The ability of Salmonella enterica serovar Tyhimurium (S. typhimurium) to adapt to and survive in refrigerated conditions plays a role in its capacity to cause food-borne disease. Exposure to low temperature elicits a classic stress response in many bacteria, a major feature of which is high level induction of CspA homologues. The exact role of these proteins at low temperatures is unknown. In the present study, mutation of S. typhimurium cspA, which encodes the major cold shock protein, was attempted using a variety of strategies. However, none of these resulted in recovery of mutants. This may have been due to the essential nature of S. typhimurium cspA, or alternatively, to transformation inefficiency.
Reporter studies showed that expression of a plasmid-based S. typhimurium cspA-lacZ fusion increased 8-fold over a period of 5 hours, when exponentially growing cells were shifted from 37°C to 15°C. This level of induction was lower than observed for the level of S. typhimurium CspA protein directly at 10°C and was less than that reported for similar studies with E. coli cspA. This discrepancy may indicate either the absence of a putative enhancer element, termed the downstream box, in the lacZ fusion construct or the absence of a chromosome-based cis acting element.
Regulation of expression of S. typhimurium cspB was examined at low temperatures using a bioluminescent reporter system. Expression of cspB was not detected at 30°C, but was highly induced at 10°C and to a lesser extent at 4°C, when the culture was in exponential phase. Expression of cspB was growth phase-dependent, such that bioluminescence from the cspBy.Mudlux fusion was approximately 10-fold lower at 10°C when the cells were in stationary phase cells, compared to an exponential phase culture. Expression of cspB at 4°C was minimal when the culture was in stationary phase. The alternative sigma factor, gs, 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.x
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, gs, 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. typhimuriumcells 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 or 4°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.