ClpXP proteases positively regulate alginate overexpression and mucoid conversion in Pseudomonas aeruginosa

• Published in: Microbiology (Society for General Microbiology) Vol. 154; no. 7; pp. 2119 - 2130
• Main Authors: Qiu, Dongru, Eisinger, Vonya M, Head, Nathan E, Pier, Gerald B, Yu, Hongwei D
• Format: Journal Article
• Language: English
• Published: Reading Soc General Microbiol 01.07.2008; Society for General Microbiology
• Subjects: Alginates - metabolism; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Bacteriology; Biological and medical sciences; Cystic Fibrosis - microbiology; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Bacterial; Genetics; Glucuronic Acid - metabolism; Glycosaminoglycans - metabolism; Hexuronic Acids - metabolism; Humans; Microbiology; Molecular Sequence Data; Mutagenesis, Insertional; Peptide Hydrolases - genetics; Peptide Hydrolases - metabolism; Phenotype; Point Mutation; Pseudomonas aeruginosa; Pseudomonas aeruginosa - genetics; Pseudomonas aeruginosa - metabolism; Pseudomonas Infections - microbiology; Sigma Factor - genetics; Sigma Factor - metabolism; Pseudomonadales; Enzyme; Galactosidase; Electroporation; Alginates; Glycosylases; Peptidases; Regulation(control); Glycosidases; Blotting assay; Immunoblotting assay; Bacteria; Hydrolases; Pseudomonadaceae; Pseudomonas aeruginosa; Conjugation; Southern blotting
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/mic.0.2008/017368-0

1 Department of Biochemistry and Microbiology, Joan C. Edwards School of Medicine at Marshall University, Huntington, WV 25755-9320, USA 2 Channing Laboratory, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA 3 Department of Pediatrics, Joan C. Edwards School of Medicine at Marshall University, Huntington, WV 25701-3655, USA Correspondence Hongwei D. Yu yuh{at}marshall.edu Overproduction of the exopolysaccharide alginate and conversion to a mucoid phenotype in Pseudomonas aeruginosa are markers for the onset of chronic lung infection in cystic fibrosis (CF). Alginate production is regulated by the extracytoplasmic function (ECF) factor AlgU/T and the cognate anti- factor MucA. Many clinical mucoid isolates carry loss-of-function mutations in mucA . These mutations, including the most common mucA22 allele, cause C-terminal truncations in MucA, indicating that an inability to regulate AlgU activity by MucA is associated with conversion to the mucoid phenotype. Here we report that a mutation in a stable mucoid strain derived from the parental strain PAO1, designated PAO581, that does not contain the mucA 22 allele, was due to a single-base deletion in mucA ( T180), generating another type of C-terminal truncation. A global mariner transposon screen in PAO581 for non-mucoid isolates led to the identification of three regulators of alginate production, clpP (PA1801), clpX (PA1802), and a clpP paralogue (PA3326, designated clpP 2). The PAO581 null mutants of clpP , clpX and clpP 2 showed decreased AlgU transcriptional activity and an accumulation of haemagglutinin (HA)-tagged N-terminal MucA protein with an apparent molecular mass of 15 kDa. The clpP and clpX mutants of a CF mucoid isolate revert to the non-mucoid phenotype. The ClpXP and ClpP2 proteins appear to be part of a proteolytic network that degrades the cytoplasmic portion of truncated MucA proteins to release the sequestered AlgU, which drives alginate biosynthesis. Abbreviations: CF, cystic fibrosis; HA, haemagglutinin The GenBank/EMBL/DDBJ accession number for the algUmucABCD sequence of Pseudomonas aeruginosa PAO581 is EF635219. A supplementary figure showing genomic profiles of PAO1 and the isogenic strain PAO581 (PAO1 mucA25 ), and a supplementary table listing mariner transposon insertions leading to the non-mucoid phenotype in the stable mucoid strain, are available with the online version of this paper.