Rapid, Transient, and Proportional Activation of σ ^B in Response to Osmotic Stress in Listeria monocytogenes

Abstract

The osmotic activation of sigma B ( B ) in Listeria monocytogenes was studied by monitoring expression of four known B -dependent genes, opuCA, lmo2230, lmo2085, and sigB. Activation was found to be rapid, transient, and proportional to the magnitude of the osmotic stress applied, features that underpin the adaptability of this pathogen.

Development and optimization of an EGFP-based reporter for measuring the general stress response inListeria monocytogenes

“…Expression of lmo2230 was shown to be rapid and transient both after heat shock at 48°C, with a 10-fold induction observed after 3 min of heat stress 32 and also 15 min after osmotic upshock when more than 160-fold higher levels of lmo2230 in wild-type were reported comparing to unstressed cells at time zero. 31 Taken together these findings give an opportunity for monitoring activation of s B in real time by following lmo2230-promoter-driven expression after a sudden change of environmental conditions. However, there are also some limitations in terms of reporting transient activation of s B with the EGFP-based reporter system caused by the high stability of the fluorescent protein under most of the experimental conditions.…”

“…However, there are also some limitations in terms of reporting transient activation of s B with the EGFP-based reporter system caused by the high stability of the fluorescent protein under most of the experimental conditions. The long half-life of EGFP (estimated at greater than 24 h) 42 makes it impossible to observe a drop in s B activity after the removal of stress, while this decrease can be demonstrated by monitoring the transcript levels of lmo2230 and other s B -regulated genes under similar conditions 31 and after heat shock. 32 Future versions of this reporter could include less stable GFP variants (with ssrA RNA tags recognized by housekeeping proteases) 42 in order to overcome this limitation.…”

“…32 Future versions of this reporter could include less stable GFP variants (with ssrA RNA tags recognized by housekeeping proteases) 42 in order to overcome this limitation. When the time scale for s B activation was compared between the RNA based approach described earlier 31 and the P lmo2230 ::egfp reporter (Fig. 5B), the maximal s B activation was evident approximately 15 min earlier when monitoring mRNA levels.…”

“…The data presented are in line with our previous transcriptomic reports of s B activity being proportional to the extent of NaCl stress. 31 The heterogeneity of fluorescence within the wild-type EGFP expressing population was significant; with~38% of the population exposed to 0.9 M NaCl failing to induce fluorescence levels that fell within the predetermined gate (Fig. 6A).…”

“…To determine whether our reporter system is able to detect quick changes of s B activity reported by earlier studies, 31,32 the relative fluorescence was investigated after osmotic upshock. In contrast to steady-state growth experiments, cells were grown without NaCl supplementation up to OD 600 = 0.6 then osmotically shocked by addition of solid NaCl (0.5 M) and samples were taken at suitable intervals from vigorously shaken cultures for microscopic observation (Fig.…”