Membrane-to-cytosol redistribution of ECF sigma factor AlgU and conversionto mucoidy in Pseudomonas aeruginosa isolates from cystic fibrosis patients

The conversion to mucoid phenotype in Pseudomonas aeruginosa during chronic infections in cystic fibrosis (CF) is due to mutations in the algU mucABCD gene cluster. This cluster encodes an extreme stress response system conse rved in Gram-negative bacteria. The system includes an ECF sigma factor, Al gU (sigma(E)), an inner membrane protein, MucA, which inhibits AlgU activit y, and MucB, a periplasmic protein that negatively controls AlgU. In this w ork, we investigated whether and how these factor interact to transduce sig nals between different cellular compartments. The mutation mucA Delta G440, which renders a large fraction of P. aeruginosa CF isolates mucoid, did no t abrogate AlgU-MucA interactions, although it eliminated MucA-MucB interac tions in the yeast two-hybrid system. The mucA Delta G440 truncation of the periplasmic C-terminal tail of MucA destabilized the molecule resulting in low or undetectable steady-state levels in P. aeruginosa. Somewhat reduced levels of MucA were also seen in cells with inactivated mucB or with the m ucACF53 allele carrying the missense P184S mutation, which mildly affected interactions with MucB. The events downstream from MucA destabilization wer e also investigated. AlgU was found to associate with inner membranes in mu cA(+) cells. In mutants destabilizing MucA, a limited redistribution of Alg U from the membrane to the cytosol was observed. The redistribution was spo ntaneous in mucA Delta G440 cells, while in mucB and mucACF53 mutants it re quired additional signals. Despite a large reduction in MucA levels in mucA Delta G440 cells, only a small fraction of AlgU was redistributed to the c ytosol and a significant portion of this sigma factor remained membrane bou nd and behaved as a peripheral inner membrane protein. The fraction of AlgU that depended on MucA for association with the membrane also brought RNA p olymerase into this compartment. These results are consistent with a model in which MucB-MucA-AlgU-RNA polymerase interactions at the membrane allow t ransduction of potentially lethal stress signals with both rapid reaction t imes of the preassembled complexes and efficient resupply at the membrane f rom the prebound components.