Biology subjects

Wang, J. D.

Publications and source records attributed to Wang, J. D..

11 recordsLinked to original sources

bioRxiv2026

Starvation-independent alarmone production inhibits translation through GTP depletion

Bacteria produce the alarmone nucleotides (p)ppGpp during stress to affect replication, transcription, translation, and metabolism. Recently, pGpp was identified as a third alarmone that is produced from the hydrolysis of (p)ppGpp. Although pGpp is a major component of bacterial stress responses, its precise role in mediating these responses is poorly understood. ppGpp and pppGpp bind translation GTPases and therefore directly affect translation to conserve resources during periods of stress. Here, we show that while pGpp is a weaker inhibitor of protein synthesis than ppGpp and pppGpp in vitro, pGpp production in the model Gram-positive bacterium Bacillus subtilis leads to faster translation inhibition in vivo. Faster translation inhibition is accompanied by greater levels of disengaged ribosomal subunits and hibernating ribosome dimers, suggesting that translation initiation is strongly inhibited. We show that alarmone production in vivo causes a severe depletion of GTP, which is sufficient for translation inhibition. Finally, we find that pGpp production also causes more robust transcriptome remodeling than (p)ppGpp production. This work supports a model that implicates all three alarmones in translation inhibition via GTP depletion.

microbiology↗

bioRxiv2024

Pyruvate Kinase Directly Generates GTP in Glycolysis, Supporting Growth and Contributing to Guanosine Toxicity

Guanosine triphosphate (GTP) is essential for macromolecular biosynthesis, and its intracellular levels are tightly regulated in bacteria. Loss of the alarmone (p)ppGpp disrupts GTP regulation in Bacillus subtilis, causing cell death in the presence of exogenous guanosine and underscoring the critical importance of GTP homeostasis. To investigate the basis of guanosine toxicity, we performed a genetic selection for spontaneous mutations that suppress this effect, uncovering an unexpected link between GTP synthesis and glycolysis. In particular, we identified suppressor mutations in pyk, which encodes pyruvate kinase, a glycolytic enzyme. Metabolomic analysis revealed that inactivating pyruvate kinase prevents guanosine toxicity by reducing GTP levels. Although traditionally associated with ATP generation via substrate-level phosphorylation, B. subtilis pyruvate kinase in vitro was found to produce GTP and UTP approximately ten and three times more efficiently than ATP, respectively. This efficient GTP/UTP synthesis extends to Enterococcus faecalis and Listeria monocytogenes, challenging the conventional understanding of pyruvate kinase's primary role in ATP production. These findings support a model in which glycolysis directly contributes to GTP synthesis, fueling energy-demanding processes such as protein translation. Finally, we observed a synergistic essentiality of the {Delta}ndk {Delta}pyk double mutant specifically on glucose, indicating that pyruvate kinase and nucleoside diphosphate kinase are the major contributors of NTP production and complement each other during glycolysis. Our work highlights the critical role of nucleotide selectivity in pyruvate kinase and its broader implications in cellular physiology.

microbiology↗

bioRxiv2024

Allosteric Regulation of Pyruvate Kinase Enables Efficient and Robust Gluconeogenesis by Preventing Metabolic Conflicts and Carbon Overflow

Glycolysis and gluconeogenesis are reciprocal metabolic pathways that utilize different carbon sources. Pyruvate kinase catalyzes the irreversible final step of glycolysis, yet the physiological function of its regulation is poorly understood. Through metabolomics and enzyme kinetics studies, we discovered that pyruvate kinase activity is inhibited during gluconeogenesis in the soil bacterium Bacillus subtilis. This regulation involves an extra C-terminal domain (ECTD) of pyruvate kinase, which is essential for autoinhibition and regulation by metabolic effectors. Introducing a pyruvate kinase mutant lacking the ECTD into B. subtilis resulted in defects specifically under gluconeogenic conditions, including inefficient carbon utilization, slower growth, and decreased resistance to the herbicide glyphosate. These defects are not caused by the phosphoenolpyruvate-pyruvate-oxaloacetate futile cycle. Instead, we identified two significant metabolic consequences of pyruvate kinase dysregulation during gluconeogenesis: increased carbon overflow into the medium and failure to expand glycolytic intermediates such as phosphoenolpyruvate (PEP). In silico analysis revealed that in wild-type cells, an expanded PEP pool enabled by pyruvate kinase regulation is critical for the thermodynamic feasibility of gluconeogenesis. Our findings underscore the importance of allosteric regulation during gluconeogenesis in coordinating metabolic flux, efficient energy utilization, and antimicrobial resistance.

microbiology↗

bioRxiv2023

RNase H genes cause distinct impacts on RNA:DNA hybrid formation and mutagenesis genome-wide

RNA:DNA hybrids such as R-loops affect genome integrity and DNA replication fork progression. The overall impacts of naturally occurring RNA:DNA hybrids on genome integrity, and the relative contributions of ribonucleases H to mitigating the negative effects of hybrids, remain unknown. Here, we investigate the contributions of RNases HII (RnhB) and HIII (RnhC) to hybrid removal, DNA replication, and mutagenesis genome-wide. Deletion of either rnhB or rnhC triggers RNA:DNA hybrid accumulation, but with distinct patterns of mutagenesis and hybrid accumulation. Across all cells, hybrids accumulate most strongly in non-coding RNAs and 5'-UTRs of coding sequences. For {Delta}rnhB, hybrids accumulate preferentially in untranslated regions and early in coding sequences. Hybrid accumulation is particularly sensitive to gene expression in {Delta}rnhC; in cells lacking RnhC, DNA replication is disrupted leading to transversions and structural variation. Our results resolve the outstanding question of how hybrids in native genomic contexts interact with replication to cause mutagenesis and shape genome organization.

genetics↗

bioRxiv2023

Mitochondrial GTP Metabolism Regulates Reproductive Aging

Healthy mitochondria are critical for reproduction. During aging, both reproductive fitness and mitochondrial homeostasis decline. Mitochondrial metabolism and dynamics are key factors in supporting mitochondrial homeostasis. However, how they are coupled to control reproductive health remains unclear. We report that mitochondrial GTP metabolism acts through mitochondrial dynamics factors to regulate reproductive aging. We discovered that germline-only inactivation of GTP- but not ATP-specific succinyl-CoA synthetase (SCS), promotes reproductive longevity in Caenorhabditis elegans. We further revealed an age-associated increase in mitochondrial clustering surrounding oocyte nuclei, which is attenuated by the GTP-specific SCS inactivation. Germline-only induction of mitochondrial fission factors sufficiently promotes mitochondrial dispersion and reproductive longevity. Moreover, we discovered that bacterial inputs affect mitochondrial GTP and dynamics factors to modulate reproductive aging. These results demonstrate the significance of mitochondrial GTP metabolism in regulating oocyte mitochondrial homeostasis and reproductive longevity and reveal mitochondrial fission induction as an effective strategy to improve reproductive health.

cell biology↗

bioRxiv2020

The nucleotide messenger (p)ppGpp is a co-repressor of the purine synthesis transcription regulator PurR in Firmicutes

The nucleotide messenger (p)ppGpp allows bacteria to adapt to fluctuating environments by reprogramming the transcriptome. Yet despite its well-recognized role in gene regulation, (p)ppGpp is only known to directly affect transcription in Proteobacteria. Here we reveal a different mechanism of gene regulation by (p)ppGpp in Firmicutes from soil bacteria to pathogens: (p)ppGpp serves as a co-repressor of the transcription factor PurR to downregulate purine biosynthesis. We identified PurR as a receptor of (p)ppGpp in Bacillus anthracis and revealed that (p)ppGpp strongly enhances PurR binding to its regulon in the Bacillus subtilis genome. A co-structure reveals that (p)ppGpp binds to a PurR pocket reminiscent of the active site of PRT enzymes that has been repurposed to serve a purely regulatory role, where the effectors (p)ppGpp and PRPP compete to allosterically control transcription. PRPP inhibits PurR DNA binding to induce transcription of purine synthesis genes, whereas (p)ppGpp antagonizes PRPP to enhance PurR DNA binding and repress transcription. A (p)ppGpp-refractory purR mutant fails to downregulate purine synthesis genes upon starvation. Our work establishes precedent of (p)ppGpp as a classical transcription co-repressor and reveals the key function of (p)ppGpp in regulating nucleotide synthesis through gene regulation, from the human intestinal tract to host-pathogen interfaces.

microbiology↗

bioRxiv2020

Systemic characterization of pppGpp, ppGpp and pGpp targets in Bacillus reveal SatA converts (p)ppGpp to pGpp to regulate alarmone composition and signaling

The alarmones pppGpp and ppGpp (collectively (p)ppGpp) protect bacterial cells from nutritional and other stresses. Here we demonstrate the physiological presence of pGpp as a third closely related alarmone in bacterial cells and also characterize and compare the proteomic targets of pGpp, ppGpp and pppGpp in Gram-positive Bacillus species. We revealed two regulatory pathways for ppGpp and pppGpp that are highly conserved across bacterial species: inhibition of purine nucleotide biosynthesis and control of ribosome assembly/activity through GTPases. Strikingly, pGpp potently regulates the purine biosynthesis pathway but does not interact with the GTPases. Importantly, we identified a key enzyme NahA that efficiently produces pGpp by hydrolyzing (p)ppGpp, thus tuning alarmone composition to uncouple the regulatory modules of the alarmones. Correspondingly, a nahA mutant displays significantly reduced pGpp levels and elevated (p)ppGpp levels, slower growth recovery from nutrient downshift, and loss of competitive fitness. These cellular consequences for regulating alarmone composition strongly implicate an expanded repertoire of alarmones in a new strategy of stress response in Bacillus and its relatives.

microbiology↗

bioRxiv2020

Molecular mechanism of regulation of the purine salvage enzyme XPRT by the alarmones pppGpp, ppGpp, and pGpp

The alarmones pppGpp and ppGpp mediate starvation response and maintain purine homeostasis to protect bacterial species. Xanthine phosphoribosyltransferase (XPRT) is a purine salvage enzyme that produces the nucleotide XMP from PRPP and xanthine. Combining structural, biochemical and genetic analyses, we show that pppGpp and ppGpp, as well as a third putative alarmone pGpp, all directly interact with XPRT and inhibit XPRT activity by competing with its substrate PRPP. Structural analysis reveals that ppGpp binds the PRPP binding motif within the XPRT active site. This motif is present in another (p)ppGpp target, the purine salvage enzyme HPRT, suggesting evolutionary conservation in different enzymes. However, XPRT oligomeric interaction is distinct from HPRT in that XPRT forms a symmetric dimer with two (p)ppGpp binding sites at the dimer interface. This results in two distinct regulatory features. First, XPRT cooperatively binds (p)ppGpp with a Hill coefficient of 2. Also, XPRT displays differential regulation by the alarmones as it is potently inhibited by both ppGpp and pGpp, but only modestly by pppGpp. Lastly, we demonstrate that the alarmones are necessary for protecting GTP homeostasis against excess environmental xanthine in Bacillus subtilis, suggesting that regulation of XPRT is key for regulating the purine salvage pathway.

microbiology↗

bioRxiv2019

Mother machine image analysis with MM3

The mother machine is a microfluidic device for high-throughput time-lapse imaging of microbes. Here, we present MM3, a complete and modular image analysis pipeline. MM3 turns raw mother machine images, both phase contrast and fluorescence, into a data structure containing cells with their measured features. MM3 employs machine learning and non-learning algorithms, and is implemented in Python. MM3 is easy to run as a command line tool with the occasional graphical user interface on a PC or Mac. A typical mother machine experiment can be analyzed within one day. It has been extensively tested, is well documented and publicly available via Github.

systems biology↗

bioRxiv2019

Evolution of (p)ppGpp-HPRT regulation through diversification of an allosteric oligomeric interaction

The signaling ligand (p)ppGpp binds diverse targets across bacteria, yet the mechanistic and evolutionary basis underlying these ligand-protein interactions remains poorly understood. Here we identify a novel (p)ppGpp binding motif in the enzyme HPRT, where (p)ppGpp shares identical binding residues for PRPP and nucleobase substrates to regulate purine homeostasis. Intriguingly, HPRTs across species share the conserved binding site yet strongly differ in ligand binding, from strong inhibition by basal (p)ppGpp levels to weak regulation at induced concentrations. Surprisingly, strong ligand binding requires an HPRT dimer-dimer interaction that allosterically opens the (p)ppGpp pocket. This dimer-dimer interaction is absent in the common ancestor but evolved to favor (p)ppGpp binding in the vast majority of bacteria. We propose that the evolutionary plasticity of oligomeric interfaces enables allosteric adjustment of ligand regulation, bypassing constraints of the ligand binding site. Since most ligands bind near protein-protein interfaces, this principle likely extends to other protein-ligand interactions.

biochemistry↗

bioRxiv2016

Flux through lipid synthesis dictates bacterial cell size.

Nutrients--and by extension biosynthetic capacity--positively impact cell size in organisms throughout the tree of life. In bacteria, cell size is reduced three-fold in response to nutrient starvation or accumulation of the alarmone ppGpp, a global inhibitor of biosynthesis. However, whether biosynthetic capacity as a whole determines cell size or if particular anabolic pathways are more important than others remains an open question. Utilizing a top-down approach, here we identify flux through lipid synthesis as the primary biosynthetic determinant of Escherichia coli cell size. Altering flux through lipid synthesis recapitulated the impact of altering nutrients on cell size and morphology, while defects in other biosynthetic pathways either did not impact size or altered size in a lipid-dependent manner. Together our findings support a model in which lipid availability dictates cell envelope capacity and ppGpp functions as a linchpin linking surface area expansion with cytoplasmic volume to maintain cellular integrity.

microbiology↗