ChIP analysis.

In order to determine whether BrlR binds to the promoter region of PA1874-77 (P_PA1874) in vivo, 3-day-old biofilms of P. aeruginosa PAO1/pMJT-brlR-V5/His₆ bearing V5/His₆-tagged BrlR were subjected to chromatin immunoprecipitation (ChIP) analysis as previously described (24, 27). P. aeruginosa PAO1 expressing untagged brlR was used as a control. Briefly, in vivo DNA-protein cross-linking, performed using 1% formaldehyde for 10 min at 37°C, and immunoprecipitation, performed using anti-V5 antibodies (Invitrogen Corp.), were done essentially as previously described (66,–68). Following immunoprecipitation, DNA was liberated by reversing the cross-linking via incubation with 0.5 M NaCl in TE (Tris-EDTA) at 65°C for 4 h. Purified DNA from PAO1/pJN-brlR and PAO1/pMJT-brlR-V5/His₆ samples was subjected to qPCR using the primers listed in Table 3. The promoter region of pscEF was used as a control in qPCRs using primers pscEF_for and pscEF_rev (Table 3). Relative transcript quantitation was accomplished using ep realplex software (Eppendorf AG) first by normalization of the abundance of the transcripts (on the basis of the C_T value) to that of the mreB transcript, followed by determination of transcript abundance ratios. Melting curve analyses were employed to verify the specific amplification of a single product.