Simultaneous analysis of seven 16S rRNA hypervariable gene regions increases efficiency in marine bacterial diversity detection.

Environmental DNA sequencing is the gold standard to reveal microbial community structures. In most applications, a one-fragment PCR approach is applied to amplify a taxonomic marker gene, usually a hypervariable region of the 16S rRNA gene. We used a new reverse complement (RC)-PCR-based assay that amplifies seven out of the nine hypervariable regions of the 16S rRNA gene, to interrogate bacterial communities in sediment samples collected from different coastal marine sites with an impact gradient. In parallel, we employed a traditional one-fragment analysis of the hypervariable V3-V4 region to investigate whether the RC-PCR reveals more of the 'unseen' diversity obtained by the one-fragment approach. As a benchmark for the full deck of diversity, we subjected the samples to PCR-free metagenomic sequencing. None of the two PCR-based approaches recorded the full taxonomic repertoire obtained from the metagenomics datasets. However, the RC-PCR approach detected 2.8 times more bacterial genera compared to the near-saturation sequenced V3-V4 samples. RC-PCR is an ideal compromise between the standard one-fragment approach and metagenomics sequencing and may guide future environmental sequencing studies, in which bacterial diversity is a central subject.

Revisiting the mercury cycle in marine sediments: A potential multifaceted role for Desulfobacterota.

Marine sediments impacted by urban and industrial pollutants are typically exposed to reducing conditions and represent major reservoirs of toxic mercury species. Mercury methylation mediated by anaerobic microorganisms is favored under such conditions, yet little is known about potential microbial mechanisms for mercury detoxification. We used culture-independent (metagenomics, metabarcoding) and culture-dependent approaches in anoxic marine sediments to identify microbial indicators of mercury pollution and analyze the distribution of genes involved in mercury reduction (merA) and demethylation (merB). While none of the isolates featured merB genes, 52 isolates, predominantly affiliated with Gammaproteobacteria, were merA positive. In contrast, merA genes detected in metagenomes were assigned to different phyla, including Desulfobacterota, Actinomycetota, Gemmatimonadota, Nitrospirota, and Pseudomonadota. This indicates a widespread capacity for mercury reduction in anoxic sediment microbiomes. Notably, merA genes were predominately identified in Desulfobacterota, a phylum previously associated only with mercury methylation. Marker genes involved in the latter process (hgcAB) were also mainly assigned to Desulfobacterota, implying a potential central and multifaceted role of this phylum in the mercury cycle. Network analysis revealed that Desulfobacterota were associated with anaerobic fermenters, methanogens and sulfur-oxidizers, indicating potential interactions between key players of the carbon, sulfur and mercury cycling in anoxic marine sediments.