2. Material and Methods
3.1. Bacterial growth and pH conditions
3.2. Protein extraction and Mini SDS-PAGE
3.3. Resolution of whole cell extracts by two-dimensional (2D) polyacrylamide gel electrophoresis
Escherichia coli inhabits the mammalian gastrointestinal tract anaerobically at high osmolarity as well as the soil aerobically where it is faced with rapid variations in osmolarities. Fermentation technology using E. coli followed by IEF-2D-PAGE was implied to visualize global proteome alterations under normal aerobic conditions and conditions of high osmolarity (0.4M NaCl). The protein profile revealed an up-regulation in the expression of ProX, HchA, OsmY and OtsB in osmotic stress induced cells, as well a down- regulation of two proteins named FliC and MetF in cells under hyper-osmotic conditions. Another protein, GlnA seemed to be expressed at equal rates under both conditions.
Escherichia coli is a gram negative microorganism which inhabits the mammalian gastrointestinal tract under anaerobic and high osmolarity conditions, but also the soil, aerobically while being subjected to high variations in osmolarity. E. coli is a facultative anaerobic bacterium which can use alternate electron acceptors (nitrate, fumarate) for anaerobic respiration (Weber et al., 2006).
Most organisms are faced during their lifetime with osmotic stress either due to changing external osmolarities, or due to desiccation or freezing. Therefore, in order to survive the cells must continuously sense and respond to these changes and adapt accordingly. Bacteria have developed strategies to survive in such environments with increased osmolarity.
While a low environmental osmolarity triggers cellular water influx and, as a result, cell swelling, the increase in osmolarity induces cellular dehydration and cell lysis.
Two basic strategies have been developed in order to cope with such changes. The salt-in strategy is mainly employed by some family members of Halobacteriacea which maintain a high intracellular salt concentration, whereas other prokaryotic organisms respond by a salt-out strategy. The latter represents a bi-phasic response, in which the primary response is an increase in K+- concentration and its counter-ion, glutamate, which is then followed by the accumulation (by synthesis and/or uptake) of organic low-molecular weight osmolytes, termed compatible solutes which are compatible with cellular processes at high concentrations and also have a stabilizing effect on proteins (Heerman and Jung, 2004).
The response to osmotic stress in bacteria always occurs at two different levels. An immediate response in the time frame of seconds to a few minutes is mediated by enzymes and transport systems which react on the level of protein activity by either increasing or decreasing their functional activity. A secondary response, on a longer time scale of several minutes to hours, involves the transcription/translation level by de novo synthesis of enzymes, transport systems and cell wall components in order to re-establish a new physiological steady-state situation in the cell (Krämer, 2010). A well described system of an osmosensitive two component system is the EnvZ/OmpR in E. coli. It regulates the expression of the genes encoding two outer membrane proteins (OMP), ompC and ompF in response to external increase of osmolarity (Krämer, 2010).
The aim of the following work was a comparative qualitative analysis of the proteome of E. coli grown under normal and high osmolarity conditions by employing fermentation technology and 2D- electrophoresis for protein analysis. Following the hypothesis that it is necessary for bacteria to up-regulate de expression of certain proteins under osmotic stress conditions, the underlying experiments were aimed at identifying global alterations in protein expression under two different environmental conditions.
2. Material and Methods
Bacterial strains and growth conditions
E. coli MG1655 over-night cultures (optical density at 600 nm [OD600]= 0.05) were aerobically grown in “Minifors” fermenters in 1 liter K10 Minimal Medium supplemented with 0.4% (w/v) glucose (Epstein and Kim, 1971) at 37°C, 200 rpm agitation, until an optical density at 600 nm [OD600] of 0.4 was reached. At this point NaCl was added to the medium to a final concentration of 0.4 M (cells exposed to hyperosmotic stress), or the cells were left untreated (cells not exposed to hyperosmotic stress). The pH measurement were carried out throughout the entire experiment using calibrated pH electrodes.
Bacterial growth curve
In order to obtain the bacterial growth curve, samples (5 ml) were harvested at the following time points: t=30, t=60, t=90, t=120, t=150, t=180, t=240 min respectively. Optical density at 600 nm was determined and absorbance values were plotted to obtain the growth curve.
Preparation of crude cell extract
Samples (250 ml) were harvested 30 minutes after the induction of the hyperosmotic stress. The same harvesting time point was applied in the case of the untreated cells. Samples were treated with chloramphenicol (0.1 mg/ml) and centrifuged (10 min at 3,500x g at 4°C). Pellet was washed with 1/5 volume Wash buffer
(100 mM Tris/HCl pH 7.5; 0.01 mg/ml chloramphenicol) and centrifugation step was repeated (10 min at 3,500x g at 4°C).
Cells were disrupted by sonication on ice in 3 mL Extraction buffer
(10 mM Tris/HCl pH 7.5; 1 mg/mL MgCl2x 6H2O; 1.39 mM PMSF; 50μg/mL DNase; 50 μg/mL RNase; 100 μg/mL Lysozyme) and the crude protein extract was separated from unbroken cells or cell debris by centrifugation (10 min at 16,100 x g at 4°C). Supernatant was collected in fresh tubes. Protein quantification was performed according to the modified Lowry method. Standard curve was performed using bovine serum albumin (BSA) and protein concentration was calculated accordingly.
Mini SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
Protein samples were qualitatively evaluated by loading them on a SDS-PAGE system under denaturing and reducing conditions. 12% polyacrylamide gels were prepared according to Laemmli (Laemmli, 1970). 15 μg and 30 μg protein from each sample were mixed with 2x Laemmli buffer (125mM Tris/HCl pH 6.8; 4% SDS;
25% glycerol; 0.01% bromophenol blue) and 5% 2-mercaptoethanol. Samples were run in 1x Tris-Glycine buffer at 20 mA per gel for ca. one hour. Gels were stained in
a 0.1% Coomassie solution for 30 minutes and destained in destaining solution
(30% ethanol; 10% acetic acid) for one hour.
Resolution of whole cell extracts by two-dimensional (2D) polyacrylamide gel
In preparation for the isolectric focusing (IEF), 700 μg protein sample was lyophilized. IEF was carried out using immobilized pH gradient strips with a linear pH gradient between 4 and 7 (17 cm) on an IPGphor system (GE Healthcare).
700 μg protein for each sample was rehydrated in the rehydration buffer (provided by course instructors). Samples were added to an IEF focusing tray, strips were place on top and covered with mineral oil. The IEF focusing tray was placed in the PROTEAN IEF cell. IEF was carried out over-night, at a maximum voltage of 10,000V/17 cm, using a default cell temperature of 20°C. Equilibration of the strips was carried out in
2 mL equilibration buffer (6 M urea; 0.375 M Tris/HCl pH 8.8; 2% SDS; 20% glycerol; 2% (v/v) 2-mercaptoethanol), at room temperature, for 20 min.
Second-dimension analysis was performed using a 12% resolving polyacrylamide gel as previously described. Gels were run over-night at 50V constant voltage. Gels were Coomassie blue stained for one hour and destained for 3 hours in destaining solution.
A 10% acetic acid destaining step was performed thereafter until background was clear.
Stained gels were scanned and 2D protein patterns were analyzed by direct visual comparison to prior MALDI-TOF MS results for samples treated under the exact same conditions.
- Quote paper
- Eva Maria Kalbhenn (Author), 2014, Bacterial adaptive response to osmotic stress. Proteome alterations, Munich, GRIN Verlag, https://www.grin.com/document/374796