Mechanisms of Integrative and Conjugative Elements in Oral Bacteria - Project Summary Streptococcus mutans resides in the oral cavity where it is known to initiate the development of dental caries. For this pathogen to thrive in the dynamic and variable environment of the mouth, S. mutans must be able to adapt to both short- and long-term ecological pressures. The horizontal acquisition of new DNA, which may encode for new beneficial traits, is a critical aspect of S. mutans adaptability. In addition, regulation of the processes involved in the acquisition of DNA are intertwined with the basic physiology and stress responses of S. mutans. Mobile genetic elements often mediate HGT and include integrative and conjugative elements (ICEs), widespread among oral bacteria. ICEs are mobile elements that are integrated on the genome and possess the ability to excise and subsequently transfer to nearby bacteria via conjugation. Importantly, these elements often contain “cargo” genes that provide some benefit to the host bacterial cells (including antibiotic resistance, metabolism, and virulence). In recent work supported by R03 DE029882 and F32 DE032551, we undertook the first characterization of TnSmu1 as a model of HGT within this critical oral pathogen. We discovered that TnSmu1 is a functional ICE, capable of transfer. Further, we have made significant progress investigating a regulatory system that controls activation of TnSmu1 and showed that TnSmu1 causes a host cell defect in cell division. Further, when TnSmu1 excision is induced, the S. mutans Type I-C CRISPR-Cas system is up-regulated, for reasons that are currently unclear but possibly suggesting an interaction between TnSmu1 and this bacterial defense mechanism. Together, this indicates TnSmu1 shares a complex relationship with its host bacterial cell. Despite the prevalence of ICEs in oral bacteria and the significant impact of TnSmu1 on host cell physiology, detailed studies of their molecular mechanisms and their impact on oral communities are lacking. To address this, we propose to use TnSmu1 as a model of conjugal transfer in the following Specific Aims: Aim 1: Dissect the regulatory mechanisms that control the switch from the integrated to excised state of TnSmu1. Aim 2: Elucidating interactions of TnSmu1, mobile genetic elements, and CRISPR-Cas defense in bacterial hosts. Aim 3: Investigating the fitness cost(s) of TnSmu1 acquisition on recipient strains. Taken together, these studies will provide a wealth of information for an understudied mechanism of gene transfer in the oral microbiome. Elucidating mechanisms of gene transfer is critical for understanding of S. mutans adaptability and the ability to combat oral disease. At the conclusion of these studies, this work will have created a new understanding of HGT, evolution, and pathogenesis of S. mutans within the oral microbiome.
