Bacterial Stress Response Research Articles

Xanthine oxidase-lactoperoxidase system: Dose-dependent antibacterial effects and global gene expression changes in infant oral microbiota.

Xanthine oxidase (XO) and lactoperoxidase (LPO) are highly abundant enzymes in milk. Their substrates, xanthine and thiocyanate, are found in elevated amounts in infant saliva, leading to a proposed interaction between milk and saliva referred to as the XO-LPO system. This system is suggested to generate reactive oxygen and nitrogen species with potential antibacterial effects. The antibacterial activity of the XO-LPO system was assessed on bacteria cultured from the oral cavities of five infants, where a reduction in bacterial growth rate was observed at 40µg mL-1 of each enzyme and with complete inhibition achieved at 200µg mL-1. Gene expression analysis showed that XO-LPO treatment led to downregulation of several reactive oxygen species-related genes, suggesting a transient bacterial stress response. The study also observed downregulation of key glycolytic enzymes, indicating that XO-LPO treatment affects bacterial metabolism at transcriptional level, suggesting a possible mechanism of action for the XO-LPO system. Collectively, these findings offer new insights into the XO-LPO system, revealing novel aspects of the interaction between lactation and microbiome influence.

(P)ppGpp synthetase Rel facilitates cellulose formation of biofilm by regulating glycosyltransferase in Brucella abortus.

Biofilms are complex adhesive structures that establish chronic infection and allow robust protection from external stressors such as antibiotics. Cellulose as one of the compositions of bacteria biofilm which protect bacteria from stress, host immune responses and resistance to antibiotics. Bacterial stress responses are regulated via guanosine pentaphosphate and tetraphosphate (p)ppGpp. This molecule has been a target of research efforts to counteract biofilm formation in pathogenic bacteria. However, a role for (p)ppGpp synthetase Rel influencing in biofilms and its cellulose formation has not been identified in Brucella. Firstly, rel mutant significantly decreased biofilm biomass and rendered biofilms more susceptible to most antibiotics. The rel mutant also showed greatly decreased biofilm architectures including exopolysaccharide, extracellular DNA, and lipid. Remarkably, we found rel mutant significantly decreased biofilm cellulose formation. We further combined proteomic analysis to explore the key proteins involved in cellulose regulation of Rel in Brucella biofilm formation. 287 differentially expressed proteins (DEPs) were identified and enriched in diverse metabolic pathway between WT and Δrel strains including purine and sulfur metabolism, transcription factors and glycosyltransferases which may be related to cellulose formation. The Q-PCR showed that mRNA levels of only glycosyltransferase (WP_006161578.1) of the 12 down-DFPs had significantly upregulated in rel mutant contrast to WT strain and β-galactosidase assay showed a negative regulatory in rel mutant. Furthermore, Rel-dependent biofilms cellulose was also restored and accompanied by an increase in glycosyltransferase (WP_006161578.1) when glucose was added in TSB medium. Overall, this work expands the role of (p)ppGpp synthetase Rel as an important regulator in biofilm and cellulose formation that is tightly linked with pathogenicity and chronic persistent infections in Brucella.

Molecular Evolution of Paralogous Cold Shock Proteins in E. coli: A Study of Asymmetric Divergence and Protein Functional Networks.

Nine homologous Cold Shock Proteins (Csps) have been recognized in the E.coli Cold Shock Domain gene family. These Csps function as RNA chaperones. This study aims to establish the evolutionary relationships among these genes by identifying and classifying their paralogous counterparts. It focuses on the physicochemical, structural, and functional analysis of the genes to explore the phylogeny of the Csp gene family. Computational tools were employed for protein molecular modeling, conformational analysis, functional studies, and duplication-divergence assessments. The research also examined amino acid conservation, protein mutations, domain-motif patterns, and evolutionary residue communities to better understand residual interactions, evolutionary coupling, and co-evolution. H33, M5, W11 and F53 residues were highly conserved within the protein family. It was further seen that residues M5, G17, G58, G61, P62, A64, V67 were intolerant to any kind of mutation whereas G3, D40, G41, Y42, S44, T54, T68, S69 were most tolerable towards substitutions. The study of residue communities displayed that the strongest residue coupling was observed in N13, F18, S27, F31, and W11. It was observed that all the gene pairs except CspF/CspH had new motifs generated over time. It was ascertained that all the gene pairs underwent asymmetric expression divergence after duplication. The Ka/ Ks ratio also revealed that all residues undertook neutral and purifying selection pressure. New functions were seen to develop in gene pairs evident from generation of new motifs. The discovery of new motifs and functions in Csps highlights their adaptive versatility, crucial for E. coli's resilience to environmental stressors and valuable for understanding bacterial stress response mechanisms. These findings will pave the way for future investigations into Csp evolution, with potential applications in microbial ecology and antimicrobial strategy development.

Tracking the Geometric and Positional Isomerization of Lipid C═C Bonds in the Bacterial Stress Responses by Mass Spectrometry.

The position and configuration of the C═C bond have a significant impact on the spatial conformation of unsaturated lipids, which subsequently affects their biological functions. Double bond isomerization of lipids is an important mechanism of bacterial stress response, but its in-depth mechanistic study still lacks effective analytical tools. Here, we developed a visible-light-activated dual-pathway reaction system that enables simultaneous [2 + 2] cycloaddition and catalytic cis-trans isomerization of the C═C bond of unsaturated lipids via directly excited anthraquinone radicals. Density functional theory calculations revealed the oxygen radical addition transition state and the addition-elimination isomerization mechanism of the reaction. A full-dimensional resolution method for C═C bond position and configuration was developed based on the bifunctional reaction and liquid chromatography-mass spectrometry. This method was then applied to the study of bacterial environmental stress response mechanisms. The C═C bond cis-trans and positional isomerization patterns of Pseudomonas membrane lipids under temperature stress were discovered, and the effect of temperature stress on fatty acid biosynthesis was also revealed. This study not only provides an effective tool and key information for the study of bacterial stress response mechanisms, but also enriches the toolbox of visible light chemical reactions.

Exploiting the fitness cost of metallo-β-lactamase expression can overcome antibiotic resistance in bacterial pathogens.

Carbapenems are last-resort antibiotics for treating bacterial infections. The widespread acquisition of metallo-β-lactamases, such as VIM-2, contributes to the emergence of carbapenem-resistant pathogens, and currently, no metallo-β-lactamase inhibitors are available in the clinic. Here we show that bacteria expressing VIM-2 have impaired growth in zinc-deprived environments, including human serum and murine infection models. Using transcriptomic, genomic and chemical probes, we identified molecular pathways critical for VIM-2 expression under zinc limitation. In particular, disruption of envelope stress response pathways reduced the growth of VIM-2-expressing bacteria in vitro and in vivo. Furthermore, we showed that VIM-2 expression disrupts the integrity of the outer membrane, rendering VIM-2-expressing bacteria more susceptible to azithromycin. Using a systemic murine infection model, we showed azithromycin's therapeutic potential against VIM-2-expressing pathogens. In all, our findings provide a framework to exploit the fitness trade-offs of resistance, potentially accelerating the discovery of additional treatments for infections caused by multidrug-resistant bacteria.

RcsF-independent mechanisms of signaling within the Rcs phosphorelay.

The Rcs (regulator of capsule synthesis) phosphorelay is a conserved cell envelope stress response mechanism in enterobacteria. It responds to perturbations at the cell surface and the peptidoglycan layer from a variety of sources, including antimicrobial peptides, beta-lactams, and changes in osmolarity. RcsF, an outer membrane lipoprotein, is the sensor for this pathway and activates the phosphorelay by interacting with an inner membrane protein IgaA. IgaA is essential; it negatively regulates the signaling by interacting with the phosphotransferase RcsD. We previously showed that RcsF-dependent signaling does not require the periplasmic domain of the histidine kinase RcsC and identified a dominant negative mutant of RcsD that can block signaling via increased interactions with IgaA. However, how the inducing signals are sensed and how signal is transduced to activate the transcription of the Rcs regulon remains unclear. In this study, we investigated how the Rcs cascade functions without its only known sensor, RcsF, and characterized the underlying mechanisms for three distinct RcsF-independent inducers. Previous reports showed that Rcs activity can be induced in the absence of RcsF by a loss of function mutation in the periplasmic oxidoreductase DsbA or by overexpression of the DnaK cochaperone DjlA. We identified an inner membrane protein, DrpB, as a multicopy RcsF-independent Rcs activator in E. coli. The loss of the periplasmic oxidoreductase DsbA and the overexpression of the DnaK cochaperone DjlA each trigger the Rcs cascade in the absence of RcsF by weakening IgaA-RcsD interactions in different ways. In contrast, the cell-division associated protein DrpB uniquely requires the RcsC periplasmic domain for activation; this domain is not needed for RcsF-dependent signaling. This suggests the possibility that the RcsC periplasmic domain acts as a sensor for some Rcs signals. Overall, the results add new understanding to how this complex phosphorelay can be activated by diverse mechanisms.

Bioisosteric replacement of pyridoxal‐5′‐phosphate to pyridoxal‐5′‐tetrazole targeting Bacillus subtilisGabR

AbstractAntimicrobial resistance is a significant cause of mortality globally due to infections, a trend that is expected to continue to rise. As existing treatments fail and new drug discovery slows, the urgency to develop novel antimicrobial therapeutics grows stronger. One promising strategy involves targeting bacterial systems exclusive to pathogens, such as the transcription regulator protein GabR. Expressed in diverse bacteria including Escherichia coli, Bordetella pertussis, and Klebsiella pneumoniae, GabR has no homolog in eukaryotes, making it an ideal therapeutic target. Bacillus subtilis GabR (bsGabR), the most studied variant, regulates its own transcription and activates genes for GABA aminotransferase (GabT) and succinic semialdehyde dehydrogenase (GabD). This intricate regulatory system presents a compelling antimicrobial target with the potential for agonistic intervention to disrupt bacterial gene expression and induce cellular dysfunction, especially in bacterial stress responses. To explore manipulation of this system and the potential of this protein as an antimicrobial target, an in‐depth understanding of the unique PLP‐dependent transcription regulation is critical. Herein, we report the successful structural modification of the cofactor PLP and demonstrate the biochemical reactivity of the PLP analog pyridoxal‐5′‐tetrazole (PLT). Through both spectrophotometric and X‐ray crystallographic analyses, we explore the interaction between bsGabR and PLT, together with a synthesized GABA derivative (S)‐4‐amino‐5‐phenoxypentanoate (4‐phenoxymethyl‐GABA or 4PMG). Most notably, we present a crystal structure of the condensed, external aldimine complex within bsGabR. While PLT alone is not a drug candidate, it can act as a probe to study the detailed mechanism of GabR‐mediated function. PLT employs a tetrazole moiety as a bioisosteric replacement for phosphate in PLP. In addition, the PLP‐4PMG adduct observed in the structure may serve as a novel chemical scaffold for subsequent structure‐based antimicrobial design.

AcfA Regulates the Virulence and Cell Envelope Stress Response of Vibrio parahaemolyticus.

Vibrio parahaemolyticus is a ubiquitous inhabitant of estuarine and marine environments that causes vibriosis in aquatic animals and food poisoning in humans. Accessory colonizing factor (ACF) is employed by Vibrio to assist in the colonization and invasion of host cells leading to subsequent illnesses. In this work, ΔacfA, an in-frame deletion mutant strain lacking the 4th to the 645th nucleotides of the open reading frame (ORF) of the acfA gene, and the complementary strain acfA+ were constructed to decipher the function of AcfA in V. parahaemolyticus. The deletion of acfA had no effect on bacterial growth but resulted in a significant reduction in biofilm formation, hemolytic activity, mucus adhesion, and the accumulated mortality of zebrafish, compared to the wild-type strain and the complementary strain acfA+. Additionally, AcfA was involved in adapting to stressors, such as H2O2, EDTA, and acid, in V. parahaemolyticus. Furthermore, RNA-Seq transcriptome analysis was conducted to identify global gene transcription alterations resulting from deletion of the acfA gene. A total of 416 differentially expressed genes were identified in the ΔacfA vs. wild-type comparison, with 238 up-regulated genes and 178 down-regulated genes. The expression of genes associated with the type III secretion system, type VI secretion system, and oligopeptide permeases system were significantly reduced, and yet the expression of genes associated with cell envelope biosynthesis and response regulation system were enhanced dramatically in the absence of the acfA gene compared to the wild-type strain. These findings suggest that AcfA may play a role in the overall success of pathogenesis and the cell envelope stress response of V. parahaemolyticus.

A microaerobically induced small heat shock protein contributes to Rhizobium leguminosarum/Pisum sativum symbiosis and interacts with a wide range of bacteroid proteins.

During the establishment of the symbiosis with legume plants, rhizobia are exposed to hostile physical and chemical microenvironments to which adaptations are required. Stress response proteins including small heat shock proteins (sHSPs) were previously shown to be differentially regulated in bacteroids induced by Rhizobium leguminosarum bv. viciae UPM791 in different hosts. In this work, we undertook a functional analysis of the host-dependent sHSP RLV_1399. A rlv_1399-deleted mutant strain was impaired in the symbiotic performance with peas but not with lentil plants. Expression of rlv_1399 gene was induced under microaerobic conditions in a FnrN-dependent manner consistent with the presence of an anaerobox in its regulatory region. Overexpression of this sHSP improves the viability of bacterial cultures following exposure to hydrogen peroxide and to cationic nodule-specific cysteine-rich (NCR) antimicrobial peptides. Co-purification experiments have identified proteins related to nitrogenase synthesis, stress response, carbon and nitrogen metabolism, and to other relevant cellular functions as potential substrates for RLV_1399 in pea bacteroids. These results, along with the presence of unusually high number of copies of shsp genes in rhizobial genomes, indicate that sHSPs might play a relevant role in the adaptation of the bacteria against stress conditions inside their host.IMPORTANCEThe identification and analysis of the mechanisms involved in host-dependent bacterial stress response is important to develop optimal Rhizobium/legume combinations to maximize nitrogen fixation for inoculant development and might have also applications to extend nitrogen fixation to other crops. The data presented in this work indicate that sHSP RLV_1399 is part of the bacterial stress response to face specific stress conditions offered by each legume host. The identification of a wide diversity of sHSP potential targets reveals the potential of this protein to protect essential bacteroid functions. The finding that nitrogenase is the most abundant RLV_1399 substrate suggests that this protein is required to obtain an optimal nitrogen-fixing symbiosis.

The BfmRS stress response protects Acinetobacter baumannii against defects in outer membrane lipoprotein biogenesis.

As the cell's surface, the outer membrane (OM) of bacteria, such as Acinetobacter baumannii, is continuously under assault from the environment or host. OM integrity is needed for cell survival, and envelope stress responses (ESRs) act to detect and repair any defects. ESRs are well-defined in Escherichia coli but are poorly conserved. We sought to identify an ESR for the essential process of OM lipoprotein biogenesis in A. baumannii. We found that the BfmRS two-component system performs this function and does so without relying on its NlpE sensor homolog, suggesting a novel mechanism of stress sensing is involved in A. baumannii. Our work identifies a key cellular role for BfmRS.

Proteomic Insights into the Regulatory Role of CobQ Deacetylase in Aeromonas hydrophila.

Post-translational modifications are crucial in regulating biological functions across both prokaryotes and eukaryotes. In Aeromonas hydrophila, CobQ, a recently identified novel deacetylase, plays a significant role in lysine deacetylation, influencing bacterial metabolism and stress responses. The present study utilized quantitative proteomics to investigate the impact of cobQ deletion on the global protein expression profile in A. hydrophila. Through data-independent acquisition mass spectrometry, we identified 233 upregulated and 41 downregulated proteins in the cobQ deletion mutant (ΔahcobQ) strain compared to the wild-type (WT) strain. Key differentially expressed proteins were involved in oxidative phosphorylation, bacterial secretion, and ribosomal function. Additionally, phenotypic assays demonstrated that the ΔahcobQ strain exhibited an increased resistance to oxidative phosphorylation inhibitors, suggesting a pivotal role for AhCobQ in energy metabolism. Outer membrane proteins and efflux pumps also showed altered expression, indicating potential implications for membrane permeability and antibiotic resistance. These results suggested that AhCobQ plays a vital regulatory role in maintaining metabolic homeostasis and responding to environmental stress, highlighting its potential as a target for therapeutic interventions against A. hydrophila infections.

Combining multiple stressors blocks bacterial migration and growth.

In nature, organisms experience combinations of stressors. However, laboratory studies use batch cultures, which simplify reality and focus on population-level responses to individual stressors.1,2,3,4,5 In recent years, bacterial stress responses have been examined with single-cell resolution using microfluidics.6,7,8,9,10,11,12 Here, we use a microfluidic approach to simultaneously provide a physical stressor (shear flow) and a chemical stressor (H2O2) to the human pathogen Pseudomonas aeruginosa. By treating cells with levels of flow and H2O2 that commonly co-occur in human host tissues,13,14,15,16,17,18 we discover that previous reports significantly overestimate the H2O2 levels required to block bacterial growth. Specifically, we establish that flow increases H2O2 effectiveness 50-fold, explaining why previous studies lacking flow required much higher concentrations. Using natural H2O2 levels, we identify the core H2O2 regulon, characterize OxyR-mediated dynamic regulation, and demonstrate that multiple H2O2 scavenging systems have redundant roles. By examining single-cell behavior, we serendipitously discover that the combined effects of H2O2 and flow block pilus-driven surface migration. Thus, our results counter previous studies and reveal that natural levels of H2O2 and flow synergize to restrict bacterial motility and survival. By studying two stressors at once, our research highlights the limitations of oversimplifying nature and demonstrates that physical and chemical stress can combine to yield unpredictable effects.

Unraveling the genetic basis of Rhizobium rhizogenes-mediated transformation and hairy root formation in rose using a genome-wide association study

Key MessageMultiple QTLs reveal the polygenic nature of R. rhizogenes-mediated transformation and hairy root formation in roses, with five key regions explaining 12.0–26.9% of trait variability and transformation-related candidate genes identified.Understanding genetic mechanisms of plant transformation remains crucial for biotechnology. This is particularly relevant for roses and other woody ornamentals that exhibit recalcitrant behavior in transformation procedures. Rhizobium rhizogenes-mediated transformation leading to hairy root (HR) formation provides an excellent model system to study transformation processes and host–pathogen interactions. Therefore, this study aimed to identify quantitative trait loci (QTLs) associated with HR formation and explore their relationship with adventitious root (AR) formation in rose as a model for woody ornamentals. A diversity panel of 104 in vitro grown rose genotypes was transformed with R. rhizogenes strain ATCC 15834 carrying a green fluorescent protein reporter gene. Phenotypic data on callus and root formation were collected for laminae and petioles. A genome-wide association study using 23,419 single-nucleotide polymorphism markers revealed significant QTLs on chromosomes one and two for root formation traits. Five key genomic regions explained 12.0–26.9% of trait variability, with some peaks overlapping previously reported QTLs for AR formation. This genetic overlap was supported by weak to moderate correlations between HR and AR formation traits, particularly in petioles. Candidate gene identification through literature review and transcriptomic data analysis revealed ten candidate genes involved in bacterial response, hormone signaling, and stress responses. Our findings provide new insights into the genetic control of HR formation in roses and highlight potential targets for improving transformation efficiency in ornamental crops, thereby facilitating future research and breeding applications.

Using In Silico Methods to Identify Protein Tyrosine Kinase A (PtkA) Homolog in Non-Tuberculous Mycobacteria (NTM)

Non-tuberculous mycobacteria (NTM) represent a diverse group of mycobacterial species known for causing opportunistic infections, especially in individuals with underlying health conditions. Unlike Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, NTM species exhibit different pathogenic characteristics and drug resistance mechanisms, making them increasingly relevant in clinical settings. PtkA is a crucial protein tyrosine kinase that regulates bacterial growth, stress response, and virulence by phosphorylating various substrates in Mtb. Understanding whether PtkA homologs exist in NTM could provide insights into their virulence and resistance mechanisms. In silico approaches, which utilize computational tools for sequence alignment, structure prediction, and functional annotation, offer a powerful means to identify homologous proteins across different species. In this article, we have employed tools like BLAST (Basic Local Alignment Search Tool), protein structure databases, and the NTM database to identify PtkA homologs in NTM genomes, providing a foundation for further studies.

C-di-AMP accumulation disrupts glutathione metabolism in Listeria monocytogenes.

C-di-AMP homeostasis is critical for bacterial stress response, cell wall integrity, and virulence. Except for osmotic stress response, the molecular mechanisms underlying other processes are not well defined. A Listeria monocytogenes mutant lacking both c-di-AMP phosphodiesterases, denoted as the ΔPDE mutant, is significantly attenuated in the mouse model of systemic infection. We utilized the ΔPDE mutant to define the molecular functions of c-di-AMP. RNAseq revealed that the ΔPDE mutant is significantly impaired for the expression of virulence genes regulated by the master transcription factor PrfA, which is activated by reduced glutathione (GSH) during infection. Subsequent quantitative gene expression analyses revealed that the ΔPDE strain is defective for PrfA-regulated gene expression both at the basal level and upon activation by GSH. We further found the ΔPDE strain to be significantly depleted for cytoplasmic GSH and impaired for GSH uptake. The ΔPDE strain was also deficient in GSH under conditions that activate GSH synthesis by the synthase GshF and upon constitutive expression of gshF, suggesting that c-di-AMP accumulation inhibits GSH synthesis activity or promotes GSH catabolism. A constitutively active PrfA* variant restored virulence gene expression in ΔPDE in broth cultures supplemented with GSH but did not rescue virulence defect in vivo. Therefore, virulence attenuation at high c-di-AMP is likely associated with defects outside of the PrfA regulon. For instance, the ΔPDE strain was sensitive to oxidative stress, a phenotype exacerbated in the absence of GshF. Our data reveal GSH metabolism as another pathway that is regulated by c-di-AMP.IMPORTANCEC-di-AMP regulates both bacterial pathogenesis and interactions with the host. Although c-di-AMP is essential in many bacteria, its accumulation also attenuates the virulence of many bacterial pathogens. Therefore, disrupting c-di-AMP homeostasis is a promising antibacterial treatment strategy and has inspired several studies that screened for chemical inhibitors of c-di-AMP phosphodiesterases. However, the molecular functions of c-di-AMP are still not fully defined, and the underlying mechanisms for attenuated virulence at high c-di-AMP levels are unclear. Our analyses in Listeria monocytogenes indicate that virulence-related defects are likely outside of the virulence gene regulon. We found c-di-AMP accumulation to impair L. monocytogenes virulence gene expression and disrupt GSH metabolism. Further studies are necessary to establish the relative contributions of these regulations to virulence and host adaptation.

An Optimized Method for Reconstruction of Transcriptional Regulatory Networks in Bacteria Using ChIP-exo and RNA-seq Datasets.

Transcriptional regulatory networks (TRNs) in bacteria are crucial for elucidating the mechanisms that regulate gene expression and cellular responses to environmental stimuli. These networks delineate the interactions between transcription factors (TFs) and their target genes, thereby uncovering the regulatory processes that modulate gene expression under varying environmental conditions. Analyzing TRNs offers valuable insights into bacterial adaptation, stress responses, and metabolic optimization from an evolutionary standpoint. Additionally, understanding TRNs can drive the development of novel antimicrobial therapies and the engineering of microbial strains for biofuel and bioproduct production. This protocol integrates advanced data analysis pipelines, including ChEAP, DEOCSU, and DESeq2, to analyze omics datasets that encompass genome-wide TF binding sites and transcriptome profiles derived from ChIP-exo and RNA-seq experiments. This approach minimizes both the time required and the risk of bias, making it accessible to non-expert users. Key steps in the protocol include preprocessing and peak calling from ChIP-exo data, differential expression analysis of RNA-seq data, and motif and regulon analysis. This method offers a comprehensive and efficient framework for TRN reconstruction across various bacterial strains, enhancing both the accuracy and reliability of the analysis while providing valuable insights for basic and applied research.

Regulatory Mechanisms and Synergistic Enhancement of the Diiron YtfE Protein in Nitric Oxide Reduction.

The diiron-containing YtfE protein in Escherichia coli is pivotal in counteracting nitrosative stress, a critical barrier to bacterial viability. This study delves into the biochemical complexity governing YtfE's conversion of nitric oxide (NO) to nitrous oxide, a key process for alleviating nitrosative stress. Through site-directed mutagenesis, we explored YtfE's molecular structure, with a particular focus on two internal transport tunnels important for its activity. Our findings illuminate Tunnel 1 as the primary conduit for substrate transport, regulated by conformational shifts within the N-terminal domain that enable substrate access to the diiron core in the C-terminal domain. Tunnel 2 emerges as a secondary, supportive route, activated when Tunnel 1 is compromised. This result challenges a previous model of distinct tunnels for substrate entry and product exit, suggesting both tunnels are capable of transporting substrates and products. Our engineering efforts enhanced the role of Tunnel 2, enabling a synergistic operation with Tunnel 1 and tripling YtfE's enzymatic activity compared to its wild-type form. This research not only deepens our understanding of YtfE's regulatory mechanism for NO reduction but also introduces a strategy to amplify its enzymatic efficiency. The outcomes offer new ravenues for modulating bacterial stress responses.

The master regulator OxyR orchestrates bacterial oxidative stress response genes in space and time

Bacteria employ diverse gene regulatory networks to survive stress, but deciphering the underlying logic of these complex networks has proved challenging. Here, we use time-resolved single-cell imaging to explore the functioning of the E.coli regulatory response to oxidative stress. We observe diverse gene expression dynamics within the network. However, by controlling for stress-induced growth-rate changes, we show that these patterns involve just three classes of regulation: downregulated genes, upregulated pulsatile genes, and gradually upregulated genes. The two upregulated classes are distinguished by differences in the binding of the transcription factor, OxyR, and appear to play distinct roles during stress protection. Pulsatile genes activate transiently in a few cells for initial protection of a group of cells, whereas gradually upregulated genes induce evenly, generating a lasting protection involving many cells. Our study shows how bacterial populations use simple regulatory principles to coordinate stress responses in space and time. A record of this paper's transparent peer review process is included in the supplemental information.

Evaluation of Choudhary et al.: Single-cell gene expression dynamics in the E. coli oxidative stress response network

One snapshot of the peer review process for "The master regulator OxyR orchestrates bacterial oxidative stress response genes in space and time" (Choudhary etal., 2024).1.

Breaking Barriers: Exploiting Envelope Biogenesis and Stress Responses to Develop Novel Antimicrobial Strategies in Gram-Negative Bacteria.

Antimicrobial resistance (AMR) has emerged as a global health threat, necessitating immediate actions to develop novel antimicrobial strategies and enforce strong stewardship of existing antibiotics to manage the emergence of drug-resistant strains. This issue is particularly concerning when it comes to Gram-negative bacteria, which possess an almost impenetrable outer membrane (OM) that acts as a formidable barrier to existing antimicrobial compounds. This OM is an asymmetric structure, composed of various components that confer stability, fluidity, and integrity to the bacterial cell. The maintenance and restoration of membrane integrity are regulated by envelope stress response systems (ESRs), which monitor its assembly and detect damages caused by external insults. Bacterial communities encounter a wide range of environmental niches to which they must respond and adapt for survival, sustenance, and virulence. ESRs play crucial roles in coordinating the expression of virulence factors, adaptive physiological behaviors, and antibiotic resistance determinants. Given their role in regulating bacterial cell physiology and maintaining membrane homeostasis, ESRs present promising targets for drug development. Considering numerous studies highlighting the involvement of ESRs in virulence, antibiotic resistance, and alternative resistance mechanisms in pathogens, this review aims to present these systems as potential drug targets, thereby encouraging further research in this direction.