, 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 [17] 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 [45]. 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 [11]. 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 [11]. 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) [11], 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 [11]. 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 [11]. 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.).