Biology subjects

Dworkin, J.

Publications and source records attributed to Dworkin, J..

13 recordsLinked to original sources

bioRxiv2026

Mechanistic insights into E. coli recovery from growth arrest

Bacteria survive hostile conditions in clinically relevant conditions by shutting down protein synthesis, but how they restart growth remains poorly understood. Here, we use an E. coli {Delta}rimM strain, which exhibits a prolonged growth arrest, as a model to investigate how bacteria recover from this arrested state and restore protein synthesis. RimM is a conserved ribosome maturation factor for the 3-major (head) domain of the 16S rRNA within the bacterial 30S subunit. The loss of RimM causes a significantly longer delay in recovery than other 30S maturation factors, including RbfA - the presumed primary factor in 30S maturation. Cryo-EM analysis of {Delta}rimM ribosomes revealed a delayed recruitment of ribosomal proteins to the 30S head domain and increased occupancy of the initiation factors IF1 and IF3, as well as recruitment of the silencing factor RsfS to the 50S subunit. These coordinated changes provide a safeguarding mechanism to block the assembly of premature 70S ribosomes. Notably, while the delayed 30S assembly in {Delta}rimM reduces the activity of global protein synthesis during the recovery phase, bacteria attempt to compensate for this deficiency by producing higher levels of the ribosomal machinery, indicating a programmatic change in energy allocation to generate the ribosome machinery. These findings highlight the importance of the RimM-assisted assembly of the ribosomal head domain for bacterial recovery from growth arrest.

molecular biology↗

bioRxiv2025

(p)ppGpp-dependent activation of gene expression during nutrient limitation

As rapidly growing bacteria begin to exhaust nutrients, their growth rate slows, ultimately leading to stasis or quiescence. Adaptation to nutrient limitation requires widespread metabolic remodeling that leads to lower cellular energy consumption. Examples of such changes include attenuated transcription of genes encoding ribosome components, in part mediated by the phosphorylated nucleotides guanosine tetra- and penta-phosphate, collectively (p)ppGpp. In addition, genes encoding proteins that facilitate survival nutrient limitation exhibit increased expression. An example is the hpf gene, encoding a broadly conserved protein responsible for protecting the ribosome from degradation under conditions limiting for ribosome synthesis. Here we show that (p)ppGpp plays a key role in the transcriptional activation of hpf as B. subtilis cells exit rapid growth. Specifically, we demonstrate that hpf transcription during nutrient limitation requires an RNA polymerase holoenzyme containing the alternative sigma factor {sigma}H, encoded by sigH, whose expression is normally inhibited by the AbrB repressor. However, when global protein synthesis decreases, in part dependent on (p)ppGpp, AbrB levels fall, leading to increased sigH transcription and, consequently, hpf activation. This mechanism couples a key physiological consequence of nutrient limitation - reduced protein synthesis - with specific gene activation, thereby linking transcriptional and translational regulation. Finally, we demonstrate that (p)ppGpp is necessary for the gene expression underlying the elaboration of developmental fates including sporulation and genetic competence. ImportanceBacteria often experience nutrient limitation and, in response, they attenuate energetically costly metabolic processes like protein synthesis. At the same time, however, they stimulate the expression of a subset of proteins that facilitate survival under this condition. This study identifies a new molecular mechanism in the model Gram-positive bacterium Bacillus subtilis responsible for gene expression in response to nutrient limitation that couples reduced global protein synthesis with increased transcription of specific genes. This mechanism mediates the elaboration of developmental fates including sporulation and genetic competence that are known responses to nutrient limitation in this organism.

microbiology↗

bioRxiv2025

Simultaneous measurements of translation rate and transcriptome in single bacterial cells by scTR-seq uncovers linked regulation within a specialized active cell population.

Cell-to-cell variation within clonal bacterial populations provides bacterial communities important advantages including opportunities for bet-hedging and metabolic division of labor. In recent years the extent of bacterial heterogeneity has been documented both at the transcriptome level and with physiological measurements of cell growth rate and translation rate. However, methods that link physiological parameters to a single cells full transcriptomic state are lacking, making it difficult to identify the regulatory mechanisms that couple physiology and transcriptional output. Here we introduce a method that combines click-chemistry enabled labeling of nascent polypeptides to measure translation rates in single cells alongside microfluidic encapsulation and single cell transcriptomic measurements, providing a tandem measurement of translation rate and transcriptome in thousands of single Bacillus subtilis cells. In a culture experiencing nutrient limitation, we identified a subpopulation of cells with a higher rate of protein translation that uniquely overexpresses genes for several metabolic processes including acetoin production and arginine synthesis. Using a genetic approach informed by the gene expression in this subpopulation, we identified a regulatory mechanism that couples the increase in protein abundance of a transcriptional regulator AlsR with expression of alsR regulated genes in this subpopulation.

microbiology↗

bioRxiv2025

Analysis of (p)ppGpp metabolism and signaling using a dynamic luminescent reporter

As rapidly growing bacteria begin to exhaust nutrients, their growth rate slows, ultimately leading to the non-replicative state of quiescence. Adaptation to nutrient limitation requires widespread metabolic remodeling that is in part mediated by the phosphorylated nucleotides guanosine tetra- and penta-phosphate, collectively (p)ppGpp. We have developed a novel reporter of (p)ppGpp abundance in the Gram-positive bacterium Bacillus subtilis based on the recent identification of a riboswitch that binds (p)ppGpp and modulates transcription via regulation of a transcriptional terminator. Placement of an unstable reporter, firefly luciferase, downstream of the riboswitch allows for sensitive and dynamic assessment of (p)ppGpp. We first confirm that the reporter accurately reflects (p)ppGpp abundance in a variety of well-established conditions. We then proceed to use it to demonstrate the physiological importance of several mechanisms of regulation of (p)ppGpp metabolism previously observed only in vitro including allosteric interactions between (p)ppGpp synthesis enzymes and the hydrolytic activity of a (p)ppGpp synthase. (p)ppGpp signaling has been implicated in the regulation of gene expression, and we demonstrate a close temporal association between gene expression and (p)ppGpp abundance, indicating a rapid, and therefore likely direct mechanism of (p)ppGpp dependent gene activation. Thus, this reporter provides a new, comprehensive analysis of (p)ppGpp signaling in vivo and offers the potential ability to sensitively monitor the temporal dynamics of (p)ppGpp abundance under diverse environmental conditions. Author SummaryMost bacteria adapt to stressful conditions such as nutrient limitation by synthesizing a signaling molecule, known as ppGpp, that consists of a hyper-phosphorylated GTP. Synthesis of ppGpp affects most aspects of cellular physiology including replication, transcription and translation. We present here a method that for the first time allows measurement of ppGpp abundance in living cells, greatly facilitating investigation into ppGpp metabolism.

microbiology↗

bioRxiv2021

Crosstalk between guanosine nucleotides regulates cellular heterogeneity in protein synthesis during nutrient limitation

Phenotypic heterogeneity of microbial populations can facilitate survival in dynamic environments by generating sub-populations of cells that may have differential fitness in a future environment. Bacillus subtilis cultures experiencing nutrient limitation contain distinct sub-populations of cells exhibiting either comparatively high or low protein synthesis activity. This heterogeneity requires the production of phosphorylated guanosine nucleotides (pp)ppGpp by three synthases: SasA, SasB, and RelA. Here we show that these enzymes differentially affect this bimodality: RelA and SasB are necessary to generate the sub-population of cells exhibiting low protein synthesis whereas SasA is necessary to generate cells exhibiting comparatively higher protein synthesis. The RelA product (pppGpp) allosterically activates SasB and we find, in contrast, that the SasA product (pGpp) competitively inhibits this activation. Finally, we provide in vivo evidence that this antagonistic interaction mediates the observed heterogeneity in protein synthesis. This work therefore identifies the mechanism underlying phenotypic heterogeneity in the central physiological process of protein synthesis. Author SummaryUpon encountering conditions that are unfavorable to growth, such as nutrient limitation, bacteria enter into a quiescent phenotype that is mediated by group of guanosine nucleotides collectively known as (pp)pGpp. These nucleotides direct the down-regulation of energy intensive processes and are essential for a striking heterogeneity in protein synthesis observed during exit from rapid growth. Here, we show that a network of (pp)pGpp synthases is responsible for this heterogeneity and describe a mechanism that allows for the integration of multiple signals into the decision to down regulate the most energy intensive process in a cell.

microbiology↗

bioRxiv2021

Ser/Thr phospho-regulation by PknB and Stp mediates bacterial quiescence and antibiotic persistence in Staphylococcus aureus

Staphylococcus aureus colonizes 30 to 50% of healthy adults and can cause a variety of diseases, ranging from superficial to life-threatening invasive infections such as bacteraemia and endocarditis. Often, these infections are chronic and difficult-to-treat despite adequate antibiotic therapy. Most antibiotics act on metabolically active bacteria in order to eradicate them. Thus, bacteria with minimized energy consumption resulting in metabolic quiescence, have increased tolerance to antibiotics. The most energy intensive process in cells - protein synthesis - is attenuated in bacteria entering into quiescence. Eukaryote-like serine/threonine kinases (STKs) and phosphatases (STPs) can fine-tune essential cellular processes, thereby enabling bacteria to quickly respond to environmental changes and to modulate quiescence. Here, we show that deletion of the only annotated functional STP, named Stp, in S. aureus leads to increased bacterial lag-phase and phenotypic heterogeneity under different stress challenges, including acidic pH, intracellular milieu and in vivo abscess environment. This growth delay was associated with reduced intracellular ATP levels and increased antibiotic persistence. Using phosphopeptide enrichment and mass spectrometry-based proteomics, we identified possible targets of Ser/Thr phosphorylation that regulate cellular processes and bacterial growth, such as ribosomal proteins including the essential translation elongation factor EF-G. Finally, we show that acid stress leads to a reduced translational activity in the stp deletion mutant indicating metabolic quiescence correlating with increased antibiotic persistence. One-sentence summaryPhospho-regulation mediates quiescence and antibiotic persistence in Staphylococcus aureus.

microbiology↗

bioRxiv2020

Bioluminescence dynamics in single germinating bacterial spores reveal metabolic heterogeneity

Spore-forming bacteria modulate their metabolic rate by over 5 orders of magnitude as they transition between dormant spores and vegetative cells, and thus represent an extreme case of phenotypic variation. During environmental changes in nutrient availability, clonal populations of spore-forming bacteria exhibit individual differences in cell fate, timing of phenotypic transitions, and gene expression. One potential source of this variability is metabolic heterogeneity, but this has not yet been measured, as existing single-cell methods are not easily applicable to spores due to their small size and strong autofluorescence. Here, we use the bacterial bioluminescence system and a highly sensitive microscope to measure metabolic dynamics in thousands of B. subtilis spores as they germinate. We observe and quantitate large variations in the bioluminescence dynamics across individual spores that can be decomposed into contributions from variability in germination timing, the amount of endogenously produced luminescence substrate, and the intracellular reducing power. This work shows that quantitative measurement of spore metabolism is possible and thus it opens venues for future study of the thermodynamic nature of dormant states.

biophysics↗

bioRxiv2019

Identification and biochemical characterization of a novel eukaryotic-like Ser/Thr kinase in E. coli

microbiology↗

bioRxiv2019

(p)ppGpp directly regulates translation initiation during entry into quiescence

Many bacteria exist in a state of metabolic quiescence where they must minimize energy consumption so as to maximize available resources over a potentially extended period of time. As protein synthesis is the most energy intensive metabolic process in a bacterial cell, it would be an appropriate target for downregulation during the transition from growth to quiescence. We find that when Bacillus subtilis exits growth, a subpopulation of cells emerges with very low levels of protein synthesis dependent on synthesis of the nucleotides (p)ppGpp. We show that (p)ppGpp inhibits protein synthesis in vivo and in vitro by preventing the allosteric activation of the essential GTPase Initiation Factor 2 (IF2) during translation initiation. Finally, we demonstrate that IF2 is an authentic in vivo target of (p)ppGpp during the entry into quiescence, thus providing a mechanistic basis for the observed attenuation of protein synthesis.

microbiology↗

bioRxiv2019

Ribosome dimerization prevents loss of essential ribosomal proteins during quiescence

The formation of ribosome dimers during periods of quiescence is widespread among bacteria and some higher eukaryotes. However, the mechanistic importance of dimerization is not well understood. In bacteria ribosome dimerization is mediated by the Hibernation Promoting Factor (HPF). Here, we report that HPF from the Gram-positive bacterium Bacillus subtilis preserves active ribosomes by preventing the loss of essential ribosomal proteins. Ribosomes isolated from strains either lacking HPF ({Delta}hpf) or encoding a mutant allele of HPF that binds the ribosome but does not mediate dimerization were substantially depleted of the small subunit proteins S2 and S3. Strikingly, these proteins are located at the ribosome dimer interface. We used single particle cryo-EM to further characterize ribosomes isolated from a {Delta}hpf mutant strain and observed that many were missing S2, S3, or both. These data support a model in which the ribosome dimerization activity of HPF evolved to protect labile proteins that are essential for ribosome function.\n\nSignificance StatementWhen nutrients become scarce, many bacterial species enter an extended state of quiescence. A major challenge of this state is how to attenuate protein synthesis, the most energy consuming cellular process, while preserving ribosomes for the return to favorable conditions. Here, we show that the ribosome-binding protein HPF which dimerizes ribosomes functions to protect essential ribosomal proteins at the dimer interface. HPF is almost universally conserved in bacteria and HPF deletions in diverse species exhibit decreased viability under nutrient limitation. Our data provide mechanistic insight into this phenotype and establish a role for HPF in maintaining translationally competent ribosomes during quiescence.

microbiology↗

bioRxiv2019

Clickable methionine as a universal probe for labelling intracellular bacteria

Despite their clinical and biological importance, the cell biology of obligate intracellular bacteria is less well understood than that of many free-living model organisms. One reason for this is that they are mostly genetically intractable. As a consequence, it is not possible to engineer strains expressing fluorescent proteins and therefore fluorescence light microscopy - a key tool in host-pathogen cell biology studies - is difficult. Strain diversity limits the universality of antibody-based immunofluorescence approaches. Here, we have developed a universal labelling protocol for intracellular bacteria based on a clickable methionine analog. Whilst we have applied this to obligate intracellular bacteria, we expect it to be useful for labelling free living bacteria as well as other intracellular pathogens.

microbiology↗

bioRxiv2018

Multisite phosphorylation regulates phenotypic variability in antibiotic tolerance

Isogenic populations of cells exhibit phenotypic variability that has specific physiological consequences. For example, individual bacteria within a population can differ in their sensitivity to an antibiotic, but whether this variability can be regulated or is generally an unavoidable consequence of stochastic fluctuations is unclear. We observed that a bacterial stress response gene, the (p)ppGpp synthetase sasA, exhibits high levels of extrinsic noise in expression, suggestive of a regulatory process. We traced this variability to the convergence of two signaling systems that together control the multisite phosphorylation of a transcription factor, an event largely unexplored in bacteria, This regulatory intersection between a Ser/Thr kinase and a prototypical two component system is crucial for controlling the appearance of outliers, rare cells with unusually high levels of sasA expression. Additionally, by examining the full distributions of gene expression we calculated the contribution of the additional Ser/Thr kinase-dependent phosphorylation in setting the relative abundance of cells with a given a level of SasA. We then created a predictive model for the probability of a given cell surviving antibiotic treatment as a function of sasA expression. Therefore, our data show that multisite phosphorylation can be used to strongly regulate variations in phenotypes across a bacterial population.

microbiology↗

bioRxiv2018

Identification and biochemical characterization of a novel PP2C-like Ser/Thr phosphatase in E. coli

In bacteria, signaling phosphorylation is thought to occur primarily on His and Asp residues. However, phosphoproteomic surveys in phylogenetically diverse bacteria over the past decade have identified numerous proteins that are phosphorylated on Ser and/or Thr residues. Consistently, genes encoding Ser/Thr kinases are present in many bacterial genomes such as E. coli, which encodes at least three Ser/Thr kinases. Since Ser/Thr phosphorylation is a stable modification, a dedicated phosphatase is necessary to allow reversible regulation. Ser/Thr phosphatases belonging to several conserved families are found in bacteria. One family of particular interest are Ser/Thr phosphatases which have extensive sequence and structural homology to eukaryotic Ser/Thr PP2C phosphatases. These proteins, called eSTPs (eukaryotic-like Ser/Thr phosphatases), have been identified in a number of bacteria, but not in E. coli. Here, we describe a previously unknown eSTP encoded by an E. coli ORF, yegK, and characterize its biochemical properties including its kinetics, substrate specificity and sensitivity to known phosphatase inhibitors. We investigate differences in the activity of this protein in closely related E. colistrains. Finally, we demonstrate that this eSTP acts to dephosphorylate a novel Ser/Thr kinase which is encoded in the same operon.\n\nImportanceRegulatory protein phosphorylation is a conserved mechanism of signaling in all biological systems. Recent phosphoproteomic analyses of phylogenetically diverse bacteria including the model Gram-negative bacterium E. coli demonstrate that many proteins are phosphorylated on serine or threonine residues. In contrast to phosphorylation on histidine or aspartate residues, phosphorylation of serine and threonine residues is stable and requires the action of a partner Ser/Thr phosphatase to remove the modification. Although a number of Ser/Thr kinases have been reported in E. coli, no partner Ser/Thrphosphatases have been identified. Here, we biochemically characterize a novel Ser/Thr phosphatase that acts to dephosphorylate a Ser/Thr kinase that is encoded in the same operon.

microbiology↗