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

The chromatin remodeller ACF acts as a dimeric motor to space nucleosomes.

Evenly spaced nucleosomes directly correlate with condensed chromatin and gene silencing. The ATP-dependent chromatin assembly factor (ACF) forms such structures in vitro and is required for silencing in vivo. ACF generates and maintains nucleosome spacing by constantly moving a nucleosome towards the longer flanking DNA faster than the shorter flanking DNA. How the enzyme rapidly moves back and forth between both sides of a nucleosome to accomplish bidirectional movement is unknown. Here we show that nucleosome movement depends cooperatively on two ACF molecules, indicating that ACF functions as a dimer of ATPases. Further, the nucleotide state determines whether the dimer closely engages one or both sides of the nucleosome. Three-dimensional reconstruction by single-particle electron microscopy of the ATPase-nucleosome complex in an activated ATP state reveals a dimer architecture in which the two ATPases face each other. Our results indicate a model in which the two ATPases work in a coordinated manner, taking turns to engage either side of a nucleosome, thereby allowing processive bidirectional movement. This novel dimeric motor mechanism differs from that of dimeric motors such as kinesin and dimeric helicases that processively translocate unidirectionally and reflects the unique challenges faced by motors that move nucleosomes

The chromatin remodeller ACF acts as a dimer and exploits the histone H4 tail and flanking DNA substrate cues to move nucleosomes

A fundamental mode of gene regulation in eukaryotes is to alter access of the nuclear machinery to DNA through packaging into chromatin. Human ACF, a member of the ISWI-family of chromatin remodeling enzymes, is an ideal model system to study the basic mechanism of moving nucleosomes because it is a small complex and makes one class of products, evenly spaced nucleosomes, thought to be important for heterochromatin formation. ACF kinetically distinguishes between different flanking DNA lengths on either side of a nucleosome, moving the nucleosome toward the longerflanking DNA faster than toward the shorter DNA.Using electron microscopy and enzymatic assays, we observe that ACF can bind and function as a cooperative homodimer to move nucleosomes. The dimeric partners bind the nucleosome near the N-terminal tails of H4 and face in opposing directions. This unusual architecture raises new questions about how the protomers collaborate rather than compete in a tug of war. We observe nucleotide-dependent changes in contacts of the enzyme with the two H4 tails. In the presence of ADP, the enzyme complex contacts one H4 tail, whereas with a nucleotide analogue thought to mimic an activated ATP state, both H4 tails are immobilized. These conformational states support a model for allosteric communication between the dimeric partners.We next focused on how the enzyme interprets and integrates two critical components of the nucleosome that we term `substrate cues:' the H4 tail and flanking DNA. We find that the H4 tail and flanking DNA stimulate remodeling activity nonadditively, suggesting that the two cues may either function in two different rate limiting steps, or that the two cues function in a coupled manner in one rate limiting step. Using spin-labeled ATP, we observe that the H4 tail but not flanking DNA is important for formation of a restricted conformation of the nucleotide-binding pocket. The H4 tail and flanking DNA both have larger effects on nucleosome remodeling than on ATP hydrolysis. The H4 tails and flanking DNAs play synergistic roles on remodeling and may be important for coupling ATP hydrolysis to nucleosome remodeling

The Histone H4 Tail Regulates the Conformation of the ATP-Binding Pocket in the SNF2h Chromatin Remodeling Enzyme

The chromatin remodeling complex ACF helps establish the appropriate nucleosome spacing for generating repressed chromatin states. ACF activity is stimulated by two defining features of the nucleosomal substrate: a basic patch on the histone H4 N-terminal tail and the specific length of flanking DNA. However, the mechanisms by which these two substrate cues function in the ACF remodeling reaction is not well understood. Using electron paramagnetic resonance spectroscopy with spin-labeled ATP analogs to probe the structure of the ATP active site under physiological solution conditions, we identify a closed state of the ATP-binding pocket that correlates with ATPase activity. We find that the H4 tail promotes pocket closure. We further show that ATPase stimulation by the H4 tail does not require a specific structure connecting the H4 tail and the globular domain. In the case of many DNA helicases, closure of the ATP-binding pocket is regulated by specific DNA substrates. Pocket closure by the H4 tail may analogously provide a mechanism to directly couple substrate recognition to activity. Surprisingly, the flanking DNA, which also stimulates ATP hydrolysis, does not promote pocket closure, suggesting that the H4 tail and flanking DNA may be recognized in different reaction steps

The chromatin remodeller ACF acts as a dimeric motor to space nucleosomes.

Evenly spaced nucleosomes directly correlate with condensed chromatin and gene silencing. The ATP-dependent chromatin assembly factor (ACF) forms such structures in vitro and is required for silencing in vivo. ACF generates and maintains nucleosome spacing by constantly moving a nucleosome towards the longer flanking DNA faster than the shorter flanking DNA. How the enzyme rapidly moves back and forth between both sides of a nucleosome to accomplish bidirectional movement is unknown. Here we show that nucleosome movement depends cooperatively on two ACF molecules, indicating that ACF functions as a dimer of ATPases. Further, the nucleotide state determines whether the dimer closely engages one or both sides of the nucleosome. Three-dimensional reconstruction by single-particle electron microscopy of the ATPase-nucleosome complex in an activated ATP state reveals a dimer architecture in which the two ATPases face each other. Our results indicate a model in which the two ATPases work in a coordinated manner, taking turns to engage either side of a nucleosome, thereby allowing processive bidirectional movement. This novel dimeric motor mechanism differs from that of dimeric motors such as kinesin and dimeric helicases that processively translocate unidirectionally and reflects the unique challenges faced by motors that move nucleosomes

The Histone H4 Tail Regulates the Conformation of the ATP-Binding Pocket in the SNF2h Chromatin Remodeling Enzyme

The chromatin remodeling complex ACF helps establish the appropriate nucleosome spacing for generating repressed chromatin states. ACF activity is stimulated by two defining features of the nucleosomal substrate: a basic patch on the histone H4 N-terminal tail and the specific length of flanking DNA. Yet the mechanisms by which these two substrate cues function in the ACF remodeling reaction is not well understood. Using electron paramagnetic resonance spectroscopy with spin-labeled ATP analogs to probe the structure of the ATP active site under physiological solution conditions, we identify a closed state of the ATP-binding pocket that correlates with ATPase activity. We find that the H4 tail promotes pocket closure. We further show that ATPase stimulation by the H4 tail does not require a specific structure connecting the H4 tail and the globular domain. In the case of many DNA helicases, closure of the ATP- binding pocket is regulated by specific DNA substrates. Pocket closure by the H4 tail may analogously provide a mechanism to directly couple substrate recognition to activity. Surprisingly, the flanking DNA, which also stimulates ATP hydrolysis, does not promote pocket closure, suggesting that the H4 tail and flanking DNA may be recognized in different reaction steps