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对根瘤菌分类学者提的建议

已有 6609 次阅读 2007-11-14 16:45 |个人分类:科研工作

对根瘤菌分类及确定根瘤菌新种的研究者来说,用多少方法才能符合要求,到现在还没绝对的标准,但根瘤菌分类分委员会委员Vineusa于2007-1-14提出的建议值得大家借鉴。大家如果按照这些方法做的话,相信发表会更顺利些。

下面是我翻译的:

定一个根瘤菌新种时至少需要多少菌株?
       
至少三个明显不同的菌株,这些不同指的是IGS-PCR-RFLPs, rep-PCR or AFLP genomic fingerprints, sequence data等,菌株应属于不同的地理来源或不同的宿主来源。总之,只有一个菌株来定根瘤菌新种绝对不行。

多少个分子标记方法要采用?
      
种内要用高分辩率的方法,如rep-PCRAFLP等进行区分,并用至少3个编码蛋白序列的基因进行比较分析。recA rpoB是必须分析的两个基因。再加上至少一个共生基因,如nifH, nodAnodC等。16S rDNA序列是必须要的,并且是几乎全长的序列。

(博主补充:关于rpoB基因,我查了一下文献,它是RNA聚合酶beta亚单位的基因,基因的长度为4150bp左右,测起来相当费事,需要设计多对引物才能将其完全测定出来。rpoB已用于一些细菌的鉴定研究,在根瘤菌上用得还不是很多,值得探索。随着基因序列测定的快速化,相信测定多个基因来比较细菌之间的关系的日子不会太远了。)

表型测定要做多少?
       
做尽量多的交叉结瘤实验。 菌株的生长温度范围、pH,对盐的耐受度,对CN的利用,对抗生素的抗性,脂肪酸分析。

DNA杂交?
       
能做最好,将新单元的几株菌之间做杂交,看它们之间的相似性。做与最相关的参比菌株的杂交。我们更希望通过多位点分析结合表型特征来定一个新种。

Tips and suggestions for the practising rhizobial taxonomist

Submitted by vinuesa on Sun, 2007-01-14 06:57.

General recommendations for the description of symbiotic rhizobia:

From: http://edzna.ccg.unam.mx/rhizobial-taxonomy/node/12

 

  • Consider the recommendations found in the Report of the ad hoc committee for the re-evaluation of the species definition in bacteriology (Stackebrandt et al. 2002). See below a selected list of key papers on the nature of prokaryotic species.

  • 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.

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., Delaere, M., Coopman, R., De Vos, P., Gillis, M. & Willems, A. (2007). Multilocus sequence analysis of Ensifer and related taxa. Int J Syst Evol Microbiol 57, 489-503.

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.

 



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