Fluorescence Tools Adapted for Real-Time Monitoring of the Behaviors of Streptococcus Species.

Tagging of bacteria with fluorescent proteins has become an essential component of modern microbiology. Fluorescent proteins can be used to monitor gene expression and biofilm growth and to visualize host-pathogen interactions. Here, we developed a collection of fluorescent protein reporter plasmids for Streptococcus mutans UA159 and other oral streptococci. Using superfolder green fluorescent protein (sfGFP) as a reporter for transcriptional activity, we were able to characterize four strong constitutive promoters in S. mutans These promoter-sfgfp fusions worked both for single-copy chromosomal integration and on a multicopy plasmid, with the latter being segregationally stable in the absence of selective pressure under the conditions tested. We successfully labeled S. mutans UA159, Streptococcus gordonii DL1, and Streptococcus sp. strain A12 with sfGFP, DsRed-Express2 (red), and citrine (yellow). To test these plasmids under more challenging conditions, we performed mixed-species biofilm experiments and separated fluorescent populations using fluorescence-activated cell sorting (FACS). This allowed us to visualize two streptococci at a time and quantify the amounts of each species simultaneously. These fluorescent reporter plasmids add to the genetic toolbox available for the study of oral streptococci.IMPORTANCE Oral streptococci are the most abundant bacteria in the mouth and have a major influence on oral health and disease. In this study, we designed and optimized the expression of fluorescent proteins in Streptococcus mutans and other oral streptococci. We monitored the levels of expression and noise (the variability in fluorescence across the population). We then created several fluorescent protein delivery systems (green, yellow, and red) for use in oral streptococci. The data show that we can monitor bacterial growth and interactions in situ, differentiating between different bacteria growing in biofilms, the natural state of the organisms in the human mouth. These new tools will allow researchers to study these bacteria in novel ways to create more effective diagnostic and therapeutic tools for ubiquitous infectious diseases.

Novel Probiotic Mechanisms of the Oral Bacterium Streptococcus sp. A12 as Explored with Functional Genomics.

Health-associated biofilms in the oral cavity are composed of a diverse group of microbial species that can foster an environment that is less favorable for the outgrowth of dental caries pathogens, like Streptococcus mutans A novel oral bacterium, designated Streptococcus A12, was previously isolated from supragingival dental plaque of a caries-free individual and was shown to interfere potently with the growth and virulence properties of S. mutans In this study, we applied functional genomics to begin to identify molecular mechanisms used by A12 to antagonize, and to resist the antagonistic factors of, S. mutans Using bioinformatics, genes that could encode factors that enhance the ability of A12 to compete with S. mutans were identified. Selected genes, designated potential competitive factors (pcf), were deleted. Certain mutant derivatives showed a reduced capacity to compete with S. mutans compared to that of the parental strain. The A12 pcfO mutant lost the ability to inhibit comX -inducing peptide (XIP) signaling by S. mutans, while mutants with changes in the pcfFEG locus were impaired in sensing of, and were more sensitive to, the lantibiotic nisin. Loss of PcfV, annotated as a colicin V biosynthetic protein, resulted in diminished antagonism of S. mutans Collectively, the data provide new insights into the complexities and variety of factors that affect biofilm ecology and virulence. Continued exploration of the genomic and physiological factors that distinguish commensals from truly beneficial members of the oral microbiota will lead to a better understanding of the microbiome and new approaches to promote oral health.IMPORTANCE Advances in defining the composition of health-associated biofilms have highlighted the important role of beneficial species in maintaining health. Comparatively little, however, has been done to address the genomic and physiological bases underlying the probiotic mechanisms of beneficial commensals. In this study, we explored the ability of a novel oral bacterial isolate, Streptococcus A12, to compete with the dental pathogen Streptococcus mutans using various gene products with diverse functions. A12 displayed enhanced competitiveness by (i) disrupting intercellular communication pathways of S. mutans, (ii) sensing and resisting antimicrobial peptides, and (iii) producing factors involved in the production of a putative antimicrobial compound. Research on the probiotic mechanisms employed by Streptococcus A12 is providing essential insights into how beneficial bacteria may help maintain oral health, which will aid in the development of biomarkers and therapeutics that can improve the practice of clinical dentistry.

Growth with Commensal Streptococci Alters Streptococcus mutans Behaviors.

As oral bacteria grow and persist within biofilms attached to the tooth's surface, they interact with other species to form synergistic or antagonistic exchanges that govern homeostasis for the overall population. One example are the interactions between the cariogenic species Streptococcus mutans and oral commensal streptococci. Previously, we showed that the cell-cell signaling pathways of S. mutans were inhibited during coculture with other oral streptococci species, leading us to posit that the S. mutans transcriptome and behaviors are broadly altered during growth with these species. To test this hypothesis, we performed whole transcriptome sequencing (RNA-seq) on cocultures of S. mutans with either Streptococcus gordonii, Streptococcus sanguinis, or Streptococcus oralis and a quadculture containing all 4 species in comparison to S. mutans grown alone. Our results reveal that in addition to species-dependent changes to the S. mutans transcriptome, a conserved response to oral streptococci in general can be observed. We monitored the behavior of S. mutans by both microscopy imaging of biofilms and in a bacteriocin overlay assay and verified that S. mutans acts similarly with each of these species but noted divergences in phenotypes when cocultured with another cariogenic Streptococcus (Streptococcus sobrinus) or with oral nonstreptococci species. RNA-seq with oral nonstreptococci showed lack of a consistent gene expression profile and overlap of differentially expressed genes found with commensal streptococci. Finally, we investigated the role of upregulated S. mutans genes within our data sets to determine if they provided a fitness benefit during interspecies interactions. Eleven total genes were studied, and we found that a majority impacted the fitness of S. mutans in various assays, highlighted by increased biomass of commensal streptococci in mixed-species biofilms. These results confirm a common, species-independent modification of S. mutans behaviors with oral commensal streptococci that emphasizes the need to further evaluate oral bacteria within multispecies settings.