mLife · Volume 5 · Issue 2
于2026年4月30日正式出版
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扫码或点击文末左下角阅读本期原文https://onlinelibrary.wiley.com/toc/2770100x/2026/5/2
Review
🔹 Recent advances in noncanonical inhibition mechanisms of anti-CRISPR proteins
Lingguang Yang, Rongjun Luo, Wei Zhou, Peipei Yin, Yue Feng, Yi Zhang
https://onlinelibrary.wiley.com/doi/10.1002/mlf2.70077
The CRISPR-Cas system constitutes an adaptive immune mechanism in prokaryotes that defends against mobile genetic elements. Within the perpetual co-evolutionary arms race between bacteria and their viral predators, bacteriophages encode anti-CRISPR (Acr) proteins that use sophisticated molecular strategies to sabotage CRISPR-Cas function. While canonical Acr proteins rely on steric blockade of Cas effectors, recent discoveries reveal unprecedented noncanonical mechanisms spanning CRISPR immunity stages. This review synthesizes recent mechanistic advances in this field since 2023, highlighting the expansion of noncanonical inhibition mechanisms beyond type I to include types II, V, and VI, as well as novel Acr interventions targeting multiple functional stages, such as spacer acquisition, translation-coupled inhibition, complex assembly/disassembly, and R-loop DNA binding. Structural insights demonstrate how Acr proteins achieve substoichiometric inhibition via conformational hijacking, catalytic repurposing, and molecular mimicry. Forged by the intense selective pressure of the phage–host conflict, these molecular innovations represent both remarkable evolutionary adaptations and versatile precision tools. They enable spatiotemporal control of CRISPR technologies, from engineered off-switches to diagnostic reset mechanisms, while posing critical challenges for therapeutic safety and microbiome management.
🔹Emerging role of metagenomic next-generation sequencing in infectious disease diagnostics: Clinical integration and future directions
Tingting Fang, Feifei Yuan, Yu Chen, Na Li, Yao Zhang, Haixia Liu, Xingchen Liu, Qing Miao, Bijie Hu
https://onlinelibrary.wiley.com/doi/10.1002/mlf2.70078
Infectious disease diagnostics has been transformed by metagenomic next-generation sequencing (mNGS), an unbiased approach that detects bacteria, viruses, fungi, and parasites in a single assay. By sequencing all nucleic acids in a sample, mNGS overcomes the narrow detection scope and slow turnaround of conventional tests, substantially improving pathogen detection. In conditions such as meningitis/encephalitis, sepsis, and pneumonia, mNGS frequently identifies etiologies missed by routine diagnostic tests, thereby facilitating earlier pathogen-directed therapy and, in selected settings, improving clinical management and outcomes. This approach is particularly valuable for immunocompromised, pediatric, and intensive care unit (ICU) patients with atypical infections. Currently, clinical mNGS workflows primarily rely on short-read sequencing platforms (e.g., Illumina), whereas long-read platforms (e.g., Nanopore, PacBio) offer advantages for rapid or high-resolution applications. Optimized bioinformatics and stringent quality control are essential for reliable results. Beyond clinical diagnostics, mNGS provides valuable genetic data on antimicrobial resistance (AMR) and pathogen phylogeny, supporting public health and outbreak surveillance (e.g., wastewater monitoring and variant tracking). Current challenges include distinguishing colonization from infection, interpreting sequencing data quantitatively, and reducing cost and turnaround time. Looking ahead, emerging strategies such as targeted panels, rapid automated workflows, and host‑response integration are expected to further shorten time‑to‑result and improve diagnostic specificity. Parallel progress in ethical and regulatory frameworks remains essential to ensure responsible implementation. To support clinical adoption, a standardized framework for clinical interpretation of mNGS results, together with associated training, has been developed and implemented. Overall, mNGS is likely to become an increasingly important component of infectious disease diagnostics, with ongoing innovations expected to broaden its clinical and epidemiological impact.
Original Research
🔹 Osmotic signaling governs sunscreen biosynthesis to safeguard desert cyanobacteria against desiccation
Lei Zhao, Hai-Feng Xu, Jin-Long Shang, Yong Li, Xiao-Yue Yin, Zhong-Chun Zhang, Huazhong Shi, Bao-Sheng Qiu
https://onlinelibrary.wiley.com/doi/10.1002/mlf2.70075
Mycosporine-like amino acids (MAAs) are natural sunscreens synthesized by a wide range of organisms. Although the induction of MAA production by ultraviolet radiation is well established, the signaling pathways involved and specific functions of MAAs under other stress conditions remain poorly understood. We demonstrated that MAAs serve as effective osmoprotectants for desiccation tolerance in the desert cyanobacterium Nostoc flagelliforme. Genetic disruption of genes encoding MAA biosynthetic enzymes eliminated MAA production, resulting in elevated oxidative damage, increased lipid peroxidation, and impaired photosynthesis under dehydration. Biochemical assays revealed that MAAs stabilize proteins and scavenge reactive oxygen species, indicating dual roles as osmolytes and antioxidants. Furthermore, we identified a signaling pathway Dsp1–OrrA that mediates the osmotic induction of MAA biosynthesis. Genetic disruption of either gene of Dsp1 and OrrA abolished osmotic induction and severely reduced desiccation tolerance. Phylogenomic analysis suggests that MAA biosynthesis is an ancient trait conserved in desiccation-tolerant cyanobacteria. This work deepens our understanding of microbial adaptation to extreme environments and provides a foundation for synthetic biology applications of MAAs.
🔹 Genome-wide screen reveals a universal role of ATP in ciprofloxacin tolerance among genetically distinct Escherichia coli persisters
Zhenfang Mei, Yawen Cai, Jianfeng Huang, Kedong Zhao, Zuqin Zhang, Dandan Yu, Shiyan Lu, Zeying Lai, Thomas K. Wood, Xinmiao Fu
https://onlinelibrary.wiley.com/doi/10.1002/mlf2.70072
Bacterial persisters show tolerance to bactericidal antibiotics and play essential roles in chronic infections; however, the general mechanisms underlying persister formation and antibiotic tolerance remain insufficiently characterized. In this study, the Escherichia coli Keio library was used to identify genes involved in ciprofloxacin tolerance by culturing each mutant to the late stationary phase (to induce persistence via starvation), followed by dilution into fresh medium for antibiotic exposure. This two-step, genome-wide screening approach enabled the identification of 37 ciprofloxacin-sensitive mutants with diverse biological functions and 11 ciprofloxacin-tolerant mutants related to amino acid and β-nicotinamide adenine dinucleotide (NAD⁺) biosynthesis, with 25 genes being identified as persister-related genes for the first time. Notably, sensitive mutants (ΔatpC, ΔatpF, ΔruvC, and Δrnr) were specifically sensitive to quinolone antibiotics, whereas tolerant mutants (ΔmetR, ΔleuB, and ΔnadB) showed tolerance to ampicillin and gentamicin. Importantly, adenosine triphosphate (ATP) levels were downregulated in ciprofloxacin-tolerant mutants and upregulated in ciprofloxacin-sensitive mutants, implying a negative correlation between ATP levels and ciprofloxacin tolerance among these genetically distinct persisters. This negative correlation was further observed when ATP levels in different mutants were chemically modulated using specific metabolites, nutrients, and respiration inhibitors. In addition, ciprofloxacin persistence across different mutants was found to correlate closely with antibiotic uptake and reactive oxygen species (ROS) levels. Collectively, these findings establish a universal role for ATP in the ciprofloxacin tolerance of genetically diverse persisters under varying resuscitation conditions, conceivably through the modulation of antibiotic uptake and ROS accumulation, and it is implied that the provision of abundant nutrients is potentially beneficial for anti-persister chemotherapy in clinic settings.
🔹 The synthetic estradiol analog E0703 enhances Akkermansia muciniphila growth for radiation-induced intestinal damage repair
Zhexin Ni, Ziqiao Yan, Mingyang Chang, Yangshuo Li, Zebin Liao, Tiantian Xia, Zhijie Bai, Ningning Wang, Chaoji Huangfu, Dezhi Sun, Yangyi Hu, Liangliang Zhang, Feiran Hao, Yongqi Dou, Pan Shen, Wei Zhou, Yue Gao
https://onlinelibrary.wiley.com/doi/10.1002/mlf2.70071
The development of safe and effective radioprotective agents with minimal side effects, particularly for high-dose exposure, remains a global priority. E0703, a novel steroidal compound structurally derived from estradiol, has shown promising radioprotective efficacy with limited estrogenic activity in prior pharmacodynamic studies. In this study, E0703 was found to significantly increase the abundance of Akkermansia muciniphila (AKK) in the intestines of both irradiated and non-irradiated mice. Co-administration of E0703 and AKK markedly improved the 7-day survival rate of mice exposed to a lethal 8.5 Gy dose of radiation. E0703 induced beneficial transcriptional changes in AKK, with enrichment in metabolic pathways such as amino acid biosynthesis, aminoacyl-tRNA biosynthesis, the tricarboxylic acid (TCA) cycle, and fatty acid biosynthesis. These alterations supported the production of glucosamine 6-phosphate (GlcN-6-P) by AKK, which contributed to intestinal tissue regeneration following irradiation. Single-cell transcriptomic analysis revealed that E0703 significantly increased the proportion of intestinal stem cells and goblet cells by Day 5 post irradiation. Mechanistically, E0703 modulated the oxidative phosphorylation pathway in these cell types, including regulation of Muc2 production. E0703 also enhanced AKK abundance in irradiated mice, particularly in the presence of mucin, thereby elevating the availability of GlcN-6-P—a critical substrate for intestinal organoid repair. These findings indicate that E0703 exerts direct effects on goblet cells and AKK, promoting host–microbe interactions that facilitate intestinal regeneration and improve survival following radiation exposure.
🔹 Quinolone tolerance in Escherichia coli due to defects in the adenosine ribonucleotides de novo biosynthesis pathway
Weiwei Zhu, Yuejuan Nong, Jie Su, Jingwen Yang, Lina Ma, Yunxin Xue, Dai Wang, Jianjun Niu, Karl Drlica, Xilin Zhao
https://onlinelibrary.wiley.com/doi/10.1002/mlf2.70059
Central carbon metabolism is thought to link reactive oxygen species (ROS) with antibiotic-mediated bacterial death. During enrichment screening of Escherichia coli with the first-generation quinolone oxolinic acid, unstable antibiotic-tolerant mutants containing deficiencies in purB were obtained. Examination of a stable deletion mutant of purA, a gene functionally related to purB, revealed reduced lethality of oxolinic acid and ciprofloxacin. This deletion mutation had little effect on the minimal inhibitory concentration (MIC) of quinolones, thereby demonstrating that the observed protection from killing was attributable to antibiotic tolerance. AMP synthesis was blocked by the ΔpurA mutation, and ciprofloxacin tolerance was reversed by exogenous AMP supplementation. Because AMP is a precursor of ATP, interference with ATP synthesis occurs in the ΔpurA mutant. RNA-Seq analysis showed that, prior to antibiotic stress, transcript levels of NADH:quinone oxidoreductase genes were reduced by the purA deficiency, thereby predisposing E. coli to antibiotic tolerance through reduced respiration. During ciprofloxacin exposure, the purA deficiency also suppressed the surge in expression of tricarboxylic acid (TCA) cycle and ATP synthesis genes, as well as the accumulation of intracellular ATP and ROS. Thus, wild-type PurA, and by extension the downstream enzyme PurB, directs AMP toward an antibiotic-mediated, ROS-dependent death pathway. Overall, defects in PurA/PurB-mediated adenosine ribonucleotides de novo biosynthesis reveal a novel quinolone tolerance mechanism that is initiated outside central carbon metabolism; tolerance is likely attributable to a limited supply of AMP, resulting in reduced ATP synthesis and suppression of ROS accumulation.
Method
🔹 Multi-target fluorescence staining of bacteria smears enables rapid machine learning-assisted species classification
Maxence Galvan, Michael Fujarski, Can Beslendi, Frieder Schaumburg, Julian Varghese, Johannes Liesche
https://onlinelibrary.wiley.com/doi/10.1002/mlf2.70076
Rapid identification of bacterial species from patient samples is crucial for clinical decision-making. In severe infections, such as bloodstream infections, the early start of an effective treatment is directly associated with reduced mortality rates. Current rapid species identification methods, such as matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) or multiplex PCR, require specialized hardware and extensive technical support that prevents application in resource-limited settings. Here, we present a staining and imaging procedure for bacterial smears using fluorescent dyes directed against intracellular structures and cell wall components. Data on relevant features were extracted from segmented images and used to train a machine learning (ML) model for species classification. The method was tested on clinical isolates from 126 patients. For the seven most common bacteria, the classification performance, indicated by area under the receiver operating characteristic (ROC) curve, ranged from 0.8 (Klebsiella pneumoniae) to 1 (Pseudomonas aeruginosa). Species that were not part of the training dataset, were reliably classified as unknown species. These results hold promise for the identification of further species, particularly Enterobacterales, and clinical application.
🔹 fCUT&Tag-Seq: An optimized CUT&Tag-based method for high-resolution profiling of histone modifications and chromatin-binding proteins in fungi
Haiting Wang, Yongjunlin Tan, Jiayue Ma, Jie Yang, Mengran Liu, Peng Jiang, Shan Lu, Haoxue Xia, Guangfei Tang, Wende Liu, Hui-Shan Guo, Chun-Min Shan
https://onlinelibrary.wiley.com/doi/10.1002/mlf2.70060
Histone modifications and chromatin-binding proteins play crucial roles in regulating gene expression in eukaryotes, with significant implications for fungal pathogenicity and development. However, profiling these modifications or proteins across the genome in fungi remains challenging due to the technical limitations of the traditional, widely used Chromatin Immunoprecipitation-Sequencing (ChIP-Seq) method. Here, we present an optimized fungal Cleavage Under Targets and Tagmentation-Sequencing (fCUT&Tag-Seq) protocol specifically designed for filamentous fungi and dimorphic fungi. Our approach involves the preparation of protoplasts and nuclear extraction to enhance antibody accessibility, along with formaldehyde crosslinking to improve protein-DNA binding efficiency. We then successfully applied fCUT&Tag-Seq to accurately profile multiple histone modifications like H3K9me3, H3K27me3, H3K4me3, and H3K18ac, across different plant pathogenic or model fungal species, including Verticillium dahliae, Neurospora crassa, Fusarium graminearum, and Sporisorium scitamineum, showing good signal-to-noise ratios, reproducibility, and detection sensitivity. Furthermore, we extended this method to profile chromatin-binding proteins, such as the histone acetyltransferase Gcn5. This study establishes fCUT&Tag-Seq as a robust and useful tool for fungal epigenetic research, enabling detailed exploration of chromatin dynamics and advancing our understanding of fungal gene regulation, development, and pathogenicity.
Correspondence
🔹 Creation of bioactive non-natural flavonoids via combinatorial prenylation–glycosylation cascades
Hongjiao Zhang, Anxin Zhang, Jiatong Ji, Wen-Bing Yin
https://onlinelibrary.wiley.com/doi/10.1002/mlf2.70063
Flavonoids have significant medicinal potential; however, the reliance on plant extraction for their production poses a substantial barrier to their practical application. Innovative approaches like enzymatic cascade catalysis and microbial synthesis present promising avenues for the efficient production of these structurally complex compounds. In this study, two non-natural flavonoids (compounds 7 and 8) were synthesized, with compound 7 displaying antifungal properties. This was achieved through the cascade enzymatic catalysis of naringenin, a key intermediate in flavonoid biosynthesis, using glycosyl- and prenyltransferases. Additionally, metabolic engineering was used to bolster the supply of the dimethylallyl pyrophosphate precursor via the isopentenol utilization pathway in Escherichia coli, facilitating the de novo creation of non-natural flavonoids. Consequently, the titers of compounds 7 and 8 reached 47.4 and 6.6 mg/l, respectively. This research highlights the potential of modular enzyme assembly in generating bioactive flavonoid derivatives and establishes a sustainable platform for the discovery of non-natural flavonoids.
🔹 An infection and pathogenesis mouse model of SARS-CoV-2-related pangolin coronavirus GX_P2V(short_3UTR)
Lai Wei, Shuiqing Liu, Shanshan Lu, Shengdong Luo, Xiaoping An, Huahao Fan, Erguang Li, Lihua Song
https://onlinelibrary.wiley.com/doi/10.1002/mlf2.12122
SARS-CoV-2-related pangolin coronavirus GX_P2V(short_3UTR) is highly attenuated, but can cause mortality in a specifically designed human ACE2-transgenic mouse model, making it a surrogate model for evaluating the efficacy of vaccines and drugs against SARS-CoV-2.
mLife
期刊简介
mLife是由中国科学院主管、中国科学院微生物研究所主办（中国微生物学会为合作单位）的我国微生物学领域第一本综合性高起点英文期刊。mLife瞄准全球微生物学领域高水平科研成果和前沿进展，报道内容覆盖微生物学各个学科。mLife的办刊目标是打造微生物学领域综合性国际旗舰期刊。目前，mLife已被国内外重要数据库ESCI、PubMed、Scopus、CSCD、DOAJ、CAS、中国科技核心期刊等收录。mLife 2024年度JCR影响因子为4.5，位于微生物学科Q1区。
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