title: Two Isoforms of Clp Peptidase in Pseudomonas aeruginosa Control Distinct Aspects of Cellular Physiology.
abstract: Caseinolytic peptidases (ClpPs) regulate diverse aspects of cellular physiology in bacteria. Some species have multiple ClpPs, including the opportunistic pathogenPseudomonas aeruginosa, in which there is an archetypical isoform, ClpP1, and a second isoform, ClpP2, about which little is known. Here, we use phenotypic assays to investigate the biological roles of ClpP1 and ClpP2 and biochemical assays to characterize purified ClpP1, ClpP2, ClpX, and ClpA. Interestingly, ClpP1 and ClpP2 have distinct intracellular roles for motility, pigment production, iron scavenging, and biofilm formation. Of particular interest, ClpP2, but not ClpP1, is required for microcolony organization, where multicellular organized structures first form on the pathway to biofilm production. We found that purified ClpP1 with ClpX or ClpA was enzymatically active, yet to our surprise, ClpP2 was inactive and not fully assembledin vitro; attempts to assist ClpP2 assembly and activation by mixing with the other Clp components failed to turn on ClpP2, as did solution conditions that have helped activate other ClpPsin vitroWe postulate that the active form of ClpP2 has yet to be discovered, and we present several potential models to explain its activation as well as the unique role ClpP2 plays in the development of the clinically important biofilms inP. aeruginosaIMPORTANCEPseudomonas aeruginosais responsible for severe infections of immunocompromised patients. Our work demonstrates that two different isoforms of the Clp peptidase, ClpP1 and ClpP2, control distinct aspects of cellular physiology for this organism. In particular, we identify ClpP2 as being necessary for microcolony organization. Pure active forms of ClpP1 and either ClpX or ClpA were characterized as assembled and active, and ClpP2 was incompletely assembled and inactive. By establishing both the unique biological roles of ClpP1 and ClpP2 and their initial biochemical assemblies, we have set the stage for important future work on the structure, function, and biological targets of Clp proteolytic enzymes in this important opportunistic pathogen.
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
authors.forename: Branwen M
authors.initials: BM
authors.lastname: Hall
authors.affiliation: Centre for Microbial Diseases and Immunity Research, Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: Elena B M
authors.initials: EBM
authors.lastname: Breidenstein
authors.affiliation: Centre for Microbial Diseases and Immunity Research, Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: César
authors.initials: C
authors.lastname: de la Fuente-Núñez
authors.affiliation: Centre for Microbial Diseases and Immunity Research, Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: Fany
authors.initials: F
authors.lastname: Reffuveille
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
authors.forename: Gina D
authors.initials: GD
authors.lastname: Mawla
authors.affiliation: Centre for Microbial Diseases and Immunity Research, Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: Robert E W
authors.initials: REW
authors.lastname: Hancock
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA tabaker@mit.edu.|Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
authors.forename: Tania A
authors.initials: TA
authors.lastname: Baker
keywords: Clp peptidase; ClpP1; ClpP2; Pseudomonas aeruginosa; biofilm; microcolonies