Lin Zeng

SppR is a transcriptional repressor of fructanase FruA and fructose-1-phosphatase SppA. In this study, a frameshift sppR mutant was constructed and analyzed using in vitro biofilm models and stress challenge assays for its role in S. mutans’ stress tolerance response and biofilm formation. In addition, luciferase reporter fusion assay and in vitro transcription assay were used to analyze the role of SppR in regulation of brpA . Results showed that sppR deficiency caused no major effect on planktonic growth and cell morphology in regular BHI broth, however negatively impacted growth at low pH and in the presence of oxidative stressor ( P < 0.001). Relative to the parent strain, UA159, the sppR mutant also had a reduced survival rate following acid killing (PP< 0.05), while the disruption of a putative SppR-binding site in the promoter reduced its activity by >7-fold (P136 genes by >2-fold ( P< 0.05), including 68 genes up-regulated and 68 genes reduced. Among the down-regulated genes were those belonging to DNA-repair and acid tolerance response. These results suggest that SppR plays an important role in S. mutans physiology including acid and oxidative stress tolerance response and regulation of BrpA expression.

From its discovery in the late 1800s to the present day, Streptococcus mutans research has been at the forefront of dental caries investigations, providing the foundation for our current understanding of the microbiological determinants of this disease. In addition, research on S. mutans has greatly advanced key areas of microbiology, establishing the organism as a notably useful model for understanding the biology of other streptococcal species, as well as Gram-positive bacteria more broadly. In this piece, we provide a retrospective of how S. mutans has served this dual role, highlighting specific areas that have benefited most from S. mutans –driven discoveries. We close by outlining emerging areas of S. mutans research that are likely to influence future breakthroughs in both oral health science and basic microbiology.

Current understanding in arginine metabolism by streptococci is limited in species and strains. We compared the ability of several abundant oral streptococci to synthesize and assimilate arginine. Multiple species of oral streptococci and their genetic mutants were characterized for growth using synthetic media with specified amounts of arginine and related amino acids. Expression of relevant genes was quantified by RT-qPCR. An intact arginine deiminase system (ADS) was required for S. sanguinis to grow with little or no arginine, a capacity substantially hindered by oxygen. Importantly, nearly all oral streptococci tested required arginine, ornithine or citrulline for growth, displaying much heterogeneity. S. mutans, while lacking ADS, grew without exogenous arginine; and presence of ornithine significantly affected its growth dynamics with strain-specific outcomes. Furthermore, loss of an ornithine transcarbamylase in S. mutans abolished ornithine-dependent effects and the ability to synthesize arginine. Expression of biosynthetic arg genes in both S. sanguinis and S. mutans was influenced, albeit differentially, by exogenous arginine, ornithine, oxygen, carbohydrates, and integrity of ADS and the major glucose transporter (EII^Man). Our study suggested that arginine is an essential nutrient for most oral streptococci, excluding S. mutans, and identified ornithine and citrulline as potential metabolic signals in oral microbiome.

Fructose catabolism by Streptococcus mutans is initiated by three phosphotransferase (PTS) transporters yielding fructose-1-phosphate (F-1-P) or fructose-6-phosphate. Deletion of one such F-1-P-generating PTS, fruI , was shown to reduce the cariogenicity of S. mutans in rats fed a high-sucrose diet. Moreover, a recent study linked fructose metabolism in S. mutans to a reactive electrophile species methylglyoxal. Here, we conducted a comparative transcriptomic analysis of S. mutans treated briefly with 50 mM fructose, 50 mM glucose, 5 mM methylglyoxal, or 0.5 mM hydrogen peroxide (H ₂ O ₂ ). The results revealed a striking overlap between the fructose and methylglyoxal transcriptomes, totaling 176 genes, 61 of which were also shared with the H ₂ O ₂ transcriptome. This core of 61 genes encompassed many of the same pathways affected by exposure to low pH or zinc intoxication. Consistent with these findings, fructose negatively impacted the metal homeostasis of a mutant deficient in zinc expulsion and the growth of a mutant of the major oxidative stress regulator SpxA1. Importantly, fructose metabolism lowered culture pH at a faster pace, allowed better survival under acidic and nutrient-depleted conditions, and enhanced the competitiveness of S. mutans against Streptococcus sanguinis , although a moderated level of F-1-P might further boost some of these benefits. Conversely, several commensal streptococcal species displayed a greater sensitivity to fructose that may negatively affect their persistence and competitiveness in dental biofilm. In conclusion, fructose metabolism is integrated into the stress core of S. mutans and regulates critical functions required for survival and its ability to induce dysbiosis in the oral cavity. IMPORTANCE: Fructose is a common monosaccharide in the biosphere, yet its overconsumption has been linked to various health problems in humans including insulin resistance, obesity, diabetes, non-alcoholic liver diseases, and even cancer. These effects are in large part attributable to the unique biochemical characteristics and metabolic responses associated with the degradation of fructose. Yet, an understanding of the effects of fructose on the physiology of bacteria and its implications for the human microbiome is severely lacking. Here, we performed a series of analyses on the gene regulation of a dental pathogen Streptococcus mutans by exposing it to fructose and other important stress agents. Further supported by growth, persistence, and competition assays, our findings revealed the ability of fructose to activate a set of stress-related functions that may prove critical to the ability of the bacterium to persist and cause diseases both within and without the oral cavity.

Previous work reported that deletion of the Enzyme IIAB subunits (EIIAB ^Man and manL ) of the glucose phosphotransferase system (PTS) (glucose-PTS, manLMNO ) in Streptococcus sanguinis impacted carbon catabolite repression and bacterial fitness. Here, a single-nucleotide polymorphism in ManN, ManNA91E, produced the unusual phenotype of increased excretion of organic acids and H ₂ O ₂ yet elevated PTS activities. To characterize the contributions of each component of the glucose-PTS to bacterial fitness, we performed genetic analyses by deleting from S. sanguinis SK36 the entire operon and each EII ^Man subunit individually; and genes encoding the catabolite control protein A (Δ ccpA ) and the redox regulator Rex (Δ rex ) for comparison. Deletion of each subunit incurred a growth defect on glucose partly due to elevated excretion of H ₂ O ₂ ; when supplemented with catalase, this defect was rescued, instead resulting in a significantly higher yield than the parent. All glucose-PTS deletion mutants presented an increased antagonism against the oral pathobiont Streptococcus mutans , a phenotype absent in Δ ccpA despite increased H ₂ O ₂ output. A shift in the pyruvate node toward mixed acid fermentation and increased arginine deiminase activity enhanced pH homeostasis in glucose-PTS mutants but not Δ ccpA . Despite the purported ability of Rex to regulate central carbon metabolism, deletion of rex had no significant impact on most of the phenotypes discussed here. These findings place glucose-PTS in the pivotal position of controlling central carbon flux in streptococci, with critical outcomes impacting acidogenicity, aciduricity, pH homeostasis, and antagonism, highlighting its potential as a therapeutic target for treating diseases with a dysbiotic microbiome. IMPORTANCE: Management of carbohydrate metabolism and environmental stress is key to the survival of oral commensal species such as S. sanguinis . Antagonism of oral pathobionts and modulation of the environmental pH and oxidative potential by commensals are crucial to the maintenance of microbial homeostasis and prevention of oral diseases including dental caries. It is therefore vital to understand how these species regulate sugar fermentation, production of acids and ammonia, and stress management in an environment known for a feast-and-famine cycle of carbohydrates and similar fluctuations in pH and oxygen tension. Here, we detail that genetic alterations of the glucose-PTS transporter in S. sanguinis can significantly affect the regulation of factors required for bacterial fitness and homeostatic ability independent of known catabolic regulators. It is then discussed how these changes may impact the survival of streptococcal species and affect caries onset.

Fructose catabolism by Streptococcus mutans is initiated by three PTS transporters yielding fructose-1-phoshate (F-1-P) or fructose-6-phosphate. Deletion of one such F-1-P-generating PTS, fruI , was shown to reduce the cariogenicity of S. mutans in rats fed a high-sucrose diet. Moreover, a recent study linked fructose metabolism in S. mutans to a reactive electrophile species (RES) methylglyoxal. Here, we conducted a comparative transcriptomic analysis of S. mutans treated briefly with 50 mM fructose, 50 mM glucose, 5 mM methylglyoxal, or 0.5 mM hydrogen peroxide (H ₂ O ₂ ). The results revealed a striking overlap between the fructose and methylglyoxal transcriptomes, totaling 176 genes, 61 of which were also shared with the H ₂ O ₂ transcriptome. This core of 61 genes encompassed many of the same pathways affected by exposure to low pH or zinc intoxication. Consistent with these findings, fructose negatively impacted metal homeostasis of a mutant deficient in zinc expulsion and the growth of a mutant of the major oxidative stress regulator SpxA1. Importantly, fructose metabolism lowered culture pH at a faster pace, allowed better survival under acidic and nutrient-depleted conditions, and enhanced the competitiveness of S. mutans against Streptococcus sanguinis , although a moderated level of F-1-P might further boost some of these benefits. Conversely, several commensal streptococcal species displayed a greater sensitivity to fructose that may negatively affect their persistence and competitiveness in dental biofilm. In conclusion, fructose metabolism is integrated into the stress core of S. mutans and regulates critical functions required for survival and its ability to induce dysbiosis in the oral cavity. Importance.: Fructose is a common monosaccharide in the biosphere, yet its overconsumption has been linked to various health problems in humans including insulin resistance, obesity, diabetes, non-alcoholic liver diseases, and even cancer. These effects are in large part attributable to the unique biochemical characteristics and metabolic responses associated with the degradation of fructose. Yet, an understanding of the effects of fructose on the physiology of bacteria and its implications to the human microbiome is severely lacking. Here we performed a series of analyses on the gene regulation of a dental pathogen Streptococcus mutans by exposing it to fructose and other important stress agents. Further supported by growth, persistence, and competition assays, our findings revealed the ability of fructose to activate a set of stress-related functions that may prove critical to the ability of the bacterium to persist and cause diseases both within and without of the oral cavity.

As a biological byproduct from both humans and microbes, glycerol’s contribution to microbial homeostasis in the oral cavity remains understudied. In this study, we examined glycerol metabolism by Streptococcus sanguinis, a commensal associated with oral health. Genetic mutants of glucose-PTS enzyme II ( manL ), glycerol metabolism ( glp and dha pathways), and transcriptional regulators were characterized with regard to glycerol catabolism, growth, production of hydrogen peroxide (H ₂ O ₂ ), transcription, and competition with Streptococcus mutans . Biochemical assays identified the glp pathway as a novel source for H ₂ O ₂ production by S. sanguinis that is independent of pyruvate oxidase (SpxB). Genetic analysis indicated that the glp pathway requires glycerol and a transcriptional regulator, GlpR, for expression and is negatively regulated by PTS, but not the catabolite control protein, CcpA. Conversely, deletion of either manL or ccpA increased the expression of spxB and a second, H ₂ O ₂ -non-producing glycerol metabolic pathway ( dha ), indicative of a mode of regulation consistent with conventional carbon catabolite repression (CCR). In a plate-based antagonism assay and competition assays performed with planktonic and biofilm-grown cells, glycerol greatly benefited the competitive fitness of S. sanguinis against S. mutans . The glp pathway appears to be conserved in several commensal streptococci and actively expressed in caries-free plaque samples. Our study suggests that glycerol metabolism plays a more significant role in the ecology of the oral cavity than previously understood. Commensal streptococci, though not able to use glycerol as a sole carbohydrate source for growth, benefit from the catabolism of glycerol through production of both ATP and H ₂ O ₂ . IMPORTANCE: Glycerol is an abundant carbohydrate in the oral cavity. However, little is understood regarding the metabolism of glycerol by commensal streptococci, some of the most abundant oral bacteria. This was in part because most streptococci cannot grow on glycerol as the sole carbon source. In this study, we show that Streptococcus sanguinis , a commensal associated with dental health, can degrade glycerol for persistence and competition through two pathways, one of which generates hydrogen peroxide at levels capable of inhibiting Streptococcus mutans . Preliminary studies suggest that several additional commensal streptococci are also able to catabolize glycerol, and glycerol-related genes are actively expressed in human dental plaque samples. Our findings reveal the potential of glycerol to significantly impact microbial homeostasis, which warrants further exploration.

Previous work reported that deletion of the Enzyme IIAB subunits (EIIAB ^Man , manL ) of the glucose phosphotransferase system (glucose-PTS, manLMNO) in Streptococcus sanguinis impacted carbon catabolite repression (CCR) and bacterial fitness. Here, a single nucleotide polymorphism in ManN, ManNA91E, produced the unusual phenotype of increased excretion of organic acids and H ₂ O ₂ , yet elevated PTS activities. To characterize the contributions of each component of the glucose-PTS to bacterial fitness, we performed genetic analyses by deleting from S. sanguinis SK36 the entire operon and each EII ^Man subunit individually; and genes encoding the catabolite control protein CcpA (Δ ccpA ) and the redox regulator Rex (Δ rex ) for comparison. Deletion of each subunit incurred a growth defect on glucose partly due to elevated excretion of H ₂ O ₂ ; when supplemented with catalase this defect was rescued, instead resulting in a significantly higher yield than the parent. All glucose-PTS deletion mutants presented an increased antagonism against the oral pathobiont Streptococcus mutans , a phenotype absent in Δ ccpA despite increased H ₂ O ₂ output. A shift in the pyruvate node towards mixed acid fermentation and increased arginine deiminase activity enhanced pH homeostasis in glucose-PTS mutants, but not Δ ccpA . Despite the purported ability of Rex to regulate central carbon metabolism, deletion of rex had no significant impact on most of the phenotypes discussed here. These findings place glucose-PTS in the pivotal position of controlling central carbon flux in streptococci, with critical outcomes impacting acidogenicity, aciduricity, pH homeostasis, and antagonism, highlighting its potential as a therapeutic target for treating diseases with a dysbiotic microbiome.

As a biological byproduct from both humans and microbes, glycerol’s contribution to microbial homeostasis in the oral cavity remains understudied. Here we examined glycerol metabolism by Streptococcus sanguinis, a commensal associated with oral health. Genetic mutants of glucose-PTS enzyme II ( manL ), glycerol metabolism ( glp and dha pathways), and transcriptional regulators were characterized with regard to glycerol catabolism, growth, production of hydrogen peroxide (H ₂ O ₂ ), transcription, and competition with Streptococcus mutans . Biochemical assays identified the glp pathway as a novel source of H ₂ O ₂ production by S. sanguinis that is independent of pyruvate oxidase (SpxB). Genetic analysis indicated that the glp pathway requires glycerol and a transcriptional regulator, GlpR, for expression and is negatively regulated by PTS, but not the catabolite control protein, CcpA. Conversely, deletion of either manL or ccpA increased expression of spxB and a second, H ₂ O ₂ -non-producing glycerol metabolic pathway ( dha ), indicative of a mode of regulation consistent with conventional carbon catabolite repression (CCR). In a plate-based antagonism assay and competition assays performed with planktonic and biofilm-grown cells, glycerol greatly benefited the competitive fitness of S. sanguinis against S. mutans. The glp pathway appears to be conserved in several commensal streptococci and actively expressed in caries-free plaque samples. Our study suggests that glycerol metabolism plays a more significant role in the ecology of the oral cavity than previously understood. Commensal streptococci, though not able to use glycerol as a sole carbohydrate for growth, benefit from catabolism of glycerol through production of both ATP and H ₂ O ₂ . Importance: Glycerol is an abundant carbohydrate found in oral cavity, both due to biological activities of humans and microbes, and as a common ingredient of foods and health care products. However, very little is understood regarding the metabolism of glycerol by some of the most abundant oral bacteria, commensal streptococci. This was in part because most streptococci cannot grow on glycerol as the sole carbon source. Here we show that Streptococcus sanguinis , an oral commensal associated with dental health, can degrade glycerol for persistence and competition through two independent pathways, one of which generates hydrogen peroxide at levels capable of inhibiting a dental pathobiont, Streptococcus mutans . Preliminary studies suggest that several other commensal streptococci are also able to catabolize glycerol, and glycerol-related genes are being actively expressed in human dental plaque samples. Our findings reveal the potential of glycerol to significantly impact microbial homeostasis which warrants further exploration.

To analyze contributions to microbial ecology of Reactive Electrophile Species (RES), including methylglyoxal, generated during glycolysis. Genetic analyses were performed on the glyoxalase pathway in Streptococcus mutans (SM) and Streptococcus sanguinis (SS), followed by phenotypic assays and transcription analysis. Deleting glyoxalase I (lguL) reduced RES tolerance to a far greater extent in SM than in SS, decreasing the competitiveness of SM against SS. Although SM displays a greater RES tolerance than SS, lguL-null mutants of either species showed similar tolerance; a finding consistent with the ability of methylglyoxal to induce the expression of lguL in SM, but not in SS. A novel paralogue of lguL (named gloA2) was identified in most streptococci. SM mutant ∆gloA2SM showed little change in methylglyoxal tolerance yet a significant growth defect and increased autolysis on fructose, a phenotype reversed by the addition of glutathione, or by the deletion of a fructose: phosphotransferase system (PTS) that generates fructose-1-phosphate (F-1-P). Fructose contributes to RES generation in a PTS-specific manner, and GloA2 may be required to degrade certain RES derived from F-1-P. This study reveals the critical roles of RES in fitness and interbacterial competition and the effects of PTS in modulating RES metabolism.