6 mM Na2HPO4, 10 mM glucose, 3% gelatin, 10 μM CCCP in dimethyl sulfoxide (DMSO), pH 7.2]; and PBS-G2

supplemented with amiloride (APBS-G2; 150 mM NaCl, 3.2 mM NaH2PO4, 13.6 mM Na2HPO4, 10 mM glucose, 3% gelatin, 10 μM amiloride, pH 7.2). All reagents were purchased from Sigma-Aldrich. Motility stocks were cultured in SP-4 motility medium and incubated with the desired pH (5.8, 6.8, 7.8, 8.8) and temperature (30, 37, 40 °C) in glass chamber slides. For motility analysis, 18 images were captured at 1000× magnification on a Leica DM IRB inverted phase-contrast/epifluorescence microscope at approximately 0.25-s intervals. Images were merged and analyzed for 20–25 motile cells as previously described (Hatchel et al., 2006). The GSK3 inhibitor Volasertib price temperature and pH data were

analyzed using two-factor factorial analysis of variance (anova) to examine the effects of both temperature and pH on motility speed. To determine the temperature and pH associated with maximal gliding speed, a statistical response surface model was fit to the data with an accompanying canonical analysis. The effects of energy source inhibitors on motility were analyzed by anova. All statistical analyses were performed using sas version 92 for Windows. The advantage of using a fast-gliding strain for analysis of motility-associated phenomena is that increased gliding speed allows clearer resolution of changes in speed under different conditions. High-passage M. penetrans strain HP88 glided in one direction with an average speed of 1201 ± 326 nm s−1 (n = 103), twice as fast as strain GTU-54-6A1, and > 20 times faster than strain HF-2 (Jurkovic et al., 2012). The gliding speed of this strain, which was used for all experiments, spanned a range of 158–2115 nm s−1, corresponding to 0.2–1.8 times the average gliding speed (Fig. 1). For subsequent experiments, values were normalized to the gliding speed observed at 37 °C and pH 7.8 in the appropriate control

buffers. Arsenate enters prokaryotic and eukaryotic cells via phosphate transporters (Rosen, 2002) and inhibits many reactions involving phosphate. These reactions include substrate-level phosphorylation events leading to ATP synthesis via the glycolysis (Warburg & Christian, 1939) and arginine dihydrolase (Knivett, 1954) pathways, the only two means of ATP synthesis available Sclareol to M. penetrans (Lo et al., 1992; Sasaki et al., 2002), as mycoplasma membrane ATP synthase actually hydrolyses ATP to create a proton gradient (Linker & Wilson, 1985). To confirm toxicity of arsenate to M. penetrans, cells were cultured in the presence of 10 mM sodium arsenate or sodium phosphate, pH 7.2. After 2 days of incubation at 37 °C, growth of M. penetrans was observed with added sodium phosphate, but not arsenate (not shown), confirming that M. penetrans takes up arsenate and its growth is inhibited at relatively low arsenate concentrations.