Applied and Environmental Microbiology最新文献

{"title":"Asphaltene biotransformation by a novel enzyme thiol peroxidase from <i>Micrococcus</i> sp. IITD107.","authors":"Nidhi Patil, Preeti Srivastava","doi":"10.1128/aem.00151-25","DOIUrl":"https://doi.org/10.1128/aem.00151-25","url":null,"abstract":"<p><p>Asphaltenes are known to cause high density and viscosity in heavy crude oil. The gene for thiol peroxidase was found to be present in five members of the nine-membered bacterial consortium able to biotransform about 75% of asphaltenes. Here, we report cloning, expression, purification, and detailed characterization of a thiol peroxidase enzyme from one of the consortium members, <i>Micrococcus</i> sp. IITD107. Asphaltene was treated with the crude cell lysate obtained after overexpression of thiol peroxidase from heterologous host <i>Escherichia coli</i> as well as with purified enzyme. A significant reduction in the major peaks obtained by gas chromatography mass spectrometry (GC-MS) of asphaltene was observed, corresponding to small polyaromatic hydrocarbons such as benzaldehyde, phenol-methylethylidene, benzenepropionic acid, and linear aliphatic chains such as heptadecane, tetradecane, octadecane, etc. Changes in the structure of asphaltene were also observed in the Fourier transform infrared spectroscopy and nuclear magnetic resonance spectra. The elemental analysis determined around 60% reduction in sulfur and 69% reduction in nitrogen. A decrease in aromaticity of asphaltene was also observed. Scanning electron microscopy imaging of the treated asphaltene fraction captured during the course of biotransformation revealed formation of pores in the structure. This is the first report demonstrating the use of thiol peroxidase for asphaltene biotransformation. The enzyme can also be used for the biological synthesis of porous carbon, which has not been reported to date.IMPORTANCEHeavy crude oil is abundant but contains asphaltene, which results in its high density and viscosity, making it unsuitable for commercial application. The removal of asphaltene leads to a reduction in heaviness of the oil and renders it light and suitable for commercialization. Asphaltene is a complex and large polyaromatic hydrocarbon consisting of several heteroatoms. The use of enzymatic biotransformation of asphaltene aids in breaking down the complex molecule into smaller moieties without affecting the calorific value. This study helps in identifying a novel enzyme thiol peroxidase for biotransformation of asphaltene and valorization of asphaltene to synthesize porous carbon.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0015125"},"PeriodicalIF":3.7,"publicationDate":"2025-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144939806","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":"Genetic manipulation of <i>cys</i>H/<i>cys</i>J in <i>Citrobacter</i> sp. XT1-2-2 enhanced cadmium immobilization by regulating metabolic pathways.","authors":"Zhudong Liu, Wei Cheng, Yilu Li, Shiping Shan, Shandong Wu, Xiaowu Wei, Hua Yang, Min Zhang, Dongxia Du","doi":"10.1128/aem.00856-25","DOIUrl":"https://doi.org/10.1128/aem.00856-25","url":null,"abstract":"<p><p>The <i>Citrobacter</i> sp. XT1-2-2 strain has emerged as a promising candidate for cadmium immobilization; however, the genetic basis underlying its sulfur-mediated bioremediation mechanisms remains inadequately understood. To address this gap, we concentrated on two pivotal genes, <i>cys</i>H and <i>cys</i>J, within the sulfate assimilation pathway. We constructed <i>Citrobacter</i> sp. XT1-2-2-::APS and <i>Citrobacter</i> sp. XT1-2-2-::SiR strains with overexpression of <i>cys</i>H or <i>cys</i>J for functional characterization. Transmission electron microscopy demonstrated a significant enhancement in the biosynthesis of CdS nanoparticles in both overexpression strains. This increase was attributed to the elevated production of hydrogen sulfide. Complementary physicochemical analyses, including Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and powder X-ray diffraction, further indicated that overexpression of <i>cys</i>H or <i>cys</i>J modified functional groups on the surface and enhanced the efficiency of sulfur metabolism. Microcosm experiments demonstrated that the contents of Cd²⁺ in the roots, culms, leaves, and grains inoculated with overexpression strains were significantly lower than those observed in the wild-type strain. These findings establish a critical role for <i>cys</i>H/<i>cys</i>J-mediated metabolic pathway regulation in cadmium immobilization. They provide a theoretical foundation for the exploration of novel bacterial-assisted techniques, marking a breakthrough in environmental biotechnology from fundamental research to the engineering application of genetically engineered bacteria.IMPORTANCEThe presence of cadmium in paddy soil poses a significant concern, primarily due to its potential threat to food safety and public health within the soil-plant system and the broader food chain. The genes <i>cys</i>H and <i>cys</i>J were overexpressed under the regulation of the erythromycin promoter within the sulfate assimilation pathway in the <i>Citrobacter</i> sp. XT1-2-2 strain. The resulting overexpression strains (XT1-2-2-::APS and XT1-2-2-::SiR) exhibited enhanced biosynthesis of CdS nanoparticles, attributed to increased hydrogen sulfide production. Compared to the wild-type strain, cadmium concentrations in the grains of XT1-2-2-::APS and XT1-2-2-::SiR were reduced by 36.49% and 62.56%, respectively. Furthermore, the residual cadmium content in the soil was elevated by 36.36% (<i>P</i> < 0.01) and 27.27% (<i>P</i> < 0.01), respectively. These results provided a theoretical foundation for the exploration of novel bacterial-assisted techniques aimed at cadmium remediation, marking a breakthrough in environmental biotechnology from fundamental research to the engineering application of genetically engineered bacteria.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0085625"},"PeriodicalIF":3.7,"publicationDate":"2025-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144939783","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":"Optimized genetic circuitry and reporters for sensitive whole-cell arsenic biosensors: advancing environmental monitoring.","authors":"Yan Guo, Ming-Qi Liu, Xue-Qin Yang, Ying-Yan Guo, Chang-Ye Hui","doi":"10.1128/aem.00601-25","DOIUrl":"10.1128/aem.00601-25","url":null,"abstract":"<p><p>The ubiquitous presence of arsenic pollution poses a significant threat to both ecosystem integrity and human health, necessitating the development of sensitive methods for arsenic detection. This study presents the development of innovative whole-cell biosensors that leverage the ArsR regulatory system within both naturally coupled and non-coupled genetic circuits, specifically optimized for detecting the highly toxic arsenic species (As(III)). These biosensors incorporate an indigoidine pigment as a reporting system and utilize the glycerol facilitator protein (GlpF) to enhance arsenic transport, offering a significant advantage over conventional detection methods by streamlining the detection process and eliminating the requirement for specialized instrumentation. Our findings reveal that the indigoidine-based biosensor, TOP10/pnK12-ABS-ind, in particular, exhibits an extensive linear detection range of 0.039 to 20 μM across various water matrices, effectively adhering to and exceeding the arsenic detection guidelines set by the World Health Organization and Chinese national standards. This research advances arsenic biosensing technology by developing a practical, cost-effective detection solution for arsenic in various aquatic settings. Compared to our previous work, this study demonstrates significant improvements in detection range and sensitivity while highlighting the importance of tailored genetic circuit design based on the reporter's choice.IMPORTANCEArsenic pollution poses a significant threat to global ecosystems and human health, with millions of people at risk of exposure through contaminated water sources. Detecting arsenic, especially in its highly toxic form (As(III)), is crucial for environmental monitoring and public health protection. However, conventional detection methods often require costly equipment and specialized expertise, limiting their feasibility in resource-limited regions. Our study addresses this challenge by developing whole-cell biosensors that leverage natural genetic circuits and a novel indigoidine pigment reporter. These biosensors offer a practical, cost-effective, and portable solution for arsenic detection, streamlining the process and eliminating the need for complex instrumentation. By enabling real-time monitoring and on-site analysis, our biosensors have the potential to significantly enhance environmental monitoring capabilities, facilitate timely remediation efforts, and safeguard public health in areas affected by arsenic contamination.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0060125"},"PeriodicalIF":3.7,"publicationDate":"2025-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12366313/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144599192","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":"Antagonistic mechanism of <i>Bacillus velezensis</i> HX0039 as a biocontrol agent against <i>Trichoderma virens</i>-induced \"Sanghuang\" green mold.","authors":"Ying Zhang, Wenheng Gao, Hui Zhang, Tao Sun, Huixiang Yang, Yan Liu, Xiaoyang Han, Dengke Yin, Weifang Xu","doi":"10.1128/aem.00005-25","DOIUrl":"10.1128/aem.00005-25","url":null,"abstract":"<p><p>Green mold disease caused by <i>Trichoderma</i> spp. is one of the serious challenges in the production of \"Sanghuang\" mushroom (<i>Sanghuangporus vaninii</i>). Herein, we report the biocontrol efficacy and related mechanisms of <i>Bacillus velezensis</i> HX0039, an antagonistic strain previously isolated from soil for field cultivation of the \"Sanghuang\" mushroom. Strain HX0039 exhibited broad-spectrum activity against 17 fungal pathogens, especially <i>Trichoderma virens</i> SH4 (a highly pathogenic strain causing \"Sanghuang\" green mold), and increased the cell membrane permeability of <i>T. virens in vitro</i>. In the \"Sanghuang\" mode of green mold disease, HX0039 had no negative effects on the mycelial growth or fruiting body formation of mushrooms and exhibited excellent biocontrol efficacy. Genomic analysis revealed that HX0039 possesses multiple genes that contribute to antimicrobial compound production, and iturin A, fengycin, macrolactin A, surfactin, and bacillibactin were detected through UPLC-Q-Exactive-Orbitrap-MS. Among these metabolites, iturin A may play a role in combating <i>T. virens</i> SH4. Hemolytic tests, cell experiments, and mouse studies demonstrated that HX0039 is a biosafe strain. Our results will provide insights into the antifungal mechanisms of <i>B. velezensis</i> HX0039 and will facilitate the further application of this strain as a biocontrol agent against <i>T. virens</i>-induced \"Sanghuang\" green mold.IMPORTANCE\"Sanghuang\" mushroom is a valuable edible and medicinal fungus. It provides numerous health benefits, including antitumor, anti-inflammatory, and immunity-enhancing properties. However, its growth is affected by green mold disease caused by <i>Trichoderma,</i> which severely hampers yield and quality. Conventional fungicide-based control methods have drawbacks like health and environmental risks, as well as the emergence of resistant pathogens. This study innovatively focuses on <i>Bacillus velezensis</i> HX0039, a strain proven to be safe for both \"Sanghuang\" mushrooms and mice. <i>In vivo</i> and <i>in vitro</i> experiments showed that HX0039 not only exhibits strong antifungal activity but also effectively prevents and controls the occurrence of \"Sanghuang\" green mold disease. Furthermore, the novelty of this research lies in its potential mechanism of action: HX0039 produces diverse metabolites like lipopeptides (especially iturin A), macrolactin A, and bacillibactin to exert its antagonistic activities. Therefore, this work demonstrates the great potential of <i>B. velezensis</i> HX0039 as an alternative to chemical fungicides in \"Sanghuang\" production.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0000525"},"PeriodicalIF":3.7,"publicationDate":"2025-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12366312/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144582894","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":"Nucleomodulins from gut bacteria: diverse mechanisms of translocation and interaction with host nuclear processes.","authors":"Sania Korgaonkar, Chandrani Bose, Swadha Anand","doi":"10.1128/aem.00211-25","DOIUrl":"10.1128/aem.00211-25","url":null,"abstract":"<p><p>Nucleomodulins (NMs) are bacterial nuclear-targeted effector proteins that interfere with a multitude of host cellular processes to shield pathogens from host immune responses. Recent years observed a surge in NM-related research owing to their potential role in both infectious diseases and cancer development. However, considering the complex nature of the interaction between NM and host factors, the field of \"NM-disease axis\" is still in the nascent phase. Thus, a comprehensive view of the known mechanisms of translocation of NMs to the host cell nucleus and mode of action, thereafter, is crucial toward deeper exploration of the \"NM-disease axis.\" The human gut is the major host niche to harbor bacterial cells (as part of \"gut microbiota\"). The current review provides an extensive collation of nucleomodulin-mediated mechanisms employed by opportunistic gut pathogens. The insights from the review would help in designing future experiments toward utilizing the NM-associated host-pathogen interaction modules in disease diagnostics and therapy.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0021125"},"PeriodicalIF":3.7,"publicationDate":"2025-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12366366/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144658181","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":"Amicoumacins produced by the native citrus microbiome isolate <i>Bacillus safensis</i> inhibit the Huanglongbing-associated bacterial pathogen \"<i>Candidatus</i> Liberibacter asiaticus\".","authors":"Flávia C Vieira, Kranthi K Mandadi, Manikandan Ramasamy, Amancio de Souza, Kiana Callahan, Corrie Fyle, Andrew Kamemoto, Amanda G Koontz, Christopher Yang, Robert Crowley, Kevin G M Kou, Katherine N Maloney, M Caroline Roper","doi":"10.1128/aem.00869-25","DOIUrl":"10.1128/aem.00869-25","url":null,"abstract":"<p><p>Huanglongbing (HLB) is a devastating citrus disease associated with the gram-negative, phloem-limited, and unculturable bacterium \"<i>Candidatus</i> Liberibacter asiaticus (<i>C</i>Las),\" which is transmitted by the Asian citrus psyllid <i>Diaphorina citri</i>. Despite extensive research, effective, long-term, and sustainable solutions for managing HLB remain elusive. Oxytetracycline (OTC) is currently used as an emergency measure, but there is an urgent need for alternative compounds to complement or replace OTC. In this study, we identified amicoumacins, a class of antimicrobial compounds produced by the bacterium <i>Bacillus safensis</i> CB729 isolated from the citrus microbiome, and demonstrated their ability to suppress <i>C</i>Las. Genome mining of <i>B. safensis</i> CB729, combined with metabolomic analysis and bioassay-guided fractionation, revealed the presence of amicoumacins and related derivatives in fractions inhibitory to <i>Liberibacter crescens</i>, a culturable surrogate for <i>C</i>Las. We tested commercially available synthetic amicoumacins A and B, along with a <i>B. safensis</i>-derived amicoumacin mixture, against <i>L. crescens</i> and <i>C</i>Las. We determined the MICs of amicoumacin A (1.25 µg/mL) and amicoumacin B (10 µg/mL) against <i>L. crescens</i>. Furthermore, amicoumacin B and the amicoumacin mixture significantly reduced <i>C</i>Las populations in <i>ex vivo</i> citrus hairy root assays. This study highlights the potential of amicoumacins as a promising group of natural products for the management of HLB, offering valuable insights for the development of novel and sustainable disease control strategies.IMPORTANCEFor two decades, the citrus industry has been severely impacted by Huanglongbing (HLB), a devastating disease caused by \"<i>Candidatus</i> Liberibacter asiaticus (<i>C</i>Las)\" and transmitted by the Asian citrus psyllid (<i>Diaphorina citri</i>). Despite extensive research, effective, long-term, and sustainable solutions remain unavailable for growers. Currently, medically relevant antibiotics, such as oxytetracycline (OTC), are used as an emergency response to combat HLB in Florida, the most affected citrus-producing state in the U.S. This underscores the urgent need for alternative treatments that can be used in rotation or as replacements for OTC. Here, we present amicoumacins, a group of bioactive secondary metabolites with antibiotic properties. We identified amicoumacin B and its derivatives from the culture broth of a <i>Bacillus safensis</i> isolate, native to citrus, and demonstrated their ability to inhibit <i>Liberibacter</i> spp. and reduce <i>C</i>Las populations in citrus tissue. This study highlights how microbial discovery can lead to the identification of antimicrobial compounds with potential applications in plant disease management.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0086925"},"PeriodicalIF":3.7,"publicationDate":"2025-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12366304/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144752101","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":"Deciphering the phenol degradation metabolic pathway in <i>Scedosporium apiospermum</i> HDO1.","authors":"Laura L Diaz Ortiz, David Botero-Rozo, Natalia Vargas, Sandra Ortiz, Silvia Restrepo, Martha J Vives","doi":"10.1128/aem.01038-25","DOIUrl":"10.1128/aem.01038-25","url":null,"abstract":"<p><p>The filamentous fungus <i>Scedosporium apiospermum</i> is a microorganism capable of phenol degradation. Phenol is a petroleum-derived pollutant and a compound widely used in several industries. As a result of its widespread use, phenol is commonly discarded and accumulated in soils and water bodies. In this study, overexpressed and repressed genes that produce enzymes involved in phenol metabolism were identified in <i>S. apiospermum</i> HDO1 when the fungus grows in the presence of phenol. The fungus was grown with either glucose (control) or phenol as the sole carbon source to achieve this. RNA from the mycelium was extracted and sequenced using the Illumina Hiseq-4000 platform, with paired-end libraries. Eighteen genes coding for enzymes related to catechol ortho-cleavage, catechol meta-cleavage, and hydroquinone pathways were annotated from the assembled transcriptome. In the differential gene expression analysis, 11 genes coding for phenol 2-monooxygenase, catechol 1,2-dioxygenase, 3-oxoadipate enol lactonase, hydroxyquinol 1,2-dioxygenase, and aldehyde dehydrogenase were overexpressed. In contrast, one gene coding for protocatechuate 3,4-dioxygenase was repressed. We show for the first time that phenol degradation in <i>S. apiospermum</i> occurs through one of the catechol routes, the catechol-ortho ring cleavage pathway, and through the hydroquinone A pathway. These findings are important because they improve the understanding of how eukaryotic microorganisms with the potential for bioremediation degrade organic pollutants such as phenol.IMPORTANCEIn recent years, bioremediation has emerged as one of the solutions to eliminate pollutants from the environment. <i>Scedosporium apiospermum</i> is one of the fungi capable of tolerating and degrading common pollutants such as phenol. This ability is of great interest as it highlights its potential for use, but also as an important eukaryotic model in contaminant metabolism. <i>S. apiospermum</i> has been widely studied for its clinical significance, but little is yet known about its role in natural environments and its capacity for removing organic pollutants. Using previously published biochemical data together with our differential gene expression results, we validated and completed the proposed phenol metabolic pathways.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0103825"},"PeriodicalIF":3.7,"publicationDate":"2025-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12366324/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144599188","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":"Breaking the paradigm-<i>Prototheca</i> algae occur only sporadically in soils under a temperate climate.","authors":"Mateusz Iskra, Mariusz Dyląg, Filip Paluch, Piotr Szwarczewski, Henryk Krukowski, Hanna Bis-Wencel, Jacek Bielecki, Tomasz Jagielski","doi":"10.1128/aem.00946-25","DOIUrl":"10.1128/aem.00946-25","url":null,"abstract":"<p><p><i>Prototheca</i> are unicellular, achlorophyllous, yeast-like microalgae that have been implicated as the causative agents of opportunistic infections in animals and humans, collectively referred to as protothecosis. Still, the algae are typically saprophytic in nature and mostly occur in areas of high humidity and high organic matter content. Only a few recent studies have explored the environmental reservoir of <i>Prototheca</i>. This work is the first study since the early 1980s to investigate the prevalence of <i>Prototheca</i> spp. in natural terrestrial sources. A total of 226 soil samples were collected, with only 10 yielding <i>Prototheca</i> growth. The overall isolation rate of the algae was 4.4%, with the predominant species being <i>Prototheca bovis</i>. These results show that <i>Prototheca</i> algae only sporadically occur in soils of the temperate climatic zone. Consequently, this work contradicts the ubiquity of <i>Prototheca</i> in soils, breaking the paradigm that, despite the lack of serious experimental appraisal, has long been perpetuated in the literature.IMPORTANCEThere is a scarcity of studies exploring the environmental habitat of <i>Prototheca</i>, a rare and poorly studied genus of microalgae, comprising opportunistic pathogens of humans and animals. This work focuses on the occurrence of <i>Prototheca</i> algae in soil environments. Given the potential hazard to human and animal health from exposure to environmental pathogens such as <i>Prototheca</i>, the prevalence of the microalgae in soils deserves an insightful examination. The study is the first since the 1980s to assess the prevalence of <i>Prototheca</i> spp. in natural terrestrial sources and the first ever performed to approach this topic by molecular methods. Soils have been considered to be ubiquitously inhabited by <i>Prototheca</i>. This view has been perpetuated in the literature despite the lack of any serious experimental appraisal. Surprisingly, we found that the algae are very rare soil inhabitants, clearly breaking the prevailing paradigm of their ubiquity in this environment.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0094625"},"PeriodicalIF":3.7,"publicationDate":"2025-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12366364/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144574707","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":"A droplet microfluidic strategy for cultivation, investigation, and high-throughput isolation of mouse gut microbiome bacteria.","authors":"Sundar Hengoju, Ketema Abdissa, Santiago T Boto, Ashkan Samimi, Karin Martin, Ilse D Jacobsen, Miriam A Rosenbaum","doi":"10.1128/aem.00695-25","DOIUrl":"10.1128/aem.00695-25","url":null,"abstract":"<p><p>Understanding the gut microbiome's intricate dynamics and its impact on host health necessitates the cultivation and isolation of its constituent microorganisms. Traditional culturing techniques often fall short in capturing the diversity of the gut microbiota, particularly for rare and slow-growing species. In this study, we present a droplet microfluidic platform as a high-throughput and efficient method for the cultivation and isolation of mouse gut microorganisms. Droplets, each encapsulating a single cell, were incubated under both aerobic and anaerobic conditions, thereby providing individual microenvironments without nutrient competition and facilitating the growth of a wide range of microorganisms. We validated the platform by successfully cultivating and isolating a diverse array of gut microorganisms, including strains with probiotic potential. A comparative analysis with traditional agar plating techniques revealed a higher number of unique isolates from the droplet cultivation method, demonstrating its enhanced capability to capture the cultivable fraction of the gut microbiome. Beyond isolation, 16S rDNA amplicon sequencing of the diverse microbial cultures in droplets demonstrated that our system reflects changes in microbial diversity induced by dietary interventions in mice. Droplet microfluidics offers a powerful and scalable tool for the high-throughput cultivation, evaluation, and isolation of gut microorganisms, paving the way for deeper insights into the gut microbiome's role in health and disease.IMPORTANCEThe gut microbiome plays a crucial role in health and disease, yet many of its microbial members remain difficult to cultivate using traditional methods. In this study, we present a droplet microfluidic platform that advances our ability to cultivate, isolate, and analyze mouse gut microorganisms. By providing individual microenvironments for single cells, this high-throughput method overcomes limitations of traditional culturing techniques, enhancing microbial diversity recovery compared to standard techniques. Furthermore, this platform can reflect changes in microbial diversity in response to dietary changes in mice, highlighting its potential for studying gut microbial dynamics.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0069525"},"PeriodicalIF":3.7,"publicationDate":"2025-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12366301/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144599187","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":"Multiplexed PCR to measure <i>in situ</i> growth rates of uropathogenic <i>E. coli</i> during experimental urinary tract infection.","authors":"Santosh Paudel, Geoffrey B Severin, Ali Pirani, Melanie M Pearson, Mark T Anderson, Evan S Snitkin, Harry L T Mobley","doi":"10.1128/aem.02382-24","DOIUrl":"10.1128/aem.02382-24","url":null,"abstract":"<p><p>Measuring bacterial growth rates <i>in vitro</i> is routine; however, determining growth rates during infection within a host has been more challenging. Peak-to-trough ratio (PTR) is a technique for studying microbial growth dynamics, calculated using the ratio of replication origin (<i>ori</i>) copies to those of the terminus (<i>ter</i>), as originally defined by whole-genome sequencing (WGS). WGS presents significant challenges in terms of expense and data analysis complexity due to the presence of host DNA in the samples. Here, we used multiplexed PCR with fluorescent probes to estimate bacterial growth rates based on the abundance of <i>ori</i>- and <i>ter</i>-adjacent loci, without the need for WGS. We establish the utility of this approach by comparing growth rates of the uropathogenic <i>Escherichia coli</i> (UPEC) strain HM86 by WGS (PTR) and qPCR to measure the equivalent <i>ori:ter</i> (O:T^PCR). We found that PTR and O:T^PCR were highly correlated and that O:T^PCR reliably predicted growth rates calculated by conventional methods. O:T^PCR was then used to calculate the <i>in situ E. coli</i> growth rates in urine, bladder, and kidneys collected over the course of a week from a murine model of urinary tract infection (UTI). These analyses revealed that the growth rate of UPEC strains gradually increased during the early stages of infection (0-6 h), followed by a slow decrease in growth rates during later time points (1-7 days). This rapid and convenient method provides valuable insights into bacterial growth dynamics during infection and can be applied to other bacterial species in both animal models and clinical infections.</p><p><strong>Importance: </strong>Accurately measuring bacterial growth rates in the host, which plays a crucial role in determining the success of pathogens in establishing infections, poses significant challenges. To address this, bacterial replication rate has been measured as a proxy for the growth rate estimation. While whole-genome sequencing (WGS) has been used for this purpose, it comes with drawbacks such as high costs and difficulties in analyzing bacterial sequences due to the overwhelming presence of host DNA. In this study, we validate a more accessible PCR-based approach compared to the established WGS method and confirmed the reliability of our PCR-based technique. We then applied it to measure the growth rate of <i>Escherichia coli</i> during experimental urinary tract infection in a mouse model. This study provides a cost-effective and efficient alternative to WGS for studying bacterial replication dynamics during infection, potentially offering new insights into pathogen behavior and host-microbe interactions.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0238224"},"PeriodicalIF":3.7,"publicationDate":"2025-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12366353/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144752106","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}