This enabled us to distinguish between

the proteolytic effect of ClpP on misfolded proteins, and how this affected growth at low temperature, and the indirect effect of ClpP caused through degradation of RpoS. Similar to the clpP mutant, we have previously shown that a mutant in the carbon starvation regulator protein gene, csrA, cause accumulation of high levels of RpoS [13]. Since we demonstrate in the current study that high level of RpoS in a clpP mutant appears to affect growth at low temperature, we hypothesised that a csrA mutant in a similar way would be growth attenuated, and included an investigation of this gene as well. Result and discussion A clpP LY3023414 mutant is impaired for growth at low this website temperature Growth of the clpP mutant was impaired on LB agar at 10°C (Figure 1A), whereas colony formation was delayed but resulted in normal size colonies at 15 and 21°C (Figure 1A). The temperature of 10°C was selected to represent the lower part of the temperature growth

range of S. Typhimurium and still allow growth experiments to be carried out within a reasonable time. With increasing incubation time at 10°C, two growth phenotypes of the clpP mutant appeared: normal sized colonies and pin-point colonies. To test if the pin-point colonies were just small due to longer doubling time, the plate with the clpP mutant was transferred to 37°C after 12 days at 10°C, grown overnight and compared with wild type strain that had also grown overnight. Normal sized colonies were formed and the cell density corresponded to the wild type strain Autophagy phosphorylation (Figure 1B). This showed that the clpP mutant was able

to restore normal growth even after a long period at 10°C. Figure 1 ClpP and CsrA are important for growth at low temperature. A) S. Typhimurium C5 and isogenic mutants were grown exponentially in LB at 37°C up to an OD600 of 0.4. The cultures were then serially diluted (10−1-, 10−2-, 10−3-, Loperamide and 10−4-fold), and 10 μl of each dilution was spotted onto LB plates. The plates were incubated at 10, 15, 21 and 37°C. The result presented is representative at least two experiments. B) The clpP are diluted as in a) and grown first at 10°C for 12 days and then transferred to 37°C for 1 day. A culture grown at 37°C for 1 day is included as control. The lag phase of the wild type C5 strain was 2.04 ± 0.66 days when grown in LB broth at 10°C, whereas the clpP mutant had a significantly longer lag phase of 9.97 ± 1.94 days (p = 0.002) (Figure 2A). The growth rate of the clpP mutant in exponential phase was 0.45 ± 0.03 days, which was a 29% reduction compared to the wildtype. The maximal density of the clpP mutant (8.29 log10 CFU/ml) was comparable to that of the wild type (8.74 log10 CFU/ml) after prolonged incubation (Figure 2B).