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

Abstract

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.

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

“…Pyruvate-formate lyase (pfl, SSA_0342) and a related pyruvate formate lyase-activating protein (pflA, SSA_1749) were upregulated 11-and 6-fold, respectively, and the pyruvate dehydrogenase operon (SSA_1137 to SSA_1140) was upregulated 2-to 3-fold. These changes in gene regulation were consistent with increased H2O2 excretion and reduced lactate production by the manL mutant (32), indicative of a shift from reductive to oxidative metabolism of pyruvate and production of alternative metabolites, including Ac-P, acetyl-CoA, formate, and acetate.…”

“…The copyright holder for this preprint this version posted September 22, 2022. ; https://doi.org/10.1101/2022.09.21.508965 doi: bioRxiv preprint optical density, and reduced eDNA levels. It appeared that DL1/manL had reduced autolytic activity, as seen in SK36/manL (32). The cultures of DL1/manL also showed significantly higher final pH (Fig.…”

“…The copyright holder for this preprint this version posted September 22, 2022. ; https://doi.org/10.1101/2022.09. 21.508965 doi: bioRxiv preprint to inhibit the growth of S. mutans (32). To understand the molecular basis for these observations, we conducted transcriptomic and metabolomic analyses of a manL mutant of SK36.…”

“…We recently identified in subpopulations of two separate stocks of S. sanguinis SK36 mutations of the gene encoding the EIIAB (ManL) components of the primary glucose-PTS permease (ManLMNO) that resulted in truncations of the ManL protein (32). Despite having a defect in carbohydrate transport, these mutants showed enhanced capacity in persisting and fermenting sugars at lower pH values than the parental strain.…”

“…Despite having a defect in carbohydrate transport, these mutants showed enhanced capacity in persisting and fermenting sugars at lower pH values than the parental strain. The mutants also had increased production of H 2 O 2 , which conferred a greater capacity to inhibit the growth of S. mutans (32). To understand the molecular basis for these observations, we conducted transcriptomic and metabolomic analyses of a manL mutant of SK36.…”