Characterization of a glutamate transporter operon, glnQHMP, in Streptococcus mutans and its role in acid tolerance

Abstract

Glutamate contributes to the acid tolerance response (ATR) of many Gram-negative and Gram-positive bacteria, but its role in the ATR of the oral bacterium Streptococcus mutans is unknown. This study describes the discovery and characterization of a glutamate transporter operon designated glnQHMP (Smu.1519 to Smu.1522) and investigates its potential role in acid tolerance. Deletion of glnQHMP resulted in a 95% reduction in transport of radiolabeled glutamate compared to the wild-type UA159 strain. The addition of glutamate to metabolizing UA159 cells resulted in an increased production of acidic end products, whereas the glnQHMP mutant produced less lactic acid than UA159, suggesting a link between glutamate metabolism and acid production and possible acid tolerance. To investigate this possibility, we conducted a microarray analysis with glutamate and under pH 5.5 and pH 7.5 conditions which showed that expression of the glnQHMP operon was downregulated by both glutamate and mild acid. We also measured the growth kinetics of UA159 and its glnQHMP-negative derivative at pH 5.5 and found that the mutant doubled at a much slower rate than the parent strain but survived at pH 3.5 significantly better than the wild type. Taken together, these findings support the involvement of the glutamate transporter operon glnQHMP in the acid tolerance response in S. mutans.

FIG. 1.

Map of the glutamate transporter operon Smu.1519 to Smu.1522. The transporter operon is located upstream of the TCSTS operon VicRKX (A). Analysis of the glnQ promoter region identified the putative −10 box, −35 box, transcriptional start site, and ribosome binding site (RBS) (all underlined in bold) as well as the putative termination sequence (underlined) (B).

FIG. 2.

Uptake of L-[U-¹⁴C]glutamic acid by S. mutans UA159 (▪) and SmuGLT (□). The maximal rate of glutamate uptake (measured after 2 min of incubation with labeled glutamate) for UA159 was over 18 times higher than that of SmuGLT (P < 0.001; n = 3).

FIG. 3.

Uptake of L-[U-¹⁴C]glutamic acid by S. mutans UA159 in the presence of a 10× excess of competing amino acids. Maximal uptake was measured after 2 min of incubation with labeled glutamic acid and unlabeled competing amino acids and then normalized to the maximal uptake in the absence of competing amino acids. Uptake was almost completely inhibited by an excess of glutamate, partially inhibited by an excess of aspartate and cysteine, and somewhat inhibited by an excess of glutamine. ***, P < 0.05 (n = 7).

FIG. 4.

Uptake of L-[U-¹⁴C]glutamic acid by S. mutans UA159 in buffer of various pHs and with different metal ions. Maximal uptake was measured after 2 min of incubation with labeled glutamic acid in the presence of potassium ions and then normalized to the maximal uptake in buffer at pH 6.0. Uptake was significantly decreased under acidic assay conditions and in the presence of sodium ions. ***, P < 0.05 (n = 6).

FIG. 5.

Glycolytic rates of S. mutans UA159 (gray bars) and SmuGLT (white bars). Glycolytic rates were monitored by measuring the rate of addition of 10 mM KOH to the cell suspension following the addition of 200 mM glucose and subsequently increasing the amounts of glutamate at pH 7.0 (n = 3).

FIG. 6.

Lactate production of S. mutans UA159 (gray bars) and SmuGLT (white bars) cultures grown in MM in the presence of 10 mM glutamate. Amounts were normalized to corresponding OD₆₀₀ values for each culture. SmuGLT produced significantly less lactate during than UA159. ***, P < 0.001 (n = 5).

FIG. 7.

Acid tolerance responses of S. mutans UA159 and SmuGLT. Cells were grown in TYE (pH 7.5) supplemented with glucose and were exposed to TYE pH 3.5 (unadapted UA159 and SmuGLT [white bars]) or incubated in TYE pH 5.5 for 2 h and then exposed to TYE pH 3.5 (adapted UA159 and SmuGLT [gray bars]). Results are shown as the percentage of the original cell count surviving after incubation at the lethal pH of 3.5 for 3 h. Glutamate significantly decreased the ATR for UA159. ***, P < 0.01 (n = 5). SmuGLT had a significantly increased ATR compared to UA159. †††, P < 0.05 (n = 5).