Listeria monocytogenes is a foodborne pathogen that causes listeriosis, a disease associated with a high mortality rate. The organism responds to a variety of stresses by activating stress-response pathways and adjusting its membrane fluidity by altering its fatty acid composition. We hypothesize that the F0F1 ATPase plays a central role in L. monocytogenes' response to multiple stresses. Wild-type and mutant cells were used to investigate the response to cold, acid, and nisin. The branched-chain fatty acid deficient, cold-sensitive mutant strain, cld1, showed significantly higher membrane rigidity (r = 0.175) compared to its wild-type 10403s (r = 0.121) when grown at 30°C, but not at 15°C (r = 0.124 and 0.116, respectively). Strain cld1 adjusted its membrane fluidity to cold stress. The F0F1 ATPase activity in strain cld1 was 3-fold higher than that of wild-type strain 10403s (0.0267 vs 0.0097 μmole Pi/min.mg protein, respectively) when cells were grown at 30°C. Supplementing cld1's growth medium with the precursor to branched fatty acid, 2-methylbutyrate, restored its fluidity but not the F0F1 ATPase activity to wild-type levels. The acid tolerance response examined differences in initial acid-sensitivity and development of tolerance to lactic acid. Strain cld1 had decreased viability when directly exposed to pH 3.5 (2.64 log CFU/ml), but gained increased viability at pH 3.5 after exposure to pH 5.5 (8.62 log CFU/ml) compared to the wild-type. Finally, a genetic approach examined the F0F1 ATPase c-subunit (atpE) expression in strains 10403s and cld1 using real-time PCR. Strain cld1 showed a 10-fold lower atpE mRNA transcript compared to 10403s. When examining the generated data, we observed that strain cld1 has higher F0F1 ATPase activity, higher initial acid sensitivity, increased protection due to the acid tolerance response, and lower c-subunit mRNA compared to strain 10403s in addition to a rigid membrane. These results support the hypothesis that adjustments took place at the level of the F0F1 ATPase. The various data were pooled in a model in which the mutant strain cld1 has a smaller c-subunit carousel compared to its wild-type 10403s, and highlights the important role of the ATPase in microbial stress response.Ph.D.Includes bibliographical references (p. 116-130)by Mohamed Z. Badaoui Najja
