Construction And Response To Acid Stress Of Salmonella Typhimurium â–³pat/â–³cobB Mutant Strain

In order to invade the host intestinal epithelial cells, as intestinal bacteria, Salmonella Typhimurium (Salmonella spp.) must overcome the acidic environment of the stomach.Previous studies showed that Salmonella have evolved a variety of response mechanisms to enhance their survival in the acidic environment. Acid tolerance response (ATR) played a major role in acid resistance in Salmonella. ATR refers to the phenomenon that a weak acid adaptation enhances viability of the bacteria under following lethal acidic conditions. Thus, after the ATR, Salmonella can better withstand stomach acid, increase the ability of invasion and virulence, resulting in the elevated probability of illness in human or other host.Currently, there are two known pH-dependent ATRs, the log phase ATR and the station phase ATR. In these two processes, many acid shock proteins (ASPs) were induced to protect and restore macromolecules under acid stress, they play an important role in the different growth stages of Salmonella. At the same time, Salmonella still have lysine decarboxylation system and arginine decarboxylation system. These amino acid transporters pump the intracellular hydrion out of cell to maintain intracellular pH relatively stable under acid conditions.Protein acetylation in prokaryotes is mainly from studies in Salmonella. The cobB in Salmonella encodes Sir2 homolog protein, exercises the function of deacetylation; pat encodes acetyltransferase (YfiQ), exercise the function of acetylation. Based on the phenomenon that a large number of proteins are acetylated in Salmonella, and acetylated proteins are involved in various physiological activities, we speculate that acetylation/ deacetylation modification system may be involved in acid stress response in Salmonella Typhimurium.Therefore, firstly we used X-Red homologous recombination system to construct pat and cobB mutant strains in the backgroud of Salmonella enterica serovar Typhimurium 14028s followed by growth rate measurement. Cfu counting was usd to detect the differences of acid tolerance response and acid resistance among three strains. Western blot was used to detect protein acetylation differences under acidic culture conditions and normal culture conditions in Salmonella Typhimurium. What’s more, the intracellular pH in three strains. At last, real-time PCR was employed to measure the transcription of acid stress related genes. Finally, in order to eveluate whether the paired acetylation/ deacetylation enzymes play an important role in acid tolerance response and acid resistance.Our results showed that X-Red homologous recombination system can be effectively used for gene knockout in Salmonella. Compared with the wild-type strain, pat or cobB mutant did not show any significant difference in growth rate, indicating that missing the genes which exercise the function of acetylation/deacetylation does not affect the growth of bacteria. ATR experiments failed to show significant survival difference in the three strains in both log phase and station phase, but bacteria in the station phase is significantly more resistant to acid stress than bacteria in log phase.These results indicated that in Salmonella the acetylation/deacetylation enzymes do not play an important role in log phase ATR and station phase ATR. But in acid resistance assay, pat mutant have a higher survival rate than wild type and cobB mutant in log phase, cobB mutant have a lower survival rate than wild type and pat mutant in station phase. Though, we found that acid treatment did not change the whole cell protein acetylation spectrum and the pHi was the same in three strains. Finally, we used real-time PCR to detect the transcriptional level of several acid stress response related genes and found that the transcription of phoP is significantly up.In summary, these results indicated that acetylase Pat and deacetylase CobB are not involved in acid tolerance response in Salmonella Typhimurium, but it seems to play an important role in acid resistance, and this regulation is mediated by key transcriptional factor PhoP probably.