Biology subjects

Wolfe, A.

Publications and source records attributed to Wolfe, A..

At least 19 recordsLinked to original sources

bioRxiv2026

A Multisensor Framework Reveals Redox Constraints on Glycolysis in vivo

Genetically encoded biosensors have transformed the study of metabolism, yet measurements of individual metabolites often provide an incomplete view of pathway regulation. Here, we develop a multisensor framework in Caenorhabditis elegans neurons to interpret glycolytic dynamics and redox state in vivo. We combine biosensors for NADH/NAD, fructose-1,6-bisphosphate, lactate, and pyruvate to resolve metabolic responses during hypoxia and redox perturbation. To causally test how redox state modulates glycolysis in vivo, we cell-specifically expressed the NADH-producing enzyme EcSTH and the NADH oxidase LbNOX to bidirectionally tune neuronal NADH/NAD balance. These perturbations revealed that redox modulation is sufficient to constrain or relieve lower glycolytic activity. Elevation of NADH/NAD promoted accumulation of upper glycolytic intermediates while suppressing lower glycolytic responses during energetic stress, consistent with inhibition at the NAD-dependent GAPDH step. Conversely, oxidation of NADH relieved this constraint and shifted metabolite pools consistent with enhanced lower glycolytic activity. Elevated NADH/NAD ratios also impaired synaptic vesicle organization, linking redox-mediated glycolytic inhibition to neuronal function. As a case study for how integrated biosensor approaches can provide semi-quantitative insight into pathway-level metabolic regulation, we genetically perturbed endogenous NADH recycling pathways. These experiments revealed a hierarchical organization of neuronal redox buffering, with lactate dehydrogenase (LDH-1) serving as the dominant route for NAD regeneration during hypoxia and glycerol-3-phosphate dehydrogenase (GPDH-2) providing a secondary compensatory pathway. Graded impairment of NADH recycling resulted in corresponding increases in fructose-1,6-bisphosphate accumulation and synaptic defects, consistent with progressive inhibition of lower glycolysis. Together, these results establish a tractable in vivo system to probe causal relationships between redox state, glycolytic dynamics, and cellular physiology.

Molecular Biology↗

bioRxiv2026

Cross-study metagenomics analysis reveals distinct microbial signatures of urinary tract infections

BackgroundUrinary tract infections (UTIs) represent a major public health concern, increasingly complicated by rising antibiotic resistance, diminishing treatment efficacy, and increasing prevalence of recurrence. The urinary tract microbiome (urobiome) remains poorly characterized, despite its potential role in UTIs. ResultsTo provide a comprehensive overview of the urobiome, we here integrated seven publicly available shotgun metagenomics studies, linking microbial composition to clinical infection status or diagnostics. Community-level analyses revealed distinct urobiome clusters, defined by one predominant bacterial taxon. Genome-resolved metagenomics allowed recovery of high-quality metagenome-assembled genomes (MAGs), enabling phylogenetic reconstruction, prediction of pathogenic potential, and profiling of antimicrobial resistance genes across multiple taxa. We then analyzed the pangenome of the clinically significant Escherichia coli species and found that its genomic variation is driven more by phylotype than isolation source or UTI status, supporting a model of opportunistic infection. ConclusionsTaken together, our analyses represent a systematic, cross-study view of the urobiome that emphasizes the ecological complexity of the urobiome and the importance of integrating functional and phylogenetic information when studying UTIs.

microbiology↗

bioRxiv2026

Carbamylome analysis reveals regulatory roles for lysine carbamylation in β-cell glycolytic and insulin-processing enzymes

In type 1 diabetes (T1D), insulin-producing {beta} cells are destroyed by an autoimmune response driven by pro-inflammatory cytokines, including interferons. {beta}-cell cytokine signaling is mediated in part by post-translational modifications, such as phosphorylation and acetylation. However, the role of other post-translational modifications in {beta}-cell cytokine signaling represents an important knowledge gap. In the context of autoimmune diseases, lysine carbamylation has gained attention for its role in pathogenesis. Here, we investigate the role of carbamylation in T1D. We found that pancreatic islet cells from the T1D model, non-obese diabetic (NOD) mice, exhibit 11% carbamylation-positive cells, whereas non-diabetic CD1 mice have only 5%. Proteomics analysis of the MIN6 insulin-producing cell line treated with a cocktail of three pro-inflammatory cytokines IFN{gamma} + IL-1{beta} + TNF identified 284 carbamylated peptides from 222 proteins impacted by the cytokine treatment. Integration of carbamylation and acetylation provided a deep view of the cytokine-regulated PTMs and potential points of interplay. A functional-enrichment analysis revealed that carbamylation was enriched in pathways related to autoimmune diseases, metabolism, DNA replication, and protein translation. Moreover, functional testing demonstrated that carbamylation inhibits the glycolytic enzyme aldolase A and the insulin-processing enzyme carboxypeptidase E, identifying a possible role for cytokine-induced {beta}-cell dysfunction. In summary, protein carbamylation is elevated in islets from NOD mice, and pro-inflammatory cytokine treatment regulates protein carbamylation in MIN6 cells. These data identify carbamylation as a potential regulatory mechanism for {beta}-cell metabolism and insulin production in the context of islet inflammation.

biochemistry↗

bioRxiv2026

PTMOverlay: A Proteomic Tool to Visualize Post-Translational Modifications Across Evolution

Evolutionary conservation has been considered a hallmark of essential basic functions in cells. Therefore, the study of evolutionarily conserved post-translational modifications (PTMs) can provide insight into their role in protein function. In this context, mass spectrometry can identify and quantify thousands of PTM sites. However, a major bottleneck lies in analyzing the large amounts of data collected by the mass spectrometer. Here we address the need for a protein sequence alignment tool for multiple PTMs across several species. We developed a tool named PTMOverlay that takes peptide identification output files and overlays PTM sites onto multiple protein sequence alignments. Examining 31 bacteria isolates, we combined their protein sequences with select PTM types, including acetylation, phosphorylation, monomethylation, dimethylation, and trimethylation. The tool revealed a variety of conserved modification sites on the bacterial central carbon metabolism. Further structural analysis revealed possible interactions between methylated arginine and lysine residues with phosphothreonine/serine sites on the homodimer interface of enolase. Overall, this tool can parse large amounts of mass spectrometry data and allows for more informed and efficient selection of sites for future studies of protein function.

systems biology↗

bioRxiv2025

Enterococcus faecalis Influences the Transcription and Metabolism of Pathogenic Escherichia coli when Grown in Co-Culture

UTI involves bacterial growth in the disparate environments of the bladder and within uroepithelial cells. The bladder is a low-nutrient environment that nonetheless supports rapid growth. Phylogenetic group B2 Escherichia coli (Ec) is frequently isolated from urinary tract infection (UTI) patients. Non-B2 strains have also been isolated and are often co-isolated with Enterococcus faecalis (Ef). We characterized the interaction between three co-isolated Ec-Ef pairs and other Ec-Ef combinations in a nutrient-rich medium which was intended to emulate the rapid growth condition of the bladder. In this medium, Ef had little effect on Ec growth but resulted in major transcriptome differences. Ef affected the non-B2 and B2 strains differently. For the non-B2 Ec strains, Ef induced transcript for genes whose products degrade ornithine via putrescine to succinate which is subsequently metabolized by the TCA cycle. For a control B2 Ec strain, Ef induced transcripts for growth rate-associated genes of macromolecular synthesis, and for similar metabolic enzymes, except for those that degrade putrescine to succinate. Unexpectedly, Ef induced transcripts for glyoxylate shunt enzymes in both non-B2 and B2 Ec strains. The bacterial disparate and constantly changing environments during UTI suggest the potential for different types of Ec-Ef interactions. Our results provide evidence for nutrient cross-feeding as one type of Ec-Ef interaction.

microbiology↗

bioRxiv2025

Evidence for an indigenous female mouse urobiome

Mice have been used as a valuable model of understanding pathophysiological mechanisms of urinary tract infection for almost six decades. Mice offer many advantages including genetic manipulation to test the role of genes and mechanisms, the availability of germ-free mice, and similarities to humans in innate immune defenses and the strain-dependent presence of vesicoureteral reflux. However, like with humans, the mouse bladder urine above the urinary sphincter has generally been assumed to be sterile. Yet, given the presence of urobiomes in other mammals and the emerging role of the human urobiome in the defense of the urinary bladder and upper urinary tract, the existence of a mouse urobiome should be critically examined as indigenous microbiota may influence experimental results. To determine if an indigenous murine urobiome exists, we obtained expressed urine from two sets of female C57BL/6J mice during three different intervals using two different extraction and sequencing methods and analyzed them simultaneously by a single method. For one set, we also obtained urine by suprapubic aspiration, which we compared to the paired expressed urine samples. We conclude that an indigenous murine urobiome exists and that expressed urine contains post-urethral microbes.

microbiology↗

bioRxiv2025

A Toolkit for In Vivo Mapping and Modulating Neurotransmission at Single-Cell Resolution

Understanding the organization and regulation of neurotransmission at the level of individual neurons and synapses requires tools that can track and manipulate transmitter-specific vesicles in vivo. Here, we present SynaptoTagMe, a suite of genetic tools in Caenorhabditis elegans to fluorescently label and conditionally ablate the vesicular transporters for glutamate, GABA, acetylcholine, and monoamines. Using a structure-guided approach informed by protein topology and evolutionary conservation, we engineered endogenously tagged versions for each transporter that maintain their physiological function while allowing for cell-specific, bright, and stable visualization. We also developed conditional knockout strains that enable targeted disruption of neurotransmitter synthesis or packaging in single neurons. We applied this toolkit to map co-expression of vesicular transporters across the C. elegans nervous system, revealing that over 10% of neurons exhibit co-transmission. Using the ADF sensory neuron as a case study, we demonstrate that serotonin and acetylcholine are trafficked in partially distinct vesicle pools. Our approach provides a powerful platform for mapping, monitoring, and manipulating neurotransmitter identity and use in vivo. The molecular strategies described here are likely applicable across species, offering a generalizable approach to dissect synaptic communication in vivo.

neuroscience↗

bioRxiv2023

Regulation of bacterial stringent response by an evolutionary conserved ribosomal protein L11 methylation

Lysine and arginine methylation is an important regulator of enzyme activity and transcription in eukaryotes. However, little is known about this covalent modification in bacteria. In this work, we investigated the role of methylation in bacteria. By reanalyzing a large phyloproteomics dataset from 48 bacterial strains representing 6 phyla, we found that almost a quarter of the bacterial proteome is methylated. Many of these methylated proteins are conserved across diverse bacterial lineages, including those involved in central carbon metabolism and translation. Among the proteins with the most conserved methylation sites is ribosomal protein L11 (bL11). bL11 methylation has been a mystery for five decades, as the deletion of its methyltransferase PrmA causes no cell growth defects. A comparative proteomics analysis combined with a guanosine polyphosphate assay of the {Delta}prmA mutant in Escherichia coli revealed that bL11 methylation is important for stringent response signaling. Moreover, we show that the {Delta}prmA mutant has an abnormal polysome profile, suggesting a role in ribosomal homeostasis during stationary growth phase. Overall, our investigation demonstrates that the evolutionary conserved bL11 methylation is important for stringent response signaling and ribosomal homeostasis. ImportanceProtein methylation in bacteria was first identified over sixty years ago. Since then, its functional role has been identified for only a few proteins. To better understand the functional role of methylation in bacteria, we analyzed a large phyloproteomics dataset encompassing 48 diverse bacteria. Our analysis revealed that ribosomal proteins are often methylated at conserved residues, suggesting that methylation of these sites may have a functional role in translation. Further analysis revealed that methylation of ribosomal protein L11 is important for stringent response signaling and ribosomal homeostasis.

systems biology↗

bioRxiv2023

Cataloging the Phylogenetic Diversity of Human Bladder Bacterial Isolates

Although the human bladder is reported to harbor unique microbiota, our understanding of how these microbial communities interact with their human hosts is limited, mostly owing to the lack of isolates to test mechanistic hypotheses. Niche-specific bacterial collections and associated reference genome databases have been instrumental in expanding knowledge of the microbiota of other anatomical sites, e.g., the gut and oral cavity. To facilitate genomic, functional, and experimental analyses of the human bladder microbiota, here we present a bladder-specific bacterial reference collection comprised of 1134 genomes. These genomes were culled from bacterial isolates obtained by a metaculturomic method from bladder urine collected by transurethral catheterization. This bladder-specific bacterial reference collection includes 196 different species, including representatives of major aerobes and facultative anaerobes, as well as some anaerobes. It captures 72.2 % of the genera found when we reexamined previously published 16S rRNA gene sequencing of 392 adult female bladder urine samples. Comparative genomic analysis found that the taxonomies and functions of the bladder microbiota shared more similarities with the vaginal microbiota than the gut microbiota. Whole-genome phylogenetic and functional analyses of 186 bladder E. coli isolates and 387 gut E. coli isolates supports the hypothesis that phylogroup distribution and functions of E. coli strains differ dramatically between these two very different niches. This bladder-specific bacterial reference collection is a unique resource that will enable hypothesis-driven bladder microbiota research and comparison to isolates from other anatomical sites.

microbiology↗

bioRxiv2022

Extracellular vimentin is sufficient to promote cell attachment, spreading, and motility by a mechanism involving N-acetyl glucosamine-containing structures

Vimentin intermediate filaments form part of the cytoskeleton of mesenchymal cells, but under pathological conditions often associated with inflammation, vimentin filaments depolymerize as the result of phosphorylation or citrullination, and vimentin oligomers are secreted or released into the extracellular environment. In the extracellular space, vimentin can bind surfaces of other cells and the extracellular matrix, and the interaction between extracellular vimentin and other cell types can trigger changes in cellular functions, such as activation of fibroblasts to a fibrotic phenotype. The mechanism by which extracellular vimentin binds external cell membranes and whether vimentin alone can act as an adhesive anchor for cells is largely uncharacterized. Here, we show that various cell types (normal and vimentin null fibroblasts, mesenchymal stem cells, A549 lung carcinoma cells) attach to and spread on polyacrylamide hydrogel substrates covalently linked to vimentin. Using traction force microscopy and spheroid expansion assays, we characterize how different cell types respond to extracellular vimentin. Cell attachment to and spreading on vimentin-coated surfaces is inhibited by hyaluronic acid (HA) degrading enzymes, HA synthase inhibitors, soluble heparin, or N-acetyl glucosamine, treatments that have little or no effect on the same cell types binding to collagen-coated hydrogels. These studies highlight the effectiveness of substrate-bound vimentin as a ligand for cells and suggest that carbohydrate structures, including the glycocalyx and glycosylated cell surface proteins that contain N-acetyl glucosamine, form a novel class of adhesion receptors for extracellular vimentin.

biophysics↗

bioRxiv2022

Spatially controlled microtubule nucleation and organization from crosslinker MAP65 condensates

Microtubule organization in cells is essential for the internal structure and coordination of events of intracellular transport, mitosis, and cell motility. For many cell types, microtubule organization is dominated by centrosomal nucleation that use gamma-tubulin to template filaments. Yet, some cell types lack centrosomes or centrioles, such as plant cells. Instead, microtubules nucleate from regions with high concentrations of microtubule binding and nucleating proteins. A mechanism that can drive high local concentrations of nucleators is liquid-liquid phase separation of proteins with intrinsically disordered regions. Here, we report that the plant microtubule nucleator and crosslinking protein, MAP65-1, can form phase separated condensates at physiological salt and temperature without extra crowding agents. These condensates are liquid at first and can mature to gel-like phases over time and with different environmental conditions. We show that these condensates can nucleate and grow microtubule bundles that form asters, regardless of the viscoelasticity of the condensate. When gel-like droplets nucleate and grow asters from a shell of tubulin at the surface, the microtubules are able to re-fluidize the MAP65 condensate. Condensate-induced cytoskeletal formation could be a universal mechanism for organization of the microtubule and actin cytoskeletons in all cell types, especially cells without centrosomes.

biophysics↗

bioRxiv2022

Profiling the plasmid conjugation potential of urinary E. coli

Escherichia coli is often associated with urinary tract infection (UTI). Antibiotic resistance in E. coli is an ongoing challenge in managing UTI. Extrachromosomal elements - plasmids - are vectors for clinically relevant traits, such as antibiotic resistance, with conjugation being one of the main methods for horizontal propagation of plasmids in bacterial populations. Targeting of conjugation components has been proposed as a strategy to curb spread of plasmid-borne antibiotic resistance. Understanding the types of conjugative systems present in urinary E. coli isolates is fundamental to assessing the viability of these strategies. In this study, we profile the two well studied conjugation systems (F-type and P-type) in the draft genomes of 65 urinary isolates of E. coli obtained from bladder urine of adult women with and without UTI-like symptoms. Most of these isolates contained plasmids and we found that conjugation genes are abundant/ubiquitous, diverse, and often associated with IncF plasmids. To validate conjugation of these urinary plasmids, we conjugated the plasmids from two urinary isolates, UMB1223 (predicted to have F-type genes) and UMB1284 (predicted to have P-type genes), into the K-12 E. coli strain MG1655. Overall, the findings of this study support the notion that care should be taken in targeting any individual component of a urinary E. coli isolates conjugation system, given the inherent mechanistic redundancy, gene diversity, and different types of conjugation systems in this population.

microbiology↗

bioRxiv2022

Normal Reproductive function in male mice lacking pituitary kisspeptin receptor.

The anterior pituitary secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) regulate gonadal development, gametogenesis and the secretion of the gonadal steroid hormones. The gonadotroph is primarily regulated by hypothalamic secretion of gonadotropin-releasing hormone (GnRH) from neurons of the rostral hypothalamus and is mediated by GnRH receptor signaling. Kisspeptin (KISS1)/kisspeptin receptor (KISS1R) signaling in GnRH neurons plays an essential role in reproductive function. As the kisspeptin receptor is present in the pituitary, kisspeptin signaling via the Kiss1r may regulate reproductive function at the level of pituitary. Using Cre/Lox technology, we deleted the Kiss1r gene in pituitary gonadotropes (PKiRKO). PKiRKO male and females have normal genital development, puberty onset, and fertility. Females have normal LH, FSH and estradiol while males had significantly increased basal serum FSH levels with no differences in basal serum LH, or testosterone levels. Overall, these findings indicate that the pituitary KISS1R does not play a role in male reproduction.

physiology↗

bioRxiv2021

A rare human centenarian variant of SIRT6 enhances genome stability and interaction with Lamin A

Sirtuin 6 (SIRT6) is a deacylase and mono-ADP ribosyl transferase (mADPr) enzyme involved in multiple cellular pathways implicated in the regulation of aging and metabolism. Targeted sequencing identified a SIRT6 allele containing two linked substitutions (N308K/A313S) as enriched in Ashkenazi Jewish (AJ) centenarians as compared to AJ control individuals. Characterization of this SIRT6 (centSIRT6) allele demonstrated it to be a stronger suppressor of LINE1 retrotransposons, confer enhanced stimulation of DNA double strand break repair, and more robust cancer cell killing compared to the wild type. Surprisingly, centSIRT6 displayed weaker deacetylase activity, but stronger mADPr activity, over a range of NAD+ concentrations and substrates. Additionally, centSIRT6 displayed a stronger interaction with Lamin A/C (LMNA), which correlated with enhanced ribosylation of LMNA. Our results suggest that enhanced SIRT6 function contributes to human longevity by improving genome maintenance via increased mADPr activity and enhanced interaction with LMNA.

molecular biology↗

bioRxiv2021

Hepatocyte androgen receptor in females mediates androgen-induced hepatocellular glucose mishandling and systemic insulin resistance

Androgen excess is one of the most common endocrine disorders of reproductive-aged women, affecting up to 20% of this population. Women with elevated androgens often exhibit hyperinsulinemia and insulin resistance. The mechanisms of how elevated androgens affect metabolic function are not clear. Hyperandrogenemia in a dihydrotestosterone (DHT)-treated female mouse model induces whole body insulin resistance possibly through activation of the hepatic androgen receptor (AR). We investigated the role of hepatocyte AR in hyperandrogenemia-induced metabolic dysfunction by using several approaches to delete hepatic AR via animal-, cell-, and clinical-based methodologies. We conditionally disrupted hepatocyte AR in female mice developmentally (LivARKO) or acutely by tail vein injection of an adeno-associated virus with a liver-specific promoter for Cre expression in ARfl/fl mice (adLivARKO). We observed normal metabolic function in littermate female Control (ARfl/fl) and LivARKO (ARfl/fl; Cre+/-) mice. Following chronic DHT treatment, female Control mice treated with DHT (Con-DHT) developed impaired glucose tolerance, pyruvate tolerance, and insulin tolerance, not observed in LivARKO mice treated with DHT (LivARKO-DHT). Further, during an euglycemic hyperinsulinemic clamp, the glucose infusion rate was improved in LivARKO-DHT mice compared to Con-DHT mice. Liver from LivARKO, and primary hepatocytes derived from LivARKO, and adLivARKO mice were protected from DHT-induced insulin resistance and increased gluconeogenesis. These data support a paradigm in which elevated androgens in females disrupt metabolic function via hepatic AR and insulin sensitivity was restored by deletion of hepatic AR.

molecular biology↗

bioRxiv2021

Phylogenomic study of Staphylococcus aureus and Staphylococcus haemolyticus clinical isolates from Egypt

Antibiotic resistant Staphylococcus infections are a global concern, with increasing cases of resistant Staphylococcus aureus and Staphylococcus haemolyticus found circulating in the Middle East. While extensive surveys have described the prevalence of resistant infections in Europe, Asia, and North America, the population structure of resistant staphylococcal Middle Eastern clinical isolates is poorly characterized. We performed whole genome sequencing of 56 S. aureus and 10 S. haemolyticus isolates from Alexandria Main University Hospital. Supplemented with additional publicly available genomes from the region (34 S. aureus and 6 S. haemolyticus), we present the largest genomic study of staphylococcal Middle Eastern isolates. These genomes include 20 S. aureus multilocus sequence typing (MLST) types and 9 S. haemolyticus MLSTs, including 3 and 1 new MLSTs, respectively. Phylogenomic analyses of each species core genome largely mirrored MLSTs, irrespective of geographical origin. The hospital-acquired spa t037/SCCmec III/MLST CC8 clone represented the largest clade, comprising 22% of S. aureus isolates. Similar to other regional genome surveys of S. aureus, the Middle Eastern isolates have an open pangenome, a strong indicator of gene exchange of virulence factors and antibiotic resistance genes with other reservoirs. We recommend stricter implementation of antibiotic stewardship and infection control plans in the region. Impact StatementStaphylococci are under-studied despite their prevalence within the Middle East. Methicillin-resistant Staphylococcus aureus (MRSA) is endemic to hospitals in this region, as are other antibiotic-resistant strains of S. aureus and S. haemolyticus. To provide insight into the strains currently in circulation within Egypt, we performed whole genome sequencing of 56 S. aureus and 10 S. haemolyticus isolates from Alexandria Main University Hospital (AMUH). Through analysis of these genomes, as well as other genomes of isolates from the Middle East, we were able to produce a more complete picture of the current diversity than traditional molecular typing strategies. Furthermore, the S. haemolyticus genome analyses provide the first insight into strains found in Egypt. Our analysis of resistance and virulence mechanisms carried by these strains provides invaluable insight into future plans of antibiotic stewardship and infection control within the region. Data SummaryRaw sequencing reads and assembled genomes can be found at BioProject Accession number PRJNA648411 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA648411).

microbiology↗

bioRxiv2019

Ablation of tanycytes of the arcuate nucleus and median eminence increases visceral adiposity and decreases insulin sensitivity in male mice.

Tanycytes are radial glial cells located in the mediobasal hypothalamus. Recent studies have proposed that tanycytes play an important role in hypothalamic control of energy homeostasis, although this has not been directly tested. Here, we report the phenotype of mice in which tanycytes of the arcuate nucleus and median eminence were conditionally ablated. Although the CSF-hypothalamic barrier was rendered more permeable, the blood-hypothalamic barrier was not altered. The metabolic effects of tanycyte ablation were likewise moderate. However, we consistently observed a significant increase in visceral fat distribution accompanying insulin insensitivity, but only in male mice, and without an effect on either body weight or food intake. A high-fat diet accelerated overall body weight gain in tanycyte-ablated mice, but the development of visceral adiposity and insulin insensitivity was attenuated. These results clarify the extent to which tanycytes regulate energy metabolism, and indicate a role for tanycytes in controlling body adiposity.

physiology↗

bioRxiv2018

The spermidine acetyltransferase SpeG regulates transcription of the small RNA RprA

Spermidine N-acetyltransferase (SpeG) acetylates and thus neutralizes toxic polyamines. Studies indicate that SpeG plays an important role in virulence and pathogenicity of many bacteria, which have evolved SpeG-dependent strategies to control polyamine concentrations and survive in their hosts. In Escherichia coli, the two-component response regulator RcsB is reported to be subject to N{varepsilon}-acetylation on several lysine residues, resulting in reduced DNA binding affinity and reduced transcription of the small RNA rprA; however, the physiological acetylation mechanism responsible for this behavior has not been fully determined. Here, we performed an acetyltransferase screen and found that SpeG inhibits rprA promoter activity in an acetylation-independent manner. Surface plasmon resonance analysis revealed that SpeG can physically interact with the DNA-binding carboxyl domain of RcsB. We hypothesize that SpeG interacts with the DNA-binding domain of RcsB and that this interaction might be responsible for SpeG-dependent inhibition of RcsB-dependent rprA transcription. This work provides a model for SpeG as a modulator of E. coli transcription through its ability to interact with the transcription factor RcsB. This is the first study to provide evidence that an enzyme involved in polyamine metabolism can influence the function of the global regulator RcsB, which integrates information concerning envelope stresses and central metabolic status to regulate diverse behaviors.

microbiology↗