, Danbury CT) until microscopic examination confirmed that all the cells were completely disrupted. The samples were cleared by centrifugation at 12000 × g for 30 min at 4°C, and the K+ ion concentration of the supernatants was measured by potassium electrode  at SRL Co.
(Tokyo Japan). RNA preparation and detection Two ml of whole cell culture were quickly mixed with 150 μl of 5% (v/v) water-saturated phenol in ethanol to prevent RNA degradation . virF and invE mRNAs were purified and analysed using a Titan™ one tube RT-PCR kit (Roche, Indianapolis IN) and Perfect Real-time™ (Takara Bio Co., Shiga Japan), as described previously . For the detection of virF mRNA by real-time PCR, virFc-314F (5′-GGAGACGTTTATTTGTATATTTCGCTCTA-3′, 120 nM) and virFc-398R (5′-GACGGTTAGCTCAGGCAATGAT-3′, 120 nM) NVP-LDE225 in vitro primers and the fluorescent probe virFc-345T (5′-FAM-AAAGCAATTTGCCCTTCATCGAT-TAMRA-3′, 32 nM) were designed by ABI primer design software (Applied Biosystems Inc., Foster CA) and synthesized see more by ABI Japan (Tokyo). Real-time PCR analysis
was performed using an ABI PRISM 2000 Thermal Cycler, as described previously . RNA preparation and real-time PCR analysis were repeated at least 3 times with similar results. Gel-shift assay The labelled RNA probe (20 fmoles), corresponding to 140 nucleotides of the invE gene (starting from the transcription start site at +1) , and purified Hfq protein (0, 1, 2, 4, 8, or 16 nM Hfq hexamer) were mixed in a volume of 10 μl in one of two RNA binding buffers (40 mM NH4Cl, 10 mM Tris-HCl pH7.5, 5 mM magnesium acetate, 0.1 mM dithiothreitol; or 100 mM NH4Cl, 10 mM Tris-HCl pH7.5, 5 mM magnesium acetate, 0.1 mM dithiothreitol) at 37°C for 10 min. Gel-shift analysis was performed at 37°C as described previously . Surface Plasmon Resonance (Biacore Analysis) Surface plasmon resonance was performed with Biacore 2000 optical sensor device using the same 140 nucleotide invE RNA
probe for the gel-shift assay ZD1839 as described previously . The probe was immobilized onto a sensor tip SA (GE Healthcare Co., Piscataway NJ), causing a change of nearly 150 resonance units. Purified Hfq protein was diluted to a final concentration of 0, 1, 2, 4 or 8 nM (Hfq hexamer) in one of two RNA binding buffers, as described for gel-shift assays, and then injected for 180 seconds through two flow cells (flow cell 1, blank; flow cell 2, invE RNA) at a flow rate of 20 ml/min at 37°C. Non-specific proteins were dissociated from the chip by washing (for 700 seconds). Bound Hfq protein was subsequently removed with 2 M NaCl. The response value of the reference cell (flow cell 1, blank) was subtracted from the response value of flow cell 2 (invE RNA) to correct for nonspecific binding, and the results are expressed as difference units (D.U.).