Effect on β-galactosidase synthesis and burden on growth of osmotic stress in Escherichia coli
© malakar et al.; licensee Springer. 2014
Received: 31 August 2014
Accepted: 11 December 2014
Published: 17 December 2014
Osmotic Shock is known to negatively affect growth rate along with an extended lag phase. The reduction in growth rate can be characterized as burden due to the osmotic stress. Studies have shown that production of unnecessary protein also burdens cellular growth. This has been demonstrated by growing Escherichia coli on glycerol in the presence of Isopropyl-β-D-1-thiogalactopyranoside (IPTG) to induce β-galactosidase synthesis which does not offer any benefit towards growth. The trade off between osmotic stress and burden on growth due to unnecessary gene expression has not been enumerated. The influence of osmotic stress on β-galactosidase synthesis and activity is not clearly understood. Here, we study the effect of salt concentration on β-galactosidase activity and burden on growth due to unnecessary gene expression in E.coli. We characterize the burden on growth in presence of varying concentrations of salt in the presence of IPTG using three strains, namely wild type, ∆lacI and ∆lacIlacZ mutant strains. We demonstrate that the salt concentrations, sensitively inhibits enzyme synthesis thereby influencing the burden on growth. In a wild type strain, addition of lactose into the medium demonstrated growth benefit at low salt concentration but not at higher concentrations. The extent of burden due to osmotic shock was higher in a lactose M9 medium than in a glycerol M9 medium. A linear relationship was observed between enzyme activity and burden on growth in various media types studied.
KeywordsEscherichia coli Osmotic stress Unnecessary Gene Expression IPTG Growth rate
External stress, such as osmotic shock, is known to burden the growth of the cell, wherein resources are channeled towards adaptation, thereby reducing the growth rate (Csonka 1989; Record et al. 1998). Osmotic stress is known to affect the phenotypic properties of cells such as metabolism, growth and protein synthesis. An increase in the external osmolarity causes loss of water from the cell resulting in shrinkage and arrest in the cell division (Record et al. 1998). The cells synthesize internal osmolytes, such as glycerol or trehalose, to restore cell volume and resumes cell growth post adaptation (Shabala et al. 2009). Thus adaptation is defined based on the restoration of cell division after an initial extended lag in growth on exposure to salt concentration resulting in osmotic stress. While the effect of salt concentration on growth and metabolism is well studied, the effect of salt concentration on enzyme synthesis and activity has not been characterized. There is a study reported wherein the authors demonstrate that viable but non-culturable cells of Escherichia coli retain enzyme activity and enteropathogenicity when exposed to sea water (Davies et al. 1995; Pommepuy et al. 1996). To address this issue, we focus our study on the effect of salt concentration on burden to growth and β-galactosidase synthesis or activity in E.coli. Further, we characterize the effect of salt concentration on cost phenomenon in a glycerol medium by inducing β-galactosidase synthesis using IPTG. Note that the synthesis of β-galactosidase in a glycerol medium does not offer any growth advantage and is an unnecessary protein in the absence of lactose (Malakar 2014; Malakar & Venkatesh 2013; Malakar & Venkatesh 2012).
It has been shown that the production of unnecessary protein burdens the cell and reduces the growth rate (Novick & Weiner 1957; Horiuchi et al. 1962; Andrews & Hegeman 1976; Koch 1988; Nguyen et al. 1989; Dong et al. 1995; Dekel & Alon 2005; Alon 2006; Malakar & Venkatesh 2014). This reduction in growth rate characterizes the cost to the cell, where the resources are channeled for the synthesis of the unnecessary protein (Maaloe & Ole 1966; Vind et al. 1993; Alon 2006). Studies have indicated that the reduction in growth rate due to unnecessary gene expression or the cost phenomenon depends on the transcriptional efficiency, ribosomal capacity and the quality of medium used for growth (Scott et al. 2010; Malakar & Venkatesh 2012; Malakar 2014). However, if the enzyme synthesized helps in the metabolism of a substrate, the enzyme synthesis provides benefit to the cell. Thus the organism has to balance the cost and benefit due to the synthesis of protein. The trade off between cost and benefit is a fundamental aspect of selection by evolution (Dekel & Alon 2005; Alon 2006). This trade off also determines which regulatory circuit will be selected in a given environmental condition (Dekel et al. 2005; Babu & Aravind 2006; Camas et al. 2006; Zaslaver et al. 2006; Kalisky et al. 2007; TÇŽnase-Nicola & Ten Wolde 2008). Understanding protein cost and adaptation is also important in biotechnology industries where micro-organisms are used to produce gratuitous proteins (Shachrai et al. 2010). Expression of unnecessary genes is one type of stress, which a cell faces within itself. Apart from this cells are exposed to a variety of environmental fluctuations. Cost benefit Analysis is an analysis of energy diversion. The impact on growth of unnecessary protein synthesis (cost) and necessary protein synthesis (benefit) under osmotic stress condition had not been reported in the literature.
It has been demonstrated that in Escherichia coli, the lac operon operates optimally to a given lactose concentration to achieve a balance between cost and benefit (Dekel & Alon 2005). The lac operon in E.coli, a well characterized system, is commonly used to characterize the impact of unnecessary gene expression on growth. E.coli is grown on a medium containing glycerol with IPTG, a non-metabolizable inducer of lac operon, synthesizing β-galactosidase which offers the burden on growth without any benefit.
In the current study, we address the effect of salt concentration on β-galactosidase synthesis and activity in Escherichia coli and the burden on growth. Characterization of burden on growth due to unnecessary protein production under osmotic shock will provide insights into the effect of protein synthesis and salt concentration on burden. We further analyze the affect of salt concentration on the growth of E.coli on lactose, thereby characterizing the benefit experienced by the cells. The study demonstrated that protein synthesis and activity is severely affected by osmotic stress thereby influencing the burden. Comparison of growth in media with and without salt yielded individual contributions of protein synthesis and salt on burden to growth. The stability of synthesized GFP at various salt concentrations, clearly demonstrated that osmotic stress repressed enzyme synthesis and not the activity. Further, a new perspective on adaptation based on protein synthesis is obtained instead of a definition based on cell division.
Materials and methods
Strains, media and reagents
The strain E.coli MG1655 (WT) CGSC 6300 was a kind gift from Dr. Manjula Reddy, CCMB, India (Samaluru et al. 2007). DMS269 with genotype lacI − and DMS1346 with genotype lacI − ∆lacZ was obtained from Daniel Stoebel (Stoebel et al. 2008). BL21(DE3) which is tagged with GFP on the lacZ promoter was also used (Davies et al. 1995). All the experiments were done in M9 defined medium consisting of M9 salts, 1 mM MgSO 4 , 0.1 mM CaCl 2 , 0.1% glycerol, 0.200 mM IPTG and specified concentrations of glycerol (Merck). The Z Buffer (pH 7.0) contained: 60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, 50 mM 2-mercaptoethanol. ONPG (pH 7.0) contained: 40 mg ONPG dissolved in 10.0 ml of 0.1 M potassium phosphate buffer. For the cost experiment specified concentrations of IPTG obtained from (Invitrogen) were used. Lactose Monohydrate obtained from Himedia was also used in some experiments.
Growth rate measurements
Where dX/dt = the growth rate of the biomass mg/L t-1
X = the concentration of biomass, mg/L
μ = the maximum specific growth rate constant, t-1
This relationship applies for the log-growth phase, when there are sufficient nutrients for growth and when the bacteria have been acclimated to the system. A minimum of four time points in exponential growth phase was used to calculate the specific growth rate. The slope of the linear fit gave growth rate.
Beta galactosidase assay
Cells were grown on M9 medium with glycerol as the carbon source. Aliquots of culture were taken at fixed OD. The cells were centrifuged and resuspend in 1 ml Z-buffer and were later placed on ice. The OD of cell suspension was measured at 600 nm. 80 μl of 0.1% SDS and 160 μl of chloroform were added to each tube. The tube was vortexed for 15 seconds. The reaction mixture was incubated at 30° C for 15 minutes. 160 μl of 4 mg/ml ONPG was added and vortexed well for 10 sec and further incubated at 30° C and timed. The reaction tube was removed after about 10 minutes. The reaction was quenched by adding 400 μl of 1 M sodium carbonate. The cell debris was spinned down. The O.D. of the aliquot was measured at 420 nm. The Miller Units was calculated using the following formula: U = 1000 x [(OD420) / [(Time) x (Vol) x OD595] where Vol is volume of the culture used in the assay in mls, and Time is in minutes (Miller 1972). The data is a mean of 3 or 4 experiments and the maximum error in β galactosidase measurement was 10%.
Determination of burden on growth (ф)
Where, μ GI is the growth rate in a glycerol media containing both salt and saturating amount of IPTG (200 μM).
Where, μ L is the growth rate on medium containing lactose and glycerol and μ G is the growth rate on medium with glycerol in the medium at various salt concentrations.
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.
Growth experiments were conducted to determine the burden on growth due to osmotic shock and addition of IPTG leading to β-galactosidase synthesis, an unnecessary enzyme in a glycerol media. Osmotic shock, due to the addition of salt (NaCl), resulted in a reduced growth rate which was highly sensitive indicated by a Hill coefficient close to three. Further, addition of IPTG decreased the growth further due to the burden caused by the synthesis of unnecessary protein. It was observed that enzyme activity was drastically affected due to osmotic shock beyond 0.4 M of salt concentration. Experiments using mutant strains of E.coli, namely ∆lacI clearly demonstrated that salt concentrations not only inhibit β-galactosidase activity but also repress its synthesis. The enzyme activity was not affected upto 0.6 M salt concentration. Thus, beyond 0.6 M salt concentration, combination of activity inhibition and repression of synthesis yielded a switch like response of β-galactosidase activity to salt concentration in the WT. The stability of synthesized GFP at various salt concentration clearly demonstrated that the synthesis was repressed and not its activity.
Interestingly, in a ∆lacI strain, although β-galactosidase is constitutively synthesized, the effect of salt was more severe as the repression of synthesis was observed even before 0.6 M salt concentration since the activity is inhibited only beyond 0.6 M salt concentration. This offers a new perspective in the definition of adaptation to osmotic shock. Adaptation is characterized by an extended lag phase before the growth is resumed post adaptation (Parmar et al. 2009). Our experiments using WT and mutant strains of E.coli suggests that the enzyme synthesis machinery is strongly affected due to osmotic shock, thus causing a drastic drop in growth rate at higher osmotic shock (beyond 0.6 M salt concentration). Thus, one can infer that the enzyme synthesis may also be a parameter to characterize the extent of adaptation to osmotic shock.
Experiments were also conducted using media containing lactose and various salt concentrations. It was noted that lactose provided benefit only upto 0.6 M of salt, correlating with the normal enzyme activity observed for less than 0.6 M salt concentration. However, beyond 0.6 M salt, due to impaired enzyme synthesis and activity, the growth on lactose reduced. In glycerol environment, expression of beta-galactosidase is unnecessary but in lactose environment expression of beta-galactosidase is necessary for lactose utilization. In the medium with both glycerol and lactose the growth rate was higher than medium with only glycerol at low salt concentrations but at higher salt concentrations the effect is reversed. This effect at higher salt concentration may be due to the effect of beta-galactosidase over expression induced by lactose on the growth rate.
Thus, at higher salt concentration, both salt and the enzyme synthesis caused a cumulative negative effect on growth. The burden at low salt concentration and higher burden at higher salt concentration yielded a highly sensitive response, with a Hill coefficient of 3.6, to growth on lactose in relation to salt concentration. Scott et al. (2010) have reported a phenomenological model relating the effect of transcriptional efficiency, ribosomal capacity and quality of the medium to growth rate (Scott et al. 2010). Their study predicted and experimentally demonstrated a linear relationship between unnecessary enzyme synthesis to extent of burden characterized by lowering of growth rate. Our results confirm the linear relationship between burden on growth due to unnecessary gene expression and enzyme activity under various osmotic stress conditions (see Figure 7). This indicated a limiting ribosomal capacity, when unnecessary protein synthesis is synthesized thereby reducing the proteomic capacity necessary for growth. It appears that the osmotic shock and the unnecessary enzyme synthesis influenced negatively the ribosomal capacity needed for growth.
In summary, the study demonstrated that the burden due to osmotic shock is highly sensitive mainly being affected by limitation in the enzyme synthesis. The effect on enzyme synthesis, which may be limited due to ribosomal capacity, by salt, was switch like. This yielded a new perspective on adaptation, which could be based on enzyme synthesis rather than on the resumption of growth. Further, the benefit by lactose was noted only at low shock levels, namely less than 0.6 M salt concentration. The linear relationship between burden and enzyme activity demonstrated that the net proteomic fraction needed for growth was reduced due to higher osmotic shock. Thus, an analysis of burden on growth in presence of osmotic shock can provide phenomenological insights into the relationship between enzyme synthesis and growth.
Authors acknowledge the support provided by Council of Scientific and Industrial Research (CSIR), India for their research fellowship. They also convey their thanks to Prof. Manjula Reddy (CCMB, India) and Prof. N. S. Punekar (IIT Bombay, India) for providing the strains K-12 MG1655 (CGSC No.6300) and BL21(DE3) respectively.
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