Transcriptomic and phenotypic responses of Listeria monocytogenes strains possessing different growth efficiencies under acidic conditions

Abstract

In an experiment delineating aciduric strains, food and clinical Listeria monocytogenes isolates tended to produce the most biomass whereas ovine and avian strains produced comparatively less biomass when exposed to high levels of sodium diacetate (SD) and potassium sorbate. Compared to reference strains that exhibited greater acid sensitivity, representative food isolates with comparatively good growth capacities in the presence of 21 mM SD at pH 5.0 accumulated reduced levels of acetate anion and K(+) ion. The aciduric nature of SD-resistant strains was also reflected by comparatively high tolerance to pH 2.4 (HCl) acid challenges, a property boosted by the presence of SD. Exposure to elevated levels of SD (21 mM SD at pH 5.0) was found to have broad effects on gene expression, as differentiated from effects caused by mildly acidic conditions (pH 5.0). SD-resistant strain FW04/0025 was more responsive to elevated SD, increasing the expression of 222 genes (>2-fold change [P < 0.05]), compared to the more sensitive EGD reference strain, which exhibited increases in expression of 112 genes. Key differences between the strains in relation to SD-enhanced transcripts were notably associated with the cell envelope, oxidative stress management, and intermediary metabolism. SD thus appears to differentially influence growth efficiency and survival of strains, under conditions relevant to acidic foods, that could be due to altered cell wall and metabolic phenotypes.

FIG. 1.

Box plots showing the interquartile ranges (boxes contain 50% of values) and standard deviations of absolute net growth of L. monocytogenes in the presence of different organic acids (in BHIB adjusted to pH 5.0). Graph A indicates the distribution of growth differences between genetic lineages I, II, and II in response to SD and potassium sorbate; graphs B and C show growth responses in relation to strain origin. Open circles represent outlier values. The horizontal lines within the boxes represent mean values, while error bars represent standard deviations. Genetic and source distributions of strain tests are shown in Table S1 in the supplemental material.

FIG. 2.

Growth rates and maximum growth levels observed for L. monocytogenes strains grown in BHI initially adjusted to a range of different pH values (pH 5.0 to 8.0) in the presence of different levels of SD. The final pH levels (±0.03 pH between strains) indicate the results of acidification of broth due to end product formation during growth. Strains tested included FW04/0025 (closed squares) and EGD (open circles). Data were determined by fitting a logistic D-model (4) to optical density data (collected at 600 nm), with error bars indicating the standard error associated with fitting the model. Model fits had r² values of >0.98. An A₆₀₀ of 0.1 equated approximately 2 × 10⁸ CFU ml⁻¹.

FIG. 3.

Intracellular levels of acetate and K⁺ ion in L. monocytogenes strains grown to the early-stationary-growth phase in BHI broth initially at pH 7.3 (white bars), pH 5.0 (hatched bars), pH 5.0 + 10 mM SD (shaded bars), and pH 5.0 + 20 mM SD (black bars). The pH values for the four different sets of conditions when cells were harvested were pH 6.6, 4.7, 5.0, and 5.0, respectively. Error bars represent standard deviations derived from the results of 3 to 4 separate biological replicate experiments.

FIG. 4.

CFU log reduction of L. monocytogenes strain numbers after acid challenge tests in BHI broth acidified to pH 2.0 with HCl and a 120 min of exposure. Cultures were diluted to approximately 10⁸ CFU ml⁻¹ before acid shock was applied after being grown in BHI at a range of initial pH values (pH 5.0 to 7.0) in the absence (open symbols) and presence (closed symbols) of 20 mM SD and tested at the mid-exponential-growth phase (upper graph) and during the early-stationary-growth phase (lower graph). Strains tested included EGD (squares) and FW04/0025 (circles).

FIG. 5.

Four-way Venn diagram showing the distribution of significantly upregulated genes, defined as having increased ≥2.0-fold (P < 0.05) in expression, for strains EGD and FW04/0025 grown to the exponential-growth phase and including a comparison of levels of gene expression in cultures grown at pH 5.0 and pH 7.3 and in cultures grown at pH 5.0 + 21 mM SD and pH 5.0. The numbers in intersections represent the number of genes shared between the different treatments for the two strains. Circled numbers indicate accumulated numbers of genes showing SD-influenced expression responses.

FIG. 6.

Metabolic pathway map indicating the steps of end product formation by L. monocytogenes (based on data from the KEGG database). End product compounds that may be formed during the growth of L. monocytogenes are indicated in boxes. Genes coding corresponding enzymes conducting biochemical transformation are indicated. Thick, black-arrowed lines indicate that the expression of gene(s) for the given enzymatic step was increased by SD treatment. Arrowed lines in gray indicate that the expression of gene(s) that was increased by SD treatment for the given enzymatic step was strain dependent (Table 4). The dashed line indicates an enzymatic process suppressed by SD treatment; thin lines indicate processes unaffected or only weakly affected by SD treatment. BCAA, branched-chain amino acids.