FtsH protease and ClpG disaggregase confer fitness advantages to the worldwide prevalent Pseudomonas aeruginosa clone C

Abstract: Pseudomonas aeruginosa is an environmental bacterium and a frequent nosocomial pathogen causing a wide range of opportunistic infections, especially in immunocompromised patients. Clone C is one of the most prevalent groups of closely related strains distributed worldwide in the environment, such as in natural aquatic habitats, and the clinical settings, such as in patients with an underlying functional impairment of the cystic fibrosis transmembrane conductance regulator. Clone C strains specifically harbor the horizontally-transferred genomic island PACGI-1. One border of the genomic island contains the transmissible locus of protein quality control (TLPQC), alternatively called the Locus of Heat Resistance (LHR) in other bacteria, predominantly encoding stress-related gene products such as proteins involved in proteostasis. Among those gene products is a xenolog of the gene encoding the AAA+ (ATPase associated with diverse cellular activities) membrane-bound protease FtsH termed FtsH2 and the AAA+ disaggregase ClpG termed ClpGGI. In clone C isolates, ftsH2 and clpGGI encoded on TLPQC exist in addition to the core genome homologs ftsH1 and clpG. This thesis investigates the divergent and convergent roles of the genomic island and core genome copies of ftsH and clpG in fitness and prevalence of the aquatic clone C isolate P. aeruginosa SG17M. Paper I identifies ftsH1 as a pleiotropic gene in P. aeruginosa SG17M, affecting a multitude of phenotypes related to fitness and adaptation such as growth, motility, biofilm formation, antibiotic resistance, autolysis, secondary metabolite secretion and oxidative and heat shock stress. In the absence of ftsH1, the TLPQC locus copy ftsH2 backs up ftsH1 functionality. FtsH1 and FtsH2 share highly conserved functional AAA+ ATPase and protease domains and form homo- and hetero-oligomers with FtsH2 distinctively produced in the late stationary phase. However, mainly FtsH1 controls the levels of the heat-shock transcription factor RpoH (σ32). Using FtsH trap variants in an in vivo crosslinking/in vitro pull-down experiment shows that the phenazine biosynthesis protein PhzC is a novel substrate for FtsH1 in P. aeruginosa SG17M. Paper II investigates the molecular basis of the differential in vivo functionality of FtsH1 and FtsH2 in P. aeruginosa SG17M. We show that the N-terminal 151 amino acids of FtsH1 are required for FtsH1 functionality and, consequently, optimal growth of P. aeruginosa SG17M. The periplasmic domain and the short glycine-rich cytoplasmic linker connecting the Nterminus to the AAA+ module are particularly crucial for the optimal functionality of FtsH1. Moreover, in vitro biochemical analysis of the purified FtsH proteases shows that FtsH1 and FtsH2 are homo-hexamers and active ATPases with differential degradation activity towards model substrates such as FITC-casein and Arc-st11-ssrA. Paper III studies the role of the ClpG/ClpGGI disaggregases in protein quality control and thermotolerance of P. aeruginosa SG17M. ClpG-type disaggregases confer superior heat tolerance through their high basal ATPase activity coupled to an efficient disaggregase activity. In addition, ClpG/ClpGGI bind aggregates independently without the involvement of the co-chaperone system via a unique N-terminal extension, which contrasts the established ClpB/DnaK co-chaperone system. Paper IV describes the role of the TLPQC/LHR encoding dna-shsp20GI-clpGGI operon in thermotolerance in the human commensal E. coli Fec10 isolate, a close homolog of E. coli K- 12. The horizontally acquired heat tolerance locus, in particular ClpGGI, is a major determinant of tolerance to a lethal temperature upshift to 65 ºC. Biochemically, ClpGGI robustly disaggregates heat-denatured model substrates such as malate dehydrogenase (MDH) and firefly luciferase without the aid of co-chaperone factors. Moreover, ClpGGI shows high intrinsic basal ATPase activity and superior thermal stability compared to the ClpB disaggregase. Paper V reports on the instant double crossover recombination frequencies upon suicide vector integration into chromosomes of the most prevalent P. aeruginosa clone C and PA14 strains. As a result, the genomic engineering of these prevalent clones can be facilitated by omitting the counterselection step. Altogether, two AAA+ proteins, the FtsH protease and the ClpG disaggregases, encoded on the clone C specific genomic island PACGI-1/TLPQC and the core genome, contribute to proteostasis and confer general fitness advantages to the worldwide prevalent P. aeruginosa clone C.