Biology subjects

Gray, M. J.

Publications and source records attributed to Gray, M. J..

14 recordsLinked to original sources

bioRxiv2026

The roles and synthesis of inorganic polyphosphate in Bacillus cereus

Inorganic polyphosphate (polyP) is a universally conserved biopolymer central to bacterial stress survival, yet understanding of its roles derives almost entirely from Gram-negative models in which polyP accumulates intracellularly following nutrient downshift. We examined polyP metabolism in the Gram-positive spore-forming bacterium Bacillus cereus using deletions of the polyP kinases PPK1 and PPK2 and the exopolyphosphatase PPX. Intracellular polyP synthesis required PPK1 and was opposed by PPX and PPK2: ppx mutants accumulated polyP in sporulation medium by 24 hours, ppx ppk2 double mutants accumulated more, and no ppk1 mutant accumulated any. A ppk1 ppx double mutant could not be generated, suggesting that unopposed PPK2 activity is lethal. Unlike Escherichia coli and Pseudomonas aeruginosa, B. cereus did not accumulate polyP after shift to minimal medium, increasing only modestly in stationary phase. Fluorescence and transmission electron microscopy localized intracellular polyP to electron-dense granules within ribosome-depleted cytoplasm. Cells bearing these granules remained membrane-intact yet failed to resume growth over 8 hours in rich medium, leading us to propose that polyP drives ribosome sequestration into condensates and a hibernation-like state. Unexpectedly, B. cereus also released close to 100{micro}M polyP extracellularly during late stationary phase, even in a ppk1 ppk2 mutant lacking both known synthetases. Extracellular polyP resisted hydrolysis by purified PPX even after deproteinization, indicating an atypical structure. Bacillus thuringiensis and Bacillus anthracis released similar amounts of extracellular polyP. Together these results identify two distinct polyP pools in the B. cereus group: a PPK1-dependent intracellular pool and an extracellular pool made by an uncharacterized pathway. ImportanceBacillus cereus is a spore-forming bacterium that causes foodborne illness and persists in soil and food-processing environments, where survival depends on managing phosphate and energy reserves during starvation. Inorganic polyphosphate (polyP), an ancient polymer used by nearly all cells to withstand stress, has been studied almost entirely as a molecule stored inside bacteria. We show that Bacillus cereus maintains two separate polyP pools. The internal pool is made by a known enzyme (PPK1) and is associated with dormant cells whose protein-making machinery appears to be packed away. The external pool is made without any known polyP-synthesizing enzyme, pointing to a novel polyP synthesis pathway that is shared with the close relatives Bacillus thuringiensis and Bacillus anthracis.

microbiology↗

bioRxiv2025

Probiotic biogeography and sepsis prevention in the neonatal intestine

Neonatal infection is one of the leading causes of neonatal morbidity and mortality worldwide, particularly in those born prematurely or with low birth weight. Probiotic bacteria have been demonstrated to protect against the development of neonatal intestinal dysbiosis and are widely used in peri- and post-natal clinical settings. However, formulations and efficacy are highly variable, highlighting a critical gap in the current understanding of the mechanistic underpinnings of successful probiotic interventions in this population. Furthermore, current studies on probiotic efficacy largely rely on indirect or relative readouts of intestinal bacterial burden. Herein, we directly mapped the biogeography of intestinal colonization and quantify the probiotic effects of Escherichia coli Nissle 1917 (EcN) and Ligilactobacillus murinus strain V10 against Klebsiella pneumoniae dysbiosis across the span of the neonatal murine intestine. Despite substantial differences in biogeography within the intestine, both EcN and L. murinus V10 significantly reduced K. pneumoniae colonization and mortality from K. pneumoniae sepsis, with EcN doing so much more robustly. EcNs probiotic effect was partially dependent on its ability to respire oxygen. Contrary to the dominant paradigm and practice in the probiotic field, combining multiple probiotic strains did not necessarily increase efficacy. Simultaneous treatment with EcN and L. murinus V10 was less effective than EcN treatment alone at preventing death from sepsis. These results highlight important variables which must be taken into account in the design of effective future probiotic intervention strategies. IMPORTANCEIn this work we use a mouse model of late-onset neonatal sepsis (LOS) to rigorously test fundamental assumptions that underlie the current paradigm for understanding the impact of probiotics on intestinal disease. We demonstrate that two distantly related probiotic bacteria (Escherichia coli Nissle 1917 and Ligilactobacillus murinus V10) can each effectively reduce both intestinal colonization and death caused by the LOS pathobiont Klebsiella pneumoniae, acting by distinct ecological and molecular mechanisms. Our results provide new evidence that will be critical for designing and implementing safe and effective probiotic treatment regimens for LOS, a devastating and difficult to treat disease. More broadly, our results show that ecological principles are key to understanding how interventions that modulate the gut microbiome work, and that some of the assumptions underlying current interventions need to be reevaluated, especially when it comes to combining multiple probiotic strains and species.

microbiology↗

bioRxiv2025

Identification of polyphosphate-binding proteins in E. coli uncovers targets involved in translation control and ribosome biogenesis

In many bacteria, polyphosphate kinase (PPK) enzymes use ATP to synthesize polyphosphate (polyP) in response to cellular stress. These chains of inorganic phosphates are joined by high-energy bonds and can reach hundreds of residues in length. PolyP plays diverse functions in helping bacteria adjust to changing environmental conditions. However, the molecular mechanisms underlying these functions are poorly understood. In eukaryotic cells, polyacidic serine- and lysine-rich (PASK) motifs of proteins can mediate binding to polyP chains. Whereas PASK motifs are relatively common in yeast and human cells, we report that these sequences are rare in bacteria commonly used for polyP research. Thus, to identify novel polyP-binding proteins in Escherichia coli, we carried out an untargeted screen and identified 7 novel targets with links to translation control and ribosome biogenesis. For two targets, the GTPase activating protein YihI and the ribonuclease Rnr, we mapped the regions of polyP interaction to non-PASK sequences and identified lysine residues critical for binding. We found that deletion of rnr suppressed the slow growth phenotype of {Delta}ppk mutants grown on minimal media. Conversely, ppk deletion resulted in decreased Rnr protein expression. These phenotypes were dependent on the polyP binding region of Rnr but independent of polyP binding itself, suggesting a complex interplay between PPK and Rnr function in E. coli. Overall, our work provides new insights into the scope of polyP binding proteins and extends the connections between polyP and the regulation of protein translation in E. coli.

molecular biology↗

bioRxiv2024

Genome-wide characterization of hypothiocyanite stress response of Escherichia coli reveals a potential role for RclB and RclC in lowering cell envelope permeability

Oxidative stress is one of the major methods of microbial population control and pathogen clearing by the mammalian immune system. The methods by which bacteria are able to escape damage by host-derived oxidants such as hydrogen peroxide (H2O2) and hypochlorous acid (HOCl) have been relatively well described, while other oxidants effect on bacteria and their genetic responses are not as well understood. Hypothiocyanite/hypothiocyanous acid (-OSCN/HOSCN) is one such antimicrobial oxidant. In this study, we used RNA-sequencing to characterize the global transcriptional response of Escherichia coli to treatment with HOSCN and observed that the response is different from the responses of E. coli to other oxidants such as H2O2, superoxide, or HOCl, and distinct from the responses of other bacteria such as Streptococcus pneumoniae and Pseudomonas aeruginosa to HOSCN. Furthermore, we found that deletion of the genes encoded downstream of HOSCN reductase rclA in E. coli, rclB and rclC, has a transcriptional effect on ompC and may play a role in membrane permeability to HOSCN. IMPORTANCEUnderstanding how bacteria sense and respond to oxidative stress provides insights into how our bodies interact with the microbial population within us. In this study, we have characterized the genetic response of E. coli to important immune oxidant hypothiocyanite and investigated the role of the rclABC genes in that response.

microbiology↗

bioRxiv2024

Inorganic polyphosphate and the stringent response coordinately control cell division and cell morphology in Escherichia coli

Bacteria encounter numerous stressors in their constantly changing environments and have evolved many methods to deal with stressors quickly and effectively. One well known and broadly conserved stress response in bacteria is the stringent response, mediated by the alarmone (p)ppGpp. (p)ppGpp is produced in response to amino acid starvation and other nutrient limitations and stresses and regulates both the activity of proteins and expression of genes. Escherichia coli also makes inorganic polyphosphate (polyP), an ancient molecule evolutionary conserved across most bacteria and other cells, in response to a variety of stress conditions, including amino acid starvation. PolyP can act as an energy and phosphate storage pool, metal chelator, regulatory signal, and chaperone, among other functions. Here we report that E. coli lacking both (p)ppGpp and polyP have a complex phenotype indicating previously unknown overlapping roles for (p)ppGpp and polyP in regulating cell division, cell morphology, and metabolism. Disruption of either (p)ppGpp or polyP synthesis led to formation of filamentous cells, but simultaneous disruption of both pathways resulted in cells with heterogenous cell morphologies, including highly branched cells, severely mislocalized Z-rings, and cells containing substantial void spaces. These mutants also failed to grow when nutrients were limited, even when amino acids were added. These results provide new insights into the relationship between polyP synthesis and the stringent response in bacteria and point towards their having a joint role in controlling metabolism, cell division, and cell growth. IMPORTANCECell division is a fundamental biological process, and the mechanisms that control it in Escherichia coli have been the subject of intense research scrutiny for many decades. Similarly, both the (p)ppGpp-dependent stringent response and inorganic polyphosphate (polyP) synthesis are well-studied, evolutionarily ancient, and widely conserved pathways in diverse bacteria. Our results indicate that these systems, normally studied as stress-response mechanisms, play a coordinated and novel role in regulating cell division, morphology, and metabolism even under non-stress conditions.

microbiology↗

bioRxiv2024

The role of metals in hypothiocyanite resistance in Escherichia coli

The innate immune system employs a variety of antimicrobial oxidants to control and kill host-associated bacteria. Hypothiocyanite/hypothiocyanous acid (-OSCN/HOSCN) is one such antimicrobial oxidant that is synthesized by lactoperoxidase, myeloperoxidase, and eosinophil peroxidase at sites throughout the human body. HOSCN has potent antibacterial activity while being largely non-toxic towards human cells. The molecular mechanisms by which bacteria sense and defend themselves against HOSCN have only recently begun to be elaborated, notably by the discovery of bacterial HOSCN reductase (RclA), an HOSCN-degrading enzyme widely conserved among bacteria that live on epithelial surfaces. In this paper, I show that Ni2+ sensitizes Escherichia coli to HOSCN by inhibiting glutathione reductase, and that inorganic polyphosphate protects E. coli against this effect, probably by chelating Ni2+ ions. I also found that RclA is very sensitive to inhibition by Cu2+ and Zn2+, metals that are accumulated to high levels by innate immune cells, and that, surprisingly, thioredoxin and thioredoxin reductase are not involved in HOSCN stress resistance in E. coli. These results advance our understanding of the contribution of different oxidative stress response and redox buffering pathways to HOSCN resistance in E. coli and illustrate important interactions between metal ions and the enzymes bacteria use to defend themselves against oxidative stress. IMPORTANCEHypothiocyanite (HOSCN) is an antimicrobial oxidant produced by the innate immune system. The molecular mechanisms by which host-associated bacteria defend themselves against HOSCN have only recently begun to be understood. The results in this paper are significant because they show that the redox buffer glutathione and enzyme glutathione reductase are critical components of the Escherichia coli HOSCN response, working by a mechanism distinct from that of the HOSCN-specific defenses provided by the RclA, RclB, and RclC proteins, and that metal ions (including nickel, copper, and zinc) may impact the ability of bacteria to resist HOSCN by inhibiting specific defensive enzymes (e.g. glutathione reductase or RclA).

microbiology↗

bioRxiv2021

The nitrogen phosphotransferase regulator PtsN (EIIANtr) regulates inorganic polyphosphate production in Escherichia coli

Inorganic polyphosphate (polyP) is synthesized by bacteria under stressful environmental conditions and acts by a variety of mechanisms to promote cell survival. While the kinase that synthesizes polyP (PPK, enocoded by the ppk gene) is well known, little is understood about how environmental stress signals lead to activation of this enzyme. Previous work has shown that the transcriptional regulators DksA, RpoN ({sigma}54), and RpoE ({sigma}24) positively regulate polyP production, but not ppk transcription, in Escherichia coli. In this work, we set out to examine the role of the alternative sigma factor RpoN and nitrogen starvation stress response pathways in controlling polyP synthesis in more detail. In the course of these experiments, we identified GlnG, GlrR, PhoP, PhoQ, RapZ, and GlmS as proteins that affect polyP production, and uncovered a central role for the nitrogen phosphotransferase regulator PtsN (EIIANtr) in a polyP regulatory pathway, acting upstream of DksA, downstream of RpoN, and apparently independently of RpoE. However, none of these regulators appears to act directly on PPK, and the mechanism(s) by which they modulate polyP production remain unclear. Unexpectedly, we also found that the pathways that regulate polyP production vary depending not only on the stress condition applied, but also on the composition of the media in which the cells were grown before exposure to polyP-inducing stress. These results constitute substantial progress towards deciphering the regulatory networks driving polyP production under stress, but highlight the remarkable complexity of this regulation and its connections to a broad range of stress-sensing pathways. IMPORTANCEBacteria respond to changes in their environments with a complex regulatory network that controls the expression and activity of a wide range of effectors important for their survival. This stress response network is critical for the virulence of pathogenic bacteria and for the ability of all bacteria to grow in natural environments. Inorganic polyphosphate (polyP) is an evolutionarily ancient and almost universally conserved stress response effector that plays multiple roles in virulence, stress response, and survival in diverse organisms. This work provides new insights into the connections between well characterized nitrogen starvation and cell envelope stress response signaling pathways and the production of polyP in Escherichia coli.

microbiology↗

bioRxiv2020

Experimental methodologies can affect pathogenicity of Batrachochytrium salamandrivorans infections

Controlled experiments are one approach to understanding the pathogenicity of etiologic agents to susceptible hosts. The recently discovered fungal pathogen, Batrachochytrium salamandrivorans (Bsal), has resulted in a surge of experimental investigations because of its potential to impact global salamander biodiversity. However, variation in experimental methodologies could thwart knowledge advancement by introducing confounding factors that make comparisons difficult among studies. Thus, our objective was to evaluate if variation in experimental methods changed inferences made on the pathogenicity of Bsal. We tested whether passage duration of Bsal culture, exposure method of the host to Bsal (water bath vs. skin inoculation), Bsal culturing method (liquid vs. plated), host husbandry conditions (aquatic vs. terrestrial), and skin swabbing frequency influenced diseased-induced mortality in a susceptible host species, the eastern newt (Notophthalmus viridescens). We found that disease-induced mortality was faster for eastern newts when exposed to a low passage isolate, when newts were housed in terrestrial environments, and if exposure to zoospores occurred via water bath. We did not detect differences in disease-induced mortality between culturing methods or swabbing frequencies. Our results illustrate the need to standardize methods among Bsal experiments; we provide suggestions for future experiments in the context of hypothesis testing.

ecology↗

bioRxiv2020

Complex interactions between DksA and stress-responsive alternative sigma factors control inorganic polyphosphate accumulation in Escherichia coli

Bacteria synthesize inorganic polyphosphate (polyP) in response to a variety of different stress conditions. PolyP protects bacteria by acting as a protein-stabilizing chaperone, metal chelator, or regulator of protein function, among other mechanisms. However, little is known about how stress signals are transmitted in the cell to lead to increased polyP accumulation. Previous work in the model enterobacterium Escherichia coli has indicated that the RNA polymerase-binding regulatory protein DksA is required for polyP synthesis in response to nutrient limitation stress. In this work, I set out to characterize the role of DksA in polyP regulation in more detail. I found that overexpression of DksA increases cellular polyP content (explaining the long-mysterious phenotype of dksA overexpression rescuing growth of a dnaK mutant at high temperature) and characterized the roles of known functional residues of DksA in this process, finding that binding to RNA polymerase is required, but none of the other functions of DksA appear to be necessary. Transcriptomics revealed genome-wide transcriptional changes upon nutrient limitation, many of which were affected by DksA, and follow-up experiments identified complex interactions between DksA and the stress-sensing alternative sigma factors FliA, RpoN, and RpoE that impact polyP production, indicating that regulation of polyP synthesis is deeply entwined in the multifactorial stress response network of E. coli. IMPORTANCEInorganic polyphosphate (polyP) is an evolutionarily ancient, widely conserved biopolymer required for stress resistance and pathogenesis in diverse bacteria, but we do not understand how its synthesis is regulated. In this work, I gained new insights into this process by characterizing the role of the transcriptional regulator DksA in polyP regulation in Escherichia coli and identifying previously unknown links between polyP synthesis and the stress-responsive alternative sigma factors FliA, RpoN, and RpoE.

microbiology↗

bioRxiv2019

Phosphate transporter PstSCAB of Campylobacter jejuni is essential for lactate-dependent growth and colonization in chickens

Campylobacter jejuni causes acute gastroenteritis world-wide and is transmitted primarily through poultry, in which it is often a commensal member of the intestinal microbiota. Previous RNASeq experiments showed that transcripts from an operon encoding a high affinity phosphate transporter (PstSCAB) of C. jejuni were among the most abundant when grown in chickens. Elevated levels of the pstSCAB mRNA were also identified in an RNASeq experiment from human infection studies. In this study, we explore the role of PstSCAB in the biology and colonization potential of C. jejuni. Our experimental results demonstrate that cells lacking PstSCAB survive poorly in stationary phase, nutrient-limiting media, and under osmotic conditions reflective of those in the chicken. Polyphosphate levels in the mutant cells were elevated at stationary phase, consistent with alterations in expression of polyphosphate metabolism genes. C. jejuni were highly attenuated in colonization of newly hatched chicks, recovered at levels several orders of magnitude below wild type. Mutant and wild type grew similarly in complex media but the pstSCAB mutant exhibited a significant growth defect in minimal media supplemented with L-lactate, postulated as a carbon source in vivo. Poor growth in lactate correlated with diminished expression of acetogenesis pathway genes previously demonstrated as important for colonizing chickens. The phosphate transport system is thus essential for diverse aspects of C. jejuni physiology and in vivo fitness and survival. ImportanceC. jejuni causes millions of gastrointestinal infections annually worldwide. Poultry and poultry products are major sources of C. jejuni infection to human as the microbe is a commensal colonizer of the chicken gastrointestinal tract. Due to the emergence of multi-drug resistance in C. jejuni, there is need to identify alternative ways to control this pathogen. Genes encoding the high-affinity phosphate transporter PstSCAB were highly expressed during colonization of C. jejuni in chicken and human. In this study, we address the role this high-affinity phosphate transporter PstSCAB of C. jejuni on chicken colonization and for its general physiology. PstSCAB is required for colonization in chicken, metabolism and survival under different stress responses and during growth on lactate, a potential substrate for growth of C. jejuni in chickens. Our study highlights that PstSCAB may be an effective target to develop mechanisms to control the bacterial burden in both chicken and human.

microbiology↗

bioRxiv2019

Isolation and maintenance of Batrachochytrium salamandrivorans cultures

Discovered in 2013, Batrachochytrium salamandrivorans (Bsal) is an emerging amphibian pathogen that causes ulcerative skin lesions and multifocal erosion. A closely related pathogen, Batrachochytrium dendrobatidis (Bd), has devastated amphibian populations worldwide, suggesting that Bsal poses a significant threat to global salamander biodiversity. To expedite research into this emerging threat, we seek to standardize protocols across the field so that results of laboratory studies are reproducible and comparable. We have collated data and experience from multiple labs to standardize culturing practices of Bsal. Here we outline common culture practices including a media for optimal Bsal growth, standard culture protocols, and a method for isolating Bsal from infected tissue.

microbiology↗

bioRxiv2019

RclA is a thermostable copper (II) reductase required for reactive chlorine resistance and host colonization in Escherichia coli.

Inflammatory bowel diseases (IBDs) are a growing health concern. Enterobacteria, including Escherichia coli, bloom to high levels in the gut during inflammation and strongly contribute to the pathology of IBDs. To survive in the inflamed gut, E. coli must tolerate high levels of antimicrobial compounds produced by the immune system, including toxic metals like copper and reactive chlorine oxidants like hypochlorous acid (HOCl). In this work, we show that the widely-conserved bacterial HOCl resistance enzyme RclA catalyzes the reduction of copper (II) to copper (I), and specifically protects E. coli against the combination of HOCl and intracellular copper, probably by preventing Cu(III) accumulation. E. coli lacking RclA were highly sensitive to HOCl and were defective in colonizing an animal host. Our results indicate unexpected complexity in the interactions between antimicrobial toxins produced by innate immune cells and suggest an important and previously unsuspected role for copper redox reactions during inflammation.

microbiology↗

bioRxiv2019

Complex responses to inflammatory oxidants by the probiotic bacterium Lactobacillus reuteri

Inflammatory diseases of the gut are associated with increased intestinal oxygen concentrations and high levels of inflammatory oxidants, including hydrogen peroxide (H2O2) and hypochlorous acid (HOCl), which are antimicrobial compounds produced by the innate immune system. This contributes to dysbiotic changes in the gut microbiome, including increased populations of pro-inflammatory enterobacteria (Escherichia coli and related species) and decreased levels of health-associated anaerobic Firmicutes and Bacteroidetes. The pathways for H2O2 and HOCl resistance in E. coli have been well-studied, but little is known about how commensal and probiotic bacteria respond to inflammatory oxidants. In this work, we have characterized the transcriptomic response of the anti-inflammatory, gut-colonizing Gram-positive probiotic Lactobacillus reuteri to both H2O2 and HOCl. L. reuteri mounts distinct responses to each of these stressors, and both gene expression and survival were strongly affected by the presence or absence of oxygen. Oxidative stress response in L. reuteri required several factors not found in enterobacteria, including the small heat shock protein Lo18, polyphosphate kinase 2, and RsiR, an L. reuteri-specific regulator of anti-inflammatory mechanisms. These results raise the intriguing possibility of developing treatments for inflammatory gut diseases that could sensitize pro-inflammatory enterobacteria to killing by the immune system while sparing anti-inflammatory, health-associated species.\n\nIMPORTANCEIt is becoming increasingly clear that effective treatment of inflammatory gut diseases will require modulation of the gut microbiota. Preventing pro-inflammatory bacteria from blooming while also preserving anti-inflammatory and commensal species is a considerable challenge, but our results suggest that it may be possible to take advantage of differences in the way different species of gut bacteria resist inflammatory oxidants to accomplish this goal.

microbiology↗

bioRxiv2019

Individual alpha frequency increases during a task but is unchanged by alpha-band flicker

Visual perception fluctuates in-synch with ongoing neural oscillations in the delta, theta, and alpha frequency bands of the human EEG. Supporting the relationship between alpha and perceptual sampling, recent work has demonstrated that variations in individual alpha frequency (IAF) correlate with the ability to discriminate one from two stimuli presented briefly in the same location. Other studies have found that after being presented with a flickering stimulus at alpha frequencies, perception of near-threshold stimuli fluctuates for a short time at the same frequency. Motivated by previous work, we were interested in whether this alpha entrainment involves shifts in IAF. While recording EEG, we tested whether two-flash discrimination (a behavioral correlate of IAF) can be influenced by [~]1s of rhythmic visual stimulation at two different alpha frequencies (8.3hz and 12.5hz). Speaking against the bottom-up malleability of IAF, we found no change in IAF during stimulation and no change in two-flash discrimination immediately afterwards. We also found synchronous activity that persisted after 12.5hz stimulation, which suggests that a separate source of alpha was entrained. Importantly, we replicated the correlation between IAF and two-flash discrimination in a no-stimulation condition, demonstrating the sensitivity of our behavioral measure. We additionally found that IAF increased during the task compared to rest, which demonstrates that IAF is influenced by top-down factors but is not involved in entrainment. In the framework of existing findings, we suggest that visual entrainment may involve ongoing perceptually-relevant oscillations from the delta to alpha frequency bands, serving to maintain rhythmic temporal expectations.

neuroscience↗