The DnaK (Hsp70) protein is an essential ATP‐dependent chaperone foldase and holdase found in most organisms. In this study, combining multiple experimental approaches we determined FliC as major interaction partner of DnaK in the opportunistic bacterial pathogen Pseudomonas aeruginosa. Implementing immunofluorescence microscopy and electron microscopy techniques DnaK was found extracellularly associated to the assembled filament in a regular pattern. dnaK repression led to intracellular FliC accumulation and motility impairment, highlighting DnaK essentiality for FliC export and flagellum assembly. SPOT–membrane peptide arrays coupled with artificial intelligence analyses suggested a highly dynamic DnaK–FliC interaction landscape involving multiple domains and transient complexes formation. Remarkably, in vitro fast relaxation imaging (FReI) experiments mimicking ATP‐deprived extracellular environment conditions exhibited DnaK ATP‐independent holdase activity, regardless of its co‐chaperone DnaJ and its nucleotide exchange factor GrpE. We present a model for the DnaK‐FliC interactions involving dynamic states throughout the flagellum assembly stages. These results expand the classical view of DnaK chaperone functioning and introduce a new participant in the Pseudomonas flagellar system, an important trait for bacterial colonisation and virulence.
