Transcription factors DksA and PsrA are synergistic contributors to Legionella pneumophila virulence in Acanthamoeba castellanii protozoa

The environmental bacterium <i>Legionella pneumophila</i>, an intracellular parasite of free-living freshwater protozoa as well as an opportunistic human pathogen, has a biphasic lifestyle. The switch from the vegetative replicative form to the environmentally resilient transmissive phase form is governed by a complex stringent response-based regulatory network that includes RNA polymerase co-factor DksA. Here, we report that, through a dysfunctional DksA mutation (DksA1), a synergistic interplay was discovered between DksA and transcription regulator PsrA using the <i>Acanthamoeba castellanii</i> protozoan infection model. Surprisingly, <i>in trans</i> expression of PsrA partially rescued the growth defect of a <i>dksA1</i> strain. Whilst <i>in trans</i> expression of DksA expectantly could fully rescue the growth defect of the <i>dksA1</i> strain, it could also surprisingly rescue the growth defect of a Δ<i>psrA</i> strain. Conversely, the severe intracellular growth defect of a Δ<i>dksA</i> strain could be rescued by <i>in trans</i> expression of DksA and DksA1, but not PsrA. <i>In vitro</i> phenotypic assays show that either DksA or DksA1 was required for extended culturability of bacterial cells, but normal cell morphology and pigmentation required DksA only. Comparative structural modelling predicts that the DksA1 mutation affects the coordination of Mg²⁺ into the active site of RNAP, compromising transcription efficiency. Taken together, we propose that PsrA transcriptionally assists DksA in the expression of select transmissive phase traits. Additionally, <i>in vitro</i> evidence suggests that the long-chain fatty acid metabolic response is mediated by PsrA together with DksA, inferring a novel regulatory link to the stringent response pathway.