Investigating the chromatin remodeling functions of polyphosphate condensates in Pseudomonas aeruginosa - PROJECT SUMMARY In response to a variety of stress and starvation cues, bacteria spend ATP to make a polymer, polyphosphate (polyP), consisting of long chains of phosphate groups. PolyP synthesis is important for fitness during stress and starvation, and promotes virulence in many human pathogens, including the opportunistic pathogen Pseudomonas aeruginosa. The molecular mechanisms by which polyP exerts its pleiotropic effects on bacterial physiology and virulence remain poorly understood, in part because of the difficulty in identifying bona fide protein interactions with this simple polyanion. While lacking specificity at the primary level of organization, polyP chains come together to form granular superstructures, membraneless condensates which are spatially organized in some species. In P. aeruginosa, polyP granules form in the nucleoid region and become evenly spaced on the cell’s long axis during nitrogen starvation. We recently discovered that the histone H1-like DNA binding protein AlgP facilitates polyP granule spacing in P. aeruginosa, and that DNA alone can modulate the biophysical properties of polyP condensates in vitro. Together with other observations, these findings lead us to propose our central hypothesis, that polyP granules remodel bacterial chromatin during stress in P. aeruginosa. We will test our central hypothesis through three aims: (1) Define the epistatic relationship between polyP and algP as regulators of gene expression and virulence under clinically relevant conditions, (2) Evaluate how polyP and polyP condensates affect interactions of AlgP with DNA in vitro, and (3) Determine how polyP condensates affect functional AlgP-chromosome interactions in cells. This proposal’s innovation lies in its integrative approach to explore an important emerging theme in the polyP field, that this polymer may be an important part of bacterial chromatin, in the context of a DNA-binding protein that may be under positive genetic selection in chronic infections. This proposal integrates detailed in vitro biophysical and quantitative cell biological studies of the disordered histone H1-like C-terminus of AlgP and its interactions with polyP and DNA with quantitative analysis of genetic variability of this domain in clinical isolates from chronic CF infections and in vivo cell and murine lung infection models. The expected outcome of this study is an understanding of how these two polyanions, polyP and DNA, work together during stress and starvation states, as well as establishing the functional significance of the genetically variable protein AlgP in the evolutionary trajectory of chronic infections. The significance of the proposed research is both in establishing the molecular function of polyP in stress responses and virulence, and contributing to our larger understanding of the role of phase separation in subcellular organization in bacteria, an emerging theme in bacterial cell biology.
