| Popis: |
The alternative sigma factor, ss, did not appear to play a significant role in cspB expression at low temperature. In contrast, Fis appeared to act as a positive regulator of cspB in stationary phase cultures. Cell survival assays (measured by ability to form colony forming units on nutrient agar plates) showed that 4% of cells that were in early exponential phase survived a rapid cold shock to 4°C, although survival was almost complete when cells were in lag phase or in late exponential phase. The alternative sigma factor, ss, did not appear to play a significant role in cell survival in response to a rapid temperature reduction to 4°C. The addition of an osmoprotectant, 0.3 M sucrose, protected against loss of plating viability to some degree for early exponential phase cells, when diluted to 4°C. 2-D PAGE analysis showed that the response of exponential phase S. typhimurium cells incubated at 10°C consisted of an adaptive phase followed by an acclimation phase, in agreement with previous reports for E. coli. Identification of CspA was verified by N-terminal sequencing. The response was delayed at 4°C and recovery of protein synthesis in the acclimation phase was not as extensive, as observed at 10°C. CspA was synthesised throughout the period of incubation at 4°C. Growth phase was found to severely affect de novo protein synthesis at low temperature. Incubation of stationary phase cells at 10°C or 4°C resulted in repression of the synthesis of the majority of proteins, although a small set of proteins was induced. CspA was not detected at 37°C, but was highly induced at 10°C. However, prolonged incubation at 4°C led to complete repression of protein synthesis, except for CspA. This study has shown that S. typhimurium adapts to low temperature in a dynamic fashion and expression of CspA is a major feature of the response. Furthermore, it appears that exponential phase S. typhimurium cells are metabolically active even after 4 days at refrigeration temperatures. |
