Regulated proteolysis controls mucoid conversion in Pseudomonas aeruginosa

Abstract

Overproduction of the exopolysaccharide alginate causes mucoid conversion in Pseudomonas aeruginosa and is a poor prognosticator in cystic fibrosis. The ECF factor AlgU and its cognate antifactor MucA are two principal regulators of alginate production. Here, we report the identification of three positive regulators of alginate biosynthesis: PA4033 (designated mucE), PA3649 (designated mucP), and algW. MucE, a small protein (9.5 kDa), was identified as part of a global mariner transposon screen for new regulators of alginate production. A transposon located in its promoter caused the overexpression of MucE and mucoid conversion in P. aeruginosa strains PAO1 and PA14. Accumulation of MucE in the envelope resulted in increased AlgU activity and reduced MucA levels. Three critical amino acid residues at the C terminus of MucE (WVF) were required for mucoid conversion via two predicted proteases AlgW (DegS) and MucP (RseP/YaeL). Moreover, as in Escherichia coli, the PDZ domain of AlgW was required for signal transduction. These results suggest that AlgU is regulated similarly to E. coli E except that the amino acid triad signals from MucE and other envelope proteins that activate AlgW are slightly different from those activating DegS.alginate ͉ anti-factor ͉ PDZ domain ͉ protease cascade ͉ signal specificity

Escherichia coli transcription factor YncC (McbR) regulates colanic acid and biofilm formation by repressing expression of periplasmic protein YbiM (McbA)

“…As excessive production of bacterial EPS is a common cause of mucoidy in E. coli and other Gram-negative bacteria (Zinkewich-Peotti and Fraser, 1988;Qiu et al, 2007), we collected EPS from mucoid MG1655 yncC culture and evaluated bacterial EPS production using an anthrone-H 2 SO 4 assay. The normalized EPS from the mucoid MG1655 yncC culture was 10±4-fold higher than that from the non-mucoid wild-type MG1655 culture.…”

Truncated type IV pilin PilA108 activates the intramembrane protease AlgW to cleave MucA and PilA108 itself in vitro

“…AlgU is 66 % identical to the ECF σ factor RpoE (Yu et al 1995) which is regulated via the regulated intramembrane proteolysis of the cognate anti-σ factor RseA in response to envelope stress signals in E. coli (Alba et al 2001(Alba et al , 2002Helmann 2002;Kanehara et al 2003;Walsh et al 2003). This mechanism of regulation is consistent in P. aeruginosa (Qiu et al 2007(Qiu et al , 2008Cezairliyan and Sauer 2009;Wood and Ohman 2009). The MucA (RseA) proteolytic cascade comprises AlgW (DegS), MucP (RseP) and ClpXP (Cezairliyan and Sauer 2009;Damron and Goldberg 2012).…”

“…MucB is a negative regulator of mucoid conversion as it binds MucA and inhibits its cleavage by AlgW (Cezairliyan and Sauer 2009). The activated protease AlgW cleaves the carboxyl domain of MucA on the periplasmic face (Cezairliyan and Sauer 2009), followed by a second cleavage by the inner membrane-associated zinc metalloprotease MucP (Qiu et al 2007;Wood and Ohman 2009;Damron and Goldberg 2012). Cytoplasmic proteases ClpXP then degrade the cytoplasmic portion of MucA to release AlgU which is sequestered to the cytoplasmic membrane via the N-terminal of its cognate antisigma factor MucA, causing mucoid conversion (Xie et al 1996;Hughes and Mathee 1998;Qiu et al 2008).…”

“…In Escherichia coli, some environmental stresses cause accumulation of misfolded outer membrane proteins (OMPs) in the periplasm (Qiu et al 2007). AlgW orthologue DegS is activated by binding of its PDZ domain to the C-terminal YQF sequence present on many OMPs such as OmpC (Walsh et al 2003).…”

Identification of Novel Genes Associated with Alginate Production in <em>Pseudomonas aeruginosa</em> Using Mini-<em>himar1</em> Mariner Transposon-mediated Mutagenesis

“…AlgU/T is a positive regulator, while mucAB-D are classified as negative regulators of alginate production [6][7][8] . Additionally, transcriptional regulators, such as AlgB, AlgQ, AlgR, and RpoN, as well as post-transcriptional and post-translational modification by catabolite repression control, kinase activity (KinB) and intramembrane proteolysis, have also been shown to be involved in alginate regulation [9][10][11][12][13][14] .…”