These systems have different induction patterns and substrate spe

These systems have different induction patterns and substrate specificities. A Selleckchem TPCA-1 driving force for both systems is transmembrane electrochemical potential, and proton is involved in acetate transport. A structural comparison of the competing solutes suggests that the size of the molecule is a determinant

factor for recognition. Future work on identification and characterization of the transporter protein is required to understand the systems comprehensively. Methods Bacterial strains and culture conditions Burkholderia species MBA4 and mutant Ins-4p-p2 were grown at 30°C in Luria Bertani medium without NaCl (LB–, 1% tryptone, 0.5% yeast extract) or in defined minimal medium [1] with 0.5 g carbon liter-1 of pyruvate, acetate, MCA, MBA, propionate, 2MCPA,

butyrate, or valerate. Transport assays MBA4 was cultured in minimal medium with pyruvate, acetate, MCA, MBA, propionate, 2MCPA, butyrate, or valerate to late logarithmic phase, with an optical density value (OD600) of 1.0-1.2, 0.9-1.1, 0.5-0.7, 0.7-0.9, 0.9-1.1, 0.1-0.2, 0.9-1.1 or 0.9-1.1, respectively. Cells were harvested by centrifugation, washed twice with phosphate buffered saline (PBS, Fluka), and adjusted to an OD600 of around 0.4. For standard transport assays, 30 μl of [2-14C]MCA (Sigma-Aldrich, diluted to 0.25 mM in PBS) or [2-14C]acetate (Sigma-Aldrich, diluted to 0.25 Selleckchem KU55933 mM in PBS) were added to 120 μl of prepared cells, mixed, and 30 μl samples were taken at various time points. Filtration and washing of cells, determinations of total protein and trapped [2-14C]MCA Fluorouracil research buy or [2-14C]acetate were carried out as previously described [12]. To determine the substrate specificity, diluted [2-14C]MCA or [2-14C]acetate was mixed with 10× competing solutes in PBS before adding to the prepared cells. Percent relative uptake was calculated as (Uptake rate with competing solute/Uptake rate without competing solute) × 100%. The competing solutes included: ethanol; one-carbon monocarboxylate formate; two-carbon glycolate, acetate, MCA and MBA; three-carbon propionate,

lactate, pyruvate and 2MCPA; four-carbon butyrate, five-carbon valerate; and four-carbon dicarboxylate succinate. The skeletal formulas and space-filling models of acetic acid, MCA, MBA, propionic acid, 2MCPA, butyric acid, and valeric acid were drawn with ACD/ChemSketch (Advanced Chemistry Development, Inc.). To study the effect of protonophore on uptake assay, {Selleck Anti-infection Compound Library|Selleck Antiinfection Compound Library|Selleck Anti-infection Compound Library|Selleck Antiinfection Compound Library|Selleckchem Anti-infection Compound Library|Selleckchem Antiinfection Compound Library|Selleckchem Anti-infection Compound Library|Selleckchem Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|buy Anti-infection Compound Library|Anti-infection Compound Library ic50|Anti-infection Compound Library price|Anti-infection Compound Library cost|Anti-infection Compound Library solubility dmso|Anti-infection Compound Library purchase|Anti-infection Compound Library manufacturer|Anti-infection Compound Library research buy|Anti-infection Compound Library order|Anti-infection Compound Library mouse|Anti-infection Compound Library chemical structure|Anti-infection Compound Library mw|Anti-infection Compound Library molecular weight|Anti-infection Compound Library datasheet|Anti-infection Compound Library supplier|Anti-infection Compound Library in vitro|Anti-infection Compound Library cell line|Anti-infection Compound Library concentration|Anti-infection Compound Library nmr|Anti-infection Compound Library in vivo|Anti-infection Compound Library clinical trial|Anti-infection Compound Library cell assay|Anti-infection Compound Library screening|Anti-infection Compound Library high throughput|buy Antiinfection Compound Library|Antiinfection Compound Library ic50|Antiinfection Compound Library price|Antiinfection Compound Library cost|Antiinfection Compound Library solubility dmso|Antiinfection Compound Library purchase|Antiinfection Compound Library manufacturer|Antiinfection Compound Library research buy|Antiinfection Compound Library order|Antiinfection Compound Library chemical structure|Antiinfection Compound Library datasheet|Antiinfection Compound Library supplier|Antiinfection Compound Library in vitro|Antiinfection Compound Library cell line|Antiinfection Compound Library concentration|Antiinfection Compound Library clinical trial|Antiinfection Compound Library cell assay|Antiinfection Compound Library screening|Antiinfection Compound Library high throughput|Anti-infection Compound high throughput screening| appropriate amounts of carbonyl cyanide m-chlorophenyl hydrazone (CCCP) were mixed with prepared cells to a final concentration of 0, 5, 10, 25, and 50 μM for 30 min before transport assays were conducted. To determine the effect of pH on transport systems, 100 mM potassium phosphate buffers of different pH values (4 to 8) were used to resuspend the bacterial cells and for diluting [2-14C]MCA and [2-14C]acetate for uptake assays.

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