PlsX deletion impacts fatty acid synthesis and acid adaptation in Streptococcus mutans

Abstract

Streptococcus mutans, one of the primary causative agents of dental caries in humans, ferments dietary sugars in the mouth to produce organic acids. These acids lower local pH values, resulting in demineralization of the tooth enamel, leading to caries. To survive acidic environments, Strep. mutans employs several adaptive mechanisms, including a shift from saturated to unsaturated fatty acids in membrane phospholipids. PlsX is an acyl-ACP : phosphate transacylase that links the fatty acid synthase II (FASII) pathway to the phospholipid synthesis pathway, and is therefore central to the movement of unsaturated fatty acids into the membrane. Recently, we discovered that plsX is not essential in Strep. mutans. A plsX deletion mutant was not a fatty acid or phospholipid auxotroph. Gas chromatography of fatty acid methyl esters indicated that membrane fatty acid chain length in the plsX deletion strain differed from those detected in the parent strain, UA159. The deletion strain displayed a fatty acid shift similar to WT, but had a higher percentage of unsaturated fatty acids at low pH. The deletion strain survived significantly longer than the parent strain when cultures were subjected to an acid challenge of pH 2.5.The ΔplsX strain also exhibited elevated F-ATPase activity at pH 5.2, compared with the parent. These results indicate that the loss of plsX affects both the fatty acid synthesis pathway and the acid-adaptive response of Strep. mutans.

Fig. 1.

Growth of ΔplsX is impaired. Cultures of Strep. mutans UA159, ΔplsX and plsX ⁺ were grown to exponential phase and used to inoculate fresh media. Growth at 37 °C was monitored using a Bioscreen C growth reader. Data points represent the mean of 10–20 individual wells. (a) Growth in BHI medium buffered to pH 7.0 with 50 mM potassium phosphate buffer. (b) Growth in BHI medium titrated to pH 5.2 with HCl. (c) Growth in BHI versus FMC medium. Note the difference in the timescale compared with (a) and (b). (d) Generation times were calculated from the data in (a)–(c) using data points at the beginning and end of exponential phase and expressed in minutes ± sd. Generation times for ΔplsX and plsX ⁺ were compared with UA159 grown under the same conditions and statistical significance was determined by Student's t-test; *P ≤ 0.05.

Fig. 2.

ΔplsX is resistant to acid challenge. Cells were suspended in 0.1 M glycine (pH 2.5). Survival was measured by serial dilution and plating on BHI agar medium. Three independent cultures were used for all measurements (n = 3). Data are represented as log(c.f.u. at the time point/c.f.u. at time zero). (a) Cells harvested from steady-state cultures grown in TY+1 % (w/v) glucose medium. (b) Cells harvested from a model biofilm grown on a glass slide in BHI medium+1 % (w/v) sucrose. (c) Cells grown in suspension overnight in BHI medium. Error bars indicate ± sd.

Fig. 3.

ATPase activity is enhanced in the ΔplsX strain. ATPase activity was measured via the method of Fiske and Subbarow, a colorimetric indicator of inorganic phosphate production (Fiske & Subbarow, 1925). Permeabilized cell membranes were prepared as described in methods, and OD₆₆₀ measurements were taken after 45 min for an end point ATPase activity; n = 3. Statistical significance between pairs indicated by brackets was determined by Student's t-test; *P ≤ 0.05. (a) Activity derived from cultures of Strep. mutans UA159, ΔplsX and plsX ⁺ grown in batch cultures in BHI medium. (b) Activity derived from steady-state cultures of Strep. mutans UA159 and ΔplsX harvested at fixed pH values of 7.0 and 5.2. (c) Transcription of atpG, the A subunit of F₀F₁-ATPase, as measured by qRT-PCR. RNA was isolated from three independent 50 ml cultures of each strain, grown in TY medium+1 % (w/v) glucose. Each sample was measured in triplicate. Error bars indicate ± sd.

Fig. 4.

Membrane fatty acid composition is altered in the ΔplsX strain. Cells were harvested from three independent chemostat cultures of Strep. mutans UA159 and ΔplsX (used in the acid-challenge assays described in Fig. 2) and analysed for membrane fatty acid content by GC-FAME; n = 3. (a) Percentage of saturated, unsaturated and other membrane fatty acids ( ± sd) from cells grown in continuous culture. Values in the ΔplsX strain that were significantly different from the parent strain grown under the same condition were determined by Student's t-test; *P ≤ 0.05. (b) Percentage of each of the fatty acid chain lengths, as measured by number of carbon atoms, from samples harvested from continuous cultures. Data represent all types of fatty acids, i.e. saturated, unsaturated, and others. All pairwise comparisons of the results in panel (b) are significantly different (P ≤ 0.05 using Student's t-test), except for the comparisons indicated by Ø. Error bars indicate ± sd.

Fig. 5.

ΔplsX is sensitive to membrane stress agents. Cultures of Strep. mutans UA159, ΔplsX and plsX ⁺ were used to inoculate 300 μl medium, and growth at 37 °C was monitored using a Bioscreen C growth reader. Data points represent the mean of 10–20 individual wells. (a) Growth in BHI medium supplemented with 1 or 2 % (v/v) ethanol (EtOH). (b) Growth in BHI medium supplemented with 100 or 150 μM SDS. (c) Growth in BHI medium supplemented with 50 or 250 μM myristic acid (C14 : 0).