Deficiency of RgpG Causes Major Defects in Cell Division and Biofilm Formation, and Deficiency of LytR-CpsA-Psr Family Proteins Leads to Accumulation of Cell Wall Antigens in Culture Medium by Streptococcus mutans

Abstract

ABSTRACTStreptococcus mutansis known to possess rhamnose-glucose polysaccharide (RGP), a major cell wall antigen.S. mutansstrains deficient inrgpG, encoding the first enzyme of the RGP biosynthesis pathway, were constructed by allelic exchange. ThergpGdeficiency had no effect on growth rate but caused major defects in cell division and altered cell morphology. Unlike the coccoid wild type, thergpGmutant existed primarily in chains of swollen, “squarish” dividing cells. Deficiency ofrgpGalso causes significant reduction in biofilm formation (P< 0.01). Double and triple mutants with deficiency inbrpAand/orpsr, genes coding for the LytR-CpsA-Psr family proteins BrpA and Psr, which were previously shown to play important roles in cell envelope biogenesis, were constructed using thergpGmutant. There were no major differences in growth rates between the wild-type strain and thergpG brpAandrgpG psrdouble mutants, but the growth rate of thergpG brpA psrtriple mutant was reduced drastically (P< 0.001). Under transmission electron microscopy, both double mutants resembled thergpGmutant, while the triple mutant existed as giant cells with multiple asymmetric septa. When analyzed by immunoblotting, thergpGmutant displayed major reductions in cell wall antigens compared to the wild type, while little or no signal was detected with the double and triple mutants and thebrpAandpsrsingle mutants. These results suggest that RgpG inS. mutansplays a critical role in cell division and biofilm formation and that BrpA and Psr may be responsible for attachment of cell wall antigens to the cell envelope.IMPORTANCEStreptococcus mutans, a major etiological agent of human dental caries, produces rhamnose-glucose polysaccharide (RGP) as the major cell wall antigen. This study provides direct evidence that deficiency of RgpG, the first enzyme of the RGP biosynthesis pathway, caused major defects in cell division and morphology and reduced biofilm formation byS. mutans, indicative of a significant role of RGP in cell division and biofilm formation inS. mutans. These results are novel not only inS. mutans, but also other streptococci that produce RGP. This study also shows that the LytR-CpsA-Psr family proteins BrpA and Psr inS. mutansare involved in attachment of RGP and probably other cell wall glycopolymers to the peptidoglycan. In addition, the results also suggest that BrpA and Psr may play a direct role in cell division and biofilm formation inS. mutans. This study reveals new potential targets to develop anticaries therapeutics.