title: Truncation of type IV pilin induces mucoidy in Pseudomonas aeruginosa strain PAO579.
abstract: Pseudomonas aeruginosa is a Gram negative, opportunistic pathogen that uses the overproduction of alginate, a surface polysaccharide, to form biofilms in vivo. Overproduction of alginate, also known as mucoidy, affords the bacterium protection from the host's defenses and facilitates the establishment of chronic lung infections in individuals with cystic fibrosis. Expression of the alginate biosynthetic operon is primarily controlled by the alternative sigma factor AlgU (AlgT/σ(22) ). In a nonmucoid strain, AlgU is sequestered by the transmembrane antisigma factor MucA to the cytoplasmic membrane. AlgU can be released from MucA via regulated intramembrane proteolysis by proteases AlgW and MucP causing the conversion to mucoidy. Pseudomonas aeruginosa strain PAO579, a derivative of the nonmucoid strain PAO1, is mucoid due to an unidentified mutation (muc-23). Using whole genome sequencing, we identified 16 nonsynonymous and 15 synonymous single nucleotide polymorphisms (SNP). We then identified three tandem single point mutations in the pilA gene (PA4525), as the cause of mucoidy in PAO579. These tandem mutations generate a premature stop codon resulting in a truncated version of PilA (PilA(108) ), with a C-terminal motif of phenylalanine-threonine-phenylalanine (FTF). Inactivation of pilA(108) confirmed it was required for mucoidy. Additionally, algW and algU were also required for mucoidy of PAO579. Western blot analysis indicated that MucA was less stable in PAO579 than nonmucoid PAO1 or PAO381. The mucoid phenotype and high PalgU and PalgD promoter activities of PAO579 require pilA(108) , algW, algU, and rpoN encoding the alternative sigma factor σ(54) . We also observed that RpoN regulates expression of algW and pilA in PAO579. Together, these results suggest that truncation in type IV pilin in P. aeruginosa strain PAO579 can induce mucoidy through an AlgW/AlgU-dependent pathway.
authors.affiliation: Department of Biochemistry and Microbiology, Joan C. Edwards School of Medicine at Marshall University, Huntington, West Virginia 25755-9320, USA.
authors.forename: T
authors.initials: T
authors.lastname: Ryan Withers
authors.forename: F
authors.initials: F
authors.lastname: Heath Damron
authors.forename: Yeshi
authors.initials: Y
authors.lastname: Yin
authors.forename: Hongwei D
authors.initials: HD
authors.lastname: Yu
mesh_terms: D000464:Alginates / Q000378:metabolism*; D004252:DNA Mutational Analysis; D036781:Fimbriae Proteins / Q000235:genetics*; D055786:Gene Knockout Techniques; D016680:Genome, Bacterial; D020723:Glucuronic Acid / Q000378:metabolism; D006603:Hexuronic Acids / Q000378:metabolism; D017354:Point Mutation; D020641:Polymorphism, Single Nucleotide; D011550:Pseudomonas aeruginosa / Q000235:genetics* / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion
chemical_list: D000464:Alginates; D006603:Hexuronic Acids; D036781:Fimbriae Proteins; D020723:Glucuronic Acid