In order to characterize the effect of salt concentration on growth, E.coli was grown in a medium containing different salt concentrations and the growth rate was estimated (see Figure 1a). As expected, due to osmotic stress, the growth rate decreased with increasing salt concentration. A Hill equation fit demonstrated that the effect on growth was highly sensitive with a Hill coefficient of 2.7 and a half saturation constant of 0.57 M salt concentration. Experiments were further performed in media containing salt and saturated amounts of IPTG. In this case, in addition to the effect of salt on growth, the effect of unnecessary protein synthesis due to β-galactosidase synthesis also influences growth (see Figure 1a). It can be noted that the growth rate was lower than that observed for growth in a medium with salt alone. A Hill equation fit demonstrated a similar sensitive response with a Hill coefficient of 2.6; however, there was a decrease in both the maximum growth rate from 0.42 h−1 to 0.37 h−1 and in the half saturation constant from 0.57 M to 0.52 M salt concentration. This indicated that unnecessary protein synthesis was imparting additional burden on growth in osmotic stress condition. Figure 1b shows the β-galactosidase activity observed at various salt concentrations when grown on a medium containing saturating amounts of IPTG. It is clear from the plot that the enzyme activity is strongly inhibited due to the osmotic stress. Normal amounts of β-galactosidase activity was observed upto 0.4 M salt concentration and decreased steeply beyond 0.4 M salt concentration. A Hill equation fit characterizing the response of enzyme activity indicated a highly ultrasensitive response with a Hill coefficient of 9 and a half saturation constant of 0.63 M salt concentration.
The quantification of the growth rate can be used to determine the burden defined as the relative decrease in the growth rate relative to the maximum observed in absence of both the salt and IPTG concentrations (see Materials and Methods section). The ф
s
calculated for medium containing various salt concentrations without IPTG showed an increasing trend with increasing salt concentrations before saturating to a value one indicating no growth at high salt concentrations (see Figure 1c). A samilar trend was observed for ф
t
the burden noted for growth on a medium with both salt and saturating amount of IPTG, though with a higher value indicating that the burden due to unnecessary protein synthesis was over and above that from salt alone. The value of фs obtained from experiments using media containing salt alone was subtracted from that containing both salt and IPTG (ф
t
), to determine the contribution of burden due to IPTG alone (ф
u
). The burden due to the synthesis of unnecessary protein, ф
U
, was about 12% in the absence of salt and remained at 12% until 0.6 M salt concentration (see Figure 2a). The contribution of unnecessary protein synthesis to burden decreased beyond 0.6 M salt concentration and was zero beyond 1 M salt concentration (see Figure 2a). This indicated that the cell could adapt perfectly upto 0.6 M salt concentration beyond which the enzyme activity was perturbed due to osmotic stress. This was also confirmed by plotting the burden due to IPTG alone with respect to the normalized β-galactosidase activity. The burden due to enzyme synthesis was about 12% until 50% of the maximum enzyme activity beyond which the burden decreased at higher salt concentrations (see Figure 2b).
A question that arises from the above analysis is whether the enzyme activity alone is affected by the salt concentration or the enzyme synthesis machinery is also disturbed due to osmotic stress. To address this issue, a mutant E.coli cell lacking the lac repressor protein (i.e. ∆lacI strain) was grown on a glycerol medium. This ensures that the β-galactosidase synthesis is constitutive and is independent of IPTG concentrations in ∆lacI strain. As control, a mutant strain lacking both lac repressor and β-galactosidase (∆lacI lacZ) strain was also grown at various salt concentrations. Figure 3a shows the growth rate of the two mutant strains on glycerol at various salt concentrations. The ∆lacI lacZ strain demonstrated a similar behavior as that of a Wild type with a Hill Coefficient of 2.8 and half saturation constant of 0.57 M salt concentration. Note that this was the same value as that for the wild type. This is expected as the ∆lacI lacZ strain would not synthesize
β-galactosidase and only the effect of salt would be observed as in a WT strain. The ∆lacI strain, on the other hand, demonstrated a lower growth rate in the absence of salt. Note that there is no requirement of IPTG (an inducer) in this case as ∆lacI strain constitutively synthesizes β-galactosidase. Although the trend was similar to the WT, the ∆lacI strain demonstrated lower growth rate relative to that observed for WT at all salt concentrations (Figure 3b). A Hill Coefficient of 3.73 and a half saturation constant of 0.6 M salt concentration was noted with a maximum specific growth rate of 0.34 h−1. Figure 3c shows the β-galactosidase activity for the constitutive synthesis of the enzyme by ∆lacI strain at various salt concentrations. The constitutive expression of β-galactosidase in ∆lacI synthesized about 40% excess β-galactosidase over the maximum enzyme activity observed in the WT strain. It was noted that the enzyme activity decreased with salt concentration with a Hill Coefficient of 1 and half saturation constant of 0.3 M salt concentration. This indicated that the observed drop in the enzyme activity in ∆lacI was not as steep as that in the WT strain. However, the 50% decrease in enzyme activity was observed at a lower salt concentration in the mutant strain than in the WT strain. To ascertain, if the drop in the activity is due to the synthesis or due to the inactivation of the synthesized protein, β-galactosidase was extracted from ∆lacI cells grown in glycerol in the absence of salt and its activity was measured by exposing it to different salt concentrations for half an hour. Figure 3c shows the deactivation of the enzyme at various salt concentrations with a Hill Coefficient of 4 and half saturation constant of 0.8 M of salt. Thus, the activity of the enzyme remains unaffected until 0.6 M salt concentration and decreases steeply beyond 0.6 M concentration. This clearly demonstrates that the osmotic stress affects both the synthesis of β-galactosidase and its activity.
In order to provide direct evidence for the activity inhibition by osmotic stress or repressed enzyme synthesis from high salt concentrations, a GFP intergrated Wild Type E.coli strain was used. To characterize the effect of salt concentration on protein synthesis, a GFP intergrated Wild Type E.coli strain was grown in a M9 medium containing glycerol and 200 μM of IPTG. Figure 4 shows the fluorescence image of cells expressing GFP. Panel (a) shows the fluorescence image for cells growing in the absence of salt concentration. It can be seen that GFP was expressed in all the cells. Panel (b) shows the fluorescence image for cells exposed to 1.5 M salt concentrations after growing the cells in a M9 glycerol medium containing 200 μM of IPTG in absence of any salt concentration. This ensured that the cells expressed GFP in a normal medium and were later exposed to salt concentration to check the effect of salt on the fluorescence. The fluorescence intensity observed after 3 h was similar to that of the cells unexposed to salt indicating that the GFP fluorescence was not affected by salt concentration. To determine the influence of the hyper-osmotic stress on the expression of GFP through the lacZ operon, cells were grown in M9 media containing glycerol, 200 μM of IPTG and 1.5 M salt concentration. Figure 4c shows the image of cells grown in a glycerol medium containg salt from the start of the experiment. It is clear that the cells express GFP only marginally in this case, indicating a strong inhibitory effect of salt on the expression system. Note that this image (i.e. shown in Figure 4c) was obtained after concentrating the cells grown under high salt concentrations in order to maintain the same number of cells in a frame as in the other images. As a control experiment, cells were grown in the absence of salt and IPTG and the images did not show any GFP expression (see Figure 4d). It can be noted that the corresponding bright field image of cells for the respective experiments shown in the right panel, indicated that the number of cells in the frame were almost identical in all the cases. Similar images analyses were obtained for different salt concentrations and a mean normalized fluorescence value were determined.
The normalized fluorescence value for cells grown in M9 medium with various salt concentrations is shown in Figure 5a. It is clear that GFP expression was strongly repressed by salt concentration. A fit of Hill equation indicated an ultrasensitive repression with a Hill coefficient of 4.2 and a half saturation concentration (K0.5) of 0.4 mol l−1 salt concentration. To quantify the effect of salt on the GFP activity, experiments were conducted with the cells exposed to 0.2 mol l−1 and 1.5 mol l−1(see the corresponding Figure 4b) salt concentrations post GFP expression in a normal medium. It was observed that the GFP intensity did not alter even after 3 h on exposure to 0.2 mol l−1 salt concentrations, while a decrease of about 15-20% in the intensity was observed on exposure to 1.5 mol l−1 salt concentrations. These experiments clearly demonstrated that the expression from the lacZ promoter was strongly inhibited by osmotic stress and it was not because of the loss in the fluorescence intensity of GFP. This implied that the protein synthesis machinery was severely affected by the osmotic stress.
The synthesis of β-galactosidase through IPTG while growing on glycerol offers burden characterizing the cost of enzyme synthesis in the presence of salt in the medium. Addition of lactose into the glycerol medium would characterize the benefit offered due to the synthesis of β-galactosidase. Therefore, experiments were performed with a medium containing lactose of 1 mM concentration and 1 g/L glycerol at different salt concentrations. The growth rate at different salt concentrations with and without 1 mM lactose in the medium is shown in Figure 6a. Lactose offered growth benefit upto 0.6 M salt concentration and a higher burden on growth beyond 0.6 M salt concentration. This observation was consistent with the enzyme activity profile in case of growth in a glycerol medium containing saturating amounts of IPTG (Figure 2b) indicating that lower amounts of β-galactosidase was synthesized beyond 0.6 M salt concentration resulting in lower growth without any benefit. This resulted in a sensitive relationship of growth on lactose to salt concentration with a Hill coefficient of 3.6 and half saturation constant of 0.53 M, which was more sensitive than that observed in the absence of lactose (2.7 and 0.57 M, respectively). Figure 6b shows the lag phase observed for the two cases with and without lactose in the medium. The lag phase needed for adaptation also correlated with the growth wherein growth on lactose demonstrated lower lag phase upto 0.6 M salt and higher beyond it. The growth rates determined from the experiments were used to quantify the burden in presence and absence of lactose by evaluating the normalized deviation of growth rate relative to growth in a glycerol medium lacking salt. The contribution to burden from β-galactosidase synthesis due to lactose was evaluating by subtracting the burden due to salt alone from the burden due to salt and lactose. Figure 6c shows the burden due to lactose (фL) as a function of salt concentration. It is clear that upto a salt concentration of 0.6 M, the cells experience benefit. The β-galactosidase activity decreases beyond 0.6 M salt concentration and the benefit reduces leading to a burden. The relationship between the burden due to lactose caused due to β-galactosidase synthesis shows a linear relationship with the β-galactosidase activity.
The overall burden experienced by E.coli cells in three different media, namely (i) medium containing glycerol and saturating IPTG along with various salt concentration, (ii) medium containing glycerol and lactose with salt and (iii) medium containing glycerol, lactose and IPTG with salt , were compared with the respective normalized β-galactosidase activity (see Figure 7). The burden demonstrated a linear relationship with enzyme activity. It was noted that saturating amounts of IPTG yielded the highest amounts of enzyme concentrations among the 3 media. The media with 1 mM lactose yielded only 40% of the maximum enzyme activity observed in presence of saturating IPTG. Addition of IPTG to lactose yielded higher enzyme activity than lactose alone. This indicated that 1 mM of lactose could not eliminate completely the repression action from lac repressor and therefore the enzyme synthesis was limited. The presence of lactose demonstrated negative burden implying benefit at low salt concentrations. The slopes of the best fit were noted to be negative with values of 0.84, 2.0, and 2.74 for growth on glycerol + IPTG, glycerol + lactose + IPTG and glycerol + lactose, respectively. This indicated that the presence of lactose indicated higher burden to salt concentration as compared to growth in a medium with glycerol alone. Thus, the media and salt concentration had a strong influence on β-galactosidase activity which further determined the extent of burden.