Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, 08193, Bellaterra, Catalunya, Spain.
Departament de Biologia Marina i Oceanografia, Institut de Ciències del Mar, ICM-CSIC, 08003, Barcelona, Catalunya, Spain.
Microb Ecol. 2023 Oct;86(3):2161-2172. doi: 10.1007/s00248-023-02220-y. Epub 2023 May 6.
Studies based on protein-coding genes are essential to describe the diversity within bacterial functional groups. In the case of aerobic anoxygenic phototrophic (AAP) bacteria, the pufM gene has been established as the genetic marker for this particular functional group, although available primers are known to have amplification biases. We review here the existing primers for pufM gene amplification, design new ones, and evaluate their phylogenetic coverage. We then use samples from contrasting marine environments to evaluate their performance. By comparing the taxonomic composition of communities retrieved with metagenomics and with different amplicon approaches, we show that the commonly used PCR primers are biased towards the Gammaproteobacteria phylum and some Alphaproteobacteria clades. The metagenomic approach, as well as the use of other combinations of the existing and newly designed primers, show that these groups are in fact less abundant than previously observed, and that a great proportion of pufM sequences are affiliated to uncultured representatives, particularly in the open ocean. Altogether, the framework developed here becomes a better alternative for future studies based on the pufM gene and, additionally, serves as a reference for primer evaluation of other functional genes.
基于蛋白质编码基因的研究对于描述细菌功能群的多样性至关重要。在好氧缺氧光养（AAP）细菌的情况下，pufM 基因已被确立为该特定功能群的遗传标记，尽管已知可用的引物存在扩增偏倚。在这里，我们回顾了现有的 pufM 基因扩增引物，设计了新的引物，并评估了它们的系统发育覆盖范围。然后，我们使用来自对比海洋环境的样本来评估它们的性能。通过比较基于宏基因组学和不同扩增子方法获得的群落的分类组成，我们表明，常用的 PCR 引物偏向于γ变形菌门和一些α变形菌目。基于宏基因组学的方法，以及使用现有和新设计引物的其他组合，表明这些群体实际上比以前观察到的要少，并且很大一部分 pufM 序列与未培养的代表有关，特别是在开阔的海洋中。总的来说，这里开发的框架成为基于 pufM 基因的未来研究的更好选择，此外，它还可以作为其他功能基因引物评估的参考。
