Design, development, and validation of new fluorescent strains for studying oral streptococci

Bacterial strains that are genetically engineered to constitutively produce fluorescent proteins have aided our study of bacterial physiology, biofilm formation, and interspecies interactions. Here, we report on the construction and utilization of new strains that produce the blue fluorescent protein mTagBFP2, the green fluorescent protein sfGFP, and the red fluorescent protein mScarlet-I3 in species Streptococcus gordonii, Streptococcus mutans, and Streptococcus sanguinis. Gene fragments, developed to contain the constitutive promoter Pveg, the fluorescent gene of interest, as well as aad9, providing resistance to the antibiotic spectinomycin, were inserted into selected open reading frames on the chromosome that were both transcriptionally silent and whose loss caused no measurable changes in fitness. All strains, except for sfGFP in S. sanguinis, were validated to produce a detectable and specific fluorescent signal. Individual stains, along with extracellular polymeric substances (EPS) within biofilms, were visualized and quantified through either widefield or super-resolution confocal microscopy approaches. Finally, to validate the ability to perform single-cell-level analysis using the strains, we imaged and analyzed a triculture mixed-species biofilm of S. gordonii, S. mutans, and S. sanguinis grown with and without the addition of human saliva. Quantification of the loss in membrane integrity using a SYTOX dye revealed that all strains had increased loss of membrane integrity with water or human saliva added to the growth media, but the proportion of the population stained by the SYTOX dye varied by species. In all, these fluorescent strains will be a valuable resource for the continued study of oral microbial ecology.IMPORTANCEStreptococci are among the earliest colonizers of the soft and hard tissues of the oral cavity and are contributors to the oral health status of the host, with involvement in dental caries, endodontic infections, periodontal disease, and the development of oral cancer. Strains genetically modified to produce fluorescent proteins that can be either visualized through microscopy imaging or quantified by their specific fluorescent intensity signal are critical tools toward the study of individual or mixed-species cultures. Our report here details the development and testing of several new strains of fluorescent oral streptococci that can be utilized in the study of microbial ecology, increasing both the availability of tools and documenting experimental approaches toward in vitro assay applications such as the study of intermicrobial interactions.