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

Abstract

ABSTRACTFluorescent 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 likeE. coli–fewer options exist for other species, includingE. faecalis, a gram-positive opportunistic pathogen. To expand the potential toolkit available forE. faecalis, 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 singleE. faecaliscells and mixed biofilm communities. To identify promising constructs for bulk-level experiments, we then measured the fluorescence spectra fromE. faecalispopulations 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 inE. faecalis.