G-quadruplexes, four-stranded DNA or RNA structures enriched in guanine bases, have long been recognized in eukaryotic genomes for their regulatory roles in transcription, replication, and genome stability. However, their presence and functional significance in bacterial pathogens, especially in the complex intracellular bacterium Mtb, have remained elusive. The intricate architecture of the Mtb genome, coupled with its notorious ability to survive hostile environments within host macrophages, presents a formidable challenge to traditional genomic analyses. The current study bridges this knowledge gap by employing CUT&Tag, a technique offering unprecedented resolution and specificity in mapping protein-DNA interactions and DNA secondary structures in situ.

In their investigation, the team subjected Mtb cultures to oxidative stress conditions mimicking the hostile environment encountered during macrophage infection. Oxidative stress, a result of reactive oxygen species generated by host immune responses, imposes a substantial threat to bacterial survival and DNA integrity. The researchers hypothesized that the bacterial genome might harbor dynamic structural adaptations, such as changes in G4 configurations, to contend with such stress. Using a G4-specific antibody in CUT&Tag assays, they profiled the genome-wide distribution of G-quadruplexes under both basal and oxidative stress conditions.

The results were illuminating. Mtb showed an unexpectedly rich landscape of G4 structures dispersed throughout its genome, but notably, the patterns shifted dramatically upon oxidative stress induction. Certain regions accumulated stabilized G-quadruplexes, suggesting that G4 formation is a responsive mechanism to oxidative DNA damage or a modulator of gene expression under stress. These stress-induced G4 foci were found in regulatory regions, including promoters of genes involved in DNA repair, stress response pathways, and essential virulence factors, underscoring the potential regulatory role of G4 structures in Mtb physiology and pathogenicity.

This dynamic adaptation challenges the classical view of bacterial genome rigidity and reveals an additional layer of gene regulation mediated by DNA secondary structure plasticity. Particularly intriguing was the discovery that non-canonical and atypical G4 motifs proliferated under oxidative stress, divergent from the well-characterized eukaryotic G4 consensus sequences. Such unconventional G4s could be uniquely tailored for bacterial survival requirements, opening the door for novel selective drug targeting that spares human host cells.

Importantly, the study’s utilization of CUT&Tag represented a technical leap forward. Traditional chromatin immunoprecipitation (ChIP)-based methods often fail to resolve secondary DNA structures due to their reliance on crosslinking and sonication steps that can disrupt fragile DNA conformations. CUT&Tag circumvents these limitations by enabling in situ tagmentation of native chromatin-bound molecules with minimal manipulation, preserving the delicate G4 architecture. The method’s heightened sensitivity and reduced background noise permitted a precise mapping of G4 elements even within Mtb’s GC-rich and complex genomic landscape.

Beyond the basic discovery, the findings have profound implications for tuberculosis (TB) treatment and drug development. Mtb’s notorious resilience against antibiotics is partly attributed to its ability to alter gene expression and survive oxidative bursts from immune cells. Targeting G-quadruplexes or their associated binding proteins could abolish this adaptive mechanism, sensitizing bacteria to both host immunity and pharmacological agents. Molecules that can selectively stabilize or destabilize bacterial G4s may emerge as adjunct therapies, enhancing the efficacy of existing antitubercular drugs.

Moreover, the study serves as a template for exploring DNA secondary structures in other prokaryotic systems. The adaptability of CUT&Tag for mapping G4 landscapes extends beyond Mtb, potentially illuminating bacterial stress responses in a wide range of pathogens. This could unravel conserved or unique genomic regulatory mechanisms, transforming our molecular understanding of infection biology and microbial survival.

Intriguingly, the authors also observed that oxidative stress not only modified the quantity but also the quality of G4 structures, inducing complex topologies and possibly promoting the formation of multimeric quadruplex assemblies. These higher-order conformations could influence genomic architecture and DNA-protein interactions more dramatically than simple G4 motifs. Such depth of structural complexity was previously only hypothesized in eukaryotic systems, suggesting a convergent evolution of DNA regulatory strategies between distant domains of life.

Equally significant was the identification of G4s overlapping with regions bound by nucleoid-associated proteins (NAPs) in Mtb. NAPs organize bacterial chromosomes and regulate gene expression, and their interplay with G4s hints at a sophisticated crosstalk between DNA secondary structure and protein-mediated chromosomal organization. This multilayered regulatory network could be crucial for rapid adaptation under fluctuating environmental stresses, including those presenting inside host cells.

From a methodological perspective, the study sets a new standard for interrogating DNA secondary structures in bacteria. The authors carefully optimized antibody specificity, reaction conditions, and sequencing pipelines to confidently distinguish bona fide G4s from potential artifacts. Their approach paves the way for integrating genome-wide structural mapping with transcriptomic and proteomic analyses to paint a comprehensive picture of stress-induced bacterial adaptation.

Further, the investigation sheds light on the evolutionary pressures shaping bacterial genome architecture. The capacity to form unconventional G4 structures suggests an intrinsic genomic plasticity that may confer advantages in maintaining genome integrity, regulating mutagenesis, or fine-tuning gene expression under oxidative duress. These findings raise provocative questions regarding the evolutionary origins and conservation of G4 motifs across diverse bacterial taxa and their role in pathogen evolution and virulence.

One of the most exciting prospects arising from this research is the translational potential. Drugs modulating G-quadruplex stability have been explored in cancer therapy, yet few efforts have targeted bacterial G4s explicitly. This study provides a rational framework to design and screen small molecules or peptides that recognize Mtb-specific G4 topologies, offering a novel class of antimicrobial agents with precisely targeted mechanisms that minimize host toxicity.

The research also invites a reevaluation of how host-pathogen interactions influence bacterial genome structure. Oxidative stress is a key battleground in the immune response to TB infection, and the discovery that this stress directly modulates bacterial DNA conformation unveils a hidden layer of molecular warfare. Understanding these dynamics could inform the development of immunomodulatory interventions or diagnostic tools based on G4 biomarker detection.

Additionally, the findings prompt a rethinking of bacterial epigenetics. While classical epigenetic modifications in bacteria, such as DNA methylation, have been intensively studied, the role of DNA secondary structures as dynamic epigenetic marks is an emerging paradigm. This study contributes compelling evidence supporting G4s as functional epigenetic-like elements modulating bacterial gene regulation in real-time environmental contexts.

In conclusion, this landmark study uncovers a previously uncharted G-quadruplex landscape within Mycobacterium tuberculosis that is responsive to oxidative stress and intimately connected to gene regulation and genome stability. The employment of CUT&Tag technology offers unparalleled insight into the dynamic structural adaptations bacteria harness to survive hostile conditions. These insights significantly broaden our understanding of bacterial genome complexity, pushing the frontier of infectious disease biology and opening transformative avenues for therapeutic innovation against tuberculosis.

Subject of Research: DNA secondary structures, specifically G-quadruplex formations, in Mycobacterium tuberculosis under oxidative stress conditions.

Article Title: CUT&Tag reveals unconventional G-quadruplex landscape in Mycobacterium tuberculosis in response to oxidative stress.

Article References:
Maurizio, I., Ruggiero, E., Zanin, I. et al. CUT&Tag reveals unconventional G-quadruplex landscape in Mycobacterium tuberculosis in response to oxidative stress. Nat Commun 16, 7253 (2025). https://doi.org/10.1038/s41467-025-62485-4

Image Credits: AI Generated

Central to this breakthrough is the development of an ambitious GFP-reporter strain library encompassing nearly 2,901 promoter regions computationally predicted across the S. Typhimurium genome. Each promoter element was fused to a green fluorescent protein gene, allowing real-time visualization and quantification of promoter activity under diverse conditions. This expansive reporter platform was leveraged to monitor transcriptional shifts during standard in vitro growth phases as well as across the complex intracellular milieu of RAW 264.7 macrophages, a widely employed murine macrophage cell line. Importantly, the study deployed both bulk fluorescence measurements and cutting-edge single-cell imaging modalities, enabling deconvolution of population-level responses into discrete subpopulations and individual-cell heterogeneity. This dual approach uncovered not just averaged promoter activities but also revealed the stochasticity and condition-specific regulation often masked in ensemble assays.

Within the intracellular environment of macrophages, bacterial pathogens encounter oxidative stress, nutrient limitation, and metal ion fluctuations. The transcriptional reprogramming of S. Typhimurium in response to these challenges is orchestrated by a suite of virulence factors, notably the Salmonella Pathogenicity Island 2 (SPI-2) locus. The reporter library confirmed intense activity within SPI-2-related promoters upon macrophage infection, consistent with prior findings that SPI-2 is critical for intracellular survival and pathogenesis. However, beyond these expected regulators, the study uncovered startling heterogeneity in promoter activity at the single-cell level. This heterogeneity suggests that within a clonal bacterial population, subpopulations may adopt divergent transcriptional states, potentially facilitating phenotypic diversification as a bet-hedging strategy in the face of fluctuating host defenses.

A particularly compelling revelation emerged in the form of 234 previously uncharacterized promoters exhibiting transcriptional activity under either in vitro or intracellular conditions. This finding significantly expands the catalog of potentially functional regulatory sequences in S. Typhimurium and points to a more complex transcriptional network than previously appreciated. Many of these novel promoters might drive expression of genes involved in metabolic adaptation and stress response that have so far eluded detection via conventional RNA-seq due to their transient or low-level expression.

Key among the metabolic adaptations uncovered was the induction of genes related to manganese homeostasis, spotlighting the small RNA mntS as an essential component in regulating intracellular manganese availability. Manganese acts as a cofactor for numerous enzymes and also mediates resistance to oxidative stress, a common weapon employed by macrophages. The insights from this study underscore the critical balance S. Typhimurium must maintain in trace metal acquisition and detoxification within the phagosomal niche, pivoting on finely tuned transcriptional circuits.

Moreover, the research illuminated the activation of the Entner–Doudoroff (ED) pathway genes within macrophages, a less commonly utilized glycolytic route in many bacteria. This pathway provides metabolic flexibility, enabling S. Typhimurium to efficiently catabolize sugars and generate energy under nutrient-restricted intracellular conditions. The data suggest that reliance on the ED pathway may represent a strategic metabolic rewiring to circumvent bottlenecks encountered during infection, highlighting the metabolic plasticity of this pathogen.

The integrative dataset generated from this extensive screening effort was made accessible through SalComKinetics, an innovative online platform for visualizing and interrogating transcriptional dynamics across conditions and time points. This resource empowers researchers worldwide to probe gene expression patterns at unprecedented resolution, facilitating systems-level analyses that could accelerate discovery of novel therapeutic targets and illuminate bacterial adaptive strategies.

Methodologically, the study represents a tour de force in combining computational prediction, genetic engineering, quantitative fluorescence assays, and live-cell microscopy. The generation of nearly three thousand individual promoter-GFP fusions required precise cloning and validation workflows, underscored by rigorous quantitative measurements to ensure reproducibility and sensitivity. The fusion of bulk and single-cell data streams allowed insights into both overarching trends and microscopic nuances of bacterial gene regulation, bridging a critical gap in current understanding of infection dynamics.

From a pathogenesis perspective, this work provides an invaluable window into how S. Typhimurium modulates its transcriptional landscape in real time during host cell invasion and residence. The finding that promoter activity is not only temporally regulated but also subject to population heterogeneity challenges simplistic models of uniform bacterial behavior inside host cells. Such heterogeneity may underlie differential survival strategies, resistance to immune clearance, or emergence of persister cells contributing to chronic infections.

Moreover, the discovery of numerous transcriptionally active yet previously unannotated promoters raises fundamental questions about the evolution and organization of bacterial genomes. It suggests that extensive layers of regulatory complexity remain to be deciphered, with potential consequences for understanding bacterial physiology, virulence, and adaptation. Such elements could encode small RNAs, alternative sigma factor-dependent transcripts, or uncharacterized open reading frames that modulate bacterial fitness under infection-related stresses.

The implications of manganese regulation through mntS also extend beyond Salmonella biology. Trace metal homeostasis is a central battlefield in host–pathogen interactions, and elucidating regulatory RNAs controlling metal acquisition could unveil novel antimicrobial intervention points. Targeting metal scavenging and detoxification pathways offers an exciting frontier in combating intracellular pathogens that have evolved sophisticated strategies to circumvent host nutritional immunity.

On the metabolic front, the elucidation of ED pathway activation shifts the conventional paradigm dominated by glycolysis toward appreciating alternative biochemical routes exploited by pathogens during infection. Understanding these shifts can inform metabolic modeling efforts aimed at predicting vulnerabilities and optimizing antimicrobial therapies that disrupt pathogen energy metabolism within host environments.

By openly sharing the promoter library and the comprehensive transcriptional datasets via SalComKinetics, this study embodies the ethos of open science and collaborative discovery. It provides a foundational toolkit not only for microbiologists investigating Salmonella but also for researchers exploring gene regulation, bacterial pathogenesis, and host–microbe interactions across diverse systems.

In summary, this landmark study redefines our understanding of Salmonella’s transcriptional responsiveness to the intracellular niche of macrophages, revealing complex, time-dependent, and heterogeneous gene expression patterns. The integration of high-throughput promoter screening with live-cell imaging charts a path forward for dissecting bacterial adaptation at an unprecedented level of detail. Such insights are poised to catalyze new avenues in infection biology, translational research, and the development of innovative antimicrobial strategies.

As infectious diseases continue to pose global health challenges, especially with rising antibiotic resistance, comprehensively mapping bacterial transcriptional dynamics in relevant host environments becomes ever more critical. The approach and findings presented herein illuminate fundamental principles of pathogen survival and open new doors for targeted interventions aimed at disarming stealthy intracellular invaders like Salmonella enterica serovar Typhimurium.

Subject of Research: Salmonella enterica serovar Typhimurium transcriptional dynamics during macrophage infection

Article Title: Profiling Salmonella transcriptional dynamics during macrophage infection using a comprehensive reporter library

Article References:
Nguyen, T.H., Wang, B.X., Diaz, O.R. et al. Profiling Salmonella transcriptional dynamics during macrophage infection using a comprehensive reporter library. Nat Microbiol 10, 1006–1023 (2025). https://doi.org/10.1038/s41564-025-01953-5

Image Credits: AI Generated

DOI: https://doi.org/10.1038/s41564-025-01953-5