Identification of Hemin-binding Protein of Oral Streptococci via Electrophoresis in SDS Polyacrylamide Gel

Background and Objectives: It has been reported that hemin binding proteins are involved in the mechanism of obtaining iron in some bacteria. Oral streptococci in the dental plaque are assumed to acquire iron through hemin or hemin compounds. The aim of this study was to identify the presence of a protein (hemin binding protein) involved in the hemin binding mechanism of oral streptococci. Methodology: In this study, we investigated the presence of proteins involved in hemin binding of oral streptococci through sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis using hemin-agarose beads. Results: As a result of SDS-PAGE analysis, similar or different sizes of hemin binding protein bands were observed depending on the strains belonging to streptococci. The molecular weight of hemin binding protein in Streptococcus gordonii, Streptococcus rattus, Streptococcus sobrinus, Streptococcus sanguis and Streptococcus oralis were approximately 95 kDa, 43 kDa, 43 kDa, 39 kDa, and 39 kDa, respectively. Conclusion: In this study, the presence of hemin binding protein in streptococci was confirmed and the proteins involved in hemin binding in different species of oral streptococci may be different.

Safety Assessment of ProBiora3, a Probiotic Mouthwash: Subchronic Toxicity Study in Rats

Streptococcus viridans are commensal bacteria that constitute a significant portion of the resident oral microflora. The objective of the present study is to investigate adverse effects, if any, of a blend of 3 natural strains, Streptococcus uberis KJ2, Streptococcus oralis KJ3, and Streptococcus rattus JH145 (probiotic mouthwash, ProBiora3). The blend is administered to rats orally once daily (5 days per week) at doses of 0, 106, or 109 colony-forming units of each strain for 14 weeks. No treatment-related adverse effects are observed in the physiological parameters during the study or in the evaluation of blood and tissue samples taken from the animals at the end. Results of an in vitro antibiotic susceptibility study demonstrate that all 3 ProBiora3 strains are susceptible to commonly used therapeutic antibiotics. The results of these investigations reveal that the no-observed-adverse-effect level of the probiotic mouthwash is 2.16 × 109 colony-forming units per strain per kilogram of body weight per day, the highest dose used.

Characterization of the Arginine Deiminase Operon of Streptococcus rattus FA-1

ABSTRACT The arginine deiminase system (ADS) is of critical importance in oral biofilm pH homeostasis and microbial ecology. The ADS consists of three enzymes. Arginine is hydrolyzed by AD (ArcA) to generate citrulline and ammonia. Citrulline is then converted to ornithine and carbamoylphosphate via ornithine carbamoyltransferase (ArcB). Finally, carbamate kinase (ArcC) transfers a phosphate from carbamoylphosphate to ADP, yielding ATP. Ammonia production from this pathway protects bacteria from lethal acidification, and ATP production provides a source of energy for the cells. The purpose of this study was to initiate a characterization of the arc operon of Streptococcus rattus, the least cariogenic and sole ADS-positive member of the mutans streptococci. Using an arcB gene fragment obtained by degenerate PCRs, the FA-1 arc operon was identified in subgenomic DNA libraries and sequence analysis was performed. Results showed that the genes encoding the AD pathway in S. rattus FA-1 are organized as an arcABCDT-adiR operon gene cluster, including the enzymes of the pathway, an arginine-ornithine antiporter (ArcD) and a putative regulatory protein (AdiR). The arcA transcriptional start site was identified by primer extension, and a σ70-like promoter was mapped 5′ to arcA. Reverse transcriptase PCR was used to establish that arcABCDT could be cotranscribed. Reporter gene fusions and AD assays demonstrated that the operon is regulated by substrate induction and catabolite repression, the latter apparently through a CcpA-dependent pathway.

Turning on and turning off the arginine deiminase system in oral streptococci

The arginine deiminase system in oral streptococci is highly regulated. It requires induction and is repressed by catabolites such as glucose or by aeration. A comparative study of regulation of the system in Streptococcus gordonii ATCC 10558, Streptococcus rattus FA-1, and Streptococcus sanguis NCTC 10904 showed an increase in activity of the system in S. sanguis of some 1467-fold associated with induction-derepression of cells previously uninduced-repressed. The activity of the system was assayed in terms of levels of arginine deiminase, the signature enzyme of the system, in permeabilized cells. Increases in enzyme levels associated with induction-derepression were less for the other two organisms, mainly because of less severe repression, especially for S. rattus FA-1, which was the least sensitive to catabolite repression or aeration. Regulation of the arginine deiminase system involving induction and catabolite repression was demonstrated also with monoorganism biofilms composed of cells of S. sanguis adherent to glass slides. Fully uninduced-repressed cells from suspension cultures or biofilms were compromised in their abilities to catabolize arginine to protect themselves against acid damage. However, it was found that the system can be rapidly turned on or turned off, although induction-derepression did appear to require cell growth. Still, the system could respond rapidly to the availability of arginine to reestablish high capacity for alkali production.Key words: arginine deiminase, oral streptococci, induction-derepression, acid damage, biofilms.