Fluorescent reporter plasmids for single-cell and bulk-level composition assays inE. faecalis

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>Fluorescent reporters are an important tool for monitoring dynamics of bacterial populations at the single cell and community level. While there are a large range of reporter constructs available–particularly for common model organisms like <jats:italic>E. coli</jats:italic>–fewer options exist for other species, including <jats:italic>E. faecalis</jats:italic>, a gram-positive opportunistic pathogen. To expand the potential toolkit available for <jats:italic>E. faecalis</jats:italic>, we modified a previously developed reporter plasmid (pBSU101) to express one of nine different fluorescent reporters and confirmed that all constructs exhibited detectable fluorescence in single <jats:italic>E. faecalis</jats:italic> cells and mixed biofilm communities. To identify promising constructs for bulk-level experiments, we then measured the fluorescence spectra from <jats:italic>E. faecalis</jats:italic> populations in microwell plate (liquid) cultures during different growth phases. Cultures showed density- and reporter-specific variations in fluorescent signal, though spectral signatures of all reporters become clear in late-exponential and stationary-phase populations. Based on these results, we identified six pairs of reporters that can be combined with simple spectral unmixing to accurately estimate population composition in 2-strain mixtures at or near stationary phase. This approach offers a simple and scalable method for selection and competition experiments in simple two-species populations. Finally, we modified the construct to express codon-optimized variants of blue (BFP) and red (RFP) reporters and show that they lead to increased fluorescence in exponentially growing cells. As a whole, the results inform the scope of application of different reporters and identify both single reporters and reporter pairs that are promising for fluorescence-based assays at bulk and single-cell levels in <jats:italic>E. faecalis</jats:italic>.</jats:p>

Spatial segregation and cooperation in radially expanding microbial colonies under antibiotic stress

“…Similarly, sensitive strains were transformed with a similar plasmid, pBSU101-DasherGFP, a pBSU101 derivative that lacks the β-lactamase insert and where eGFP is replaced by a brighter synthetic GFP (Dasher-GFP; ATUM ProteinPaintbox, https://www.atum.bio/). The reporter plasmids are described in detail in (93, 70). The plasmids also express a streptomycin resistance gene, and all media was therefore supplemented with streptomycin.…”