Biology subjects

Taylor, Z. A.

Publications and source records attributed to Taylor, Z. A..

4 recordsLinked to original sources

bioRxiv2024

Systematic Analysis of the Glucose-PTS in Streptococcus sanguinis Highlighted its Importance in Central Metabolism and Bacterial Fitness

Previous work reported that deletion of the Enzyme IIAB subunits (EIIABMan, 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 H2O2, 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 EIIMan subunit individually; and genes encoding the catabolite control protein CcpA ({Delta}ccpA) and the redox regulator Rex ({Delta}rex) for comparison. Deletion of each subunit incurred a growth defect on glucose partly due to elevated excretion of H2O2; 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 {Delta}ccpA despite increased H2O2 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 {Delta}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. ImportanceManagement 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.

microbiology↗

bioRxiv2024

Glycerol Metabolism Contributes to Competition by Oral Streptococci through Production of Hydrogen Peroxide

As a biological byproduct from both humans and microbes, glycerols 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 (H2O2), transcription, and competition with Streptococcus mutans. Biochemical assays identified the glp pathway as a novel source of H2O2 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, H2O2-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 H2O2. ImportanceGlycerol 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.

microbiology↗

bioRxiv2022

Glucose PTS Modulates Pyruvate Metabolism, Bacterial Fitness, and Microbial Ecology in Oral Streptococci

Spontaneous mutants with defects in the primary glucose phosphotransferase (PTS) permease (manLMNO) of Streptococcus sanguinis SK36 showed enhanced fitness at low pH. Transcriptomics and metabolomics with a manL deletion mutant (SK36/manL) revealed redirection of pyruvate to production of acetate and formate, rather than lactate. The observations were consistent with measurements of decreased lactic acid accumulation and increased excretion of pyruvate and H2O2. Genes showing increased expression in SK36/manL included those encoding carbohydrate transporters, extracellular glycosidases, intracellular polysaccharide (IPS) metabolism, arginine deiminase, and pathways for metabolism of acetoin, ethanolamine, ascorbate and formate; along with genes required for membrane biosynthesis and adhesion. Streptococcus mutans UA159 persisted much better in biofilm co-cultures with SK36/manL than with SK36, an effect that was further enhanced by culturing the biofilms anaerobically but dampened by adding arginine to the medium. We posited that the enhanced persistence of S. mutans with SK36/manL was in part due to excess excretion of pyruvate by the latter, as addition of pyruvate to S. mutans-S. sanguinis co-cultures increased the proportions of UA159 in the biofilms. Reduction of the buffer capacity or increasing the concentration of glucose benefited UA159 when co-cultured with SK36, but not with SK36/manL; likely due to the altered metabolism and enhanced acid tolerance of the mutant. When manL was deleted in S. mutans or Streptococcus gordonii, the mutants presented altered fitness characteristics. Our study demonstrated that PTS-dependent modulation of central metabolism can profoundly affect streptococcal fitness and metabolic interactions, revealing another dimension in commensal-pathogen relationships influencing dental caries development. ImportanceDental caries is underpinned by a dysbiotic microbiome and increased acid production. As beneficial bacteria that can antagonize oral pathobionts, oral streptococci such as S. sanguinis and S. gordonii can ferment many carbohydrates, despite their relative sensitivity to low pH. We characterized the molecular basis for why mutants of glucose transporter ManLMNO of S. sanguinis showed enhanced production of hydrogen peroxide and ammonia, and improved persistence under acidic conditions. Significant metabolic shift involving more than 300 genes required for carbohydrate transport, energy production, and envelope biogenesis was observed. Significantly, manL mutants engineered in three different oral streptococci displayed altered capacities for acid production and interspecies antagonism, highlighting the potential for targeting the glucose-PTS to modulate the pathogenicity of oral biofilms.

microbiology↗

bioRxiv2021

Spontaneous Mutants of Streptococcus sanguinis with Defects in the Glucose-PTS Show Enhanced Post-Exponential Phase Fitness

Genetic truncations in a gene encoding a putative glucose-PTS protein (manL, EIIABMan) were identified in subpopulations of two separate laboratory stocks of Streptococcus sanguinis SK36; the mutants had reduced PTS activities on glucose and other monosaccharides. Using an engineered mutant of manL and its complemented derivative, we showed that the ManL-deficient strain had improved bacterial viability in stationary phase and was better able to inhibit the growth of the dental caries pathogen Streptococcus mutans. Transcriptional analysis and biochemical assays suggested that the manL mutant underwent reprograming of central carbon metabolism that directed pyruvate away from production of lactate, increasing production of hydrogen peroxide (H2O2) and excretion of pyruvate. Addition of pyruvate to the medium enhanced the survival of SK36 in overnight cultures. Meanwhile, elevated pyruvate levels were detected in the cultures of a small, but significant percentage ([~]10%), of clinical isolates of oral commensal bacteria. Furthermore, the manL mutant showed higher expression of the arginine deiminase system than the wild type, which enhanced the ability of the mutant to raise environmental pH when arginine was present. Significant discrepancies in genome sequence were identified between strain SK36 obtained from ATCC and the sequence deposited in GenBank. As the conditions that are likely associated with the emergence of spontaneous manL mutations, i.e. excess carbohydrates and low pH, are those associated with caries development, we propose that the glucose-PTS strongly influences commensal-pathogen interactions by altering the production of ammonia, pyruvate, and H2O2. ImportanceA health-associated dental microbiome provides a potent defense against pathogens and diseases. Streptococcus sanguinis is an abundant member of a health-associated oral flora that antagonizes pathogens by producing hydrogen peroxide. There is a need for a better understanding of the mechanisms that allow bacteria to survive carbohydrate-rich and acidic environments associated with the development of dental caries. We report the isolation and characterization of spontaneous mutants of S. sanguinis with impairment in glucose transport. The resultant reprograming of central metabolism in these mutants reduced the production of lactic acid and increased pyruvate accumulation; the latter enables these bacteria to better cope with hydrogen peroxide and low pH. The implications of these discoveries in the development of dental caries are discussed.

microbiology↗