Persistence of Vibrio cholerae in waters of fluctuating salinity relies on the capacity of this facultative enteric pathogen to adapt to varying osmotic conditions. In an event of osmotic downshift, osmolytes accumulated inside the bacterium can be quickly released through tension-activated channels. With the newly established procedure of giant spheroplast preparation from V. cholerae, we performed the first patch-clamp characterization of its cytoplasmic membrane and compared tension-activated currents with those in Esherichia coli. Saturating pressure ramps revealed two waves of activation belonging to the ∼1-nS mechanosensitive channel of small conductance (MscS)-like channels and ∼3-nS mechanosensitive channel of large conductance (MscL)-like channels, with a pressure midpoint ratio p0.5MscS/p0.5MscL of 0.48. We found that MscL-like channels in V. cholerae present at a density three times higher than in E. coli, and yet, these vibrios were less tolerant to large osmotic downshocks. The Vibrio MscS-like channels exhibit characteristic inward rectification and subconductive states at depolarizing voltages; they also adapt and inactivate at subsaturating tensions and recover within 2 s upon tension release, just like E. coli MscS. Trehalose, a compatible internal osmolyte accumulated under hypertonic conditions, significantly shifts activation curves of both MscL- and MscS-like channels toward higher tensions, yet does not freely partition into the channel pore. Direct electrophysiology of V. cholerae offers new avenues for the in situ analysis of membrane components critical for osmotic survival and electrogenic transport in this pathogen.
Strong osmotic forces constantly affect all microorganisms, probably with only a few exceptions of obligatory symbionts or parasites. A cell 2 µm in diameter, surrounded by a single unarmored membrane, would burst with only a 20-mosmol inside versus outside osmotic gradient. At the same time, most gram-negative bacteria survive 600–1,000-mosmol downshocks with uncompromised viability (Britten and McClure, 1962; Csonka and Hanson, 1991; Schleyer et al., 1993; Wood et al., 2001). The early perception was that bacteria can tolerate high internal pressure because they are surrounded by a rigid cell wall, but the peptidoglycan network was shown to be stretchable (Koch and Woeste, 1992), and the more common opinion now is that the bacterium survives mostly because of its ability to rapidly dissipate excess osmolytes through a special emergency release system (Martinac et al., 1987; Levina et al., 1999). This system consists of several types of mechanosensitive channels activated at different tension thresholds (Berrier et al., 1996; Schumann et al., 2010; Naismith and Booth, 2012), with mechanosensitive channel of small conductance (MscS) and mechanosensitive channel of large conductance (MscL) mediating the bulk of osmolyte release. The success of the rescuing operation depends on one simple condition: the release of excess osmolytes must outpace the rate at which the lytic level of hydrostatic pressure inside the cell is reached. It is not surprising that expression of mechanosensitive channels is under the control of transcription factors regulating stress responses (Stokes et al., 2003), especially responses to cell wall damage. It is now obvious that bacterial mechanosensitive channels are capable of quickly dissipating ionic gradients (Boer et al., 2011), but their permeabilities to organic compatible osmolytes have not been studied. Functional properties of such channels are under intense scrutiny through the application of direct electrical recording (Martinac et al., 1987), reconstitution (Sukharev et al., 1993), and structural (Perozo, 2006; Steinbacher et al., 2007) and computational (Gullingsrud and Schulten, 2003; Anishkin et al., 2008) techniques, but limited primarily to Escherichia coli.
Facultative pathogens that spend part of their life as free-living forms face the same problem of osmotic adaptation. Vibrio cholerae populates an extremely broad range of aquatic environments, from rain water to deep sea water of high salinity (Pruzzo and Huq, 2005; Grim et al., 2010; Huq and Grim, 2011) and estuarial zones, where the salinity and osmotic pressure can broadly fluctuate. Pathogenic strains of V. cholerae are transmitted predominantly through fresh water, in which it is typically dropped from the intestines (Barcina, 1995). The fact that microorganisms survive these abrupt changes in osmolarity implies a very efficient osmoregulation system (Keymer et al., 2007; Naughton et al., 2009) that enhances environmental stability and transmission from one host to another. Extension of the electrophysiological platform to V. cholerae may uncover new aspects of its osmoregulation system that are critical for osmotic survival. Previously, biophysical properties of the outer membrane channels (porins) from V. cholerae were studied in reconstituted systems (Duret and Delcour, 2006; Duret et al., 2007; Pagel and Delcour, 2011), with the emphasis on adaptation to the intestinal environment. The membrane aspects of V. cholerae osmotic survival in fresh water have not been studied in direct electrophysiological experiments before.
Here, we adapted the giant spheroplast preparation technique to V. cholerae, previously developed for E. coli, and completed the first patch-clamp survey of its cytoplasmic membrane. We found two dominant channel activities similar to MscS and MscL in E. coli. Despite a higher overall density of mechanosensitive channels, V. cholerae exhibits lower tolerance to large osmotic shocks. We present the first comparison of channel activities between the two enteric species, emphasizing differences that suggest parameters that may define environmental fitness of these bacteria.
MATERIALS AND METHODS
Strains, spheroplast preparation, and electrophysiology
WT V. cholerae (N16691) from A. Huq’s laboratory and WT E. coli strain AW405, provided by J. Adler (University of Wisconsin, Madison, WI), were used in the study. The original procedure of giant spheroplast preparation from E. coli (Ruthe and Adler, 1985; Martinac et al., 1987) includes a 1.5–2-h incubation in the presence of 0.06 mg/ml cephalexin, during which bacteria produce long filamentous cells. The filaments are resuspended in 1 M sucrose buffer and subjected to lysozyme digestion (0.2 mg/ml) in the presence of 5 mM EDTA. Within 5–10 min of lysozyme treatment, filaments collapse into spheres of 3–6 µm in diameter, at which point the reaction is stopped with excess Mg2+. Spheroplasts are separated from the rest of the reaction and debris using quick sedimentation through a one-step sucrose gradient. Spheroplasts were stored in a high sucrose/BSA medium at −80°C. This procedure was used to make AW405 E. coli spheroplasts and was applied to N16961 V. cholerae with adaptations as described in Results.
Borosilicate glass (Drummond) was empirically shown to be suitable for tight-seal formation with bacterial membranes. Channel activities in giant spheroplasts were recorded from excised membrane patches at voltages between −80 and 80 mV. Negative pressures (suction) were applied to patches with a high speed pressure-clamp apparatus (HSPC-1; ALA Scientific Instruments). Pressure protocol programming, data acquisition, and analysis were performed using PClamp 10 software (Molecular Devices). Ensemble and single-channel currents were analyzed after Rs correction of the traces by the equation, GP = I/(V − IRS), where GP is the patch conductance and V and I are the transmembrane voltage and current, respectively. The series pipette resistance (RS ≈ 2.5–3 MΩ) was determined in each experiment after the patch broke.
To analyze the effect of trehalose on single-channel conductance, we used the macroscopic equation accounting for the pore (Rp) and access (Rs) resistances separately: Rch = Rp + Rs = (l + 0.5πa)ρ/πa2, where l and a are the length and radius of the pore (Hall, 1975; Hille, 2001). Specific resistances (ρ) of recording buffers with and without trehalose were measured using CDM230 conductivity meter (Radiometer).
The midpoint of activation was determined with 1-s saturating pressure ramp protocols, and the change in midpoint was monitored before and after the addition of trehalose to the bath. A combination of ramps of varying duration was used to visualize single-channel events to determine the current passing through a single channel. Inactivation of the MscS-like channel was examined with protocols that compared the number of channels available both before and after the patch was held under a subsaturating tension for 10 s. Specific protocols are explained in the figure legends.
Osmotic survival experiments
After observing large tension-activated conductances and anticipating corresponding osmotic permeability responses, we compared osmotic shock survival for V. cholerae and E. coli, in parallel side-by-side experiments. Both species grow well in Luria-Bertani medium (LB) plus 3% NaCl (HiLB, 1,180 mosmol). Fresh overnight cultures were diluted at 1:100 in this medium and pregrown for 2 h to OD600 of ∼0.4. The high osmolarity–adapted cultures (50-µl aliquots) were then abruptly shocked into 5 ml of diluted media (LB, LB/2, LB/4, LB/8, LB/16, and distilled water). After a 15-min incubation and quick 1:200 dilution in the medium it was shocked, each culture was plated in duplicates, and the colonies were counted the next morning. Osmolarities of media were measured using a vapor pressure osmometer (5520; Wescor Biomedical Systems).
Vibrio spheroplast preparation
The original procedure of giant spheroplast preparation from E. coli (Ruthe and Adler, 1985) was adapted by Martinac et al. (1987) for patch clamp; it includes a 1.5–2-h incubation in the presence of 0.06 mg/ml of the septation blocker cephalexin, during which bacteria grow but do not divide, producing filamentous cells. Our results show that V. cholerae is susceptible to both cephalexin and lysozyme. Filamentous forms grew in LB broth in the presence of 0.1 mg/ml cephalexin at the approximate rate of 100 µm/h. Filaments 80–150-µm long remained stable after sedimentation at 2,000 g and gentle pipetting. The concentration of lysozyme (0.4 mg/ml) and EDTA (25 mM) required for spheroplast formation in V. cholerae was twice what was needed for analogous E. coli preparations and on average took 20–30 min, which is also double the requirement of E. coli. Spheroplasts were separated from the debris by centrifugation through 1 M sucrose. Fig. 1 illustrates relative sizes of intact bacteria, filamentous forms, and giant spheroplasts. Spheroplasts often had a slightly irregular nonspherical shape, signifying remnants of undigested peptidoglycan.
Mechanoelectrical responses of the inner membrane to pressure ramps
Under sustained 20–40-mm Hg suction delivered from the pressure-clamp apparatus, V. cholerae spheroplasts formed tight seals with borosilicate glass pipettes, typically within 2–15 min. As with E. coli, patch excision was achieved by tapping on the base of the micromanipulator. Inside-out patches stimulated by triangular ramps of saturating pressure under a 30-mV hyperpolarizing voltage exhibited 2–7-nA currents in both types of preparations, which invariably displayed two-wave responses (Fig. 2 A). In E. coli, the first wave was ascribed to the MscS population, possibly with a mixture of a few MscK channels (Li et al., 2002), and the second, activating at higher tensions, represents a uniform MscL population. In V. cholerae, we observed very similar two-wave responses, usually with a lesser relative amplitude of the first wave. Relative amplitudes of the two waves were essentially independent on the speed of the pressure ramp. To avoid voltage-clamp errors caused by pipette series resistance (Rs) under high (>2-nA) currents, Rs correction was introduced (see Materials and methods). The corrected traces provided fractions of the total conductance ascribed to each type of channel and pressure midpoints for activation. The ramp responses clearly show that in both enteric bacteria, MscL is the dominant conductance responsible for the bulk of tension-activated permeability increase. MscS-like channels in Vibrio represent a considerably smaller fraction of conductance compared with MscS of E. coli; they also show no activation hysteresis (difference in midpoints on the ascending and descending legs of the triangular ramp), whereas half of E. coli MscS deactivates at a considerably lower pressure compared with the activation midpoint, producing an asymmetrical ramp response (Fig. 2 B).
To determine unitary conductances, we applied sequences of slower ramps to visualize single-channel events (Fig. 2, C and D). From multiple traces taken at different voltages, we created I-V plots and determined slope conductances at hyperpolarizing voltages (pipette positive) for single MscS-like (1.0 ± 0.1 nS) and MscL-like channels (2.6 ± 0.1 nS, or 2.9 nS after Rs correction) of V. cholerae (Fig. 3). This information permitted us to quantify the numbers and densities of channels in each preparation. Based on the previous results that the pressure midpoints for E. coli MscL correspond to ∼12-mN/m tension (Sukharev et al., 1999; Moe and Blount, 2005), we estimated the radius of curvature and area for each E. coli patch using the law of Laplace. Because we used the same 2.5–3-MOhm pipettes throughout this project, we first assumed that the average geometry of patches in both preparations was the same. The second assumption was that the tension midpoints for E. coli MscL and V. cholerae MscL-like channels are the same (Moe et al., 1998). Using these two different assumptions, we estimated patch areas and densities of channels in each preparation. The first assumption (Table 1, columns 4 and 5) gave a 20% lower estimation of channel density in V. cholerae relative to the second assumption (columns 8 and 9) because of a slight difference in estimated mean patch radius (1.37 vs. 1.50 µm).
The comparison of the ensemble currents in multiple patches (Fig. 1) reveals modest but statistically significant differences in activation midpoint ratios and substantial differences between channel numbers and densities in the membranes of E. coli and V. cholerae (Table 1). In E. coli (AW405), MscS and MscL are present on average at 3.6 and 4.3 channels/µm2, with a ratio of midpoint pressures of 0.60 (p0.5MscS/p0.5MscL). The MscS-like and MscL-like channels occur in V. cholerae (N16961) at densities of 1.2 and 11 channels/µm2, respectively, with a midpoint ratio of 0.48. Based on the data shown in Fig. 2 and Table 1, MscL-like channels are present at a 2.5 times higher density and MscS-like channels are present at a third the density in V. cholerae compared with E. coli. These differences in channels densities, confirmed in three independent batches of spheroplasts, prompted us to compare resistances of the two strains against acute osmotic downshock. The inference was that the higher overall density of channels in V. cholerae should correlate with higher osmotic survival.
Osmotic survival of V. cholerae
Survival rates of estuarial/enteric V. cholerae were quantified with that of primarily enteric E. coli in parallel osmotic dilution and plating experiments. Both cultures were pre-adapted in high osmotic LB (LB base with 0.52 M NaCl and 1,180 mosmol), where they grew for 2 h to OD600 of ∼0.5. The cultures were then abruptly diluted at 1:100 into media of different osmolarity (1,180, 425, 212, 106, 70, and 48 mosmol and distilled water). As seen from Fig. 4, both cultures easily survived a 970-mosmol downshift, after which the number of colonies for V. cholerae decreased. E. coli remained completely viable after an ∼1,100-mosmol drop, but beyond that point its survival precipitously declined as well. Remarkably, between the final osmolarity of 48 mosmol (LB/16) and distilled water, there was almost no difference in the survival rates: ∼2–4% E. coli and ∼0.3–0.1% V. cholerae. Although osmotic shock tolerance is comparable in the two enteric species, V. cholerae is clearly more sensitive despite the fact that it had the overall higher density of mechanosensitive channels. We hypothesize that this higher density of mechanosensitive channels may be a compensation for some other vulnerable traits of Vibrio, such as cell geometry, elasticity of the cell wall, or limited permeability of channels to specific osmolytes. The possible reasons for Vibrio vulnerability are presented in Discussion. The repeated observation of surviving colonies in distilled water suggests that bacterial populations are nonuniform and always contain a small fraction of highly resistant cells.
I-V relationships for the two dominant activities
To investigate how closely mechanosensitive channel activities in V. cholerae resemble those in E. coli, we measured I-V curves in a broad range for the two dominant activities. It was easier to record single MscS-like channels, which activate at a relatively low threshold, when the patch was initially silent. Conversely, to characterize MscL-like activities, we had to saturate the population of MscS-like channels first and record single MscL-like activities on top of a 1-nA current plateau. Because of reduced patch resistance in the presence of 8–10 open MscS-like channels, the MscL-like channels conducted at apparent 2.6 nS. After Rs correction (Rs = 2.9 MΩ, the open pipette resistance), the conductance was close to the 3.0-nS activity reported for MscL channels recorded in MscS-null E. coli strains (Levina et al., 1999; Chiang et al., 2004). In parallel experiments, E. coli MscL exhibited an apparent conductance of 2.4 nS when recorded at the background of ∼20 saturated MscS channels, 22% lower than the previously established unitary conductance of E. coli MscL. We also observed that the large-conductance channel from Vibrio also had a stronger propensity toward the uppermost subconductive state than E. coli MscL (compare Fig. 3, A and B).
The Vibrio MscS-like channel displayed a substantial inward rectification; the I-V curves were linear at hyperpolarizing voltages (pipette positive) but had a lower slope at depolarizing voltages. This MscS-like channel exhibited open-state noise at depolarizing (negative pipette) voltages, similar to what was observed in E. coli MscS (Akitake et al., 2005). These fast-flickering substates are likely to be the reason for rectification.
MscS-like channel inactivates and recovers
The earlier saturation of MscS-like channel population (plateau in Fig. 2 A) allowed us to choose a pressure range at which we could see activity only from this channel. Traces presented in Fig. 5 show patch responses to a pulse-step-pulse protocol, revealing the processes of adaptation and inactivation. In each protocol, the first short saturating pulse applied in the beginning revealed the entire active population, whereas the next prolonged step of pressure conditions the process of inactivation. The short saturating pulse delivered at the end tests for the remaining active population. The trace presented in Fig. 5 A shows a noticeable inactivation under a subthreshold step that did not invoke any activity. When the conditioning pressure step was at the midpoint for channel activation (determined with a 1-s ramp), inactivation was maximal (Fig. 5 B). The recovery experiment (Fig. 5 C) used a similar protocol; after the conditioning step pressure was dropped to zero, the patch was probed by a sequence of saturating pulses, tracing the process of recovery from inactivation. As illustrated in Fig. 5 D, inactivation of the MscS-like channel is tension driven. The Vibrio MscS-like channel thus behaved very much like E. coli MscS (Akitake et al., 2005; Kamaraju et al., 2011), with one subtle difference: it shows a small amount of silent inactivation at tensions that invoke no activity (Fig. 5 A).
Both MscS-like and MscL-like channels are modulated by compatible osmolytes
Ramp responses of the entire channel population in control and in the presence of 0.5 or 1 M trehalose on the cytoplasmic side of the patch are demonstrated in Fig. 6. V. cholerae (Fig. 6 A) and E. coli (Fig. 6 B) are both shown to be sensitive to the presence of this compatible osmolyte, evident by a right-shift of the activation curve and a decrease of the active population. The bottom panels (Fig. 6, C and D) show the magnified responses of MscS-like channels at the foot of the trace in both control and with 1 M trehalose traces. In four independent experiments, the average shift of MscS activation midpoint in the presence of 1 M trehalose was 24 ± 16% for V. cholerae and 12 ± 5% for E. coli. Shifts of MscL-like channels were 24 ± 4% and 11 ± 2% for V. cholerae and E. coli, respectively. It is clear that trehalose modulates channel activity, possibly through tension-driven intercalation into the bilayer (Rudolph et al., 1986; Luzardo et al., 2000; Villarreal et al., 2004) or perhaps through an osmotic or crowding effect, which has been shown to promote MscS inactivation (Grajkowski et al., 2005). We have also observed right-shifts of MscL-like channel activation in the presence of 1 M glycine-betaine, another compatible osmolyte. Glycine-betaine exerted no visible effect on the MscS-like channel of Vibrio.
I-V relationships were analyzed for the two dominant channel activities in the absence and presence of trehalose on the cytoplasmic side of the patch. Fig. 7 shows these changes upon the addition of 1 M trehalose. Data (Fig. 7 and Table 2) were treated using the macroscopic conductance equation (Hille, 2001), which accounts for pore resistance (Rp) and access resistances from each side of the channel (Rs1 and Rs2) (see Materials and methods and Fig. 7 C). Previously, it was shown that the unitary conductances of both MscL and MscS scale linearly with specific conductivity of the recording buffer, suggesting bulk-like conditions for ion transport through wide water-filled pores (Sukharev et al., 1999; Sukharev, 2002). Conductivity measurements indicated that specific resistance of the recording buffer increased three times, from 33 to 98 Ωcm in the presence of 1 M trehalose. We presumed, if trehalose freely partitioned into the channel pore, that we would observe a proportional decrease in pore conductance. Values of conductance determined from the data (Gexp) match closely with those calculated via the macroscopic conductance equation (Gcalc); variation of only Rs1 (the cytoplasmic side of the membrane patch) proportional to changes of bulk-specific resistance (ρ) caused by trehalose sufficiently explains the observed change in unitary currents. This suggests that the resistance of the pore itself (Rp) does not visibly change upon osmolyte addition, and thus, trehalose does not freely partition from the cytoplasm or accumulate in MscS-like or MscL-like pores. Prefilters on the cytoplasmic side of each channel formed by C-terminal domains (Chang et al., 1998; Anishkin et al., 2003; Steinbacher et al., 2007) may impede partitioning, whereas the exit toward the periplasm is likely to be unimpeded. The presented data do not exclude trehalose permeation through MscL or MscS pores; it simply shows that the steady-state pore occupancy with the osmolyte is low, possibly because of a slower (prefilter-limited) entry rate from the cytoplasmic side and a higher rate of exit on the periplasmic side.
V. cholerae readily produces filamentous forms and giant spheroplasts under experimental conditions similar to what was developed for E. coli. This enables direct patch-clamp characterization of the V. cholerae cytoplasmic membrane and the individual components involved in osmotolerance. The first electrophysiological survey of V. cholerae revealed a robust tension-stimulated conductance response mediated by two dominant types of mechanosensitive channel activities, similar to what was observed in E. coli: the MscS-like (0.8–1-nS) and MscL-like (2.6–3-nS) channels. The N16961 V. cholerae genome (Heidelberg et al., 2000) harbors one clear orthologue of E. coli MscL (available at RefSeq under accession no. NP_233001; 136 amino acids [aa], 65% identity) and a likely orthologue of E. coli MscS (available at RefSeq under accession no. NP_230134; 287 aa, 48% overall identity with an 84% conserved TM3-β domain region). A more distant MscS orthologue is coded by RefSeq accession number NP_232581 (291 aa, 21% identity). An additional ORF predicts a longer 569-aa protein with an N-terminal extension (available at RefSeq under accession no. NP_231387; 37% identity of the homologous C-terminal part to E. coli MscS). A putative orthologue of E. coli MscM (YbdG) (Schumann et al., 2010) is also present in the N16961 V. cholerae genome (available at RefSeq under accession no. NP_229921; 412 aa, 54% identity), but there is no obvious orthologue of MscK (KefA) (Li et al., 2002). Because these channel species have not been characterized individually, we presume that MscS-like activities can potentially be generated by a mixed channel population.
Based on the densities of active channels in excised patches (Table 1), the numbers of channels per cell (5–8 µm2 in surface area) can be estimated as 18–30 heptameric MscSs and 20–35 pentameric MscLs in AW405 E. coli. V. cholerae, respectively, should have 6–10 MscS-like and 50–90 functional MscL-like channels per cell. Our estimation of the number of E. coli MscL channels is approximately two to three times lower than previously determined in fluorescent microscopy experiments (Bialecka-Fornal et al., 2012), which estimated 300–400 MscL subunits per cell grown in MLB medium. It is possible that not all synthesized subunits form functional pentamers.
The notable difference in mechanoelectrical responses of the two dominant species is that the density of MscL-like channels is approximately three times higher whereas the density of MscS-like channels is about three times lower in V. cholerae compared with E. coli. Not only is the proportion of MscL to MscS activities different, but the pressure midpoint ratio p0.5MscS/p0.5MscL for the two dominant channel types in Vibrio is lower, at 0.48 ± 0.05 versus 0.60 ± 0.05 for E. coli. If we take the MscL-like activation midpoint as a reference, the Vibrio MscS-like channel activates earlier than its E. coli counterpart. In addition, the Vibrio MscS-like wave shows no hysteresis during a 1-s triangular ramp, signifying a faster closing rate than that in E. coli. MscS-like channels of Vibrio exhibit similar rectifying conductance and tension-dependent inactivating behavior as E. coli MscS. Vibrio MscS-like channels recover from inactivation as fast as E. coli MscS does (Akitake et al., 2007; Kamaraju et al., 2011), with a characteristic time of 1.5 s.
Despite V. cholerae N16691 having a three times larger overall density of mechanosensitive channels, it is less tolerant to strong downshocks (1,000–1,200 mosmol) compared with E. coli AW405. Vibrio alginolyticus, for instance, a strictly marine representative of this genus lacking the mscL gene, is extremely sensitive to osmotic downshifts, but expression of E. coli MscL in this microorganism was shown to dramatically increase its shock tolerance (Nakamaru et al., 1999). High density of MscL channels in V. cholerae might compensate for some other traits that make it osmotically vulnerable. The data above allow us to infer the traits to be considered.
In the event of abrupt osmotic shock, the bacterial osmolyte release system will be able to rescue the cell by releasing excess osmolytes faster than hydrostatic pressure inside the cell reaches the lytic level. In the beginning, osmotic influx of water will stretch the elastic cell wall and the outer membrane. Tension in the inner membrane during osmotic swelling will begin to develop as the membrane unfolds. The kinetics of tension buildup will be defined by the magnitude of the osmotic gradient, elasticity of the cell wall, and the membrane excess area, which requires a certain degree of cell wall distension. Once the threshold tension that activates mechanosensitive channels is reached, the rate of osmolyte release should be high enough to negate the osmotic water influx before critical rupturing tension develops. The observed lower tolerance of V. cholerae to abrupt osmotic downshocks compared with E. coli, despite a higher density of MscL, points to the necessity of better understanding the following three factors.
(1) Distribution of cell sizes in the population.
According to the law of Laplace, tension in the membrane is proportional to the pressure gradient multiplied by the radius of curvature, and thus larger cells must be more vulnerable. It has been well documented that stress conditions lead to the decrease of average size in microbial populations (Barcina, 1995; Chaiyanan et al., 2001). In exponential cultures, V. cholerae cells appear to be slightly larger than E. coli. The lower tail of size distributions in both populations may constitute the most shock-tolerant cells. In addition to outward cell geometry, the excess amount of membrane, and the mechanical strength of the retaining cell wall (peptidoglycan layer), might vary between the two species.
(2) Water permeability and relative densities of MscS and MscL.
Under hydrostatic pressure of water flooding the cytoplasm through the membrane and aquaporins, MscS is predicted to open first and dissipate osmotic gradients. If MscS’s osmolyte permeability is insufficient, building up tension will open MscL, a true emergency valve that activates near the lytic tension. Earlier activation of MscS would mediate a more gradual permeability response with tension, which might be beneficial. The density of MscS-like channels in V. cholerae under the specific conditions of this study is lower than in E. coli; thus, the first line of defense is not as strong. In E. coli, expression of both channels is under the control of stress-activated factor RpoS, and overexpression of one partially represses the other. The physiological role of densities of aquaporins, MscS, and MscL channels in both species needs to be better understood. It was proposed that a high density of MscL may increase effective elasticity of the membrane and thus dampen tension through protein expansion (Boucher et al., 2009). In addition, the clustering and distribution of channels between the cylindrical part of the rod-shaped cell and the poles (Romantsov et al., 2010), where curvatures and tensions are different, may influence the hydrostatic pressure at which threshold tension is reached.
(3) The nature of internal osmolytes and channel permeability.
The relatively large size of permeation pores in MscS and MscL may ensure that the release kinetics are comparable with the millisecond kinetics of osmotic swelling (Boer et al., 2011). For this reason, efficiency of compatible osmolyte release might be an important factor defining the speed of cellular escape from osmotic rupture. However, permeabilities of MscS or MscL for organic osmolytes have not been studied in detail. Also, the composition of preferred compatible osmolytes used by each of the bacterial species might be different (Schleyer et al., 1993; Pflughoeft et al., 2003). In this investigation, we were able to observe modulating effects of the common compatible osmolyte trehalose on the behavior of both MscS-like and MscL-like channels. Right-shifts of activation curves were accompanied with substantial inactivation of MscS-like channels. Our single-channel recordings suggest that trehalose present in the cytoplasmic compartment does not freely partition into the pore, yet these conductance measurements do not exclude permeation.
In conclusion, the establishment of V. cholerae as a system for electrophysiological analysis opens new perspectives for studies of a mechanically activated osmolyte release system and electrogenic transport in this pathogen. The first patch-clamp survey has shown that V. cholerae has lower density of MscS-like channels but higher density of MscL-like channels compared with E. coli. Both V. cholerae channels have gating and conductive properties comparable to their E. coli counterparts. This is the first report on WT V. cholerae, which shows that compatible osmolytes, especially trehalose, strongly modulate the mechanoelectrical response, shifting activation curves to the right. Trehalose does not seem to partition freely into the open MscL channel, bringing into question the actual exit path of this abundant osmolyte. The comparison of E. coli and V. cholerae’s sensitivities to osmotic downshock strongly suggests that there must be other factors that contribute to the environmental fitness of this bacterium other than just the density of mechanosensitive channels in its membrane.
The authors thank Claire Costenoble-Caherty for expert technical assistance with bacterial cultures, BLAST searches, and alignments.
This work was partially supported by the National Institutes of Health (NIH; grant RO1 A1039129-11A2) and the National Science Foundation (grant 0813066 to A. Huq and NIH grant R21 AI105655-01 to S. Sukharev and A. Huq).
Edward N. Pugh Jr. served as editor.
- Abbreviations used in this paper:
- Luria-Bertani medium
- mechanosensitive channel of large conductance
- mechanosensitive channel of small conductance
- Submitted: 4 March 2013
- Accepted: 5 June 2013
This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).