The membrane-cytoplasmic linker defines activity of FtsH proteases in Pseudomonas aeruginosa clone C.

Pandemic Pseudomonas aeruginosa clone C strains encode two inner-membrane associated ATP-dependent FtsH proteases. PaftsH1 is located on the core genome and supports cell growth and intrinsic antibiotic resistance, whereas PaftsH2, a xenolog acquired through horizontal gene transfer from a distantly related species, is unable to functionally replace PaftsH1. We show that purified PaFtsH2 degrades fewer substrates than PaFtsH1. Replacing the 31-amino acid-extended linker region of PaFtsH2 spanning from the C-terminal end of the transmembrane helix-2 to the first seven highly divergent residues of the cytosolic AAA+ ATPase module with the corresponding region of PaFtsH1 improves hybrid-enzyme substrate processing in vitro and enables PaFtsH2 to substitute for PaFtsH1 in vivo. Electron microscopy indicates that the identity of this linker sequence influences FtsH flexibility. We find membrane-cytoplasmic (MC) linker regions of PaFtsH1 characteristically glycine-rich compared to those from FtsH2. Consequently, introducing three glycines into the membrane-proximal end of PaFtsH2's MC linker is sufficient to elevate its activity in vitro and in vivo. Our findings establish that the efficiency of substrate processing by the two PaFtsH isoforms depends on MC linker identity and suggest that greater linker flexibility and/or length allows FtsH to degrade a wider spectrum of substrates. As PaFtsH2 homologs occur across bacterial phyla, we hypothesize that FtsH2 is a latent enzyme but may recognize specific substrates or is activated in specific contexts or biological niches. The identity of such linkers might thus play a more determinative role in the functionality of and physiological impact by FtsH proteases than previously thought.

ClpP1P2 peptidase activity promotes biofilm formation in Pseudomonas aeruginosa.

Caseinolytic proteases (Clp) are central to bacterial proteolysis and control cellular physiology and stress responses. They are composed of a double-ring compartmentalized peptidase (ClpP) and a AAA+ unfoldase (ClpX or ClpA/ClpC). Unlike many bacteria, the opportunistic pathogen Pseudomonas aeruginosa contains two ClpP homologs: ClpP1 and ClpP2. The specific functions of these homologs, however, are largely elusive. Here, we report that the active form of PaClpP2 is a part of a heteromeric PaClpP17 P27 tetradecamer that is required for proper biofilm development. PaClpP114 and PaClpP17 P27 complexes exhibit distinct peptide cleavage specificities and interact differentially with P. aeruginosa ClpX and ClpA. Crystal structures reveal that PaClpP2 has non-canonical features in its N- and C-terminal regions that explain its poor interaction with unfoldases. However, experiments in vivo indicate that the PaClpP2 peptidase active site uniquely contributes to biofilm development. These data strongly suggest that the specificity of different classes of ClpP peptidase subunits contributes to the biological outcome of proteolysis. This specialized role of PaClpP2 highlights it as an attractive target for developing antimicrobial agents that interfere specifically with late-stage P. aeruginosa development.

Two Isoforms of Clp Peptidase in Pseudomonas aeruginosa Control Distinct Aspects of Cellular Physiology.

Caseinolytic peptidases (ClpPs) regulate diverse aspects of cellular physiology in bacteria. Some species have multiple ClpPs, including the opportunistic pathogen Pseudomonas 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 assembled in 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 ClpPs in vitro We 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 in P. aeruginosaIMPORTANCEPseudomonas aeruginosa is 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.

A novel approach for studying histone H1 function in vivo.

In this report, we investigate the mechanisms that regulate Drosophila histone H1 expression and its association with chromatin in vivo. We show that histone H1 is subject to negative autoregulation and exploit this result to examine the effects of mutations of the main phosphorylation site of histone H1.