FIG. 4.

PAO1 kinB::aacC1 exhibits elevated levels of AlgB and AlgU, and HA-MucA degradation in PAO1 kinB::aacC1 requires algW, algB, and rpoN. Shown are representative panels of blots from three independent experiments with 40 μg of total lysate. (A) A Western blot of total cell lysate of PAO1 kinB::aacC1 shows elevated levels of AlgB and AlgU. Western blots of cell lysates were prepared from cells after 24 h growth on PIA. The membranes were probed with anti-AlgU, anti-AlgB, and anti-alpha subunit of RNA polymerase (loading control). Levels of each protein were adjusted for loading and then normalized to PAO1 levels and expressed as means ± standard deviations. Note that deletion of algU did not abolish AlgB expression. (B) Western blot analysis of N-terminally HA-tagged MucA in PAO1 and PAO1 kinB::aacC1 isogenic backgrounds. Cell lysates were prepared after 48 h of growth on PIA-carbenicillin plates supplemented with 0.1% arabinose. The membranes were immunoblotted with rat anti-HA diluted 1:1,000 (Roche). Lane 2, PAO1 pHERD20T-mucA is a negative control for background and cross-reactivity. Lanes 1 and 3 to 7, HA-mucA expressed in trans from pHERD20T. Levels of each protein were adjusted for loading and then normalized to PAO1 pHERD20T-HA-mucA levels and expressed as means ± standard deviations. Apparent molecular masses are depicted. NM and M indicate nonmucoid and mucoid phenotypes, respectively.