Biology subjects

Jalihal, A. P.

Publications and source records attributed to Jalihal, A. P..

13 recordsLinked to original sources

bioRxiv2026

Identification and deletion of the extracellular polysaccharide slime pathway across the genus Xanthobacter

Foods and commodities made by microbial bioproduction offer a sustainable alternative to those produced from our traditional petrochemical-based supply chains. Increasingly, Xanthobacter strains are used for bioproduction because of their unique metabolic properties (e.g., CO2 and N2 fixation) and wide feedstock range, including growth on agricultural products (e.g., fructose, sucrose) and electrically generated substrates (e.g., hydrogen gas, methanol, acetate, and formate). Many Xanthobacter strains, however, produce an extracellular polysaccharide (EPS) slime that makes plating, pelleting, and handling these cultures challenging and shuttles away resources from biomass and other biosynthetic pathways. While slime-free mutants of slime-producing Xanthobacter strains have been isolated, the causative mutations were unknown. Thus, slime-free mutants need to be painstakingly reisolated based on phenotypic properties in each academically and industrially relevant strain, unless we know the gene to target. Here, we identified the gene and underlying mutation that causes a slime-free phenotype in the X. autotrophicus mutant 7CSF. When we deleted this gene, xsbA, slime production was abolished in the wild-type strain. We showed that deleting xsbA homologs similarly abolished slime production in slime-producers across the genus Xanthobacter (X. versatilis, X. autotrophicus, X. flavus, and X. tagetidis). We also identified a Wzx/Wzy-dependent EPS pathway downstream of xsbA that is conserved across slime-producing Xanthobacter strains. Lastly, abolishing slime production improved colony formation and pelleting, with minimal impact on growth. We demonstrated that challenging phenotypic properties in Xanthobacter strains can be removed, improving their usability as a chassis for sustainable bioproduction.

microbiology↗

bioRxiv2025

A phase oscillator model of cell cycles reveals nuclear density control in a branched fungal network

Multinucleate cells are widespread in biology, from human skeletal muscle and placenta to many filamentous fungi, yet their basic cell biology remains poorly understood. Maintaining an appropriate nuclear-to-cytoplasmic ratio is essential across cell types for physiological function, and mechanisms of size control have been extensively studied in mononucleate cells. Much less is known about how comparable control is achieved in cells where many nuclei share a common cytoplasm. The filamentous fungus Ashbya gossypii forms a branching mycelial network in which individual nuclei divide asynchronously, while the number of nuclei per cell volume (the nuclear density) is tightly controlled. How global regulation of nuclear density coexists with local cell cycle asynchrony remains unclear. We address this by combining a mathematical model of nuclear division in a growing and branching cell with live-cell microscopy. We model nuclei as a dividing population of phase oscillators within a branching cell network and parameterize the model with measurements from Ashbya cells. The model demonstrates that asynchrony is required to prevent large density fluctuations that would result from synchronous division, and that introducing a nuclear-density checkpoint to the cell cycles leads to synchrony if it is the only mechanism of density control. We find that coupling branch formation to nuclear density both stabilizes nuclear density and prevents the emergence of synchronous cycles. Our results indicate that asynchronous nuclear cycles together with density-responsive branching maintain a constant nuclear density, revealing a strategy for regulating the nuclear-to-cytoplasmic ratio in large multinucleate cells. SignificanceMultinucleate cells appear in diverse biological contexts, from human tissues to filamentous fungi, yet many fundamental aspects of their cell biology are still unclear. Regulating the nuclear-to-cytoplasmic ratio is important across cell types, and little is known about how this is achieved in large, multinucleate fungal cells. Using mathematical modeling and live-cell imaging, we identify how the density of nuclei is controlled in a growing and branching fungal mycelial network. We find that asynchronous nuclear cycles together with density-responsive branching can stabilize the nuclear-to-cytoplasmic ratio within a growing fungal network. This illustrates how large multinucleate cells can control nuclear density even as their morphology becomes increasingly complex.

cell biology↗

bioRxiv2025

Continuous accelerated rock weathering by marine bacteria with enhanced siderophore production

Silicate mineral weathering (dissolution) is a scalable strategy for capture and storage of CO2 but is too slow for industrial deployment. Bacteria can accelerate mineral dissolution by secreting siderophores, molecules that solubilize iron released from the mineral that would otherwise passivate the mineral surface. Here, we investigated how to deploy siderophore-producing bacteria at scale to continuously enhance dissolution of the mineral olivine. We demonstrated that natural genetic regulation precludes continuous siderophore production in mineral bioreactors. To overcome this limitation, we engineered the marine bacterium Alteromonas macleodii to enhance its production of siderophores, conferring a 2.6-fold increase in the rate of olivine dissolution. Life Cycle Analysis indicated that renewable feedstocks and minimal replenishment of modified cells are critical to achieve net CO2 removal at scale. With these guidelines, we constructed pilot-scale continuous mineral bioreactors that use unprocessed seawater and a renewable acetate feedstock. In reactors with engineered cells, we directly measured removal of 0.50 g CO2 per day from the air through alkalinity generation, while a control reactor without cells did not generate alkalinity in net. These platforms and bioprocess principles will inform the design of large-scale, sustainable, unit operations for alkaline mineral processing.

systems biology↗

bioRxiv2024

Condensates act as translation hubs to coordinate multinucleate cell growth

Coordination between growth and nuclear division is a common cell feature. In some syncytia, nuclei divide asynchronously throughout the cell but growth occurs only at discrete locations, raising the question how the processes are locally regulated and globally coordinated. In the syncytial fungus Ashbya gossypii, both cell-cycle progression and hyphal elongation require condensates formed by the protein Whi3 in complex with distinct mRNA species. We show that Whi3 condensates are enriched for translation regulators and are associated with local, spatially patterned translation of specific target RNAs near nuclei and growth sites. Whi3-RNA condensates can both promote and repress mRNA translation in an RNA- and condensate size-dependent manner in vitro. Condensate-interfaces are sites of translation, tunable by condensate composition, RNA valency and protein charge-state in vitro. Together, these data suggest that Whi3 condensates can generate a continuum of translation states that vary depending on the subcellular location and resident RNA sequences.

cell biology↗

bioRxiv2024

RNA encodes physical information

Most amino acids are encoded by multiple codons, making the genetic code degenerate. Synonymous mutations affect protein translation and folding, but their impact on RNA itself is often neglected. We developed a genetic algorithm that introduces synonymous mutations to control the diversity of structures sampled by an mRNA. The behavior of the designed mRNAs reveals a physical code layered in the genetic code. We find that mRNA conformational heterogeneity directs physical properties and functional outputs of RNA-protein complexes and biomolecular condensates. The role of structure and disorder of proteins in biomolecular condensates is well appreciated, but we find that RNA conformational heterogeneity is equally important. This feature of RNA enables both evolution and engineers to build cellular structures with specific material and responsive properties.

biophysics↗

bioRxiv2024

Expansion and revision of the genus Xanthobacter and proposal of Roseixanthobacter gen. nov.

The nitrogen-fixing, chemolithoautotrophic genus Xanthobacter is found worldwide across numerous diverse environments and is an important member of many ecosystems. These species serve as model systems for their metabolic properties in academic settings and have industrial applications in bioremediation and sustainable protein, food, and fertilizer production. Despite their abundance, interest, and importance, the majority of Xanthobacter strains are without a genome sequence, and only 8 validly published species are known to date. To expand our understanding of the diversity and evolutionary history of the genus, we sequenced the genomes of 37 repository strains and 26 novel environmental strains we isolated. After performing comparative phylogenomic analyses, we expand and revise the genus Xanthobacter and propose the novel genus Roseixanthobacter gen. nov. For the Xanthobacter, we describe 10 novel species, bringing the total to 18: Xanthobacter agilis, Xanthobacter albus sp. nov., Xanthobacter aminoxidans, Xanthobacter autotrophicus, Xanthobacter cornucopiae sp. nov., Xanthobacter dioxanivorans, Xanthobacter flavus, Xanthobacter lutulentifluminis sp. nov., Xanthobacter nonsaccharivorans sp. nov., Xanthobacter oligotrophicus, Xanthobacter pseudotagetidis sp. nov., Xanthobacter sediminis sp. nov., Xanthobacter tagetidis, Xanthobacter toluenivorans sp. nov., Xanthobacter variabilis sp. nov., Xanthobacter versatilis sp. nov., Xanthobacter viscosus, and Xanthobacter wiegelii sp. nov. For the Roseixanthobacter gen. nov., we describe 5 novel species formerly classified as Xanthobacter: Roseixanthobacter finlandensis sp. nov., Roseixanthobacter glucoisosaccharinivorans sp. nov., Roseixanthobacter liquoris sp. nov., Roseixanthobacter pseudopolyaromaticivorans sp. nov., and Roseixanthobacter psychrophilus sp. nov. We characterized the phenotypic properties of these type strains, including temperature, salinity and pH ranges, carbon substrate utilization, motility, antibiotic susceptibility, slime production, autotrophic growth, and enzymatic activities. We discovered a more diverse range of phenotypes across the genus Xanthobacter than previously known and elucidated the evolutionary history within the genus. These findings and genome sequences will help further the application of Xanthobacter biology in academic, industrial, and environmental settings and provide additional insight into the unique biological properties that make these species attractive for such applications.

microbiology↗

bioRxiv2023

Dynamic superresolution by Bayesian nonparametric image processing

Assessing dynamic processes at single molecule scales is key toward capturing life at the level of its molecular actors. Widefield superresolution methods, such as STORM, PALM, and PAINT, provide nanoscale localization accuracy, even when distances between fluorescently labeled single molecules ("emitters") fall below lights diffraction limit. However, as these superresolution methods rely on rare photophysical events to distinguish emitters from both each other and background, they are largely limited to static samples. In contrast, here we leverage spatiotemporal correlations of dynamic widefield imaging data to extend superresolution to simultaneous multiple emitter tracking without relying on photodynamics even as emitter distances from one another fall below the diffraction limit. We simultaneously determine emitter numbers and their tracks (localization and linking) with the same localization accuracy per frame as widefield superresolution does for immobilized emitters under similar imaging conditions ({approx}50 nm). We demonstrate our results for both in cellulo data and, for benchmarking purposes, on synthetic data. To this end, we avoid the existing tracking paradigm relying on completely or partially separating the tasks of emitter number determination, localization of each emitter, and linking emitter positions across frames. Instead, we develop a fully joint posterior distribution over the quantities of interest, including emitter tracks and their total, otherwise unknown, number within the Bayesian nonparametric paradigm. Our posterior quantifies the full uncertainty over emitter numbers and their associated tracks propagated from origins including shot noise and camera artefacts, pixelation, stochastic background, and out-of-focus motion. Finally, it remains accurate in more crowded regimes where alternative tracking tools cannot be applied.

biophysics↗

bioRxiv2023

Dynamical control enables the formation of demixed biomolecular condensates

Macromolecular phase separation underlies the regulated formation and dissolution of biomolecular condensates. What is unclear is how condensates of distinct and shared macromolecular compositions form and coexist within cellular milieus. Here, we use theory and computation to establish thermodynamic criteria that must be satisfied to achieve compositionally distinct condensates. We applied these criteria to an archetypal ribonucleoprotein condensate and discovered that demixing into distinct protein-RNA condensates cannot be the result of purely thermodynamic considerations. Instead, demixed, compositionally distinct condensates arise due to asynchronies in timescales that emerge from differences in long-lived protein-RNA and RNA-RNA crosslinks. This type of dynamical control is also found to be active in live cells whereby asynchronous production of molecules is required for realizing demixed protein-RNA condensates. We find that interactions that exert dynamical control provide a versatile and generalizable way to influence the compositions of coexisting condensates in live cells.

biophysics↗

bioRxiv2021

Passive plasma membrane transporters play a critical role in perception of carbon availability in yeast

Recently, our lab found that the canonical glucose/galactose regulation pathway in yeast makes the decision to metabolize galactose based on the ratio of glucose to galactose concentrations in the external medium. This led to the question of where and how the ratio-sensing is achieved. Here, we consider the possibilities of an intracellular, extracellular, or membrane bound ratio sensing mechanisms. We show that hexose transporters in the plasma membrane are mainly responsible for glucose/galactose ratio-sensing in yeast. Further, while the glucose sensors Gpr1, Snf3, and Rgt2 are not required for ratio sensing, they help modulate the ratio sensing phenotype by regulating the expression of individual transporters in different environments. Our study provides an example of an unexpected, but potentially widespread, mechanism for making essential decisions.

cell biology↗

bioRxiv2020

Modeling and Analysis of the Macronutrient Signaling Network in Budding Yeast

In eukaryotes, distinct nutrient signals are relayed by specific plasma membrane receptors to signal transduction pathways that are interconnected in complex information-processing networks. The function of these networks is to govern robust cellular responses to unpredictable changes in the nutritional environment of the cell. In the budding yeast, Saccharomyces cerevisiae, these nutrient signaling pathways and their interconnections have been well characterized. However the complexity of the signaling network confounds the interpretation of the overall regulatory logic of the control system. Here, we propose a literature-curated molecular mechanism of the integrated nutrient signaling network in budding yeast, focusing on early temporal responses to carbon and nitrogen signaling. We build a computational model of this network to reconcile literature-curated quantitative experimental data with our proposed molecular mechanism. We evaluate the robustness of our estimates of the models kinetic parameter values. We test the model by comparing predictions made in mutant strains with qualitative experimental observations made in the same strains. Finally, we use the model to predict nutrient-responsive transcription factor activities in a number of mutant strains undergoing complex nutrient shifts.

systems biology↗

bioRxiv2019

Multivalent proteins rapidly and reversibly phase-separate upon osmotic cell volume change

Processing bodies (PBs) and stress granules (SGs) are prominent examples of sub-cellular, membrane-less compartments that are observed under physiological and stress conditions, respectively. We observe that the trimeric PB protein DCP1A rapidly (within [~]10 s) phase-separates in mammalian cells during hyperosmotic stress and dissolves upon isosmotic rescue (over [~]100 s) with minimal impact on cell viability even after multiple cycles of osmotic perturbation. Strikingly, this rapid intracellular hyperosmotic phase separation (HOPS) correlates with the degree of cell volume compression, distinct from SG assembly, and is exhibited broadly by homo-multimeric (valency [≥] 2) proteins across several cell types. Notably, HOPS sequesters pre-mRNA cleavage factor components from actively transcribing genomic loci, providing a mechanism for hyperosmolarity-induced global impairment of transcription termination. Together, our data suggest that the multimeric proteome rapidly responds to changes in hydration and molecular crowding, revealing an unexpected mode of globally programmed phase separation and sequestration that adapts the cell to volume change. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/748293v2_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@1ef81a5org.highwire.dtl.DTLVardef@1288c1eorg.highwire.dtl.DTLVardef@107355aorg.highwire.dtl.DTLVardef@3fb071_HPS_FORMAT_FIGEXP M_FIG C_FIG IN BRIEFCells constantly experience osmotic variation. These external changes lead to changes in cell volume, and consequently the internal state of molecular crowding. Here, Jalihal and Pitchiaya et al. show that multimeric proteins respond rapidly to such cellular changes by undergoing rapid and reversible phase separation. HIGHLIGHTSO_LIDCP1A undergoes rapid and reversible hyperosmotic phase separation (HOPS) C_LIO_LIHOPS of DCP1A depends on its trimerization domain C_LIO_LISelf-interacting multivalent proteins (valency [≥] 2) undergo HOPS C_LIO_LIHOPS of CPSF6 explains transcription termination defects during osmotic stress C_LI

cell biology↗

bioRxiv2018

Dynamic recruitment of single RNAs to processing bodies depends on RNA functionality

Cellular RNAs often colocalize with cytoplasmic, membrane-less ribonucleoprotein (RNP) granules enriched for RNA processing enzymes, termed processing bodies (PBs). Here, we track the dynamic localization of individual miRNAs, mRNAs and long non-coding RNAs (lncRNAs) to PBs using intracellular single-molecule fluorescence microscopy. We find that unused miRNAs stably bind to PBs, whereas functional miRNAs, repressed mRNAs and lncRNAs both transiently and stably localize within either the core or periphery of PBs, albeit to different extents. Consequently, translation potential and positioning of cis-regulatory elements significantly impact PB-localization dynamics of mRNAs. Using computational modeling and supporting experimental approaches we show that phase separation into large PBs attenuates mRNA silencing, suggesting that physiological mRNA turnover predominantly occurs outside of PBs. Instead, our data support a role for PBs in sequestering unused miRNAs to regulate their surveillance and provides a framework for investigating the dynamic assembly of RNP granules by phase separation at single-molecule resolution.

molecular biology↗

bioRxiv2018

Efficient Synthesis of Mutants Using Genetic Crosses

The genetic cross is a fundamental, flexible, and widely-used experimental technique to create new mutant strains from existing ones. Surprisingly, the problem of how to efficiently compute a sequence of crosses that can make a desired target mutant from a set of source mutants has received scarce attention. In this paper, we make three contributions to this question.\n\nFirst, we formulate several natural problems related to efficient synthesis of a target mutant from source mutants. Our formulations capture experimentally-useful notions of verifiability (e.g the need to confirm that a mutant contains mutations in the desired genes) and permissibility (e.g., the requirement that no intermediate mutants in the synthesis be inviable).\n\nSecond, we develop combinatorial techniques to solve these problems. We prove that checking the existence of a verifiable, permissible synthesis is NP-complete in general. We complement this result with three polynomial time or fixed-parameter tractable algorithms for optimal synthesis of a target mutant for special cases of the problem that arise in practice.\n\nThird, we apply these algorithms to simulated data and to synthetic data. We use results from simulations of a mathematical model of the cell cycle to replicate realistic experimental scenarios where a biologist may be interested in creating several mutants in order to verify model predictions. Our results show that the consideration of permissible mutants can affect the existence of a synthesis or the number of crosses in an optimal one. Our algorithms gracefully handle the restrictions that permissible mutants impose. Results on synthetic data show that our algorithms scale well with increases in the size of the input and the fixed parameters.

systems biology↗