Bacterial rotary export ATPases are allosterically regulated by the nucleotide second messenger cyclic-di-GMP.
Bottom Line: The addition of cdG was shown to inhibit FliI and HrcN ATPase activity in vitro.Finally, a combination of site-specific mutagenesis, mass spectrometry, and in silico analysis was used to predict that cdG binds to FliI in a pocket of highly conserved residues at the interface between two FliI subunits.Our results suggest a novel, fundamental role for cdG in controlling the function of multiple important bacterial export pathways, through direct allosteric control of export ATPase proteins.
Affiliation: From the Molecular Microbiology Department and.Show MeSH
Mentions: Experiments were performed as described by Nesper et al. (45). P. fluorescens cells were grown in M9 0.4% (w/v) pyruvate medium ± 0.4% (w/v) casamino acids to stationary phase and to mid-logarithmic phase, lysogenic broth to stationary phase, and Kings B medium to logarithmic phase. Cells were collected by centrifugation for 5 min at 5,000 × g. The pellet was resuspended in lysis buffer (6.7 mm MES, 6.7 mm HEPES, pH 7.5, 200 mm NaCl, 6.7 mm sodium acetate and 10 mm β-mercaptoethanol) with protease inhibitors and DNase I (Roche). Cells were lysed using a French press (3 × 20,000 p.s.i.), and lysates were centrifuged at 100,000 × g for 1 h. The supernatant was then used to identify soluble cdG binding proteins. 600 μg of the soluble protein mixture was used and was mixed with 20 μl of capture buffer (100 mm HEPES, pH 7.5, 250 mm sodium acetate, 50 mm magnesium acetate, 50% (v/v) glycerol), plus 12.5 μl of 10 mm cdG for the control samples. Volumes were adjusted to 100 μl with water, and the reactions were then incubated for 2 h at 4 °C, before UV irradiation for 4 min using a caproBox (Caprotec Bioanalytics, Berlin, Germany). Magnetic streptavidin beads (50 μl) were added with 25 μl of 5× wash buffer (250 mm Tris, pH 7.5, 5 m NaCl, 0.1% (w/v) n-octyl-β-glucopyranoside), and the samples were incubated for 45 min at 4 °C on a rotary wheel. The beads were collected with a magnet, and the samples were washed six times with 200 μl of 1× wash buffer. The beads were resuspended in 20 μl of sample buffer, incubated for 10 min at 95 °C, and separated for 10 min on a precast 12% (w/v) SDS acrylamide gel at 100 V. Protein bands were then excised and sent for mass spectrometric analysis. The same protocol was followed for the competition experiment (see Fig. 1C). Similarly to the controls, 1 mm of each nucleotide was added to a protein mixture of 10 μm and preincubated for 1 h before the addition of the capture compound (10 μm).