Saccharomyces cerevisiae SRE-sensitive strains KZ1-1C, 8A-1B, W303C, and BY4741 and SRE-resistant and lipid biosynthesis mutant strains Δerg3, Δsyr2, Δipt1, Δelo2, Δelo3, and Δfah1 were described previously (Taguchi et al., 1994; Hama et al., 2000; Stock et al., 2000). SRE-resistant strains Δerg3 (formerly Δsyr1, Taguchi et al., 1994) and Δfah1 are single-gene disruptants and isogenic to parental strains 8A-1B and BY4741, respectively. All other SRE-resistant mutants are single-gene disruptants and isogenic to parental strain W303C. C. albicans ATCC10231 was obtained from the American Type Culture Collection (Manassas, VA, USA). All yeast strains were grown at 28°C and maintained (at 5°C) with yeast extract–peptone dextrose (YPD) broth or agar medium (Hama et al., 2000).

SRE (M_r 1224) was purified from P. syringae pv. syringae strains B301D or M1 by previously described methods of Bidwai et al. (1987), (Adetuyi et al. 1995). SP22A (M_r 2142) and SP25A (M_r 2397) were purified from extracts of strains P. syringae pv. syringae B301D and M1 respectively, using methods described earlier (Bensaci and Takemoto, 2007).

For measuring growth inhibition in liquid batch cultures, S. cerevisiae strain KZ1-1C was first grown in YPD broth medium at 28°C in 125 mL capacity Erlenmeyer flasks with rotary shaking for 48 h to a density of ~8 × 10⁸ cells mL⁻¹. The cells were centrifuged and the sedimented cells suspended in YPD broth medium to give 2.4 × 10⁷ cells mL⁻¹. SRE, SP22A, or SP25A were added at designated concentrations (between 0.8 and 9.3 μM). The suspensions were incubated with rotary shaking at 28°C and samples removed hourly for direct cell counts using a light microscope and hemocytometer. Minimal inhibitory concentrations (MICs) were determined by the microbroth dilution assay according to methods of the National Committee of Clinical Laboratory Standards (NCCLS, 2002). Yeast strains were grown to a final concentration of 10⁸ colony-forming units (CFU) mL⁻¹ and suspended at a final concentration of 5 × 10⁵ CFU mL⁻¹. Cell suspensions (25 μL) were added to 25 μL aliquots of twofold serial dilutions of SRE, SP22A, and SP25B. YPD broth media were dispensed (100 μL total volume) in wells of 96-well polystyrene microtiter plates. The plates were incubated for 24 h at 28°C. The MICs were determined by visual inspection of the plate. For determination of cell viability, a suspension of 4 × 10⁵ CFU mL⁻¹ was treated with different concentrations of SRE, SP22A, and SP25A and the cell suspension was twofold serially diluted. Aliquots (100 μL) were spread-plated onto YPD agar, the agar plates incubated for 24–48 h at 28°C, and the numbers of colony-forming units per milliliter of undiluted cell suspension determined. All growth inhibitory experiments were performed in triplicate and the results reported as mean values.

Whole cell K⁺ efflux rates were determined as changes in extracellular K⁺ concentrations. Yeast strain KZ1-1C was grown in YPD broth medium with rotary shaking at 28°C to a density of 10⁸ cells mL⁻¹. Cells were suspended in 2 mM Tris/MES buffer, pH 6.5, and 0.1 M-glucose with or without SRE, SP22A, or SP25A (at concentrations of 1, 10, or 100 μM) to A₆₀₀ nm of 1 in 125 mL capacity Erlenmeyer flasks with rotary shaking (200 rpm) at 28°C (Takemoto et al., 1991). At 5 min after suspension, 1 mL samples were withdrawn, centrifuged in an Eppendorf microcentrifuge for 30 s, and the supernatant fractions were recovered. K⁺ concentrations were determined by atomic absorption spectroscopy (AA/AE spectrophotometer 457, Instrumentation Laboratories).

The net cellular uptake of ⁴⁵Ca²⁺ was measured using strain KZ1-1C as described previously (Takemoto et al., 1991). Cells were grown in YPD broth medium to a density of 1 × 10⁷ cells mL⁻¹, harvested by centrifugation, and then suspended in YPD broth medium to a density of 4 × 10⁷ cells mL⁻¹). Ten milliliter aliquots were dispensed in 125 mL capacity Erlenmeyer flasks with radioactive ⁴⁵CaCl₂ (Amersham Radiochemicals) and incubated with rotary shaking (200 rpm) at 28°C. The specific radioactivity of ⁴⁵Ca²⁺ was adjusted to 4 mCi (148 MBq) per millimole of CaCl₂ (Takemoto et al., 1991). SRE, SP22A, or SP25A were added 5 min following addition of ⁴⁵CaCl₂. At designated times, cell samples (200 μL) were collected on glass fiber filters (0.45 μM pore size), the filters were quickly washed twice with ice-cold water, and the radioactivity on the filters determined using a liquid scintillation counter.

Lipid bilayer electrophysiological experiments were performed using 1,2-dioleoyl-sn-glycero-3-phosphorylcholine (DOPC; Avanti Polar Lipids) as previously described (Blasko et al., 1998; Malev et al., 2002). Solutions of 0.1 M NaCl were buffered with 5 mM MOPS (Sigma) to pH 6. Bilayer lipid membranes were prepared by a monolayer-opposition technique (Montal and Mueller, 1972) on a 50- to 100-μm diameter aperture in the 15-μm thick Teflon film separating two (cis and trans) compartments of a Teflon chamber. SP22A, SP25A, and SRE were added to the aqueous phase of the cis-side compartment. A pair of Ag/AgCl electrodes with agarose/2 M KCl bridges was used to apply transmembrane voltages and to measure single-channel currents. All experiments were performed at room temperature (22 ± 2°C). Current measurements were carried out using an Axopatch 200B amplifier (Axon Instruments) in a voltage clamp mode. The data were filtered with a low-pass 8-pole Model 9002 Bessel filter (Frequency Devices) at 1 kHz and directly recorded into computer memory with a sampling frequency of 5 kHz. Data were analyzed using pClamp 9.2 (Axon Instruments) and Origin 7.0 (Origin Lab). Current transition histograms were generated for each tested voltage. Histogram peaks were fitted with the normal distribution function. Single-channel conductance was calculated as the mean single-channel current divided by the applied transmembrane voltage.

SRE and syringopeptins SP22A and SP25A inhibited the growth of different strains of S. cerevisiae and C. albicans ATCC 10231 (Table 1). In all cases, SP22A was ~2-fold more active than SP25A and both were less inhibitory than SRE. The same relative inhibitory capabilities (i.e., SRE > SP22A > SP25A) were observed when measuring effects on cell numbers in batch growth cultures (Table 2) and on cell viability (Figure 1). In microbroth dilution inhibition assays, growth did not resume at MIC levels of SRE, SP22A, or SP22B after 48 h incubation indicating that these compounds were fungicidal (data not shown).

Previous studies showed that certain S. cerevisiae mutants with defects in sphingolipid or sterol biosynthesis are more resistant to SRE than isogenic wild type strains (Grilley et al., 1998; Hama et al., 2000; Stock et al., 2000). In the present study, mutant strains with genes deleted for dihydrosphingosine C-4 hydroxylase (Δsyr2), mannosyl-diinositolphosphoryl-ceramide synthase (Δipt1), sphingolipid very long chain elongases (Δelo2 and Δelo3), and sterol C5,6 desaturase (Δerg3) were each less susceptible to inhibition by SP22A and SP25A at MICs for their respective isogenic wild type parental strains (Table 1; Figure 1B). Mutant strain Δfah1 defective in sphingolipid fatty acid α-hydroxylase was more susceptible to SP22A than to SRE or SP25A (Figure 1B) but nevertheless more resistant to SP22A concentrations below 2 μM that were inhibitory to isogenic wild type strain BY4741 (Table 1). The inhibitory patterns suggest influences of target membrane lipids on the fungicidal action of SP22A and SP25A similar to those for SRE (Grilley et al., 1998; Hama et al., 2000; Stock et al., 2000; Kaulin et al., 2005). Although needed at higher concentrations with the mutants, the same relative inhibitory capabilities of SRE > SP22A > SP25A were observed regardless of the lipid biosynthesis defect.

As previously observed for SRE (Takemoto et al., 1991), SP22A, and SP25A caused net K⁺ effluxes and Ca²⁺ influxes in whole cells of S. cerevisiae strain KZ1-1C (Figure 2). Higher concentrations of SP22A and SP25A were required to give the effects as compared to SRE. SRE stimulated K⁺ efflux at concentrations starting from 1 μM; while 10 and 100 μM of SP22A and SP25A, respectively, were required to achieve similar degrees of K⁺ efflux. Cellular Ca²⁺ influx was observed within 15 min after 5 μM SRE addition (data not shown). But, 40 and 120 μM of SP22A and SP25A, respectively, were required for detection of Ca²⁺ influx. At these concentrations, Ca²⁺ influx was not evident until about 20 and 30 min after addition of SP22A and SP25A, respectively.

SRE forms well-characterized voltage-dependent ion-conducting channels in lipid bilayers (Feigin et al., 1996; Kaulin et al., 1998; Malev et al., 2002). To examine whether the differences in the relative fungicidal capabilities of SRE, SP22A, and SP25A may lie in their different channel-forming properties, the conductances of planar lipid bilayers doped with the three compounds were compared (Figure 3). In all three cases, application of potentials negative from the side of lipodepsipeptide addition induced increases in macroscopic conductance. After a 5-min equilibration, the conductance reached its stationary value, which within the deviations expected for stochastically operating discrete pores, was stable for at least 10 min (Figure 3A). Application of positive voltages produced membrane conductance decreases (not shown), indicating closing of the pores. SRE produced smaller steady-state conductances at concentrations that were significantly higher (~150-fold) than those produced by the syringopeptins. SP22A induced slightly higher conductances than SP25A.

Single-channel recordings with all three lipodepsipeptides demonstrated a similar pattern (Figure 3B). In all cases, two types of single-channel conductance fluctuations were observed, small and large, differing in the levels of conductance five to sixfold as was described earlier for SRE (Malev et al., 2002; Kaulin et al., 2005). The dwell times of the large channels were longer than those of the small ones. The values of the single-channel conductance for the small channels over the range of ±200 mV were close for all three lipodepsipeptides (Figure 3C). Thus, the properties of the single transmembrane channels, formed by SRE, SP22A, and SP25A were essentially the same.