Reversible fixation of microbial cells for targeted genomics

Abstract

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.