Biology subjects

Chawla, R.

Publications and source records attributed to Chawla, R..

3 recordsLinked to original sources

bioRxiv2023

Reentrant DNA shells tune polyphosphate condensate size

The ancient, inorganic biopolymer polyphosphate (polyP) occurs in all three domains of life and affects myriad cellular processes. An intriguing feature of polyP is its frequent proximity to chromatin, and in the case of many bacteria, its occurrence in the form of magnesium-enriched condensates embedded in the nucleoid, particularly in response to stress. The physical basis of the interaction between polyP and DNA, two fundamental anionic biopolymers, and the resulting effects on the organization of both the nucleoid and polyP condensates remain poorly understood. Given the essential role of magnesium ions in the coordination of polymeric phosphate species, we hypothesized that a minimal system of polyP, magnesium ions, and DNA (polyP-Mg2+-DNA) would capture key features of the interplay between the condensates and bacterial chromatin. We find that DNA can profoundly affect polyP-Mg2+ coacervation even at concentrations several orders of magnitude lower than found in the cell. The DNA forms shells around polyP-Mg2+ condensates and these shells show reentrant behavior, primarily forming in the concentration range close to polyP-Mg2+ charge neutralization. This surface association tunes both condensate size and DNA morphology in a manner dependent on DNA properties, including length and concentration. Our work identifies three components that could form the basis of a central and tunable interaction hub that interfaces with cellular interactors. These studies will inform future efforts to understand the basis of polyP granule composition and consolidation, as well as the potential capacity of these mesoscale assemblies to remodel chromatin in response to diverse stressors at different length and time scales.

biophysics↗

bioRxiv2021

The Histone H1-like protein AlgP facilitates even spacing of polyphosphate granules in Pseudomonas aeruginosa

Synthesis of polyphosphate (polyP) is an ancient and universal stress and starvation response in bacteria. In many bacteria, polyP chains come together to form granular superstructures within cells. Some species appear to regulate polyP granule subcellular organization. Despite the critical role of polyP in starvation fitness, the composition of these structures, mechanism(s) underpinning their organization, and functional significance of such organization are poorly understood. We previously determined that granules become transiently evenly spaced on the cells long axis during nitrogen starvation in the opportunistic human pathogen Pseudomonas aeruginosa. Here, we developed a granule-enrichment protocol to screen for polyP granule-localizing proteins. We identified AlgP as a protein that associates with polyP granules. We further discovered that AlgP is required for the even spacing of polyP granules. AlgP is a DNA-binding protein with a 154 amino acid C-terminal domain enriched in KPAA repeats and variants of this repeat, with an overall sequence composition similar to the C-terminal tail of eukaryotic histone H1. Granule size, number, and spacing are significantly perturbed in the absence of AlgP, or when AlgP is truncated to remove the C-terminus. The {Delta}algP and algP{Delta}CTD mutants having fewer, larger granules. We speculate that AlgP may contribute to spacing by tethering polyP granules to the chromosome, thereby inhibiting fusion with neighboring granules. Our discovery that AlgP facilitates granule spacing allows us for the first time to directly uncouple granule biogenesis from even spacing, and will inform future efforts to explore the functional significance of granule organization on fitness during starvation. IMPORTANCEThe mechanisms underpinning polyPs pleiotropic effects on bacterial starvation physiology remain elusive. This simple polyanions lack of protein binding specificity has impeded validation of bona fide polyP-binding proteins. However, polyP forms granule superstructures with spatial specificity. Our granule enrichment protocol identified a polyP granule-associated protein in Pseudomonas aeruginosa, AlgP. AlgP was originally reported as regulator of alginate, an extracellular polysaccharide important in biofilm formation, including in cystic fibrosis (CF) chronic infections. AlgPs putative role in alginate biosynthesis has recently been called into question. We establish a distinct, previously unknown function for AlgP in modulating the subcellular organization of polyP, another polymer important for pathogenesis. In CF clinical isolates, the C-terminal repeat domain of AlgP is a hotspot for genetic rearrangements. Our finding that the C-terminus of AlgP is required for granule organization lays the groundwork for exploring the functional significance of these mutations in the evolutionary trajectory of chronic infections.

microbiology↗

bioRxiv2017

Reversed graph embedding resolves complex single-cell developmental trajectories

Organizing single cells along a developmental trajectory has emerged as a powerful tool for understanding how gene regulation governs cell fate decisions. However, learning the structure of complex single-cell trajectories with two or more branches remains a challenging computational problem. We present Monocle 2, which uses reversed graph embedding to reconstruct single-cell trajectories in a fully unsupervised manner. Monocle 2 learns an explicit principal graph to describe the data, greatly improving the robustness and accuracy of its trajectories compared to other algorithms. Monocle 2 uncovered a new, alternative cell fate in what we previously reported to be a linear trajectory for differentiating myoblasts. We also reconstruct branched trajectories for two studies of blood development, and show that loss of function mutations in key lineage transcription factors diverts cells to alternative branches on the a trajectory. Monocle 2 is thus a powerful tool for analyzing cell fate decisions with single-cell genomics.

genomics↗