Applied and Environmental Microbiology最新文献

{"title":"The APSES transcription factor Swi6B upregulates <i>CATALASE 1</i> transcription to enhance oxidative stress tolerance of <i>Ganoderma lucidum</i>.","authors":"Lingshuai Wang, Lingyan Shi, Shuhan Zhang, Jiping Ma, Cheng Zhang, Huhui Chen, Mingwen Zhao","doi":"10.1128/aem.00679-25","DOIUrl":"https://doi.org/10.1128/aem.00679-25","url":null,"abstract":"<p><p>As sessile organisms, fungi often encounter various stresses throughout their lifespan, resulting in the overproduction of reactive oxygen species, which impedes the normal growth of fungi. Previously, we revealed that the Swi6B transcription factor is involved in the stress tolerance of <i>Ganoderma lucidum</i>. However, the underlying molecular mechanism is unclear. The present study demonstrated that oxidative stress increased the levels of Swi6B. Direct binding of Swi6B to the promoter region of <i>catalase 1</i> (<i>CAT1</i>) resulted in increased transcription of <i>CAT1</i> and reduced H₂O₂ levels in <i>SWI6B</i> overexpressing strains. In addition to increased Swi6B protein, the phosphorylation level of Swi6B was increased by H₂O₂ treatment. The overexpression of <i>SLT2</i>, a mitogen-activated protein kinase that interacts with Swi6B, increased the phosphorylation level of Swi6B, and treatment with H₂O₂ further enhanced this increase. As a result, both the binding of Swi6B to the <i>CAT1</i> gene and tolerance to H₂O₂ were increased in <i>SLT2</i>-overexpressing strains. The present findings revealed that the Slt2-Swi6B-CAT1 pathway responds to oxidative stress and contributes to improving the survival of <i>G. lucidum</i> in adverse environments.IMPORTANCEIn fungi, environmental stress leads to the accumulation of intracellular reactive oxygen species and leads to oxidative stress. Here, we found that the overexpression of the APSES transcription factor <i>Swi6B</i> enhances tolerance to oxidative stress in <i>Ganoderma lucidum</i>. Swi6B binds to the promoter region of <i>CAT1</i>, which increases <i>CAT1</i> transcription and reduces the H₂O₂ levels. In addition, the phosphorylation of Swi6B by Slt2 promotes the regulation of <i>CAT1</i> by Swi6B. The Slt2-Swi6B-CAT1 pathway is important for the response of <i>G. lucidum</i> to oxidative stress.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0067925"},"PeriodicalIF":3.9,"publicationDate":"2025-06-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144324326","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Articles of Significant Interest in This Issue.","authors":"","doi":"10.1128/aem.01148-25","DOIUrl":"10.1128/aem.01148-25","url":null,"abstract":"","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":"91 6","pages":"e0114825"},"PeriodicalIF":3.9,"publicationDate":"2025-06-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12180515/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144324327","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Toward simulating offshore oilfield conditions: insights into microbiologically influenced corrosion from a dual anaerobic biofilm reactor.","authors":"Liam M Jones, Niall Hanrahan, Maria Salta, Torben Lund Skovhus, Kathryn Thomas, Timothy Illson, Julian Wharton, Jeremy S Webb","doi":"10.1128/aem.02221-24","DOIUrl":"10.1128/aem.02221-24","url":null,"abstract":"<p><p>Oilfield systems are a multifaceted ecological niche, which consistently experiences microbiologically influenced corrosion. However, simulating the environmental conditions of an offshore system within the laboratory is notoriously difficult. A novel dual anaerobic biofilm reactor protocol allowed a complex mixed-species marine biofilm to be studied. Interestingly, electroactive corrosive bacteria and fermentative electroactive bacteria growth was supported within the biofilm microenvironment. Critically, the biotic condition exhibited pits with a greater average area, which is characteristic of microbiologically influenced corrosion. This research seeks to bridge the gap between experimental and real-world scenarios, ultimately enhancing the reliability of biofilm management strategies in the industry.</p><p><strong>Importance: </strong>It is becoming more widely understood that any investigation of microbiologically influenced corrosion requires a multidisciplinary focus on multiple lines of evidence. Although there are numerous standards available to guide specific types of testing, there are none that focus on integrating biofilm testing. By developing a novel dual anaerobic reactor model to study biofilms, insights into the different abiotic and biotic corrosion mechanisms under relevant environmental conditions can be gained. Using multiple lines of evidence to gain a holistic understanding, more sustainable prevention and mitigation strategies can be designed. To our knowledge, this is the first time all these metrics have been combined in one unified approach. The overall aim of this paper was to explore recent advances in biofilm testing and corrosion research and provide recommendations for future standards being drafted. However, it is important to note that this article itself is not intending to serve as a standard.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0222124"},"PeriodicalIF":3.9,"publicationDate":"2025-06-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12175496/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143539815","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Identification of factors limiting the efficiency of transplanting extracellular electron transfer chains in <i>Escherichia coli</i>.","authors":"Laura-Alina Philipp, Lukas Kneuer, Carina Mayer-Windhorst, Simon Jautelat, Nhat Quang Le, Johannes Gescher","doi":"10.1128/aem.00685-25","DOIUrl":"10.1128/aem.00685-25","url":null,"abstract":"<p><p>Research in electro-microbiology provides unique opportunities to study and exploit microbial physiology. Several efforts have been made to transplant the extracellular electron transport chain from the native exoelectrogenic model organism <i>Shewanella oneidensis</i> into <i>Escherichia coli</i>. However, systematic comparisons between donor and recipient strain configurations are largely missing. Hence, the proposed minimal protein set, consisting of the <i>c</i>-type cytochromes cytoplasmic membrane protein A (CymA), small tetraheme cytochrome (STC), MtrA, and MtrC, as well as the <i>β</i>-barrel protein MtrB, was heterologously expressed in <i>E. coli</i> in different expansion stages. These stages were compared to corresponding <i>S. oneidensis</i> strains in terms of anthraquinone-2,6-disulfonate (AQDS) and ferric citrate reduction rates. This revealed that transplantation of heterologous extracellular electron transfer (EET) chains is associated with a tremendous decrease in electron transfer rates. As the acquired electron transfer rates were not competitive to <i>S. oneidensis</i>, it was hypothesized that protein localization and maturation might be affected by heterologous expression. Hence, the type II secretion system from <i>S. oneidensis</i> was also transplanted into an <i>E. coli</i> strain. The latter allowed the secretion of the terminal reductase MtrC onto the cell surface of <i>E. coli</i> for the first time. This was correlated with significantly increased but still insufficient extracellular electron transfer rates. Further experiments suggest that the correct folding of MtrB might be a further bottleneck.IMPORTANCEResearch on transplanting extracellular electron transfer (EET) chains into non-native exoelectrogens is vital for advancing bioenergy and bioremediation technologies. Enabling these organisms to transfer electrons to external surfaces like anodes can enhance microbial fuel cell efficiency and electricity generation from organic waste. This approach can broaden the range of substrates and products for biotechnological applications, offering innovative solutions for sustainable production. Our work shows that transplanting the EET chain of <i>Shewanella oneidensis</i> into <i>Escherichia coli</i> is more complex than previously suggested. The heterologous expression of only <i>c</i>-type cytochromes and the β-barrel protein MtrB is insufficient for competitive reduction rates. Predominantly, MtrC and MtrB require specific proteins for transport and folding, necessitating co-expression and maturation. We could identify the type II secretion system of <i>S. oneidensis</i> as crucial for MtrC secretion in <i>E. coli</i>. Thereby, this work highlights the substrate specificity of bacterial type II secretion systems, suggesting methods to optimize protein production and secretion in bioelectrochemical applications.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0068525"},"PeriodicalIF":3.9,"publicationDate":"2025-06-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12175506/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143958061","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Impact of flushing procedures on drinking water biostability and invasion susceptibility in distribution systems.","authors":"Fien Waegenaar, Thomas Pluym, Elise Vermeulen, Bart De Gusseme, Nico Boon","doi":"10.1128/aem.00686-25","DOIUrl":"10.1128/aem.00686-25","url":null,"abstract":"<p><p>Ensuring high-quality drinking water remains challenging, as complaints about odors, discoloration, or contamination persist. In Belgium and beyond, traditional flushing is a common curative strategy that involves discharging large water volumes through hydrants while the network remains in use. In some cases, free chlorine (0.5 mg/L) is added, and consumers are advised not to drink the water. However, flushing can alter water biostability, potentially increasing susceptibility to microbial invasion. This study used a pilot-scale drinking water distribution system with three identical 100 m polyvinyl chloride(PVC) loops (DN 80 mm) to assess the impact of flushing with and without chlorination as practiced in chlorinated networks. Loop 1 was flushed with tap water and sodium hypochlorite (NaOCl), followed by two non-chlorinated flushes, loop 2 was unflushed, and loop 3 underwent three flushes. Biostability was assessed using online flow cytometry, and susceptibility to bacterial invasion (<i>Aeromonas media</i>, <i>Pseudomonas putida</i>, and <i>Serratia fonticola</i>) was evaluated in the days following flushing. The water had a 7-day residence time. Results showed that chlorinated flushing promoted microbial regrowth (3.8 × 10⁵ vs 2.0 × 10⁵ and 1.6 × 10⁵ cells/mL for loops 1, 2, and 3, respectively), primarily of resident <i>Sphingopyxis</i> spp. Biofilm cell densities (~4 × 10⁶ cells/cm²) remained stable across conditions. Bacterial indicators declined over time, with <i>P. pudita</i> and <i>S. fonticola</i> surviving longer (>100 hours) than <i>A. media</i> (13 hours). Decay rates were highest in chlorinated loops, likely due to increased microbial competition. For example, the decay constant of <i>S. fonticola</i> at 20°C was -0.082 h^-1, -0.042 h^-1, and -0.027 h^-1 for loops 1, 2, and 3, respectively.</p><p><strong>Importance: </strong>Traditional flushing is used as a curative strategy to solve unwanted quality issues during distribution, yet its impact on microbial biostability remains poorly understood. This study provides critical insights into how traditional flushing, both with and without chlorination, influences microbial regrowth and susceptibility to invasion. Findings reveal that chlorinated flushing promotes the regrowth of resident drinking water bacteria while accelerating the decay of introduced unwanted bacterial indicators, emphasizing the complex trade-off between microbial control and system stability. Understanding these dynamics is essential for optimizing flushing procedures, minimizing unintended consequences, and improving distribution system resilience.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0068625"},"PeriodicalIF":3.9,"publicationDate":"2025-06-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12175504/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143966500","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Citric acid impairs type B trichothecene biosynthesis of <i>Fusarium graminearum</i> but enhances its growth and pigment biosynthesis: transcriptomic and proteomic analyses.","authors":"Luzhou Cai, Ling Li, Dong Li, Yanping Wu, Jinrong Bai, Kai Zhong, Hong Gao","doi":"10.1128/aem.01531-24","DOIUrl":"10.1128/aem.01531-24","url":null,"abstract":"<p><p><i>Fusarium graminearum</i> is a pathogenic fungus that causes devastating plant diseases such as Fusarium head blight, leading to significant food waste and posing a threat to human health due to the accumulation of mycotoxins. As a soil-borne fungus, <i>F. graminearum</i> interacts closely with plants and soil, completing its life cycle in an intricate environment. In this investigation, citric acid (CA), a plant root exudate and heavy metal chelator, was investigated for its effect on <i>F. graminearum</i> at concentrations of 2.5, 5, 10, and 20 mM. The fungus treated with this CA gradient exhibited different growth conditions and mycelial colors. Integrative analysis of transcriptomics and proteomics found that the growth of the fungus was accelerated by the upregulation of carbon source metabolism-related enzymes such as phosphoenolpyruvate carboxykinase (PEPCK), Glpk, and LAI12, and the mycelial pigments were altered by polyketide biosynthetic enzymes like AurF, AurJ, and AurT, which were responsible for the production of aurofusarin and rubrofusarin. Interestingly, many <i>Tri</i> genes and their corresponding proteins along with type B trichothecene biosynthesis were significantly downregulated, though the fungal growth was promoted. In this study, the CK, 2.5, and 5 mM groups had similar expression patterns of RNA and protein, while the 10 and 20 mM groups were alike. With the validation of CK, 5, and 10 mM groups, the discovery of CA as a type B trichothecene biosynthesis inhibitor and growth promoter was expected to facilitate its reasonable application and contribute to further research for the prevention of <i>F. graminearum</i>.</p><p><strong>Importance: </strong><i>Fusarium graminearum</i> is a challenging phytopathogen that causes plant disasters worldwide, such as Fusarium head blight and ear rot in small grain crops like wheat and maize. Besides, the invasion of the fungus on plants is often accompanied by the production of health-threatening mycotoxins-trichothecenes. Therefore, the control of <i>Fusarium graminearum</i> has always been a hot area of research. However, currently, the prevalence of these plant diseases around the world and the mycotoxin accumulation beyond safety limits indicate the necessity for more related research. In the last decades, researchers have sought to identify the key substances associated with the growth, propagation, and mycotoxin production of the fungus, such as carbon and nitrogen sources. Organic acids have always been considered antifungal agents due to their abundant H+ content. However, their comprehensive impact on fungi has been rarely investigated. This research focused on the effect of citric acid, a type of crop exudate and a common heavy-metal chelator, on the metabolism of <i>Fusarium graminearum</i>. The result was expected to provide a theoretical basis for further studies on plants-soil-fungi interactions and the reasonable utilization of citric acid in agriculture.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0153124"},"PeriodicalIF":3.9,"publicationDate":"2025-06-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12175541/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143959077","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Bacterial genome-wide association studies: exploring the genetic variation underlying bacterial phenotypes.","authors":"Qiuping Yang, Xiaoqi Wang, Mengting Han, Huanjing Sheng, Yulu Sun, Li Su, Wenjing Lu, Mei Li, Siyue Wang, Jia Chen, Shenghui Cui, Bao-Wei Yang","doi":"10.1128/aem.02512-24","DOIUrl":"10.1128/aem.02512-24","url":null,"abstract":"<p><p>With the continuous advancements in high-throughput genome sequencing technologies and the development of innovative bioinformatics tools, bacterial genome-wide association studies (BGWAS) have emerged as a transformative approach for investigating the genetic variations underlying diverse bacterial phenotypes at the population genome level. This review provides a comprehensive overview of the application of BGWAS in elucidating genetic determinants of bacterial drug resistance, pathogenicity, host specificity, biofilm formation, and probiotic fermentation characteristics. We systematically summarize the BGWAS workflow, including study design, data analysis pipelines, and the bioinformatics software employed at various stages. Furthermore, we highlight specialized tools tailored for BGWAS and discuss their unique features and applications. We also discuss confounding factors that can influence the accuracy and reliability of BGWAS results, including population structure, linkage disequilibrium, and multiple testing. By incorporating recent advancements, this review serves as a comprehensive reference for researchers utilizing BGWAS to uncover the genetic basis of bacterial phenotypes.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0251224"},"PeriodicalIF":3.9,"publicationDate":"2025-06-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12175497/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144075347","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Organic particle scavenging by marine bacteria: influences of bacterial nanoscale surface properties.","authors":"Yosuke Yamada, Toshiaki Mochizuki, Nirav Patel, Farooq Azam, Hideki Fukuda, Toshi Nagata, Satoshi Mitarai","doi":"10.1128/aem.01049-25","DOIUrl":"https://doi.org/10.1128/aem.01049-25","url":null,"abstract":"<p><p>Marine bacteria contribute to biogeochemical cycles by scavenging organic nanoparticles such as cell fragments, viruses, excretions from phytoplankton and bacteria, and naturally occurring polymeric substances. Nonetheless, bacterial surface properties influencing this process remain poorly understood. A previous study found that marine bacteria exhibit significant variation in surface roughness, which affects nanoparticle attachment, influencing bacterial survival and marine biogeochemical cycles. However, cell surface characteristics such as Young's modulus and adhesiveness have rarely been measured. This study investigated bacterial nanoscale surface properties and their effects on nanoparticle attachment. Atomic force microscopy was employed to measure these parameters of 559 individual bacterial cells collected from Okinawa coastal waters. These results revealed significant variation in Young's modulus (6-21,000 kPa) and adhesiveness (86-1,200 pN). Subsequent coincubation experiments with polystyrene beads and virus-like particles, as model nanoparticles, demonstrated a significant negative correlation between Young's modulus and the attachment of virus-like particles, whereas no significant relationships were observed for other factors. Our results suggest that measuring bacterial surface properties provides novel insights into their strategies for resource utilization and their contribution to marine biogeochemical cycles.IMPORTANCESurface properties of marine bacteria are believed to influence their ability to acquire nanoparticles for nutrition. However, studies on these properties are limited, and the relationship with nanoparticle attachment remains unclear. This study measured Young's modulus and adhesiveness of marine bacteria, investigating their variability and their influence upon nanoparticle attachment. This work sheds light on biophysical mechanisms driving bacterial nanoparticle utilization, as well as ecological and biogeochemical implications of bacterial surface properties in marine environments.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0104925"},"PeriodicalIF":3.9,"publicationDate":"2025-06-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144301054","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Ultrahigh-throughput screening of environmental bacteria for proteolytic activity using droplet-based microfluidics.","authors":"Akihiro Nakamura, Yoshiyuki Suzuki, Nobuyuki Homma, Yosuke Shida, Rikako Sato, Hiroaki Takaku, Wataru Ogasawara","doi":"10.1128/aem.00109-25","DOIUrl":"https://doi.org/10.1128/aem.00109-25","url":null,"abstract":"<p><p>Exploration of diverse microbial sources, particularly environmental bacteria, is needed to identify novel and efficient peptidases/proteases that can be used in a variety of industrial applications. However, conventional function-based screening methods are inefficient and preclude the use of diverse microbial resources. This study illustrates a revolutionary approach to microbial screening using droplet-based microfluidics and fluorescence-activated droplet sorting that targets endopeptidases. Droplet-based microfluidic systems are a powerful tool for culturing microorganisms and for detecting microbial functions inside droplets with ultra-high throughput. However, droplet-based microfluidics for screening the proteolytic activity of environmental bacteria at a large scale remains largely unexplored. Here, we screened approximately 630,000 microorganisms in 6 h and obtained four species with high peptidase activity using droplet-based microfluidics. Furthermore, we isolated an Asp-specific endopeptidase from the isolated bacteria <i>Lysobacter soli</i> and showed that its activity was 2.4-fold higher than that of the related commercially available enzyme. The successful isolation of Asp-specific peptidase with superior activity in a short period of time compared to existing alternatives underscores the efficacy of droplet-based microfluidics for function-based microbial screening.</p><p><strong>Importance: </strong>As global efforts to reduce environmental impact progress, realization of the significance of biomanufacturing bio-based products has risen, increasing the demand for microbial-based manufacturing. Producing diverse bio-based products through biomanufacturing requires isolating suitable host organisms from environmental sources and screening them for essential genetic characteristics. Efficient screening methods based on microbial activity and functionality are thus essential to significantly expand the scope of bio-based products. Here, we demonstrate the development of a highly efficient screening system for functional screening of environmental bacteria using a droplet-based microfluidic device. This platform enables the streamlined isolation of microbial strains and acquisition of genetic resources from the environment and is tailored to specific microbial activities and functions. In this study, we have demonstrated the efficacy of this droplet-based method for functional screening and have shown its potential for scalability to industrial levels for advancing bio-based production.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0010925"},"PeriodicalIF":3.9,"publicationDate":"2025-06-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144282136","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Enzymatic property and stabilization mechanism of LysBT1, a novel polyextremotolerant endolysin with a C-terminal S-layer homology domain.","authors":"Yu Li, Ke Luo, Chaofeng Jiang, Yihao Zhang, Yong Yang, Yitong Yao, Huai Li, Fei Gan, Xiao-Feng Tang, Bing Tang","doi":"10.1128/aem.00867-25","DOIUrl":"https://doi.org/10.1128/aem.00867-25","url":null,"abstract":"<p><p>Phage-encoded endolysins are getting increasing attention because of their potential to serve as alternative antimicrobials to combat antibiotic-resistant bacteria. Here, we report a novel endolysin LysBT1, which is encoded by a prophage of thermophilic <i>Brevibacillus thermoruber</i> WF146 and comprises an N-acetylmuramoyl-L-alanine amidase domain and an S-layer homology (SLH) domain not found in known endolysins. LysBT1 is not only extremely thermostable, retaining more than 60% activity after 1 h incubation at 95°C, but also highly stable over a wide pH range of 4.0-11.0. Moreover, the thermostability of LysBT1 could be enhanced by EDTA or reducing agents. Although none of the seven cysteine residues of LysBT1 participate in disulfide bond formation, six of them, including the catalytic Zn²⁺-coordinating Cys156, are involved in stabilizing the enzyme at elevated temperatures. The SLH domain contributes to the thermostability of LysBT1 and mediates cell surface binding of the enzyme to facilitate enzymatic lysis of strain WF146 cells via increasing local enzyme concentration around the substrate. LysBT1 is capable of trimerization, where the SLH domains are predicted to form a three-prong spindle-like trimer similar to that in S-layer proteins. The SLH domain of LysBT1 could bind to cell surfaces of both Gram-positive and Gram-negative bacteria. LysBT1 can lyse not only Gram-positive strain WF146, <i>Geobacillus stearothermophilus</i>, and <i>Bacillus subtilis</i> but also Gram-negative <i>Escherichia coli</i> and <i>Acinetobacter baumannii</i> with the aid of EDTA or citric acid. EDTA also facilitates LysBT1 to lyse <i>Bacillus cereus</i>, probably because EDTA-induced disorganization of the S-layer allows LysBT1 to access and hydrolyze the peptidoglycan.IMPORTANCEThe emergence of antibiotic-resistant bacteria has led to an urgent requirement to develop novel antimicrobials, and endolysins are regarded as ideal alternatives to antibiotics. The thermostability of endolysins plays an important role in the feasibility of enzymatic bacteriolysis. However, reports on thermostable endolysins are limited, and little is known about their stabilization mechanisms. Our results demonstrate that the thermophile-derived prophage endolysin LysBT1 is highly thermostable and functional under polyextreme (multiple forms of stress) conditions, enabling the enzyme to lyse both Gram-positive and Gram-negative bacteria in synergy with outer membrane permeabilizer. Moreover, we found that the unique S-layer homology domain of LysBT1 contributes to the stability, activity, oligomerization, and cell-wall binding ability of the enzyme. This study not only characterizes a novel endolysin but also provides new clues about the stabilization mechanisms of endolysins.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0086725"},"PeriodicalIF":3.9,"publicationDate":"2025-06-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144282233","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}