L arabinose

The pBAD24-mazE-at, mazF-at, –mazEF-at, and -mazF-at mutants (MazF-E24N, -E24Q, -E24K, -E24D, and -E24A) were transformed into E. coli BW25113. To conduct the cell toxicity assay, transformed cells were grown on M9 agar plates with or without 0.2% L-arabinose (Merck, St. Louis, MO, USA) at 37 °C for 20 h. To construct the growth curve, transformed cells were grown in an M9 liquid medium with 0.5% glycerol at 37 °C in the presence or absence of 0.2% L-arabinose. The cell cultures were sampled at 0, 30, 60, 90, 120, 150, and 240 min after the induction.

Bacterial strains and plasmids used in this study are listed in Supplementary Table S1. P. aeruginosa strains (PA14 and PAO1) were cultivated in lysogeny broth (LB) at 37°C with shaking at 180 rpm. Alternatively, strains were cultivated under conditions, which either favored or inhibited phoB or tctD expression. Therefore strains were grown in DeMoss medium (10 g/l DL- Alanin; 20 ml/l Glycerol; 20 mM MgCl2; 0.1 M Na2SO4, 50 μM Fe(III)Citrat, pH 7.5) supplemented with low phosphate (0.8 mM K2HPO4) and high phosphate (4 mM K2HPO4), respectively, or M9 minimal medium with 10 mM citrate or 10 mM glucose. E. coli DH5α was routinely used for subcloning and propagation, E. coli S17-1 and WM3064 for bacterial conjugations. For plasmid selection and maintenance, antibiotics were added at the following final concentrations (μg/ml): for E. coli, ampicillin 100; gentamicin 15; for P. aeruginosa, carbenicillin 400; gentamicin 30. For gene expression L-arabinose (Sigma) was added to the culture medium in the induction range between 0.1% and 0.2% of L-arabinose.

Xyl crystals were obtained at 283 K using the hanging-drop vapor-diffusion method under conditions similar to those previously described [23 (link)]. In a typical experiment, 1 μl of Xyl solution was mixed with an equal volume of reservoir solution containing 0.1 M HEPES (N-(2-hydroxyethyl)piperazine-N’-(2-ethanesulfonic acid)) buffer, pH 7.0, and 5% (w/v) PEG 6000. Crystal formation was accelerated by streak seeding using available Xyl crystals as seeds. In these conditions, tetragonal Xyl crystals with dimensions of about 100 x 100 x 100 μm were formed overnight.
Prior to data collection, a Xyl crystal was immersed for 5 s in reservoir solution containing 20% (v/v) glycerol before flash-cooling in liquid nitrogen. Xyl crystals with bound L-arabinose (Xyl•arabinose), D-xylose (Xyl•xylose), or L-arabinose and D-xylose (Xyl•arabinose•xylose) were prepared by soaking Xyl crystals for 10 min in reservoir solution supplemented with 500 mM L-arabinose (Sigma-Aldrich), D-xylose (Sigma-Aldrich), or both L-arabinose and D-xylose, respectively. These monosaccharide-bound crystals were cryoprotected in a similar way as for the native crystal with solutions consisting of reservoir solution containing 35% (v/v) PEG 300 and the monosaccharide supplements. All cryoprotection and ligand soaking procedures were performed at 283 K.