P aminobenzoic acid

Isopentenyl pyrophosphate and folate + p-aminobenzoic acid rescue experiments were carried in 96-well plates over 96 h essentially as previously described for in vitro growth inhibition assays. However, for each comparison, two identical 96 h assays were performed simultaneously. For isopentenyl pyrophosphate rescue experiments one plate was supplemented with 200 µM isopentenyl pyrophosphate^{45 (link)} whereas the other was not. Clindamycin and cycloguanil were included as positive and negative controls respectively. For folate + p-aminobenzoic acid rescue experiments all assays were performed in custom RPMI 1640 containing no folate or p-aminobenzoic acid (Life Technologies; USA), one plate was then supplemented with folate 1 mg/mL and 1 mg/mL p-aminobenzoic acid, while the remaining plate was left without supplementation. Cycloguanil and chloroquine served as positive and negative controls respectively in these assays. In each case two independent assays were performed, each in triplicate wells.

Glucose and NaOH were purchased from VWR (West Chester, PA). Ca(OH)₂, p-aminobenzoic acid and CH₃COONH₄ were purchased from Alfa Aesar (Heysham, England). Thiamine was purchased from Alfa Aesar (Ward Hill, MA). Dowex 1X4 resin (chloride form) and biotin were purchased from Sigma-Aldrich (St. Louis, MO). H₂SO₄ (98%) and NaCl were purchased from VWR (West Radnor, PA). K₂HPO₄, KH₂PO₄, MgSO₄·7H₂O, MnSO₄·H₂O, FeSO₄·7H₂O were purchased from Fisher Scientific (Fair Lawn, NJ). Citric acid was purchased from Mallinckrodt Chemicals (Phillipsburg, NJ). CaCO₃ was purchased from EMD Chemicals (Gibbstown, NJ). Reinforced Clostridial Broth medium (RCM) was purchased from HIMEDIA laboratories (Mumbai, India). Cellic CTec 2 was obtained from Novozymes North America, Inc (Franklinton, UC). DI water was produced by the Barnstead Nanopure UV Ultrapure Water System (Thermo Fisher Scientific, Marietta, OH).
Clostridium acetobutylicum ATCC 824 was used for butanol production. It was routinely stored as spores at 4 °C and treated by heat shock at 75 °C for 10 min followed by cooling down in an ice bath prior to cultivation. The RCM medium was sparged with nitrogen and then autoclaved at 121 °C for 15 min. The heat-shocked cells were grown until the optical density (OD) reached 1.30 determined by an UV–vis spectrometer (Thermo Scientific, Madison, WI) at 600 nm.