A Screen for Novel Regulators of Polyphosphate Accumulation in Escherichia Coli Identifies Genes Involved in Envelope Stress, Central Metabolism, and Phosphate Homeostasis

Abstract

Polyphosphates (polyP) are chains of inorganic phosphate that are present in a wide variety of organisms such as yeast, mammals, and bacteria. Despite the interesting and diverse functions of polyP across systems, bacterial polyP is of particular interest to researchers due to its contributions to bacterial survival. Across bacterial taxa, polyP accumulation promotes resistance to diverse environmental stressors, can act as a protein chaperone, and positively regulates pathogenicity, making polyP an interesting target to help treat bacterial infections. In the gram-negative bacterium, Escherichia coli, polyP is synthesized from ATP by a single polyphosphate kinase (PPK) enzyme, and is degraded into inorganic phosphate by an exopolyphosphatase. PolyP synthesis by PPK in E. coli is stress-dependent, as there is little to no detectable polyP present in the absence of stress, followed by the rapid accumulation of polyP upon exposure to stressors such as nutrient limitation. Despite this stress-specific activity, the exact trigger(s) of PPK activity at the onset of stress remain uncharacterized, as PPK’s expression and in vitro activity do not correlate with polyP accumulation. To address this open question, this work aims to characterize novel regulators of polyP that can be leveraged to investigate the mechanism of PPK activation. To achieve this, we conducted a screen of single-gene deletion mutants targeting a highly conserved family of signalling proteins called two-component systems (TCS), which have existing links to polyP biology. In doing so we highlighted five polyP-regulators, three of which (cpxA, arcB, and arcA) have not yet been reported in the literature. Upon following up on one of our hits, the envelope stress-sensing histidine kinase CpxA, we found that activation of the CpxRA TCS promotes an increase in polyP accumulation. However, polyP regulation by this system appears to be independent of PPK or PPX expression, leaving the door open to future analyses of PPK regulation, possibly at the level of localization or oligomerization. Overall, this work contributes to our fundamental knowledge surrounding the relationship between PPK activity, polyP accumulation, and cellular stress.