Christopher A. Azaldegui

All Publications

• Polyphosphate modulates the stress-responsive formation of functional RNA-protein condensates in bacteria and mammalian cells. PLoS biology Guan, J., Hurto, R. L., Rai, A., Bhattrai, J., Azaldegui, C. A., Ortiz-Rodríguez, L. A., Liu, Q., Biteen, J. S., Freddolino, L., Jakob, U. 2026; 24 (4): e3003775

  Abstract

  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 produces long-chain polyphosphates, which scaffold the RNA chaperone Hfq into high molecular weight complexes, which eventually phase separate together with components of the RNA translation and processing machinery. The presence of polyphosphate within these condensates controls Hfq function by selectively stabilizing polyadenylated RNAs involved in transcription and protein translation and by promoting interactions with translation- and RNA-metabolism-associated proteins involved in de novo protein synthesis. Lack of polyphosphate significantly impairs condensate formation, increases cell death, and hinders recovery from N-starvation. In functional analogy, we demonstrate that polyP contributes specifically to the formation of Processing (P)-bodies in mammalian cell lines, revealing that a single, highly conserved and ancestral polyanion serves as a modulator for functional phase-separated condensate formation across the tree of life.

  View details for DOI 10.1371/journal.pbio.3003775

  View details for PubMedID 42044170

  View details for PubMedCentralID PMC13193609

• The kinetics and mobility of a ParA ATPase drive carboxysome distribution in Halothiobacillus neapolitanus. bioRxiv : the preprint server for biology Azaldegui, C. A., Swasthi, H. M., Hu, L., Pulianmackal, L. T., Rivett-Trznadel, H., Liu, J., Vecchiarelli, A. G., Biteen, J. S. 2026

  Abstract

  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 α-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.

  View details for DOI 10.64898/2026.03.03.709431

  View details for PubMedID 41867820

  View details for PubMedCentralID PMC13001409

• Stress changes the material state of a bacterial biomolecular condensate and shifts its function from mRNA decay to storage. Nature communications Ortiz-Rodríguez, L. A., Yassine, H., Hatami, A., Nandana, V., Azaldegui, C. A., Cheng, J., Zhu, Y., Schrader, J. M., Biteen, J. S. 2025; 16 (1): 10019

  Abstract

  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.

  View details for DOI 10.1038/s41467-025-65358-y

  View details for PubMedID 41238547

  View details for PubMedCentralID PMC12618670

• Nucleoid compaction influences carboxysome localization and dynamics in Synechococcus elongatus PCC 7942. mBio Dudley, C. E., Azaldegui, C. A., Foust, D. J., LaCommare, O., Biteen, J. S., Vecchiarelli, A. G. 2025; 16 (10): e0191925

  Abstract

  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 matrix for positioning but a dynamic player in spatial organization. Given the widespread role of ParA-family ATPases in the positioning of diverse cellular cargos, our study suggests that the nucleoid compaction state is a fundamental, yet underappreciated, determinant of mesoscale organization across bacteria.Bacteria can organize their internal components in specific patterns to ensure proper function and faithful inheritance after cell division. In the cyanobacterium Synechococcus elongatus, protein-based compartments called carboxysomes fix carbon dioxide and are distributed in the cell by a two-protein positioning system. Here, we discovered that when cells stop growing or face stress, these positioning proteins stop working, yet carboxysomes remain distributed in the cell. Our study shows that the bacterial chromosome, which holds genetic information, can also act as a flexible scaffold that holds carboxysomes in place when compacted. This insight reveals that the bacterial chromosome plays a key physical role in organizing the cell. Similar positioning systems are found across many types of bacteria; therefore, our findings suggest that nucleoid compaction may be a universal and underappreciated factor in maintaining spatial order in cells that are not actively growing.

  View details for DOI 10.1128/mbio.01919-25

  View details for PubMedID 40838714

  View details for PubMedCentralID PMC12506020

• Engineering Spatial Control of Bacterial Organelles. bioRxiv : the preprint server for biology Hoang, Y., Jadhav, P. V., Trettel, D. S., Dow, R. E., Kwon, S., Matej, K., Byrne, J. A., Azaldegui, C. A., Giessen, T. W., Pi, H., Mosalaganti, S., Vecchiarelli, A. G. 2025

  Abstract

  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 expressed 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.

  View details for DOI 10.1101/2025.09.22.677801

  View details for PubMedID 41040230

  View details for PubMedCentralID PMC12485843

• Brownian ratchet mechanisms for carboxysome positioning in bacteria. Current opinion in microbiology Azaldegui, C. A., Vecchiarelli, A. G., Biteen, J. S. 2025; 87: 102638

  Abstract

  ParA-type ATPases position multiple cellular cargos in bacteria. DNA partitioning by ParA is proposed to occur via a Brownian ratchet mechanism. It is unclear whether this mechanism accounts for the distribution of other ParA-positioned cargos. Among these cargos are bacterial microcompartments (BMCs), protein-based organelles that encapsulate metabolic components in bacteria. The most widely studied BMC is the carboxysome, which sequesters carbon fixation machinery and contributes significantly to global carbon fixation. Carboxysomes are spatially regulated by the Maintenance of Carboxysome Distribution (Mcd) system. Similarities between the McdA protein and the ParA ATPase family proteins have led to the proposal of a Brownian ratchet-based positioning mechanism for carboxysomes. Here, we describe ParA-type ATPases, the proposed variations of the Brownian ratchet mechanism, and how they may account for carboxysome organization.

  View details for DOI 10.1016/j.mib.2025.102638

  View details for PubMedID 40737819

  View details for PubMedCentralID PMC12459610

• Tuning the interaction of a ParA-type ATPase with its partner separates bacterial organelle positioning from partitioning. bioRxiv : the preprint server for biology Byrne, J. A., Swasthi, H. M., Hu, L., Azaldegui, C. A., Liu, J., Vecchiarelli, A. G. 2025

  Abstract

  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.

  View details for DOI 10.1101/2025.05.22.655647

  View details for PubMedID 40475624

  View details for PubMedCentralID PMC12139897

• Exploring Transient States of PAmKate to Enable Improved Cryogenic Single-Molecule Imaging. Journal of the American Chemical Society Perez, D., Dowlatshahi, D. P., Azaldegui, C. A., Ansell, T. B., Dahlberg, P. D., Moerner, W. E. 2024

  Abstract

  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 nanoscale 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) that photoactivate or photoswitch efficiently at cryogenic temperatures, but long on-times due to reduced quantum yield of photobleaching remain 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 that 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 the study of other FPs at cryogenic temperatures.

  View details for DOI 10.1021/jacs.4c05632

  View details for PubMedID 39388715

• An experimental framework to assess biomolecular condensates in bacteria. Nature communications Hoang, Y., Azaldegui, C. A., Dow, R. E., Ghalmi, M., Biteen, J. S., Vecchiarelli, A. G. 2024; 15 (1): 3222

  Abstract

  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 discover that an established marker for insoluble protein aggregates, IbpA, has different colocalization patterns with bacterial condensates and aggregates, demonstrating its potential 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.

  View details for DOI 10.1038/s41467-024-47330-4

  View details for PubMedID 38622124

  View details for PubMedCentralID PMC11018776

• Ruminococcus bromii enables the growth of proximal Bacteroides thetaiotaomicron by releasing glucose during starch degradation. Microbiology (Reading, England) Rangarajan, A. A., Chia, H. E., Azaldegui, C. A., Olszewski, M. H., Pereira, G. V., Koropatkin, N. M., Biteen, J. S. 2022; 168 (4)

  Abstract

  Complex carbohydrates shape the gut microbiota, and the collective fermentation of resistant starch by gut microbes positively affects human health through enhanced butyrate production. The keystone species Ruminococcus bromii (Rb) is a specialist in degrading resistant starch; its degradation products are used by other bacteria including Bacteroides thetaiotaomicron (Bt). We analysed the metabolic and spatial relationships between Rb and Bt during potato starch degradation and found that Bt utilizes glucose that is released from Rb upon degradation of resistant potato starch and soluble potato amylopectin. Additionally, we found that Rb produces a halo of glucose around it when grown on solid media containing potato amylopectin and that Bt cells deficient for growth on potato amylopectin (∆sus Bt) can grow within the halo. Furthermore, when these ∆sus Bt cells grow within this glucose halo, they have an elongated cell morphology. This long-cell phenotype depends on the glucose concentration in the solid media: longer Bt cells are formed at higher glucose concentrations. Together, our results indicate that starch degradation by Rb cross-feeds other bacteria in the surrounding region by releasing glucose. Our results also elucidate the adaptive morphology of Bt cells under different nutrient and physiological conditions.

  View details for DOI 10.1099/mic.0.001180

  View details for PubMedID 35471195

• Benzoic acid derivatives as luminescent sublimation dyes in cyanoacrylate fuming of latent fingerprints. Journal of forensic sciences Azaldegui, C., Aguilar, G., Enriquez, S., Madonna, C., Parish Fisher, C., Burks, R. 2021; 66 (3): 1085-1093

  Abstract

  Development of latent prints employing cyanoacrylate ester (CA) can be a multistep process including CA fuming and subsequent fluorescent staining to produce fingerprints of sufficient contrast for comparison work. To enable a single-step CA fuming-staining process, a selection of fluorophores have been developed as sublimation dyes in CA fuming. A greater array of such luminescent sublimation dyes would allow users greater flexibility in selecting a particular dye-CA combination to best suit their processing needs. Toward this end, six benzoic acid derivatives were evaluated for use as luminescent sublimation dyes under elementary CA fuming conditions using a single non-porous surface type and an inexpensive handheld UV lamp for excitation. Two benzoic acid derivatives, 2-hydroxybenzoic acid (salicylic acid) and 2-aminobenzoic acid (anthranilic acid), were identified as new potential luminescent sublimation dyes with stained fingerprints excited at 254 nm. The fluorescence intensity and stability of prints produced via the sublimation of CA with 2-hydroxybenzoic acid and 2-aminobenzoic acid were evaluated over approximately six weeks using image and statistical analysis.

  View details for DOI 10.1111/1556-4029.14678

  View details for PubMedID 33547641

• The emergence of phase separation as an organizing principle in bacteria. Biophysical journal Azaldegui, C. A., Vecchiarelli, A. G., Biteen, J. S. 2021; 120 (7): 1123-1138

  Abstract

  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 article 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.

  View details for DOI 10.1016/j.bpj.2020.09.023

  View details for PubMedID 33186556

  View details for PubMedCentralID PMC8059088