Volmer, J. G. | Search bioRxiv

bioRxiv2026

Reversible fixation of microbial cells for targeted genomics

Phylogeny- or activity-based fluorescent labelling of microorganisms coupled with fluorescence activated cell sorting (FACS) is a powerful approach to rapidly isolate cells of interest from complex communities for targeted genomics. However, the method has not been widely adopted because standard paraformaldehyde or ethanol fixation procedures hamper nucleic acid recovery and/or long-term sample storage. Here, we present dithiobis(succinimidyl propionate) (DSP) as a reversible fixative for microbial cells that enables fluorescence imaging and DNA extraction and amplification for sequencing. DSP fixation is compatible with widely used fluorescence in situ hybridization (FISH) approaches and bioorthogonal non-canonical amino acid tagging, and can be applied to diverse sample types, where it consistently performed as well as paraformaldehyde. Furthermore, high-quality genomes were obtained from individual and multiple bacterial cells selectively extracted from complex samples using FISH-FACS. Thus, DSP is a dual-use fixative for microbiome imaging and sequencing, improving targeted microbial genomics.

microbiology↗

bioRxiv2024

Metagenomic analysis of marsupial gut microbiomes provides a genetic basis for the low methane economy

The potent greenhouse gas methane is an end-product of plant biomass digestion by gut microbiota, though the amount produced and/or released varies among herbivorous animals. On a per unit of feed basis, macropodid marsupials (e.g. kangaroos) are widely thought to be low methane-emitting herbivores compared to high methane-producing ruminant livestock. How the gut microbiome contributes to the low methane status of marsupials is not well understood but of high potential value for a low methane economy. Here, we analyse the faecal metagenomes of 14 different marsupial species and 1,394 derived metagenome-assembled genomes (MAGs), focusing on the functional distinction of the bacterial and archaeal communities compared to ruminant faecal microbiomes. Though composition and function of the marsupial gut microbiome considerably varied across and within animal species, there was a clear host-associated bacterial signature for the community that differed significantly between marsupial hosts and compared to ruminants. Of particular note was a range of Bacteroidota, Campylobacterota, Desulfobacterota, Pseudomonadota and Verrucomicrobiota species that were enriched in marsupials and encode H2-uptake hydrogenases that mediate hydrogenotrophic respiration. Additionally, in support of an enrichment of electron sinks, enzymes for butyrate, propionate, and glutamate production, as well as nitrate, nitrite, and fumarate respiration were enriched in marsupials. Collectively, these data suggest that, by favoring an enrichment of alternate hydrogen sinks of bacterial origin, the low methane phenotype reported for marsupials is feasible and offers a genetic basis to pursue reductions of livestock methane emissions.

microbiology↗