TL;DR
The results suggest that AnCAN33 is associated with stress adaptation, membrane integrity, and cellular energy-related responses in A. niger under RF and natamycin stress.
Background: Tuberculosis remains a major global health threat, exacerbated by the increasing prevalence of drugresistant strains. The RNA methyltransferase mraW is predicted to play an important regulator of bacterial gene expression and cellular physiology. However, its function in mycobacteria remains unclear. Objective: This study aims to characterize the function of the mraW RNA methyltransferase in Mycobacterium smegmatis using a CRISPR interference (CRISPRi)-mediated gene knockdown. Methods: A mraW-targeting sgRNA was constructed and expressed in M. smegmatis mc²155, and knockdown effect was induced using anhydrotetracycline (ATc). Knockdown efficiency was confirmed by RT-qPCR, which showed approximately 55% reduction in mraW transcript levels. Results: Phenotypic analysis revealed that mraW knockdown resulted in ATc concentration-dependent growth inhibition, reduced colony formation, and decreased bacterial viability in both solid and liquid culture conditions. Consistent with the inducible nature of the CRISPRi system, stronger growth defects were observed at higher ATc concentrations, suggesting increased repression of mraW, while pLJR962 vector controls remained unaffected, confirming that the observed effects were specifically associated with mraW knockdown. These findings demonstrate that mraW is important for optimal growth and survival in M. smegmatis, supporting a role for RNA methyltransferases in essential mycobacterial physiological processes. Conclusion: This study further highlights the utility of CRISPRi for functional characterization of essential genes in mycobacteria and suggests that RNA methylation pathways may represent novel potential targets for future anti-tuberculosis strategies.
ABSTRACT This study used a functional genomics approach to explore the role of a xenobiotic response element (XRE)-type regulator (SMU.405c) in Streptococcus mutans physiology, including the expression of biofilm regulatory protein BrpA. Results showed that deletional mutation of xre significantly reduced the ability of the deficient mutant to grow in the presence of methyl viologen, a commonly used oxidative stressor (P < 0.001). When challenged in a hydrogen peroxide killing assay, the survival rate of the ∆xre mutant was >2-log less than the parent strain after 60 min (P < 0.001). Luciferase reporter fusion assays showed that xre deficiency had no significant effect on luciferase expression when it was under the control of the intact brpA promoter, but the reporter activity increased by >6-fold (P < 0.001) when the reporter gene was fused to a brpA promoter derivative with deletion of a putative XRE-binding box. Electrophoretic mobility shift assay (EMSA) showed that recombinant XRE interacted with the brpA promoter, resulting in an electrophoretic shift of the promoter probes. In vitro transcription assay also showed that inclusion of XRE caused transcription to fall off, significantly reducing full-length brpA transcripts. RNA-seq analysis revealed that deficiency of XRE led to altered expression of >102 genes by >2-fold (P < 0.05), including 28 with increased expression, and 74 with decreased expression. Among the down-regulated were genes for DNA repair and oxidative stress tolerance response. These results suggest that XRE (SMU.405c) in S. mutans plays an important role in brpA expression and oxidative stress tolerance response. IMPORTANCE Streptococcus mutans, a keystone pathogen in human dental caries, primarily lives in the highly diverse microbiota on tooth surfaces, where the conditions are often harsh and fluctuate frequently. Locus SMU.405c was annotated to encode a xenobiotic response element (XRE)-like transcriptional regulator, but no information is available concerning the role of this protein in S. mutans pathophysiology. This study used a functional genomics approach along with molecular and transcriptomic analysis to characterize a deletional xre mutant, and the results showed that xre deficiency in S. mutans resulted in weakened oxidative stress tolerance response and alterations in transcription of >102 genes, including those known to play an important role in cell envelope biogenesis and stress tolerance response. Reporter fusion assay, electrophoretic mobility shift assay (EMSA), and in vitro transcription further demonstrated that the XRE-like regulator encoded by SMU.405c is a repressor of brpA expression and plays an important role in oxidative stress tolerance response. Streptococcus mutans, a keystone pathogen in human dental caries, primarily lives in the highly diverse microbiota on tooth surfaces, where the conditions are often harsh and fluctuate frequently. Locus SMU.405c was annotated to encode a xenobiotic response element (XRE)-like transcriptional regulator, but no information is available concerning the role of this protein in S. mutans pathophysiology. This study used a functional genomics approach along with molecular and transcriptomic analysis to characterize a deletional xre mutant, and the results showed that xre deficiency in S. mutans resulted in weakened oxidative stress tolerance response and alterations in transcription of >102 genes, including those known to play an important role in cell envelope biogenesis and stress tolerance response. Reporter fusion assay, electrophoretic mobility shift assay (EMSA), and in vitro transcription further demonstrated that the XRE-like regulator encoded by SMU.405c is a repressor of brpA expression and plays an important role in oxidative stress tolerance response.
Findings support a working model in which iron limitation-associated physiological stress contributes to the inhibitory effects of taxifolin on C. perfringens growth and colonization-related phenotypes and support its potential use as a plant-derived approach for limiting C. perfringens intestinal colonization.
Wheat Fusarium head blight (FHB) is a globally prevalent and destructive fungal disease predominantly caused by Fusarium graminearum. Pydiflumetofen, a novel succinate dehydrogenase inhibitor (SDHI) fungicide, exhibits strong inhibitory activity against F. graminearum; however, the molecular regulatory mechanisms underlying the field-developed resistance in this pathogen remain poorly defined. In the present study, a field-evolved resistant isolate W24-039 and a sensitive isolate W24-016 were subjected to multi-omics analysis. The sequencing results identified compound mutations C89S/A93V in SdhC2, and A21T/S30F in SdhD of the resistant strain, which confer stable fungicide resistance without any detectable fitness costs. Physiological tests revealed that these target mutations sustain the homeostasis of succinate dehydrogenase (SDH) activity and intracellular ATP production. Following pydiflumetofen treatment, the sensitive isolate displayed remarkable declines in SDH activity, intracellular ATP content and deoxynivalenol (DON) biosynthesis, accompanied by markedly elevated cell membrane permeability. Transcriptomic sequencing uncovered 2221 differentially expressed genes (DEGs) in the sensitive strain under fungicide stress, and 2566 DEGs in the resistant isolate under the same conditions. The genes associated with detoxification and drug efflux, including cytochrome P450, glutathione S-transferase (GST), ABC and MFS transporters, were significantly upregulated in the resistant isolate. Metabolomic analysis indicated that differential metabolites were mainly enriched in the tricarboxylic acid (TCA) cycle, amino acid metabolism and membrane lipid biosynthesis pathways. The resistant strain maintained intact TCA cycle operation and accumulated high levels of pivotal metabolites such as phosphatidylcholine, unsaturated fatty acids and reduced glutathione. Integrated multi-omics analysis verified that the ABC transporter and glutathione metabolism pathways serve as core regulatory modules governing fungicide resistance. Collectively, F. graminearum develops resistance via the synergistic effects of SDH compound mutations, enhanced detoxification and efflux, and global metabolic remodeling, demonstrating that target-site mutation alone is not the sole driver of resistance, which is instead controlled by an intricate regulatory network involving multiple coordinated pathways. This study systematically characterizes the resistance regulatory network of F. graminearum against pydiflumetofen, and provides theoretical guidance for the rational application and sustainable field resistance management of this fungicide.
Hesperetin is a naturally occurring flavonoid with reported antimicrobial activity, but its antibacterial modes of action remain poorly understood. In this study, the antibacterial effects of hesperetin in Escherichia coli were investigated. Hesperetin treatment increased intracellular reactive oxygen species (ROS) levels, and scavenger-based experiments supported the contribution to downstream effects. Elevated ROS was associated with membrane depolarization and oxidative membrane damage, including lipid peroxidation, as indicated by increased malondialdehyde (MDA) levels. Hesperetin treatment also increased DNA damage, including chromatin condensation and DNA fragmentation. Because recA is a key regulator of the SOS DNA repair response, a recA-deficient strain was used to assess the role of SOS-associated repair signaling; DNA damage was significantly greater in the recA-deficient strain than in the wild-type strain. In addition, programmed cell death-like features, including phosphatidylserine externalization detected by Annexin V/PI staining, were observed. Collectively, these findings indicate that the antibacterial activity of hesperetin involves ROS-associated membrane injury, oxidative DNA damage, and recA-dependent SOS-associated responses, together with programmed cell death-like features in E. coli.
Biosynthetic selenium nanoparticles (SeNPs) exhibit superior bioavailability and detoxification potential compared to inorganic selenium forms. In this study, the strain Stenotrophomonas maltophilia SE5, isolated from the feces of piglets (Duroc × Large White × Landrace), was utilized as the wild-type strain and subjected to atmospheric and room temperature plasma (ARTP) mutagenesis. A high-yielding mutant, Mu537, was successfully obtained, which exhibited an enhanced tolerance to sodium selenite, with its maximum tolerable concentration increasing from 1.0 g/L to 1.5 g/L. After 48 h of cultivation, Mu537 achieved an approximate 35% increase in SeNPs conversion rate and a 40% increase in SeNPs concentration relative to the parental strain. Integrated whole-genome sequencing and transcriptomic analysis revealed that pivotal genes associated with efficient SeNPs biosynthesis, including cysB, trxA, and sufE, were significantly up-regulated. These findings provide a systematic understanding of the potential molecular mechanisms driving enhanced SeNPs biosynthesis, offering both high-quality microbial resources and theoretical support for the industrial production of SeNPs.
