Microbial Communities and Diversities in Mudflat Sediments Analyzed Using a Modified Metatranscriptomic Method

Abstract

Intertidal mudflats are land–sea interaction areas and play important roles in global nutrient cycles. However, a comprehensive understanding of microbial communities in these mudflats remains elusive. In this study, mudflat sediment samples from the Dongtan wetland of Chongming Island, the largest alluvial island in the world, were collected. Using a modified metatranscriptomic method, the depth-wise distributions of potentially active microbial communities were investigated based on small subunit ribosomal RNA (SSU rRNA) sequences. Multiple environmental factors were also measured and analyzed in conjunction with the prokaryotic composition profiles. A prokaryotic diversity analysis based on the metatranscriptome datasets revealed two or threefold higher diversity indices (associated with potentially active microbes participating in biogeochemical processes in Dongtan) compared with the diversity indices based on 16S rRNA gene amplicons. Bacteria were numerically dominant relative to archaea, and the potentially active prokaryotic taxa were mostly assigned to the bacterial phyla Chloroflexi, Acidobacteria, and Bacteroidetes and the classes Delta- and Gamma-proteobacteria, along with the archaeal lineages phylum Bathyarchaeota and the order Thermoplasmatales. The total nitrogen and carbon content of the sediment samples were environmental factors that significantly affected the depth-wise distributions of both bacterial and archaeal communities. Furthermore, the activity of potentially active taxa (including the prevalent order Desulfobacterales and family Anaerolineaceae) appeared to be significantly underestimated by PCR-based methods, notably at the DNA level, and indicates that using normal PCR amplification of DNA limits the study of potential microbial activity. This is the first study of potentially active microbial communities in depth-wise sediments from Dongtan. The improved knowledge of microbial communities in Dongtan provides a foundation for exploring biogeochemical cycling and microbial functions.

Design of targeted primers based on 16S rRNA sequences in meta-transcriptomic datasets and identification of a novel taxonomic group in the Asgard archaea

“…In the meta-transcriptional assay, the 5′ terminus of SSU rRNA was ligated to the adaptor with a tag sequence and reverse-transcribed using a random primer with a tag sequence. Therefore, most SSU rRNA sequences determined with this method contained complete 5′ termini [26]. To amplify full-length novel microbial SSU rRNA sequences, the 5′ termini of SSU rRNA sequences from this dataset were used for primer evaluation with Microbiota metagenome Primer Explorer (MIPE) software [35].…”

“…2). Although sequences related to those of Type A and Type B ( Table 2) only occupied approximately 1/20,000 of the original SSU rRNA sequences in the meta-transcriptomic datasets [26], one-third of the fulllength SSU rRNA gene sequences via PCR amplification with targeted primers designed solely based on sequences in Type A (12 sequences) and Type B (25 sequences) were affiliated with the Asgard superphylum. Microbial taxa with a relative abundance below 0.01% [36] were defined as rare biospheres, which normally may be discarded when performing microbial community analysis.…”

“…Through PacBio RS II sequencing, 557 and 1139 clean sequences were obtained after filtering chimeras separately from samples S1 and S2. Among these sequences, 45 sequences in Evaluation of universal primers for mudflat meta-transcriptomic SSU rRNA sequences [26]. Screening criteria: the sequences of this OTU are taxonomically unclassified at the domain or/and phylum level, primer-binding regions in each sequence of this OTU are complete and the mismatched bases with the universal primer ≥3, and the sequence number in the OTU ≥10.…”

“…However, single-cell genome and metagenome sequencing have shown limited capacity to discover microbial dark matter because massive data are needed for metagenome assembly and binning. Modified meta-transcriptomic methods that were used to enrich SSU rRNA without universal primers [26] provided an opportunity to discover active microbial dark matter from the "rare biosphere" not covered by universal primers.…”

“…To identify "rare biosphere" microbial dark matter from meta-transcriptomic datasets, we developed a method for designing new primers based on SSU rRNA datasets by using a modified meta-transcriptomic method [26] and amplified nearly full-length 16S rRNA genes of novel taxa by using these newly designed forward primers and universal reverse primer ( Fig. 1).…”