Comprehensive mutational analysis of sucrose-metabolizing pathways in Streptococcus mutans reveals novel roles for the sucrose phosphotransferase system permease

Abstract

Sucrose is perhaps the most efficient carbohydrate for the promotion of dental caries in humans, and the primary caries pathogen Streptococcus mutans encodes multiple enzymes involved in the metabolism of this disaccharide. Here, we engineered a series of mutants lacking individual or combinations of sucrolytic pathways to understand the control of sucrose catabolism and to determine whether as-yet-undisclosed pathways for sucrose utilization were present in S. mutans. Growth phenotypes indicated that gtfBCD (encoding glucan exopolysaccharide synthases), ftf (encoding the fructan exopolysaccharide synthase), and the scrAB pathway (sugar-phosphotransferase system [PTS] permease and sucrose-6-PO(4) hydrolase) constitute the majority of the sucrose-catabolizing activity; however, mutations in any one of these genes alone did not affect planktonic growth on sucrose. The multiple-sugar metabolism pathway (msm) contributed minimally to growth on sucrose. Notably, a mutant lacking gtfBC, which cannot produce water-insoluble glucan, displayed improved planktonic growth on sucrose. Meanwhile, loss of scrA led to growth stimulation on fructooligosaccharides, due in large part to increased expression of the fruAB (fructanase) operon. Using the LevQRST four-component signal transduction system as a model for carbohydrate-dependent gene expression in strains lacking extracellular sucrases, a PlevD-cat (EIIA(Lev)) reporter was activated by pulsing with sucrose. Interestingly, ScrA was required for activation of levD expression by sucrose through components of the LevQRST complex, but not for activation by the cognate LevQRST sugars fructose or mannose. Sucrose-dependent catabolite repression was also evident in strains containing an intact sucrose PTS. Collectively, these results reveal a novel regulatory circuitry for the control of sucrose catabolism, with a central role for ScrA.

Fig 1

Growth curves of the mutants of major sucrose-metabolizing pathways in S. mutans. All strains were grown in BHI medium overnight, subcultured in BHI to mid-exponential phase, and then diluted 1:300 into TV medium containing 0.2% sucrose. Growth was monitored using a Bioscreen C reader set at 37°C with readings (OD₆₀₀) taken every 30 min. (A) UA159 (wild type), MMZ948 [gtfD(M9stop)], MMZ945 (gtfBC), and MMZ950 (gtfBCD ftf); (B) UA159, MMZ932 (scrA), and MMZ931 (scrB); (C) MMZ950, MMZ983 (gtfBCD ftf scrAB), and MMZ984 (gtfBCD ftf fruA); (D) UA159 and MMZ1025 (gtfBC scrAB). Two isolates were included for MMZ1025, as variability in lag phase was noted.

Fig 2

Minor contributions from the Msm pathway to growth on sucrose. Growth tests were performed as described for Fig. 1. (A) Strains MMZ950 (gtfBCD ftf), MMZ966 (gtfBCD ftf scrAB fruA), MMZ967 (gtfBCD ftf scrAB fruA msmE), and MMZ968 (gtfBCD ftf scrAB fruAB msmE); (B) strains UA159 and ΔEI (ptsI).

Fig 3

Role of EII^Tre in sucrose metabolism by S. mutans. Strains UA159, MMZ952 (gtfABCD ftf fruA), MMZ993 (gtfABCD ftf fruA scrA), and MMZ997 (gtfABCD ftf fruA scrA treB) were grown in TV medium containing 0.2% sucrose.

Fig 4

Sucrose-transporting activities measured in strains UA159, MMZ952, MMZ996, and MMZ997. Cells were harvested from exponentially growing cultures in BHI, washed in Na/K-phosphate buffer, permeabilized with toluene:acetone, and tested in PTS assays. The results are the average activities measured using three independent cultures. Units are expressed as nmol NADH oxidized (mg of protein)⁻¹ (min)⁻¹ in a PEP-dependent manner. The error bars represent standard deviations, and the asterisks indicate P values of <0.05 (Student's t test).

Fig 5

Growth of UA159 and fruA and scrA mutants on fructooligosaccharides (FOS). Strains UA159, SP8 (fruA), and MMZ932 (scrA) were cultured to mid-exponential phase in BHI medium and used to inoculate TV base medium containing 0.2% of Fn-FOS (A) or GFn-FOS (B). Also compared were the growths of UA159 in the two FOS preparations (C).