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. 2009 Sep 4;5(9):e1000572. doi: 10.1371/journal.ppat.1000572

Figure 5. Analysis of VreI (PA0675) stability and activity.

Figure 5

(A) SDS-PAGE of P. aeruginosa wild-type cells (WT) and the vreR sigma factor regulator mutant bearing the pMUM3RσHA-tag plasmid coding for the VreI-HA-tagged protein. Total proteins (upper panel), and cytosol and membrane fractions (lower panel) were separated in 15% (w/v) acrylamide gel. Log phase cells were incubated 45–60 min with (+ in upper panel, and all samples in lower panel) or without (− in upper panel) 1 mM IPTG. (B) β-galactosidase activity of P. aeruginosa wild-type or PA0676 mutant cells containing the pMP0691bKm plasmid (PA0691::lacZ transcriptional fusion) and the pMMB67EH (empty), the pMUM3 (overexpressing the vreI ECF sigma factor) or the pMMB-PUMA3 (overexpressing the whole PUMA3 CSS system) plasmid. Cells were grown overnight in LB liquid medium in the presence of 1 mM IPTG. The β-galactosidase activity is expressed in Miller Units.