Bacterial Stress Response Research Articles

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.

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.

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.

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.

Aquatic environment drives the emergence of cell wall-deficient dormant forms in Listeria

Stressed bacteria can enter a dormant viable but non-culturable (VBNC) state. VBNC pathogens pose an increased health risk as they are undetectable by growth-based techniques and can wake up back into a virulent state. Although widespread in bacteria, the mechanisms governing this phenotypic switch remain elusive. Here, we investigate the VBNC state transition in the human pathogen Listeria monocytogenes. We show that bacteria starved in mineral water become VBNC by converting into osmotically stable cell wall-deficient coccoid forms, a phenomenon that occurs in other Listeria species. We reveal the bacterial stress response regulator SigB and the autolysin NamA as major actors of VBNC state transition. We lastly show that VBNC Listeria revert to a walled and virulent state after passage in chicken embryos. Our study provides more detail on the VBNC state transition mechanisms, revealing wall-free bacteria naturally arising in aquatic environments as a potential survival strategy in hypoosmotic and oligotrophic conditions.

ActA-mediated PykF acetylation negatively regulates oxidative stress adaptability of Streptococcus mutans.

Dental caries poses a significant challenge to global oral health, driven by microbial dysbiosis within dental biofilms. The pathogenicity of Streptococcus mutans, a major cariogenic bacterium, is closely linked to its ability to adapt to changing environments and cellular stresses. Our investigation into the protein acetylation mechanisms, particularly through the acetyltransferase ActA, reveals a critical pathway by which S. mutans modulates its adaptability to oxidative stress, the dominant stressor within dental biofilms. By elucidating how ActA affects the oxidative stress adaptability and competitiveness of S. mutans through the regulatory axis of ActA-PykF-pyruvate, our findings provide insights into the dynamic interplay between cariogenic and commensal bacteria within dental biofilms. This work emphasizes the significance of protein acetylation in bacterial stress response and competitiveness, opening avenues for the development of novel strategies to maintain oral microbial balance within dental biofilms.

The CpxAR signaling system confers a fitness advantage for flea gut colonization by the plague bacillus.

The bacterium Yersinia pestis is the agent of flea-borne plague. Our knowledge of the mechanisms used by the plague bacillus to infect the flea vector is limited. The up-regulation of the envelope stress response under the control of the Cpx signaling pathway was previously shown in a transcriptomic study. Here, our in vivo and in vitro approaches suggest a model in which Y. pestis uses the CpxAR phosphorelay system to sense and respond to the copper present in the flea gut, thereby optimizing the flea gut colonization. In other words, the system is essential for bacterial fitness in the flea.

Quantitative Analysis of Rhodobacter sphaeroides Storage Organelles via Cryo-Electron Tomography and Light Microscopy.

Bacterial cytoplasmic organelles are diverse and serve many varied purposes. Here, we employed Rhodobacter sphaeroides to investigate the accumulation of carbon and inorganic phosphate in the storage organelles, polyhydroxybutyrate (PHB) and polyphosphate (PP), respectively. Using cryo-electron tomography (cryo-ET), these organelles were observed to increase in size and abundance when growth was arrested by chloramphenicol treatment. The accumulation of PHB and PP was quantified from three-dimensional (3D) segmentations in cryo-tomograms and the analysis of these 3D models. The quantification of PHB using both segmentation analysis and liquid chromatography and mass spectrometry (LCMS) each demonstrated an over 10- to 20-fold accumulation of PHB. The cytoplasmic location of PHB in cells was assessed with fluorescence light microscopy using a PhaP-mNeonGreen fusion-protein construct. The subcellular location and enumeration of these organelles were correlated by comparing the cryo-ET and fluorescence microscopy data. A potential link between PHB and PP localization and possible explanations for co-localization are discussed. Finally, the study of PHB and PP granules, and their accumulation, is discussed in the context of advancing fundamental knowledge about bacterial stress response, the study of renewable sources of bioplastics, and highly energetic compounds.

Stress response in Escherichia coli following sublethal phenalene-1-one mediated antimicrobial photodynamic therapy: an RNA-Seq study

Since the molecular mechanisms behind adaptation and the bacterial stress response toward antimicrobial photodynamic therapy (aPDT) are not entirely clear yet, the aim of the present study was to investigate the transcriptomic stress response in Escherichia coli after sublethal treatment with aPDT using RNA sequencing (RNA-Seq). Planktonic cultures of stationary phase E. coli were treated with aPDT using a sublethal dose of the photosensitizer SAPYR. After treatment, RNA was extracted, and RNA-Seq was performed on the Illumina NextSeq 500. Differentially expressed genes were analyzed and validated by qRT-PCR. Furthermore, expression of specific stress response proteins was investigated using Western blot analysis.The analysis of the differential gene expression following pathway enrichment analysis revealed a considerable number of genes and pathways significantly up- or down-regulated in E. coli after sublethal treatment with aPDT. Expression of 1018 genes was up-regulated and of 648 genes was down-regulated after sublethal treatment with aPDT as compared to irradiated controls. Analysis of differentially expressed genes and significantly de-regulated pathways showed regulation of genes involved in oxidative stress response and bacterial membrane damage. In conclusion, the results show a transcriptomic stress response in E. coli upon exposure to aPDT using SAPYR and give an insight into potential molecular mechanisms that may result in development of adaptation.Graphical abstract

Differential Abundance of Protein Acylation in Mycobacterium tuberculosis Under Exposure to Nitrosative Stress.

Human macrophages generate antimicrobial reactive nitrogen species in response to infection by Mycobacterium tuberculosis (Mtb). Exposure to these redox-reactive compounds induces stress response in Mtb, which can affect posttranslational modifications (PTM). Here, we present the global analysis of the PTM acylation of Mtb proteins in response to a sublethal dose of nitrosative stress in the form of nitric oxide (NO) using label free quantification. A total of 6437 acylation events were identified on 1496 Mtb proteins, and O-acylation accounted for 92.2% of the events identified, while 7.8% were N-acylation events. About 22% of the sites identified were found to be acylated by more than one acyl-group. Furthermore, the abundance of each acyl-group decreased as their molecular weight increased. Quantitative PTM analysis revealed differential abundance of acylation in proteins involved in stress response, iron ion homeostasis, growth, energy metabolism, and antimicrobial resistance (AMR) induced by nitrosative stress over time. The results reveal a potential role of Mtb protein acylation in the bacterial stress responses and AMR. To our knowledge, this is the first report on global O-acylation profile of Mtb in response to NO. This will significantly improve our understanding of the changes in Mtb acylation under nitrosative stress, highly relevant for global health.

Defining the functional properties of cyclopropane fatty acid synthase from Pseudomonas aeruginosa PAO1

Cyclopropane fatty acid synthases (CFAS) catalyze the conversion of unsaturated fatty acids to cyclopropane fatty acids (CFAs) within bacterial membranes. This modification alters the biophysical properties of membranes and has been correlated with virulence in several human pathogens. Despite the central role played by CFAS enzymes in regulating bacterial stress responses, the mechanistic properties of the CFAS enzyme family and the consequences of CFA biosynthesis remain largely uncharacterized in most bacteria. We report the first characterization of the CFAS enzyme from Pseudomonas aeruginosa (PA) – an opportunistic human pathogen with complex membrane biology that is frequently associated with antimicrobial resistance and high tolerance to various external stressors. We demonstrate that CFAs are produced by a single enzyme in PA and that cfas gene expression is upregulated during the transition to stationary phase and in response to oxidative stress. Analysis of PA lipid extracts reveal a massive increase in CFA production as PA cells enter stationary phase and help define the optimal membrane composition for in vitro assays. The purified PA-CFAS enzyme forms a stable homodimer and preferentially modifies phosphatidylglycerol lipid substrates and membranes with a higher content of unsaturated acyl chains. Bioinformatic analysis across bacterial phyla shows highly divergent amino acid sequences within the lipid binding domain of CFAS enzymes, perhaps suggesting distinct membrane binding properties among different orthologues. This work lays an important foundation for further characterization of CFAS in Pseudomonas aeruginosa and for examining the functional differences between CFAS enzymes from different bacteria.

Microbial Primer: what is the stringent response and how does it allow bacteria to survive stress?

The stringent response is a conserved bacterial stress response that allows bacteria to alter their activity and survive under nutrient-limiting conditions. Activation of the stringent response is characterized by the production of intracellular signalling molecules, collectively termed (p)ppGpp, which interact with multiple targets inside bacterial cells. Together, these interactions induce a slow growth phenotype to aid bacterial survival by altering the transcriptomic profile of the cell, inhibiting ribosome biosynthesis and targeting enzymes involved in other key metabolic processes.

Unraveling bacterial stress responses: implications for next-generation antimicrobial solutions

The accelerated spread of antimicrobial-resistant bacteria has caused a serious health problem and rendered antimicrobial treatments ineffective. Innovative approaches are crucial to overcome the health threat posed by resistant pathogens and prevent the emergence of untreatable infections. Triggering stress responses in bacteria can diminish susceptibility to various antimicrobials by inducing resistance mechanisms. Therefore, a thorough understanding of stress response control, especially in relation to antimicrobial resistance, offers valuable perspectives for innovative and efficient therapeutic approaches to combat antimicrobial resistance. The aim of this study was to evaluate the stress responses of 8 different bacteria by analyzing reporter metabolites, around which significant alterations were observed, using a pathway-driven computational approach. For this purpose, the transcriptomic data that the bacterial pathogens were grown under 11 different stress conditions mimicking the human host environments were integrated with the genome-scale metabolic models of 8 pathogenic species (Enterococcus faecalis OG1R, Escherichia coli EPEC O127:H6 E2348/69, Escherichia coli ETEC H10407, Escherichia coli UPEC 536, Klebsiella pneumoniae MGH 78578, Pseudomonas aeruginosa PAO1, Staphylococcus aureus MRSA252, and Staphylococcus aureus MSSA476). The resulting reporter metabolites were enriched in multiple metabolic pathways, with cofactor biosynthesis being the most important. The results of this study will serve as a guide for the development of antimicrobial agents as they provide a first insight into potential drug targets.

Activation of the l-fucose utilization cluster in Campylobacter jejuni induces proteomic changes and enhances Caco-2 cell invasion and fibronectin binding

Most Campylobacter jejuni isolates carry the fucose utilization cluster (Cj0480c-Cj0489) that supports the metabolism of l-fucose and d-arabinose. In this study we quantified l-fucose and d-arabinose metabolism and metabolite production, and the impact on Caco-2 cell interaction and binding to fibronectin, using C. jejuni NCTC11168 and the closely related human isolate C. jejuni strain 286. When cultured with l-fucose and d-arabinose, both isolates showed increased survival and production of acetate, pyruvate and succinate, and the respective signature metabolites lactate and glycolic acid, in line with an overall upregulation of l-fucose cluster proteins. In vitro Caco-2 cell studies and fibronectin-binding experiments showed a trend towards higher invasion and a significantly higher fibronectin binding efficacy of C. jejuni NCTC11168 cells grown with l-fucose and d-arabinose, while no significant differences were found with C. jejuni 286. Both fibronectin binding proteins, CadF and FlpA, were detected in the two isolates, but were not significantly differentially expressed in l-fucose or d-arabinose grown cells. Comparative proteomics analysis linked the C. jejuni NCTC11168 phenotypes uniquely to the more than 135-fold upregulated protein Cj0608, putative TolC-like component MacC, which, together with the detected Cj0606 and Cj0607 proteins, forms the tripartite secretion system MacABC with putative functions in antibiotic resistance, cell envelope stress response and virulence in Gram negative pathogenic bacteria. Further studies are required to elucidate the role of the MacABC system in C. jejuni cell surface structure modulation and virulence.

Ligand-Receptor Interaction-Induced Intracellular Phase Separation: A Global Disruption Strategy for Resistance-Free Lethality of Pathogenic Bacteria.

Addressing the global challenge of bacterial resistance demands innovative approaches, among which multitargeting is a widely used strategy. Current strategies of multitargeting, typically achieved through drug combinations or single agents inherently aiming at multiple targets, face challenges such as stringent pharmacokinetic and pharmacodynamic requirements and cytotoxicity concerns. In this report, we propose a bacterial-specific global disruption approach as a vastly expanded multitargeting strategy that effectively disrupts bacterial subcellular organization. This effect is achieved through a pioneering chemical design of ligand-receptor interaction-induced aggregation of small molecules, i.e., DNA-induced aggregation of a diarginine peptidomimetic within bacterial cells. These intracellular aggregates display affinity toward various proteins and thus substantially interfere with essential bacterial functions and rupture bacterial cell membranes in an "inside-out" manner, leading to robust antibacterial activities and suppression of drug resistance. Additionally, biochemical analysis of macromolecule binding affinity, cytoplasmic localization patterns, and bacterial stress responses suggests that this bacterial-specific intracellular aggregation mechanism is fundamentally different from nonselective classic DNA or membrane binding mechanisms. These mechanistic distinctions, along with the peptidomimetic's selective permeation of bacterial membranes, contribute to its favorable biocompatibility and pharmacokinetic properties, enabling its in vivo antimicrobial efficacy in several animal models, including mice-based superficial wound models, subcutaneous abscess models, and septicemia infection models. These results highlight the great promise of ligand-receptor interaction-induced intracellular aggregation in achieving a globally disruptive multitargeting effect, thereby offering potential applications in the treatment of malignant cells, including pathogens, tumor cells, and infected tissues.

The uncharacterized PA3040-3042 operon is part of the cell envelope stress response and a tobramycin resistance determinant in a clinical isolate of Pseudomonas aeruginosa.

An important category of bacterial stress response systems is bacteriophage defense, where systems are triggered by bacteriophage infection and activate a response which may either destroy the phage genome or destroy the infected cell so that the rest of the population survives. In some bacteria, the cell envelope stress response is activated by bacteriophage infection, but it is unknown whether this contributes to the survival of the infection. We have found that a conserved uncharacterized operon (PA3040-PA3042) of the cell envelope stress regulon in Pseudomonas aeruginosa, which has very few dedicated phage defense systems, responds to phage infection and stationary phase as well as envelope stress and is important for growth and biofilm formation in a clinical isolate of P. aeruginosa, even in the absence of phages. As homologs of these genes are found in other bacteria, they may be a novel component of the general stress response.