K.A. Bagramyan Department of Biophysics, Yerevan State University, 1 Alex Manoogyan Str., 375049 Yerevan, Armenia (e-mail: KBaghramyan@ysu.am).
Aims: To study the effect of diethylsulphoxide (DESO) on Escherichia coli growth, survival and ionic exchange in comparison with dimethylsulphoxide (DMSO).
Methods and Results: Bacterial survival was estimated by counting colony-forming units and by the most probable number (five-tube) technique; the K+ and H+ transport and H2 formation were determined electrochemically. Diethylsulphoxide at concentrations between 0·01 and 0·5% (w/v) stimulated and above 5% decreased the anaerobic growth rate and survival. 2H+ : K+ exchange and H2 formation were lost at 5% DESO. At 0·05% DESO the kinetic characteristics of H+ : K+ exchange and H2 formation were typical for ΔμH+-dependent TrkA uncoupled with F0F1 under respiration.
Conclusions: Diethylsulphoxide at low concentrations serves as an electron acceptor for an anaerobic respiratory chain stimulating bacterial growth and survival through the modulation of H+ : K+ exchange and H2 formation activity. The effects of DESO were more pronounced than those of DMSO.
Significance and Impact of the Study: Diethylsulphoxide determines essential biological and therapeutic properties that make its application preferable.
Dimethylsulphoxide (DMSO) is a water-miscible solvent that has wide applications in cell biology. It influences intramembrane interactions in a way that is different from that of some other solvents that make hydrogen bonds with water molecules. There are stronger interactions between DMSO and water than between DMSO molecules themselves and this affects membrane stability (Gordeliy et al. 1998; Yu and Quinn 1998), membrane permeability and fusion (Wood and Wood 1975). This appears to be responsible for the biological and therapeutic impact of DMSO. However, there is no information about the biological activity of its nearest homologue, diethylsulphoxide (DESO). The strong interaction between DESO molecules tends to form an even more associative structure than DMSO, resulting in a number of unique physicochemical properties (Markarian and Stockhausen 2000). This structure serves to modulate the stability of biomolecular structures and often their functional organization either by direct interaction or via their interaction with solvating water molecules. From this point of view, the interaction of DESO and membrane systems may have an important influence on membrane function.
This paper presents data on the effect of DESO on the activities of key membrane transport systems, such as F0F1 ATPase and the TrkA K+ uptake system, which function in Escherichia coli as an ATP-driven pump in order to stabilize ΔμH+ (Trchounian et al. 1998) using redox couples donated by formate through the formate hydrogen lyase (FHL) (Bagramyan and Martirosov 1989). It is thus of particular interest to understand the mechanisms of biological activity of DESO and to partly explain how dialkylsulphoxides commonly act on bacterial growth and survival.
MATERIALS AND METHODS
Bacterial strain, culture media and reagents
Wild type E. coli strain K12 was used throughout. Bacteria were grown at 37°C either anaerobically or aerobically as described (Trchounian et al. 1998). The growth medium (pH 7·3–7·4) contained: (a) 2% (w/v) peptone, 0·5% (w/v) NaCl and 0·2% (w/v) glucose or (b) 50 mmol l–1 glycerol, 46 mmol l–1 K2HPO2, 23 mmol l–1 KH2PO4, 0·4 mmol l–1 MgSO4, 8 mmol l–1 (NH4)2SO4, 8 μmol l–1 FeSO4 and 22 mmol l–1 glucose or 16 mmol l–1 sodium succinate (for aerobic growth). The peptone and agar were from Difco (Detroit, MI, USA), polipeptone was from Kyokuto (Kyoto, Japan), standard pH solutions were from Radiometer (Copenhagen, Denmark) and N,N′-dicyclohexylcarbodiimide (DCCD) was from Sigma (St. Louis, MO, USA). Dimethylsulphoxide and DESO were used after their purification.
Measurement of bacterial growth and survival
Medium (b) was inoculated at 1% with an overnight culture prepared from medium (a), at approx. 104–105 colony-forming units (cfu) ml–1, and incubated at 37°C. Diethylsulphoxide or DMSO, between 0·005 and 30% (w/v), was added to medium (b) before inoculation. Bacterial growth was monitored by measuring the absorbance of the culture at 600 nm using a spectrophotometer. The specific growth rate was estimated at a period when the logarithm of the optical density (O.D.) change increased linearly with time (Rhoads et al. 1976). Survival was determined by counting cfu and comparing the results using a five-tube Most Probable Number (MPN) method. Up to five 10-fold dilutions were used to inoculate each of the five tubes for MPN determinations. These were incubated at 37°C and examined for growth after 48 h. Positive tubes from the highest dilution showing growth were streaked onto agar plates to check for contamination. The MPN values were calculated using published tables (Kaprelyants and Kell 1992). All measurements were done in triplicate.
Study of the K+ and H+ transport
The transport of H+ and K+ through the bacterial membrane was investigated by measuring [K+]ex and [H+]ex, respectively. Small changes in H+ and K+ activities were calibrated by titration with 0·05 mmol l–1 HCl and 0·02 mmol l–1 KCl. [K+]ex and [H+]ex were defined with ion-selective glass electrodes as described previously (Trchounian et al. 1998). These electrodes, together with redox electrodes, were introduced into a chamber with the assay mixture. The electrode potentials were simultaneously recorded using a multichannel potentiometer. To study K+ uptake, samples of the growth culture were removed, washed with distilled water and resuspended in a medium consisting of 100 mmol l–1 Tris-phosphate (pH 7·5), 0·4 mmol l–1 MgSO4, 1 mmol l–1 NaCl and 1 mmol l–1 KCl. K+ uptake was initiated by the addition of glucose (22 mmol l–1). Bacterial cells grown to an O.D.600 nm of 0·5 (1·8 × 107 cells ml–1), which corresponds to the midexponential growth phase, were harvested by centrifugation (5200 g for 20 min) and washed with distilled water. Cells became partially K+ containing in the presence of glucose. Such a transfer of cells into a solution of high osmolarity provided a hyper-osmotic shock for bacteria (Rhoads et al. 1976). The Km for K+ uptake was determined through the use of a Lineweaver–Burk plot. When used, bacteria of a high count (1012 cells ml–1) were incubated with 0·2 mmol l–1 DCCD 10 min before the addition of glucose. The DCCD-inhibited ion flux was the difference between the values of fluxes in the absence or presence of the reagent at a concentration of 0·05 mmol l–1. Stoichiometry was calculated as a ratio of DCCD-inhibited H+ and K+ fluxes.
Measurement of redox potential and H2 formation
Redox potential and H2 formation by whole cells were measured using a pair of the redox, titanium-silicate and platinum electrodes (Enterprise of Electrometer Equipment, Gomel, Belarus) as previously described (Trchounian et al. 1997). The production rate of H2 was determined as the difference between the initial rates of decreasing redox potentials for the platinum and titanium-silicate electrodes (mV min–1 mg–1 dry weight). H2 formation was also detected using a chemical assay (Bagramyan and Martirosov 1989) and by the Durham tube method (Barrett et al. 1984) for bacterial cultures grown to late logarithmic or stationary phase.
The effect of diethylsulphoxide on the growth and survival of bacteria
In our first exploratory experiments, we tested the effect of DESO on bacteria either anaerobically or aerobically. The growth stimulatory effect of DESO on E. coli under anaerobic conditions was apparent at concentrations between 0·05 and 1% (Fig. 1a). The growth rate increased by twofold and was higher than in cultures with DMSO. This phenomenon has been described for DMSO (Tran and Unden 1998) and suggests the operation of an anaerobic respiratory chain in E. coli with DESO as an electron acceptor resulting in an increased ATP yield. Diethylsulphoxide became toxic at concentrations above 5%; the growth rate decreased substantially at 30% DESO and gave a 2·8-fold lower growth yield than in control assays (Fig. 1a). Under aerobic conditions, O2 rather than DESO acts as a terminal electron acceptor in a respiratory chain. There was no marked stimulatory effect of low concentrations of DESO on aerobically growing E. coli (Fig. 1b). The high concentrations of DESO or DMSO both also suppressed bacterial growth under aerobic conditions: the specific growth rate of cells fell up to fourfold.
There were no significant differences between the duration of the lag phase and the growth rate (0·9 and 00·85 h–1, respectively) during exponential growth under anaerobic conditions at 0·01% DESO. The duration of the lag phase increased (13 h) and the growth rate decreased (0·1 h–1) on the addition of 5% DESO. The cell growth stopped with final cell concentrations of no more than 107 cells ml–1 when 30% DESO was added (not shown). The survival of E. coli remained higher for the assays taken from the culture medium with 0·05% DESO (initial cfu approx. 109) than was achieved in the assays with DMSO (when the maximum growth was obtained) (Fig. 2) throughout the duration of the experiment.
Effect of diethylsulphoxide on H+ : K+ exchange and H2 formation
H+ : K+ exchange and H2 formation were determined in anaerobically grown bacteria in the presence of DESO. In the presence of 5% DESO, H+ extrusion from cells was low and K+ uptake and H2 formation were absent (Table 1). This could be linked to the inactivation of H+ : K+ exchanging proteins by high concentrations of DESO due to their reversible configurational changes when DESO substitutes for water in growth and experimental media (Topolev and Krishtalik 1999). These results correlated with the effects of high concentrations of DESO on growth and survival described above. The presence of DESO under aerobic conditions also resulted in the inhibition of K+ accumulation by bacteria (not shown). The opposite action of DESO on H+ extrusion from bacteria was observed at 2% and below where it was amplified 5·1-fold (2·4-fold with DMSO) (Fig. 3b). The values of K+ fluxes after the addition of glucose were lower than those on fermentation (Table 1) (a 1·4-fold decrease in the rate of K+ uptake (2·3-fold with DMSO)). The stoichiometry of DCCD-inhibited fluxes varied between 14·4 and 11·2. The Km values for K+ uptake at 37°C were 4·1 and 3·7 mmol l–1 for DESO and DMSO, respectively. The H+ extrusion rate and its duration were increased at a low concentration of DESO (0·05%) (Fig. 3b). These data were similar to the results obtained for E. coli grown in the presence of nitrates and performing nitrate respiration (Trchounian et al. 1996).
Table 1. H2 formation and kinetic characteristics of H+ : K+ exchange by Escherichia coli K12 grown under anaerobic conditions in the presence of diethylsulphoxide (DESO) or dimethylsulphoxide (DMSO)
The relationship between the survival of bacteria and the mechanism of stabilization of the membrane has been intensively discussed. Results of X-ray diffraction experiments indicated that the decrease in the repulsive interaction between the headgroups of the phospholipid induced by low concentrations of DMSO in water plays an important role in this stabilization (Yamashita et al. 2000). Therefore, the modification of membrane structure should be reflected in the activity of membrane systems.
The observed effects of DESO on bacterial membrane systems may be explained in different ways, taking into account the properties of this substance. The increasing H+ flux and its duration observed at low concentrations of DESO on utilization of glucose is good evidence to support DESO serving as an electron acceptor for anaerobic respiration in E. coli, as described for DMSO (Weiner et al. 1992). The effect of DMSO on ATP-dependent K+ uptake observed under anaerobic conditions may be explained by the considerable variation of the energetics and ATP yields for these conditions (Tran and Unden 1998).
However, the interaction of DESO with carboxylic acid should be considered (Markarian and Beylerian 1985). The formation of H2 and CO2 from formate is catalysed by FHL in E. coli, which is expressed under fermentation but not in respiring cells (Böck and Sawers 1996). High concentrations of DESO might increase such an interaction with liberated CO2, decreasing FHL activity. The latter is coupled with the activity of F0F1 and TrkA (2H+ : K+ exchange) as proposed (Bagramyan and Martirosov 1989; Bagramyan et al. 2000). Since H+ is extruded from cells by means of the DCCD-sensitive proton pumps, F0F1 and FHL, their activities can also be suppressed by high concentrations of DESO.
Consequently, it should be assumed that there are changes in the properties of the membrane surface in the presence of DESO that are responsible for the modifications in the nature of the H+ : K+ exchange and H2 formation by FHL.
Dimethylsulphoxide is normally selected based on the criteria of low toxicity and permeability to the plasma membrane. An interesting consequence of this study is that the action of DESO on bacterial growth, survival and ion transport seems to be comparable to the effects of its homologue, DMSO. Moreover, such effects were more pronounced because of the lower concentrations of DESO used. Diethylsulphoxide appears to determine essential biological and therapeutic properties, and the relatively non-toxic nature of this compound makes its applications preferable.
This work was supported by the International Science and Technology Center (Grant No. A-199-99).