Increased ATPase activity is responsible for acid sensitivity of nisin-resistant Listeria monocytogenes ATCC 700302

Abstract

The growth of the foodborne pathogen Listeria monocytogenes can be controlled by nisin, an antimicrobial peptide. A spontaneous mutant of L. monocytogenes shows both resistance to nisin and increased acid sensitivity compared to the wild type. Changes in the cell membrane correlated with nisin resistance, but the mechanism for acid sensitivity appears unrelated. When hydrochloric or lactic acid is added to cultures, intracellular ATP levels drop significantly in the mutant (P < 0.01) compared to the results seen with the wild type. Characterization of the F(0)F(1) ATPase, which hydrolyzes ATP to pump protons from the cell cytoplasm, shows that the enzyme is more active in the mutant than in the wild type. These data support a model in which the increased activity of the mutant ATPase upon acid addition depletes the cells' supply of ATP, resulting in cell death.

FIG. 1.

Changes in intracellular (closed symbols) and extracellular (open symbols) ATP levels of the wild-type Scott A strain (A) and the nisin-resistant mutant NR 30 strain (B). Circles indicate controls, triangles represent the addition of HCl, and squares represent the addition of lactic acid. Addition of both acids resulted in a final pH of 4.3. Significant (P < 0.05) differences in intracellular ATP levels of all six conditions are indicated by different letters.

FIG. 2.

Effect of lactic acid treatment (triangles; pH 4.9) on the Scott A (A) and NR 30 (B) strains. Circles represent untreated cells. Closed symbols represent intracellular ATP, and open symbols represent extracellular ATP. Significant (P < 0.05) differences in intracellular ATP levels of the four conditions are indicated by different letters.

FIG. 3.

Influence of pH on ATP hydrolysis when the reaction mixture included 10 mM ATP and 20 mM MgCl₂. Closed circles represent strain Scott A. Open circles represent strain NR 30. Error bars represent 1 standard deviation.

FIG. 4.

DCCD inhibition of the Scott A (closed circles) or NR 30 (open circles) strain at pH 5.

FIG. 5.

Kinetics of strain Scott A (A) and strain NR 30 (B) ATPase assayed at pH 5.0 with 20 mM MgCl₂. Error bars represent 1 standard deviation.