Ribosome Profiling in the Model Diatom Thalassiosira pseudonana.

Diatoms are an important group of eukaryotic microalgae, which play key roles in marine biochemical cycling and possess significant biotechnological potential. Despite the importance of diatoms, their regulatory mechanisms of protein synthesis at the translational level remain largely unexplored. Here, we describe the detailed development of a ribosome profiling protocol to study translation in the model diatom Thalassiosira pseudonana, which can easily be adopted for other diatom species. To isolate and sequence ribosome-protected mRNA, total RNA was digested, and the ribosome-protected fragments were obtained by a combination of sucrose-cushion ultracentrifugation and polyacrylamide gel electrophoresis for size selection. To minimize rRNA contamination, a subtractive hybridization step using biotinylated oligos was employed. Subsequently, fragments were converted into sequencing libraries, enabling the global quantification and analysis of changes in protein synthesis in diatoms. The development of this novel ribosome profiling protocol represents a major expansion of the molecular toolbox available for diatoms and therefore has the potential to advance our understanding of the translational regulation in this important group of phytoplankton. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Ribosome profiling in Thalassiosira pseudonana Alternate Protocol: Ribosome profiling protocol for diatoms using sucrose gradient fractionation.

Effects of organic matter and low oxygen on the mycobenthos in a coastal lagoon.

Fungi living in sediments ('mycobenthos') are hypothesized to play a role in the degradation of organic matter deposited at the land-sea interface, but the environmental factors influencing the mycobenthos are poorly understood. We used mock community calibrated Illumina sequencing to show that the mycobenthos community structure in a coastal lagoon was significantly changed after exposure to a lignocellulose extract and subsequent development of benthic anoxia over a relatively short (10 h) incubation. Saprotrophic taxa dominated and were selected for under benthic anoxia, specifically Aquamyces (Chytridiomycota) and Orbilia (Ascomycota), implicating these genera as important benthic saprotrophs. Protein encoding genes involved in energy and biomass production from Fungi and the fungal-analogue group Labyrinthulomycetes had the highest increase in expression with the added organic matter compared with all other groups, indicating that lignocellulose stimulates metabolic activity in the mycobenthos. Flavobacteria dominated the active bacterial community that grew rapidly with the lignocellulose extract and crashed sharply upon O2 depletion. Our findings indicate that the diversity, activity and trophic potential of the mycobenthos changes rapidly in response to organic matter and decreasing O2 concentrations, which together with heterotrophic Flavobacteria, undergo 'boom and bust' dynamics during lignocellulose degradation in estuarine ecosystems.

Linking Uncultivated Microbial Populations and Benthic Carbon Turnover by Using Quantitative Stable Isotope Probing.

Benthic environments harbor highly diverse and complex microbial communities that control carbon fluxes, but the role of specific uncultivated microbial groups in organic matter turnover is poorly understood. In this study, quantitative DNA stable isotope probing (DNA-qSIP) was used for the first time to link uncultivated populations of bacteria and archaea to carbon turnover in lacustrine surface sediments. After 1-week incubations in the dark with [13C]bicarbonate, DNA-qSIP showed that ammonia-oxidizing archaea (AOA) were the dominant active chemolithoautotrophs involved in the production of new organic matter. Natural 13C-labeled organic matter was then obtained by incubating sediments in the dark for 2.5 months with [13C]bicarbonate, followed by extraction and concentration of high-molecular-weight (HMW) (>50-kDa) organic matter. qSIP showed that the labeled organic matter was turned over within 1 week by 823 microbial populations (operational taxonomic units [OTUs]) affiliated primarily with heterotrophic Proteobacteria, Chloroflexi, Verrucomicrobia, and Bacteroidetes However, several OTUs affiliated with the candidate microbial taxa Latescibacteria, Omnitrophica, Aminicentantes, Cloacimonates, AC1, Bathyarchaeota, and Woesearchaeota, groups known only from genomic signatures, also contributed to biomass turnover. Of these 823 labeled OTUs, 52% (primarily affiliated with Proteobacteria) also became labeled in 1-week incubations with [13C]bicarbonate, indicating that they turned over carbon faster than OTUs that were labeled only in incubations with 13C-labeled HMW organic matter. These taxa consisted primarily of uncultivated populations within the Firmicutes, Bacteroidetes, Verrucomicrobia, and Chloroflexi, highlighting their ecological importance. Our study helps define the role of several poorly understood, uncultivated microbial groups in the turnover of benthic carbon derived from "dark" primary production.IMPORTANCE Little is known about the ecological role of uncultivated microbial populations in carbon turnover in benthic environments. To better understand this, we used quantitative stable isotope probing (qSIP) to quantify the abundance of diverse, specific groups of uncultivated bacteria and archaea involved in autotrophy and heterotrophy in a benthic lacustrine habitat. Our results provide quantitative evidence for active heterotrophic and autotrophic metabolism of several poorly understood microbial groups, thus demonstrating their relevance for carbon turnover in benthic settings. Archaeal ammonia oxidizers were significant drivers of in situ "dark" primary production supporting the growth of heterotrophic bacteria. These findings expand our understanding of the microbial populations within benthic food webs and the role of uncultivated microbes in benthic carbon turnover.

A 16S rRNA gene sequencing and analysis protocol for the Illumina MiniSeq platform.

High-throughput sequencing of the 16S rRNA gene on the Illumina platform is commonly used to assess microbial diversity in environmental samples. The MiniSeq, Illumina's latest benchtop sequencer, enables more cost-efficient DNA sequencing relative to larger Illumina sequencing platforms (e.g., MiSeq). Here we used a modified custom primer sequencing approach to test the fidelity of the MiniSeq for high-throughput sequencing of the V4 hypervariable region of 16S rRNA genes from complex communities in environmental samples. To this end, we designed additional sequencing primers that enabled application of a dual-index barcoding method on the MiniSeq. A mock community was sequenced alongside the environmental samples in four different sequencing runs as a quality control benchmark. We were able to recapture a realistic richness of the mock community in all sequencing runs, and identify meaningful differences in alpha and beta diversity in the environmental samples. Furthermore, rarefaction analysis indicated diversity in many environmental samples was close to saturation. These results show that the MiniSeq can produce similar quantities of high-quality V4 reads compared to the MiSeq, yet is a cost-effective option for any laboratory interested in performing high-throughput 16S rRNA gene sequencing.