Cell Sorting-Directed Selection of Bacterial Cells in Bigger Sizes Analyzed by Imaging Flow Cytometry during Experimental Evolution

Abstract

Cell morphology is an essential and phenotypic trait that can be easily tracked during adaptation and evolution to environmental changes. Thanks to the rapid development of quantitative analytical techniques for large populations of cells based on their optical properties, morphology can be easily determined and tracked during experimental evolution. Furthermore, the directed evolution of new culturable morphological phenotypes can find use in synthetic biology to refine fermentation processes. It remains unknown whether and how fast we can obtain a stable mutant with distinct morphologies using fluorescence-activated cell sorting (FACS)-directed experimental evolution. Taking advantage of FACS and imaging flow cytometry (IFC), we direct the experimental evolution of the E. coli population undergoing continuous passage of sorted cells with specific optical properties. After ten rounds of sorting and culturing, a lineage with large cells resulting from incomplete closure of the division ring was obtained. Genome sequencing highlighted a stop-gain mutation in amiC, leading to a dysfunctional AmiC division protein. The combination of FACS-based selection with IFC analysis to track the evolution of the bacteria population in real-time holds promise to rapidly select and culture new morphologies and association tendencies with many potential applications.

Relationships among bacterial cell size, diversity, and taxonomy in rumen

“…We need some new methods to minimize these potential limitations. Bacteria exhibit morphological differentiation associated with different species, including coccoid, rod, spirilla types, and various other uncommon shapes ( Kysela et al, 2016 ; van Teeseling et al, 2017 ; Tian et al, 2023 ). Based on the difference in size or shape of microorganisms, size fractionation using filters with various pore sizes can be used to separate them ( Sorokin et al, 2017 ; Lewis et al, 2020 ).…”

Implementation of Fluorescent-Protein-Based Quantification Analysis in L-Form Bacteria

“…The resulting high-resolution microscopic cell images can be further processed using analytical tools like ImageJ, which offers various convenient ready-to-use plugins [6,7]. Additionally, fluorescence-activated cell sorting (FACS) and imaging flow cytometry (IFC) have been developed for applications including cell sorting [8][9][10], quantification [11,12], bacterial viability assessment [13,14], dynamic monitoring of bacterial morphology [15,16] and analysis of the heterogeneity in bacterial communities [16,17]. However, the quality and reproducibility of the results of these tools primarily rely on the employed fluorescent labeling markers for targeted cells [18,19].…”