Ampicillin

pRSET-A with Green Lindoblum was transformed into E. coli BL21 (DE3) (Merck Millipore, Burlington, MA, USA) cells and cultured in 3 mL LB medium with 50 μg/mL ampicillin (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) at 20 °C for 2 to 3 days. pRSET-A with Green Pegassos was transformed into E. coli JM109 (DE3) (Promega, Madison, WI, USA) cells and cultured in 200 mL LB medium with 50 μg/mL ampicillin (FUJIFILM Wako Pure Chemical Corporation) at 20 °C for 3 days. The cells were harvested by centrifugation at 16,000 g for 5 min at 4 °C. The pellets were suspended in phosphate-buffered saline (PBS; pH 7.4) with 40 μg/mL lysozyme (FUJIFILM Wako Pure Chemical Corporation) and 0.5% Triton X-100 (Sigma-Aldrich, for Green Lindoblum only), and lysed by ultrasonic homogenisation. After centrifugation at 16,000 g for 10 min at 4 °C of the lysate, the supernatant containing the over-expressed Green Lindoblum/Pegassos protein was added to PBS. Emission spectra of 470 nm in the absence or presence of 10 mM lactate or 1 mM pyruvate were measured using a spectro-fluorophotometer (F-2500, Hitachi, Tokyo, Japan). The dynamic range (F/F₀) was normalised to the peak in the absence of lactate or pyruvate, and the variant showing the highest F/F₀ was selected.

Portions (20 μL) of E. coli strain JM109 (1 × 10¹⁰ colony-forming units (CFU)/mL) were cultured for various times in 2 mL of 2xYT liquid medium (BD Difco, Cat# 244020) containing various concentrations of pyridoxal 5′-phosphate (pH adjusted to 7.0), after which OD₆₀₀ was measured with a GENESYS 30 visible spectrophotometer (Thermo Fisher Scientific, Cat# 840–277000). In addition, the JM109 strain was transformed with 1 μg of the pBlueScript II SK + plasmid (Invitrogen), which harbors an ampicillin resistance gene as a selection marker, and was then spread on LB agar plates containing ampicillin (100 μg/mL) (Wako, Cat# 012–23303) with or without pyridoxal 5′-phosphate (3 mg/mL) and incubated overnight.

The Gram-positive bacteria methicillin-susceptible S. aureus (MSSA; strains 209P and ATCC29213), methicillin-resistant S. aureus (MRSA; MS29202 and ATCC43300), vancomycin-susceptible E. faecalis (VSE; ATCC29212), vancomycin-resistant E. faecalis (VRE: VanB; ATCC51299), vancomycin-resistant E. faecium (VRE: VanA; ATCC700221), and B. subtilis (ATCC6633) and the Gram-negative bacteria Escherichia coli (NIHJ) and Mycobacterium bovis (PG45; ATCC25523) were used in this study. DIF derivatives (Figure 1) were synthesized as previously described [16 (link),22 (link)] and stored at −20 °C as 10 mM solutions in DMSO or ethanol (EtOH). AB0022A, Pf-1, and Pf-2 were synthesized as described [30 (link),31 (link)] and stored at −20 °C as 10 mM solutions in DMSO. A combined solution of penicillin (5000 units/mL) and streptomycin (5 mg/mL) was purchased from Gibco (ThermoFisher Scientific, Waltham, MA, USA); vancomycin and oxacillin were obtained from Sigma-Aldrich (St. Louis, MO, USA); and cefoxitin and ampicillin were bought from Wako Pure Chemical Industries (Osaka, Japan). The hydrophobic index (ClogP) of each DIF derivative was calculated by using ChemDraw 16.0 software (PerkinElmer, Inc. Waltham, MA, USA).