report of the ad hoc committee for the re-evaluation of the species definition in bacteriology. Int J Syst Evol Microbiol 52, 1043-1047.

Staley, J. T. (2006). The bacterial species dilemma and the genomic-phylogenetic species concept. Philos Trans R Soc Lond B Biol Sci 361, 1899-1909.

Tettelin, H., Masignani, V., Cieslewicz, M. J. & other authors (2005). Genome analysis of multiple pathogenic isolates of Streptococcus agalactiae: Implications for the microbial "pan-genome". Proceedings of the National Academy of Sciences 102, 13950-13955.

Tindall BJ, Rosselló-Móra R, Busse HJ, Ludwig W, Kämpfer P. Notes on the characterization of prokaryote strains for taxonomic purposes. Int J Syst Evol Microbiol. 2010 Jan;60(Pt 1):249-66. Epub 2009 Aug 21. PubMed PMID: 19700448.

van Berkum P, Elia P, Eardly BD. Multilocus sequence typing as an approach for population analysis of Medicago-nodulating rhizobia. J Bacteriol. 2006 Aug; 188(15):5570-7. PubMed PMID: 16855247; PubMed Central PMCID: PMC1540022.

van Berkum P, Elia P, Eardly BD. Application of multilocus sequence typing to study the genetic structure of megaplasmids in medicago-nodulating rhizobia. Appl Environ Microbiol. 2010 Jun;76(12):3967-77. Epub 2010 Apr 23. PubMed PMID: 20418433; PubMed Central PMCID: PMC2893487.

Vinuesa P, Rojas-Jiménez K, Contreras-Moreira B, Mahna SK, Prasad BN, Moe H, Selvaraju SB, Thierfelder H, Werner D. Multilocus sequence analysis for assessment of the biogeography and evolutionary genetics of four Bradyrhizobium species that nodulate soybeans on the asiatic continent. Appl Environ Microbiol. 2008 Nov;74(22):6987-96. Epub 2008 Sep 12. PubMed PMID: 18791003; PubMed Central PMCID: PMC2583495.

Vinuesa, P. 2010. Multilocus Sequence Analysis and Bacterial Species Phylogeny Estimation. Chapter 3, pg. 41-64, in A. Oren and R. T. Papke (ed.), Molecular Phylogeny of Microorganisms. Caister Academic Press, Norfolk, UK. Abstract. ISBN: 978-1-904455-67-7.