Polyphosphate granule biogenesis is temporally and functionally tied to cell cycle exit during starvation in Pseudomonas aeruginosa

Abstract

Polyphosphate (polyP) granule biogenesis is an ancient and ubiquitous starvation response in bacteria. Although the ability to make polyP is important for survival during quiescence and resistance to diverse environmental stresses, granule genesis is poorly understood. Using quantitative microscopy at high spatial and temporal resolution, we show that granule genesis in Pseudomonas aeruginosa is tightly organized under nitrogen starvation. Following nucleation as many microgranules throughout the nucleoid, polyP granules consolidate and become transiently spatially organized during cell cycle exit. Between 1 and 3 h after nitrogen starvation, a minority of cells have divided, yet the total granule number per cell decreases, total granule volume per cell dramatically increases, and individual granules grow to occupy diameters as large as ∼200 nm. At their peak, mature granules constitute ∼2% of the total cell volume and are evenly spaced along the long cell axis. Following cell cycle exit, granules initially retain a tight spatial organization, yet their size distribution and spacing relax deeper into starvation. Mutant cells lacking polyP elongate during starvation and contain more than one origin. PolyP promotes cell cycle exit by functioning at a step after DNA replication initiation. Together with the universal starvation alarmone (p)ppGpp, polyP has an additive effect on nucleoid dynamics and organization during starvation. Notably, cell cycle exit is temporally coupled to a net increase in polyP granule biomass, suggesting that net synthesis, rather than consumption of the polymer, is important for the mechanism by which polyP promotes completion of cell cycle exit during starvation.ost of our understanding of bacterial physiology comes from laboratory studies of bacteria growing under nutrientrich conditions. However, in many environments, bacteria face dramatic fluctuations in nutrient conditions, including long periods of scarcity when they survive in a nonproliferative stationary state. Although eukaryotic cells and some bacteria have discrete cell cycle checkpoints, many fast-growing bacterial species have uncoupled DNA replication and cell division. They use multifork DNA replication to achieve a doubling time that is faster than the time required to copy the chromosome, giving them a competitive edge in nutrient-replete conditions. This strategy requires distinct regulatory mechanisms and comes at a considerable cost when there is a rapid downshift in nutrient availability: stalled open DNA replication forks are vulnerable to potentially lethal double-stranded DNA breaks (1). Therefore, the ability to reallocate resources under such conditions to prioritize completion of DNA replication is critical for survival. Prioritizing chromosome remodeling and compaction by starvation-specific nucleoid structural proteins is also important during such transitions because resources for DNA repair become limited in deep starvation (2-4). Operating an uncoupled cell cycle, where growth, DNA replication, and cell ...

Inorganic Polyphosphate Accumulation in Escherichia coli Is Regulated by DksA but Not by (p)ppGpp

“…I doubt that the specific mutations present in the CF1693(Mϩ) strain used here were present in the strains used in those studies, but stringent alleles arise very commonly in relA spoT strains (21,70). In other bacterial species, including Caulobacter crescentus and Pseudomonas aeruginosa, mutants lacking (p)ppGpp are reported to make wild-type amounts of polyP (90,91), supporting the conclusion that, in general, (p)ppGpp production is not necessary for polyP production in bacteria. The physiological relevance of the significant activation of ppk expression by nutrient limitation in an rpoB3449 mutant ( Fig.…”

Molecular characterization of CHAD domains as inorganic polyphosphate binding modules

“…In bacteria, polyP granules are spatially restricted in the nucleoid region (3). PolyP has also been shown to accumulate in the nucleolus of myeloma cells (45).…”

Identity and functions of inorganic and inositol polyphosphates in plants

“…2). In bacteria, polyPs accumulate within granules in the nucleoid region (Racki et al, 2017) and can regulate various cellular processes, including growth, sporulation, response to nutrient deprivation, virulence, cell cycle and metal toxicity (Xie & Jakob, 2019). PolyPs also act as primordial chaperones in bacteria, binding to unfolded proteins and promoting their refolding (Gray et al, 2014).…”

“…While CDC26 is essential for Arabidopsis embryo development and regulates plant growth, AtTTM3 seems to be dispensable under favorable growth conditions (Lorenzo-Orts et al, 2019b). While the physiological roles of AtTTM3 remain unclear, it is of note that functional connections between polyPs and cell cycle regulation are well established in other prokaryotes and eukaryotes (Bru et al, 2016;Racki et al, 2017). There are at least two additional TTM proteins in plants (AtTTM1 and AtTTM2), where the TTM domain is located at the C-terminus of a uridine kinase domain (Ung et al, 2014(Ung et al, , 2017.…”