Alkali production associated with malolactic fermentation by oral streptococci and protection against acid, oxidative, or starvation damage

Abstract

Alkali production by oral streptococci is considered important for dental plaque ecology and caries moderation. Recently, malolactic fermentation (MLF) was identified as a major system for alkali production by oral streptococci, including Streptococcus mutans. Our major objectives in the work described in this paper were to further define the physiology and genetics of MLF of oral streptococci and its roles in protection against metabolic stress damage. L-Malic acid was rapidly fermented to L-lactic acid and CO(2) by induced cells of wild-type S. mutans, but not by deletion mutants for mleS (malolactic enzyme) or mleP (malate permease). Mutants for mleR (the contiguous regulator gene) had intermediate capacities for MLF. Loss of capacity to catalyze MLF resulted in loss of capacity for protection against lethal acidification. MLF was also found to be protective against oxidative and starvation damage. The capacity of S. mutans to produce alkali from malate was greater than its capacity to produce acid from glycolysis at low pH values of 4 or 5. MLF acted additively with the arginine deiminase system for alkali production by Streptococcus sanguinis, but not with urease of Streptococcus salivarius. Malolactic fermentation is clearly a major process for alkali generation by oral streptococci and for protection against environmental stresses.

Fig. 1

Organization of the malolactic gene cluster in the Streptococcus mutans UA159 genome (Ajdić et al. 2002). The genes are numbered using the Oralgen nomenclature (http://www.oralgen.lanl.gov).

Fig. 2

Malolactic fermentation of Streptococcus mutans UA159 and deletion mutants in biofilms at constant pH of 4.0 and excess l-malate (50 mmol/L). n = 3; error bars indicate SD. BDM, biofilm dry mass.

Fig. 3

Effects of deletion mutations of mleR (A), mleS (B), or mleP (C) on MLF protection against acid killing of Streptococcus mutans UA159. Cells were exposed to a constant pH of 3.0 with or without 25 mmol/L l-malate. N₀, the initial number of colony-forming units per millilitre; N, the number of colony-forming units at time t of acid exposure. Error bars indicate SD, n = 3.

Fig. 4

Malate protection of Streptococcus mutans UA159 against oxidative and starvation damage. (A) Protection against killing by 0.2% (63.2 mmol/L) hydrogen peroxide at pH 5; (B) protection against peroxide killing at pH 4.0; (C) protection against inactivation at pH 5.0 by 0.00075% (0.24 mmol/L) hydrogen peroxide of glyceraldehyde-3-phosphate dehydrogenase in cells permeabilized for the enzyme assay; (D) protection against starvation killing of cells harvested from stationary-phase suspension cultures, ,washed, and then starved at 37 °C in standard salt solution (50 mmol/L KCl plus 1 mmol/L MgCl₂) at pH 5.0. Error bars indicate SD; n = 3.

Fig. 5

Acid production from glucose (25 mmol/L) by Streptococcus mutans UA159 in suspensions with (filled triangles) or without (filled squares) neutralization through base production from MLF (25 mmol/L malate) at a constant pH value of 5 (A) or 4.0 (B). Values above 0.00 indicate acid production from glycolysis, while values below 0.00 indicate net base production from MLF plus glycolysis. Error bars indicate SD; n = 3. BDM, biofilm dry mass.

Fig. 6

Induction of malolactic fermentation by 25 mmol/L l-malate in biofilms of Streptococcus mutans UA159. Induction involved a switch from TY–sucrose to TY–malate feeding of the biofilms, as indicated in the figure. Error bars indicate SD; n = 3. Open squares indicate biofilm growth, while filled bars indicate MLF. BDM, biofilm dry mass.

Fig. 7

Alkali production at pH 5.0 by Streptococcus sanguinis ATCC10556 cells in suspensions (upper panel) or biofilms (lower panel) with no catabolite addition (filled squares), with addition of 25 mmol/L l-arginine (filled triangles), with 25 mmol/L l-malate (filled, inverted triangles), or with both 25 mmol/L l-arginine and 25 mmol/L l-malate (filled circles). Error bars indicate SD; n = 3. BDM, biofilm dry mass; CDM, cell dry mass.