237 Synthesis and discussion Decoding Microbial Activity: Metatranscriptomics and Metaproteomics In Chapter 6, we applied both metatranscriptomics (also in Chapters 4 and 5) and metaproteomics to characterize the stress response of a slow-growing microbial culture, while also addressing some of their limitations. The combination of these techniques yielded a highly sensitive RNA signal and a comprehensive overview of the cellular protein profile in the enriched culture. In the salt stress experiment of Chapter 6, we did not get a clear expression signal of the important osmolyte N(ε)-acetyl-β-L-lysine production genes kamA and ablB. Here, reverse transcriptase-quantitative PCR (RT-qPCR) with newly designed primers was required to obtain a higher sensitivity of the expression of the osmolyte production genes. This could also have been achieved by repeating the experiment with a higher-depth metatranscriptome or, by more frequent sampling immediately after the increase in salt additions. In Chapter 5, biomass for RNA was sampled right after sulfide addition (2-3 hours), giving us a better insight into the response of the cells. Another limitation of the transcriptomics was the often low mRNA quantity obtained upon stress. One way to overcome this was a rRNA depletion step, which might be selective against archaeal rRNA (Chapters 4 and 6). Furthermore, the amount of RNA replicates matters, in (Chapter 4 and 5) three and in (Chapter 6) four replicates were sequenced, which gave fewer deviations in the RNA signal. A technical difficulty of biomass sampling for mRNA is the delicate nature of the single-stranded mRNA, which needs to be sampled as fast as possible under anoxic conditions. For Chapters 4 and 6, samples were transported in an anoxic syringe to the anaerobic hood and, after spinning down and removing the supernatant, immediately snap-frozen (Chapter 4). In Chapter 6, the biomass was first freezedried overnight and then extracted. For Chapter 5, we improved the RNA-directed biomass sampling and immediately added a DNA/RNA stabilizing solution, spun down the cell combined with overnight freeze-drying. One advantage of metatranscriptomics over metaproteomics is the lower biomass requirement; with approximately 1.5–2.5 g/kg of dry weight biomass, it requires only about 2–5 ml in three or four replicates, compared to 60 ml in two replicates for metaproteomics. 8
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