Biology subjects

Azaldegui, C. A.

Publications and source records attributed to Azaldegui, C. A..

9 recordsLinked to original sources

bioRxiv2026

The kinetics and mobility of a ParA ATPase drive carboxysome distribution in Halothiobacillus neapolitanus

Carboxysomes are bacterial microcompartments that drive efficient carbon fixation in autotrophic bacteria. Critical to their function and inheritance is their spatial organization by the ParA-type ATPase, McdA, and its partner protein, McdB. Here, we investigate the -carboxysome McdAB system in Halothiobacillus neapolitanus using biochemical assays, quantitative fluorescence imaging, and mathematical modeling. We find that, unlike most ParA-type ATPases, the ATPase activity of McdA is only stimulated by DNA rather than by its partner protein McdB. Despite this difference, McdB conserves the ability to displace McdA from DNA, suggesting that ATP hydrolysis and DNA unbinding by McdA are not strictly coupled. Together with its ability to diffuse while bound to DNA, McdA forms gradients on the nucleoid that prevent carboxysome aggregation via a Brownian ratchet mechanism. Overall, these findings reveal key differences in a ParA-type ATPase that may be specific for the spatial organization of protein-based organelles in bacteria.

biophysics↗

bioRxiv2025

Engineering Spatial Control of Bacterial Organelles

Bacteria were once thought to lack organelles, but it is now clear they confine cellular reactions using an array of membrane- and protein-based compartments. A central question, however, is how bacterial organelles are organized in the cell, and whether their spatial control can be engineered. Here, we show that a two-protein system (McdAB) that positions carboxysomes - CO2-fixing organelles found in autotrophic bacteria - can be repurposed to provide programmable spatial control to diverse organelles in Escherichia coli. McdAB not only restores proper assembly and positioning of heterologously produced carboxysomes in E. coli, but can also be reprogrammed to spatially organize all other known types of bacterial organelles, including encapsulins, biomolecular condensates, and even membrane-bound organelles. Programmable spatial organization of bacterial organelles establishes a new design principle for synthetic biology, where the location of reactions is as tunable as their content. Our work paves the way for more efficient biocatalysis in engineered microbes.

microbiology↗

bioRxiv2025

Tuning the interaction of a ParA-type ATPase with its partner separates bacterial organelle positioning from partitioning

The maintenance of carboxysome distribution (Mcd) system comprises the proteins McdA and McdB, which spatially organize carboxysomes to promote efficient carbon fixation and ensure their equal inheritance during cell division. McdA, a member of the ParA/MinD family of ATPases, forms dynamic gradients on the nucleoid that position McdB-bound carboxysomes. McdB belongs to a widespread but poorly characterized class of ParA/MinD partner proteins, and the molecular basis of its interaction with McdA remains unclear. Here, we demonstrate that the N-terminal 20 residues of H. neapolitanus McdB are both necessary and sufficient for interaction with McdA. Within this region, we identify three lysine residues whose individual substitution modulates McdA binding and leads to distinct carboxysome organization phenotypes. Notably, lysine 7 (K7) is critical for McdA interaction: substitutions at this site result in the formation of a single carboxysome aggregate positioned at mid-nucleoid. This phenotype contrasts with that of an McdB deletion, in which carboxysome aggregates lose their nucleoid association and become sequestered at the cell poles. These findings suggest that weakened McdA-McdB interactions are sufficient to maintain carboxysome aggregates on the nucleoid but inadequate for partitioning individual carboxysomes across it. We propose that, within the ParA/MinD family of ATPases, cargo positioning and partitioning are mechanistically separable: weak interactions with the cognate partner can mediate positioning, whereas effective partitioning requires stronger interactions capable of overcoming cargo self-association forces.

microbiology↗

bioRxiv2025

Nucleoid Compaction Influences Carboxysome Localization and Dynamics in Synechococcus elongatus PCC 7942

The bacterial nucleoid is not just a genetic repository--it serves as a dynamic scaffold for spatially organizing key cellular components. ParA-family ATPases exploit this nucleoid matrix to position a wide range of cargos, yet how nucleoid compaction influences these positioning reactions remains poorly understood. We previously characterized the Maintenance of Carboxysome Distribution (Mcd) system in the cyanobacterium Synechococcus elongatus PCC 7942, where the ParA-like ATPase McdA binds the nucleoid and interacts with its partner protein, McdB, to generate dynamic gradients that distribute carboxysomes for optimal carbon fixation. Here, we investigate how nucleoid compaction impacts carboxysome positioning, particularly during metabolic dormancy when McdAB activity is downregulated. We demonstrate that a compacted nucleoid maintains carboxysome organization in the absence of active McdAB-driven positioning. This finding reveals that the nucleoid is not merely a passive substrate for positioning, but an active player in spatial organization. Given the widespread role of ParA-family ATPases in the positioning of diverse cellular cargos, our study suggests that nucleoid compaction state is a fundamental, yet underappreciated, determinant of mesoscale organization across bacteria.

microbiology↗

bioRxiv2025

HP-Bodies - Ancestral Condensates that Regulate RNA Turnover and Protein Translation in Bacteria

Uncovering what drives select biomolecules to form phase-separated condensates in vivo and identifying their physiological significance are topics of fundamental importance. Here we show that nitrogen-starved Escherichia coli produce long-chain polyphosphates, which scaffold the RNA chaperone Hfq into phase-separating high molecular weight complexes together with components of the RNA translation and processing machinery. The presence of polyphosphate within these condensates, which we termed HP-bodies, controls Hfq function by selectively stabilizing polyadenylated RNAs involved in transcription and protein translation, and promoting interactions with translation- and RNA-metabolism-associated proteins involved in de novo protein synthesis. Lack of polyphosphate prevents HP-body formation, which increases cell death and significantly hinders recovery from N-starvation. In functional analogy, we demonstrate that polyP contributes specifically to the formation of Processing (P)-bodies in human cell lines, revealing that a single, highly conserved and ancestral polyanion serves as the universal scaffold for functional phase-separated condensate formation across the tree of life.

microbiology↗

bioRxiv2024

Stress Changes the Bacterial Biomolecular Condensate Material State and Shifts Function from mRNA Decay to Storage

Bacterial ribonucleoprotein bodies (BR-bodies) are dynamic biomolecular condensates that play a pivotal role in RNA metabolism. We investigated how BR-bodies significantly influence mRNA fate by transitioning between liquid- and solid-like states in response to stress. With a combination of single-molecule and bulk fluorescence microscopy, biochemical assays, and quantitative analyses, we determine that BR-bodies promote efficient mRNA decay in a liquid-like condensate during exponential growth. On the other hand, BR-bodies are repurposed from sites of mRNA decay to reservoirs for mRNA storage under stress; a functional change that is enabled by their transition to a more rigid state, marked by reduced internal dynamics, increased molecular density, and prolonged residence time of ribonuclease E. Furthermore, we manipulated ATP levels and translation rates, and we conclude that the accumulation of ribosome-depleted mRNA is a key factor driving BR-body rigification, and that condensate maturation further contributes to this process. Upon nutrient replenishment, stationary-phase BR-bodies disassemble, releasing stored mRNAs for rapid translation, demonstrating that BR-body function is governed by a reversible mechanism for resource management. These findings reveal adaptive strategies by which bacteria regulate RNA metabolism through condensate-mediated control of mRNA decay and storage.

biophysics↗

bioRxiv2024

Exploring transient states of PAmKate to enable improved cryogenic single-molecule imaging

Super-resolved cryogenic correlative light and electron microscopy is a powerful approach which combines the single-molecule specificity and sensitivity of fluorescence imaging with the nano-scale resolution of cryogenic electron tomography. Key to this method is active control over the emissive state of fluorescent labels to ensure sufficient sparsity to localize individual emitters. Recent work has identified fluorescent proteins (FPs) which photoactivate or photoswitch efficiently at cryogenic temperatures, but long on-times due to reduced quantum yield of photobleaching remains a challenge for imaging structures with a high density of localizations. In this work, we explore the photophysical properties of the red photoactivatable FP PAmKate and identify a 2-color process leading to enhanced turn-off of active emitters, improving localization rate. Specifically, after excitation of ground state molecules, we find a transient state forms with a lifetime of [~]2 ms under cryogenic conditions which can be bleached by exposure to a second wavelength. We measure the response of the transient state to different wavelengths, demonstrate how this mechanism can be used to improve imaging, and provide a blueprint for study of other FPs at cryogenic temperatures.

biophysics↗

bioRxiv2023

An experimental framework to assess biomolecular condensates in bacteria

High-resolution imaging of biomolecular condensates in living cells is essential for correlating their properties to those observed through in vitro assays. However, such experiments are limited in bacteria due to resolution limitations. Here we present an experimental framework that probes the formation, reversibility, and dynamics of condensate-forming proteins in Escherichia coli as a means to determine the nature of biomolecular condensates in bacteria. We demonstrate that condensates form after passing a threshold concentration, maintain a soluble fraction, dissolve upon shifts in temperature and concentration, and exhibit dynamics consistent with internal rearrangement and exchange between condensed and soluble fractions. We also discovered that an established marker for insoluble protein aggregates, IbpA, has different colocalization patterns with bacterial condensates and aggregates, demonstrating its applicability as a reporter to differentiate the two in vivo. Overall, this framework provides a generalizable, accessible, and rigorous set of experiments to probe the nature of biomolecular condensates on the sub-micron scale in bacterial cells.

microbiology↗

bioRxiv2020

The emergence of phase separation as an organizing principle in bacteria

Recent investigations in bacteria suggest that membraneless organelles play a crucial role in the subcellular organization of bacterial cells. However, the biochemical functions and assembly mechanisms of these compartments have not yet been completely characterized. This Review assesses the current methodologies used in the study of membraneless organelles in bacteria, highlights the limitations in determining the phase of complexes in cells that are typically an order of magnitude smaller than a eukaryotic cell, and identifies gaps in our current knowledge about the functional role of membraneless organelles in bacteria. Liquid-liquid phase separation (LLPS) is one proposed mechanism for membraneless organelle assembly. Overall, we outline the framework to evaluate LLPS in vivo in bacteria, we describe the bacterial systems with proposed LLPS activity, and we comment on the general role LLPS plays in bacteria and how it may regulate cellular function. Lastly, we provide an outlook for super-resolution microscopy and single-molecule tracking as tools to assess condensates in bacteria. Statement of SignificanceThough membraneless organelles appear to play a crucial role in the subcellular organization and regulation of bacterial cells, the biochemical functions and assembly mechanisms of these compartments have not yet been completely characterized. Furthermore, liquid-liquid phase separation (LLPS) is one proposed mechanism for membraneless organelle assembly, but it is difficult to determine subcellular phases in tiny bacterial cells. Thus, we outline the framework to evaluate LLPS in vivo in bacteria and we describe the bacterial systems with proposed LLPS activity in the context of these criteria.

biophysics↗