Published April 9, 2024 | Published
Journal Article Open

Polyphosphate affects cytoplasmic and chromosomal dynamics in nitrogen-starved Pseudomonas aeruginosa

Abstract

Polyphosphate (polyP) synthesis is a ubiquitous stress and starvation response in bacteria. In diverse species, mutants unable to make polyP have a wide variety of physiological defects, but the mechanisms by which this simple polyanion exerts its effects remain unclear. One possibility is that polyP’s many functions stem from global effects on the biophysical properties of the cell. We characterize the effect of polyphosphate on cytoplasmic mobility under nitrogen-starvation conditions in the opportunistic pathogen Pseudomonas aeruginosa. Using fluorescence microscopy and particle tracking, we quantify the motion of chromosomal loci and cytoplasmic tracer particles. In the absence of polyP and upon starvation, we observe a 2- to 10-fold increase in mean cytoplasmic diffusivity. Tracer particles reveal that polyP also modulates the partitioning between a “more mobile” and a “less mobile” population: Small particles in cells unable to make polyP are more likely to be “mobile” and explore more of the cytoplasm, particularly during starvation. Concomitant with this larger freedom of motion in polyP-deficient cells, we observe decompaction of the nucleoid and an increase in the steady-state concentration of ATP. The dramatic polyP-dependent effects we observe on cytoplasmic transport properties occur under nitrogen starvation, but not carbon starvation, suggesting that polyP may have distinct functions under different types of starvation.

Copyright and License

© 2024 the Author(s). Published by PNAS. This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).

Acknowledgement

We thank the Center for Environmental Microbial Interactions (CEMI) at Caltech, where the project was conceived. We are grateful to all members of the Manley and Racki groups for insightful discussions. We thank Amanda Habel for help with optimizing the luciferase assay and optimizing both acquisition and tracking parameters for the chromosome and μNS particles. We thank Ravi Chawla for testing our tracking parameters on carbon starvation data. We thank Megan Bergkessel for helpful feedback on the manuscript. Funding was received from the NIH (DP2-GM-140918 to L.R.R.), European Research Council (ERC CoG 819823, Piko to S. Manley and S. Magkiriadou), the Swiss NSF (182429 to S. Manley and W.L.S.), and the Donald E. and Delia B. Baxter Foundation Fellowship (L.R.R.). This is manuscript #30145 from The Scripps Research Institute.

Contributions

D.K.N., S. Manley, and L.R.R. conceived of research and administered the project; S. Magkiriadou, W.L.S., D.K.N., S. Manley, and L.R.R. designed research; L.R.R. performed research and contributed new reagents; S. Magkiriadou, and W.L.S. developed software; S. Magkiriadou, W.L.S., and L.R.R. analyzed data; S. Magkiriadou, W.L.S., S. Manley, and L.R.R. validated analysis; S. Magkiriadou, W.L.S., D.K.N., S. Manley, and L.R.R. reviewed and edited the paper; S. Magkiriadou, S. Manley, and L.R.R. wrote the paper.

Data Availability

Values used in graphs; code data have been deposited in Zenodo; Github (https://doi.org/10.5281/zenodo.5772215 (45) and https://github.com/LEB-EPFL/tracking-and-diffusion (46)).

Conflict of Interest

The authors declare no competing interest.

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Additional details

Created:
April 8, 2024
Modified:
June 4, 2024