Storm 820 phosphorimager

Inhibition of lipid II synthesis was performed in vitro using membrane preparations of Micrococcus luteus DSM 1790 as described by Schneider et al. (19 (link)) with the addition of radiolabeled [¹⁴C]UDP-GlcNAc. Membranes were isolated from lysozyme-treated cells by centrifugation (40,000 × g for 60 min at 4°C), washed twice in 50 mM Tris-HCl and 10 mM MgCl₂ (pH 7.5), and stored under liquid nitrogen until use. Reaction mixtures were carried out in a final volume of 75 μl and contained 400 μg of membrane protein, 5 nmol undecaprenyl phosphate (C55-P), 50 nmol UDP-N-acetylmuramyl pentapeptide (UDP-MurNAc-PP), 50 nmol [¹⁴C]UDP-GlcNAc in 60 mM Tris-HCl (pH 8), 5 mM MgCl₂, and 0.5% (wt/vol) Triton X-100. UDP-MurNAc-PP was purified as described previously (22 (link)). C6H was added to the reaction mixture in molar ratios of 2:1 (referring to the total amount of C55-P [5 nmol]). After 1 h at 30°C, the lipids were extracted with 1 volume of n-butanol-6 M pyridine-acetate (2:1, vol/vol) (pH 4.2). The reaction products were separated by TLC (silica plates [60F254]; Merck) using chloroform-methanol-water-ammonia (88:48:10:1) as the solvent (20 (link)). Radiolabeled spots were visualized using a biomolecular imager for radioisotope detection (Storm 820 PhosphorImager; Amersham Biosciences) and the image was analyzed with ImageQuant TL v 2005 (Nonlinear Dynamics, Ltd.) software.

The lipid composition of the different E. coli strains was determined by triplicate following labeling with [1-^14C]acetate (Perkin Elmer). LB cultures (1.5 mL) were inoculated to an initial optical density at 600 nm (OD₆₀₀) of 0.1 from precultures grown in the same medium. After the addition of 1 μCi/mL [1-^14C]acetate to each culture, they were incubated for 24, 48 or 72 h. The cells were harvested by centrifugation and resuspended in 100 μL of water. The lipids were extracted according to the method of Bligh and Dyer [51 (link)]. The chloroform phase was used for lipid analysis by one-dimensional thin-layer chromatography (TLC) using high-performance TLC silica gel 60 plates (Merck) and ethyl acetate-hexane-acetic acid (60:40:5 [vol/vol/vol]) as the mobile phase. Two-dimensional TLC was performed as described previously [52 (link)]. Radioactivity was detected using a Storm 820 PhosphorImager (Amersham Biosciences). Image analysis and signal quantification were carried out using ImageQuant TL (Amersham Biosciences).

Recombinant 6XHisStABF1 (1 μg), previously phosphorylated or not, was incubated with 6xHisStCDPK3 (125 ng) or with anti-His antibody. ABRE oligonucleotides sense and antisense (aattccGGACACGTGGCGtaagct) were used as probes. 100 pmol of oligonucleotides (probes) were annealed by heating at 100°C during 5 min, followed by slow cooling to facilitate interaction. The ABRE probe was labeled using dCTP[α³²P] and T4 polynucleotide kinase (New England Biolabs, Ipswich, MA). For interaction assays, 6xHisStABF1 was incubated during 15 min at 4°C with 1.5 μg of poly (dl-dC), in 30 μl buffer containing: 10 mM Tris-HCl, pH 7.5, 2 mM MgCl₂, 100 mM NaCl, 1 mM EDTA, 4% (v/v) glycerol, and 1 mM DTT. Then, 1.5 ng of ABRE probe was added to the reaction and incubated during 15 min at room temperature. Reactions were solved in 6% acrylamide gels in buffer TBE 0.5x (45 mM Tris Base, 45 mM boric acid, 1 mM EDTA). Gels were exposed during 1 h to a cassette with an amplifying signal screen, and signal was recorded using a Storm 820 PhosphorImager (Amersham Pharmacia Biotech)