Streptococcus mutans yidC1 and yidC2 impact cell-envelope biogenesis, biofilm 1 matrix and biophysical properties 2

31 Proper envelope biogenesis of Streptococcus mutans, a biofilm-forming and 32 dental caries causing oral pathogen , requires two paralogs ( yidC1 and yidC2 ) of the 33 universally conserved YidC/Oxa1/Alb3 family of membrane integral chaperones and 34 insertases. Deletion of either paralog attenuates virulence in vivo but the mechanisms of 35 disruption remain unclear. Here, we determined whether deletion of yidC affects cell 36 surface properties, extracellular glucan production, and/or the structural organization of 37 EPS matrix and biophysical properties of S. mutans biofilm. Compared to wild type, the 38 ΔyidC 2 mutant lacked staining with vancomycin at the division septum, while the 39 ΔyidC1 mutant resembled wild-type. Additionally, deletion of either yidC1 or yidC2 40 resulted in less insoluble glucan synthesis, but produced more soluble glucans, 41 especially at early and mid-exponential growth phases. Alteration of glucan synthesis by 42 both mutants yielded biofilms with less dry-weight and insoluble EPS. In particular, 43 deletion of yidC2 resulted in significant reduction of biofilm biomass and pronounced 44 defects in the spatial organization of the EPS matrix, thus modifying the 3D biofilm architecture. The defective biofilm harbored smaller bacterial clusters with high cell density and less surrounding EPS compared to wild type, which was stiffer in compression yet more susceptible to removal by shear. Together, our results indicate that elimination of either yidC paralog results in changes to the cell envelope and glucan production that ultimately disrupts biofilm development and EPS matrix structure- composition, thereby altering the physical properties of the biofilms and facilitating their 51 removal. YidC proteins, therefore, represent potential therapeutic targets for cariogenic 52 biofilm control. are universally conserved in all bacteria, and are traditionally studied in the context of membrane protein insertion and assembly. Both YidC paralogs Streptococcus mutans are required for proper envelope biogenesis and full virulence, indicating these proteins may also contribute to optimal biofilm formation in streptococci. Here we show that deletion of either yidC results in changes to the structure and physical properties of the EPS matrix produced by S. mutans , ultimately impairing optimal biofilm development, diminishing its mechanical stability, and facilitating its removal. Importantly, the universal conservation of bacterial yidC orthologs, combined with our findings, provide a rationale for YidC as a possible drug target for anti-biofilm therapies. nm) a Leica using a Diatome diamond knife. Grids were post-stained with 1% uranyl acetate and Reynold’s lead citrate buffers and then imaged on an FEI Tecnai G2 Spirit TEM, operating at 80 keV and micrographs collected in the range of 4,000 - 100,000 X magnification. Results shown are of representative images. The analyze feature in 508 ImageJ (v. 2.0.0) was used to measure cell wall thickness and cell diameter. Images 509 were calibrated based on the number of pixels in the scale bar. For cell wall thickness, 3 separate measurements were averaged together for each cell, with a minimum of 9 511 cells measured per strain. For cell diameter, measurements were taken across the middle of each cell at the narrowest point. A minimum of 6 cells were compared for 513 each strain. One-Way ANOVA with Dunnett’s post hoc test was used to determine significant differences between mutant and wild-type strains.