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

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 mucA22 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 clpP2). The PAO581 null mutants of clpP, clpX and clpP2 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.

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.