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

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

Characterization of the Temperature-Sensitive Mutations un-7 and png-1 in Neurospora crassa

The model filamentous fungus Neurospora crassa has been studied for over fifty years and many temperature-sensitive mutants have been generated. While most of these have been mapped genetically, many remain anonymous. The mutation in the N. crassa temperature-sensitive lethal mutant un-7 was identified by a complementation based approach as being in the open reading frame designated NCU00651 on linkage group I. Other mutations in this gene have been identified that lead to a temperature-sensitive morphological phenotype called png-1. The mutations underlying un-7 result in a serine to phenylalanine change at position 273 and an isoleucine to valine change at position 390, while the mutation in png-1 was found to result in a serine to leucine change at position 279 although there were other conservative changes in this allele. The overall morphology of the strain carrying the un-7 mutation is compared to strains carrying the png-1 mutation and these mutations are evaluated in the context of other temperature-sensitive mutants in Neurospora

Temperature shift of growing tips of wild-type, <i>un-7</i> and <i>png-1</i> strains to 37°C.

<p>Panels A–C were taken after shifting overnight slide cultures incubated at room temperature to 37°C for 4 hours and panels D–F show the cultures grown on slides at room temperature. (A,D) Wild type strain 2489. (B, E) <i>png-1</i> strain 9860. (C, F) <i>un-7</i> strain 2176.</p

Neurospora TS lethal genes involved in protein production, transport, or quality control.

<p>Neurospora TS lethal genes involved in protein production, transport, or quality control.</p

Alignment of the putative amino acid sequence from the mutated region of the NCU00651 protein from wild type and two mutants as well as select fungi.

<p>The altered amino acid residues in the UN-7 and PNG-1 proteins are indicated by grey shading and amino acid residue 273 is indicated with an “*” above the sequence. Amino acid residue 279 is indicated with a “∧” above the sequence. The locus designations for other species are as follows: <i>Sordaria macrospora</i> CBI51252; <i>Aspergillus fumigatus</i> EDP54057; <i>Coccidioides immitis</i> CIMG_08062; <i>Magnaporthe grisea</i> MGG_03598; <i>Saccharomyces cerevisiae</i> EE09111; <i>Phycomyces blakesleeanus</i> 178891.</p

Cumulative growth of wild type and mutant strains at 37°.

<p>Values are the average of three measurements plus or minus the standard deviation.</p

Selected cosmids spanning the genomic region including NCU00651.

<p>Open reading frames are indicated above the line representing the genome sequence while the cosmids are indicated below the line.</p