Abstract
Microbiome studies mostly rely on total DNA extracts obtained directly from environmental samples. The total DNA consists of both intra- and extracellular DNA, which differ in terms of their ecological interpretation. In the present study, we have investigated for the first time the differences among the three DNA types using microbiome sequencing of Picea abies deadwood logs (Hunter decay classes I, III, and V). While the bacterial compositions of all DNA types were comparable in terms of more abundant organisms and mainly depended on the decay class, we found substantial differences between DNA types with regard to less abundant amplicon sequence variants (ASVs). The analysis of the sequentially extracted intra- and extracellular DNA fraction, respectively, increased the ecological depth of analysis compared to the directly extracted total DNA pool. Both DNA fractions were comparable in proportions and the extracellular DNA appeared to persist in the P. abies deadwood logs, thereby causing its masking effect. Indeed, the extracellular DNA masked the compositional dynamics of intact cells in the total DNA pool. Our results provide evidence that the choice of DNA type for analysis might benefit a study’s answer to its respective ecological question. In the deadwood environment researched here, the differential analysis of the DNA types underlined the relevance of Burkholderiales, Rhizobiales and other taxa for P. abies deadwood decomposition and revealed that the role of Acidobacteriota under this scenario might be underestimated, especially compared to Actinobacteriota.
Highlights
Before the advent of sequencing technologies, molecular microbial ecology was severely limited by low-resolution observation of microbial diversity
For an in-depth screening of the deadwood bacteriome, we compared the bacterial community analyzed by Illumina sequencing of the V4 16S rDNA of the directly extracted total DNA pool and its sequentially extracted extracellular and intracellular fractions. By analyzing these three DNA types we aimed to answer the following questions: (i) Does bacterial richness differ among DNA types and does it increase with progressing decay?; (ii) What can bacteriome information from the intra- and extracellular DNA contribute to the picture drawn by the total DNA pool?; (iii) Is there a masking effect of the extracellular DNA over the intracellular DNA?; and (iv) from our results, can we extrapolate some guidelines that may help in choosing the most suitable DNA type for molecular screening according to the specific research question?
Bacteriomes Differ Vastly Between decay classes (DCls) and to a Smaller Extent Between DNA Types Considering the main effects, there were no significant differences in amplicon sequence variants (ASVs) numbers among DNA types and DCls
Summary
Before the advent of sequencing technologies, molecular microbial ecology was severely limited by low-resolution observation of microbial diversity. We will focus on the three most common DNA types: the total DNA pool and its extracellular and intracellular DNA fractions (Nannipieri et al, 2020). It is extracted directly from the environmental samples using standard protocols based on enzymatic, physical and/or chemical cell lysis and contains both intra- and extracellular DNA. The DNA occurring in the extracellular environment is recovered by gentle washings in alkaline buffer; the remaining pellet is processed by cell-lysis inducing steps so as to extract the intracellular DNA. Thereafter, both DNA fractions are purified to obtain DNA compatible for downstream analyses. Due to the various terms and abbreviations used for these different DNA types in the literature (reviewed by Nagler et al, 2018a) and to avoid any misunderstanding and misinterpretation of the results, we will not use any acronyms throughout the text
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