Modern molecular analyses have revolutionized the study of microbial communities, yet DNA extraction and sequencing remain critical sources of bias. This study investigated the impact of seven different DNA extraction protocols and two 16S rRNA hypervariable regions (V1-V3 and V3-V4) on the profiling of a complex anaerobic fermentative biomass selected for medium-chain fatty acids production. Microscopic analysis established a baseline community dominated by Actinobacteria (53% ± 2%) and Firmicutes (47% ± 3%). The results demonstrate that Kit1 and Kit5 provided the highest DNA yields (up to 603 ng/μL) and the most effective recovery of these hard-to-lyse phyla, although they introduced a slight taxonomic bias toward Actinobacteria. In contrast, protocols relying on intensive chemical lysis without robust mechanical disruption (Kit4) significantly underestimated total bacterial abundance and showed the lowest purity. 16S rRNA gene sequencing revealed that the V3-V4 region provided higher alpha-diversity and a more balanced representation of the community core compared to V1-V3, which was more susceptible to extraction-related variability and overrepresented the genus Olsenella. Our multi methodological approach reveals significant biases introduced by both extraction technique and 16S rRNA gene region. This evidence highlights that protocol optimization is mandatory for achieving an accurate and comprehensive characterization of microbial ecosystems.
Developing N2-fixing partnerships between diazotrophs and non-legumes can enhance soil fertility and reduce dependence on synthetic fertilisers. Unlike legumes, non-legumes lack the genetic ability to form root nodule symbiosis with rhizobia but can form facultative associations with free-living diazotrophs. Engineering these microbes by transferring key traits underlying efficient nodule formation and N2-fixation from well-characterised rhizobia represents a central aim in synthetic biology to enhance biological nitrogen fixation in non-legumes. However, the lack of effective tools for identifying compatible and engineerable microbial partners is a key challenge. To address this, we have developed nodulation (nod) gene reporters to screen both rhizobia and non-rhizobia capable of expressing Sinorhizobium meliloti nod genes, which encode bacterial signals initiating nodule formation in legumes. The biosensors include a superfolder GFP reporter controlled by the inducible nod box promoter (PnodA), plant signal-dependent activators nodD1 and nodD2, and a constitutively mScarlet-I marker, named nodD1-PnodA and nodD2-PnodA. Their functionality was validated across diverse rhizobia and non-rhizobia using in vitro and in planta induction assays. This reporter system enables high-throughput identification of novel bacteria capable of recognising and responding to legume signalling molecules that coordinate symbiotic nitrogen fixation.
The growing research into the archaeal lipidome has uncovered a remarkable structural diversity in isoprenoidal glycerol dialkyl glycerol tetraethers (iGDGTs) and revealed complex membrane adaptations, especially in extreme environments. We performed a comprehensive analysis of the lipidome from the subsurface aquifer of the CO2-rich, cold-water Geyser Andernach (Germany), using ultra-high-resolution mass spectrometry. We detected iGDGT-0, presumably derived from the dominant community member Candidatus Altiarchaeum, providing supporting evidence for its ability to synthesise tetraethers, as previously predicted from metagenomic data. Beyond the typical iGDGT-0 and acyclic glycerol trialkyl glycerol tetraether (iGTGT-0), we discovered novel structural derivatives, here referred to as extended iGDGTs and iGTGTs, characterised by the asymmetrical addition of up to two isoprenoid units to only one of their hydrocarbon side chains, analogous to those found in extended archaeols. The apparent absence of GDGT ring synthase A and B genes in the corresponding metagenome-assembled genome raises the possibility that the producing archaea may utilise extended iGDGTs as a membrane adaptation to cope with the nutrient-depleted conditions of the geyser environment, highlighting the adaptive flexibility of archaea to extreme physicochemical conditions.
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