BACKGROUND: To respond and adapt to environmental challenges, prokaryotes regulate cellular processes rapidly and reversibly through protein phosphorylation and dephosphorylation. This study investigates the intracellular proteome and Ser/Thr/Tyr phosphoproteome of the oral commensal Streptococcus gordonii. Intracellular proteins from planktonic cells of S. gordonii DL1 were extracted and subjected to 2D-gel electrophoresis. Proteins in general were visualized using Coomassie Brilliant Blue and T-Rex staining. Phosphorylated proteins were visualized with Pro-Q Diamond Phosphoprotein Gel Stain. Proteins were identified by LC-MS/MS and sequence analysis.

RESULTS: In total, sixty-one intracellular proteins were identified in S. gordonii DL1, many of which occurred at multiple isoelectric points. Nineteen of these proteins were present as one or more Ser/Thr/Tyr phosphorylated form. The identified phosphoproteins turned out to be involved in a variety of cellular processes.

CONCLUSION: Nineteen phosphoproteins involved in various cellular functions were identified in S. gordonii. This is the first time the global intracellular Ser/Thr/Tyr phosphorylation profile has been analysed in an oral streptococcus. Comparison with phosphoproteomes of other species from previous studies showed many similarities. Proteins that are consistently found in a phosphorylated state across several species and growth conditions may represent a core phosphoproteome profile shared by many bacteria.

Background Salivary mucin MUC5B seems to promote biodiversity in dental biofilms, and thereby oral health, for example, by inducing synergistic 'mucolytic' activities in a variety of microbial species that need to cooperate for the release of nutrients from the complex glycoprotein. Knowledge of how early colonizers interact with host salivary proteins is integral to better understand the maturation of putatively harmful oral biofilms and could provide key insights into biofilm physiology. Methods The early oral colonizer Streptococcus gordonii DL1 was grown planktonically and in biofilm flow cell systems with uncoated, MUC5B or low-density salivary protein (LDP) coated surfaces. Bacterial cell proteins were extracted and analyzed using a quantitative mass spectrometry-based workflow, and differentially expressed proteins were identified. Results and conclusions Overall, the proteomic profiles of S. gordonii DL1 were similar across conditions. Six novel biofilm cell proteins and three planktonic proteins absent in all biofilm cultures were identified. These differences may provide insights into mechanisms for adaptation to biofilm growth in this species. Salivary MUC5B also elicited specific responses in the biofilm cell proteome. These regulations may represent mechanisms by which this mucin could promote colonization of the commensal S. gordonii in oral biofilms.

Bacterial metabolism in oral biofilms is comprised of complex networks of nutritional chains and biochemical regulations. These processes involve both intraspecies and interspecies networks as well as interactions with components from host saliva, gingival crevicular fluid, and dietary intake. In a previous paper, a large salivary glycoprotein, mucin MUC5B, was suggested to promote a dental health-related phenotype in the oral type strain of Streptococcus gordonii DL1, by regulating bacterial adhesion and protein expression. In this study, nuclear magnetic resonance-based metabolomics was used to examine the effects on the metabolic output of monospecies compared to dual species early biofilms of two clinical strains of oral commensal bacteria, S. gordonii and Actinomyces naeslundii, in the presence of MUC5B. The presence of S. gordonii increased colonization of A. naeslundii on salivary MUC5B, and both commensals were able to utilize MUC5B as a sole nutrient source during early biofilm formation. The metabolomes suggested that the bacteria were able to release mucin carbohydrates from oligosaccharide side chains as well as amino acids from the protein core. Synergistic effects were also seen in the dual species biofilm metabolome compared to the monospecies, indicating that A. naeslundii and S. gordonii cooperated in the degradation of salivary MUC5B. A better understanding of bacterial interactions and salivary-mediated regulation of early dental biofilm activity is meaningful for understanding oral biofilm physiology and may contribute to the development of future prevention strategies for biofilm-induced oral disease.

IMPORTANCE: The study of bacterial interactions and salivary-mediated regulation of early dental biofilm activity is of interest for understanding oral microbial adaptation to environmental cues and biofilm maturation. Findings in oral commensals can prove useful from the perspectives of both oral and systemic health of the host, as well as the understanding of general microbial biofilm physiology. The knowledge may provide a basis for the development of prognostic biomarkers, or development of new treatment strategies, related to oral health and disease and possibly also to other biofilm-induced conditions. The study is also an important step toward developing the methodology for similar studies in other species and/or growth conditions.