Applied and Environmental Microbiology最新文献

{"title":"Comparative analysis of environmental persistence of SARS-CoV-2 variants and seasonal coronaviruses.","authors":"Geun Woo Park, Boris Reija, Azaibi Tamin, Heather Hicks, Matthew Hayden Flanders, John M Metz, Shufang Fan, Jennifer L Harcourt, Jennifer M Folster, Natalie Thornburg, Jan Vinjé","doi":"10.1128/aem.01688-24","DOIUrl":"https://doi.org/10.1128/aem.01688-24","url":null,"abstract":"<p><p>Conducting persistence studies of infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on environmental surfaces may require a biosafety level 3 (BSL-3) laboratory. We aimed to compare the environmental persistence of BSL-2 level human coronaviruses (229E, NL63, and OC43) and bovine coronavirus (BoCoV) with three SARS-CoV-2 variants (WA-1, Delta, and Omicron). OC43 (1.8 TCID₅₀/mL) and BoCoV (1.0 TCID₅₀/mL) had lower detection thresholds in cell culture assays compared to 229E (150 TCID₅₀/mL) and NL63 (2,670 TCID₅₀/mL) and were used for persistence tests at room temperature. Viable OC43 became undetectable (>5.2log₁₀) after 48 hours on stainless steel and plastic coupons but exhibited extended persistence up to 72 hours on touchscreen glass coupons. In contrast, BoCoV remained viable for up to 120 hours with <1.8 log₁₀ infectivity loss. Both OC43 and BoCoV showed a reduction of >5 log₁₀ on vinyl coupons after 48 hours. On stainless steel coupons, the viability of all three SARS-CoV-2 variants became undetectable (>2.3 log₁₀ reduction) after 48 hours, with minor differences in reduction levels at 24 hours, whereas on touchscreen glass coupons, the viable virus could be detected for up to 48 hours for WA-1 and Omicron and 72 hours for the Delta variant. Regardless of coupon or virus type, viral RNA titers increased <4.5 Ct values after 120 hours. Our data demonstrate distinct persistence characteristics between BoCoV and OC43, with neither fully mimicking SARS-CoV-2 variants. This variability along with the impact of surface types on viral persistence underscores the need for caution when using these viruses as surrogates for SARS-CoV-2.IMPORTANCEIn this study, we evaluated three human seasonal coronaviruses (OC43, NL63, and 229E) and one bovine coronavirus (BoCoV) as potential surrogate viruses for SARS-CoV-2. Our data suggest that among the four surrogate viruses tested, OC43 and BoCoV were the most promising candidates due to their assay sensitivity, ease of handling, and high genetic similarity to SARS-CoV-2. However, neither BoCoV nor OC43 fully mimicked the environmental persistence characteristics of SARS-CoV-2 variants highlighting the potential limitations of using surrogate viruses.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0168824"},"PeriodicalIF":3.9,"publicationDate":"2025-03-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143565838","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":"Deletion of <i>Re</i>-citrate synthase allows for analysis of contributions of tricarboxylic acid cycle directionality to the growth of <i>Heliomicrobium modesticaldum</i>.","authors":"Alexandria M Layton, Christopher McCauley, Kevin E Redding","doi":"10.1128/aem.01772-24","DOIUrl":"https://doi.org/10.1128/aem.01772-24","url":null,"abstract":"<p><p><i>Heliomicrobium modesticaldum,</i> a phototrophic member of the phylum Firmicutes and family Clostridiales, possesses most of the enzymes specific to the reductive tricarboxylic acid (rTCA) cycle, except for the key enzyme, ATP-citrate lyase. It is thought to utilize a split TCA cycle when growing on pyruvate as a carbon source, in which the oxidative TCA (oTCA) direction generates most of the 2-ketoglutarate, but some can be produced in the reductive direction. Although a typical <i>Si</i>-citrate synthase gene is not found in the genome, it was suggested that gene HM1_2993, annotated as homocitrate synthase, actually encodes <i>Re</i>-citrate synthase, which would function as the initial enzyme of the oTCA cycle. We deleted this gene to test this hypothesis and, if true, see what effect severing access to the oTCA cycle would have on this organism. The endogenous CRISPR-Cas system was used to replace the open reading frame with a selectable marker. The deletion mutants could grow on pyruvate but were unable to grow phototrophically on acetate + CO₂ as carbon source. Growth on acetate could be rescued by the addition of different electron sources (formate or ascorbate), suggesting that the oTCA cycle is used to oxidize acetate to generate electrons required to drive the carboxylation of acetyl-CoA. The deletion mutants were capable of growing in acetate minimal media without additional organic supplements beyond formate, demonstrating that the rTCA cycle can be employed to support sufficient 2-ketoglutarate production in this organism, unlike citrate synthase mutants in several chemoheterotrophic organisms utilizing the oTCA cycle.</p><p><strong>Importance: </strong>Heliobacteria are a unique group of phototrophic bacteria that are obligate anaerobes and possess a rudimentary system to use light as a source of energy. They do not make oxygen or fix carbon dioxide. Here, we explore their fundamental carbon metabolism to understand the role and operation of the central TCA cycle. This work shows both the role and operation of this cycle under different growth modes and explains how these organisms can obtain electrons to drive their biosynthetic metabolism. This foundational knowledge will be crucial in the future when attempts are made to use this organism as a platform for oxygen-sensitive synthesis of compounds in an anaerobe that can use light as its energy source.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0177224"},"PeriodicalIF":3.9,"publicationDate":"2025-03-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143565839","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":"Effects of post-adulthood environmental hygiene improvement on gut microbiota and immune tolerance in mice.","authors":"Na Li, Mengjie Li, Honglin Zhang, Zhimao Bai, Zhongjie Fei, Yangyang Dong, Xinting Zhang, Pengfeng Xiao, Xiao Sun, Dongrui Zhou","doi":"10.1128/aem.02477-24","DOIUrl":"https://doi.org/10.1128/aem.02477-24","url":null,"abstract":"<p><p>Changes in diet, cleanliness, stress, and exercise patterns may contribute to the disappearance of various gut microbes in humans who relocate to developed countries from developing countries. To explore the impact of environmental cleanliness on the gut microbiota, adult mice housed in a general animal room were divided into three groups. The control group was subjected to an unchanged living environment, SPF mice were moved to a specific pathogen-free (SPF) animal room with higher environmental cleanliness, and SPFL (specific pathogen-free specific with a fecal leakage grid) mice were moved to the SPF animal room and reared in cages with the function of preventing mice from eating feces as much as possible. Metagenome sequencing results showed that the gut microbial diversity decreased after the environmental change, accompanied by a substantial loss in gut microbiota, including genera known to have protective effects against allergies and those involved in short-chain fatty acid production. Additionally, the abundance of functional genes involved in short-chain fatty acid metabolism, amino acid synthesis, vitamin metabolism, flagellar assembly, and bacterial chemotaxis decreased. The environmental hygiene improvement also resulted in significant increases in total serum IgE, IL-4, IL-5, and IL-13 levels in mice with artificially induced chronic inflammatory dermatosis. Compared with SPF mice, preventing mice from eating feces as much as possible decreased the gut microbial diversity but did not markedly change functional gene expression or total serum cytokine levels.</p><p><strong>Importance: </strong>Research has indicated that the human gut microbial diversity gradually decreases, while the prevalence of allergic diseases increases after movement from developing countries to developed countries. A healthy gut microbiota is necessary for proper human immune function. Movement from undeveloped to developed regions is often accompanied by an increase in environmental cleanliness. However, whether changes in environmental cleanliness are an important factor contributing to the decreased gut microbial diversity and increased prevalence of allergic diseases has not been reported. This study demonstrates the impact of increased environmental cleanliness on gut microbiota and susceptibility to allergic diseases and contributes to a better understanding of the increased incidence rate of various chronic diseases.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0247724"},"PeriodicalIF":3.9,"publicationDate":"2025-03-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143565840","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":"Sulfate assimilation regulates antioxidant defense response of the cyanobacterium <i>Synechococcus elongatus</i> PCC 7942 to high concentrations of carbon dioxide.","authors":"Yujie Mu, Huiting Chen, Jianwei Li, Pei Han, Zhen Yan","doi":"10.1128/aem.00115-25","DOIUrl":"https://doi.org/10.1128/aem.00115-25","url":null,"abstract":"<p><p>The adaptive evolution of cyanobacteria over a prolonged period has allowed them to utilize carbon dioxide (CO₂) at the low concentrations found in the atmosphere (0.04% CO₂) for growth. However, whether the exposure of cyanobacteria to high concentrations of CO₂ results in oxidative stress and the activation of antioxidant defense response remains unknown, albeit fluctuations in other culture conditions have been reported to exert these effects. The current study reveals the physiological regulation of the model cyanobacterium <i>Synechococcus elongatus</i> PCC 7942 upon exposure to 1% CO₂ and the underlying mechanism. Exposure to 1% CO₂ was demonstrated to induce oxidative stress and activate antioxidant defense responses in <i>S. elongatus</i>. Further analysis of variations in metabolism between <i>S. elongatus</i> cells grown at 0.04% CO₂ and exposed to 1% CO₂ revealed that sulfate assimilation was enhanced after the exposure to 1% CO₂. A strain of <i>S. elongatus</i> lacking the gene <i>cysR</i>, encoding a global transcriptional regulator for genes involved in sulfate assimilation, was generated by deleting the gene from the genomic DNA. A comparative analysis of the wild-type and <i>cysR</i>-null strains indicated the regulation of the antioxidant response by sulfate assimilation. In addition, lines of evidence were presented that suggest a role of degradation of phycobilisome in the antioxidant response of <i>S. elongatus</i> under conditions of 1% CO₂ and sulfate limitation. This study sheds light on the <i>in situ</i> effects of high CO₂-induced stress on the ecophysiology of cyanobacteria upon exposure to diverse scenarios from a biotechnological and ecological perspective.IMPORTANCECyanobacteria that grow autotrophically with CO₂ as the sole carbon source can be subject to high-CO₂ stress in a variety of biotechnological and ecological scenarios. However, physiological regulation of cyanobacteria in response to high-CO₂ stress remains elusive. Here, we employed microbial physiological, biochemical, and genetic techniques to reveal the regulatory strategies of cyanobacteria in response to high-CO₂ stress. This study, albeit physiological, provides a biotechnological enterprise for manipulating cyanobacteria as the chassis for CO₂ conversion and sheds light on the <i>in situ</i> ecological effects of high CO₂ on cyanobacteria.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0011525"},"PeriodicalIF":3.9,"publicationDate":"2025-03-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143565841","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":"Hemolysin III drives neuroinvasion and meningitis while impacting desiccation resilience in <i>Cronobacter sakazakii</i>.","authors":"Jiawei Liu, Qianquan Wan, Daizong Gaoxin, Zhanhui Song, Ke Mao, Fengzhu Wu, Xiangyu Liu, Linsen Mu","doi":"10.1128/aem.00339-25","DOIUrl":"https://doi.org/10.1128/aem.00339-25","url":null,"abstract":"<p><p><i>Cronobacter sakazakii</i> is an emerging pathogen associated with severe neonatal diseases, including meningitis. Its ability to cross the blood-brain barrier (BBB) is critical for meningitis development, yet the underlying mechanisms remain unclear. This study investigates the role of Hemolysin III (Hly III), encoded by the <i>ESA_00432</i>, in neuroinvasion and environmental resilience. A markerless deletion of <i>ESA_00432</i> (Δ<i>ESA_00432</i>) revealed that the mutant exhibited unaltered biofilm formation, increased hydrophilicity, and enhanced desiccation resistance compared to the wild type, suggesting that Hly III imposes a fitness cost on <i>C. sakazakii</i> under non-invasive conditions. In a rat infection model, the Δ<i>ESA_00432</i> strain demonstrated significantly reduced brain colonization without affecting bacterial loads in blood, liver, or spleen, underscoring the specific importance of Hly III in neuroinvasion. Cellular assays further revealed that, although the mutant maintained similar levels of adherence and invasion in Caco-2 cells and comparable adhesion to human brain microvascular endothelial cells (HBMECs) as the wild type, its ability to invade HBMECs was markedly diminished. These results suggest that Hly III is crucial for efficient neuroinvasion and BBB translocation while imposing a trade-off on environmental resilience, providing insights into the balance between virulence, and environmental adaptability in <i>C. sakazakii</i>.</p><p><strong>Importance: </strong>The ability of <i>Cronobacter sakazakii</i> to cause severe neonatal infections, particularly meningitis, presents a significant public health concern, yet the molecular mechanisms that enable its neuroinvasion remain poorly understood. In this study, we identify Hemolysin III (Hly III), encoded by the ESA_00432 gene, as a key factor in the bacterium's ability to cross the blood-brain barrier (BBB) and initiate meningitis. Our findings demonstrate that Hly III is essential for efficient invasion of human brain microvascular endothelial cells (HBMECs) and subsequent brain colonization in a rat model, underscoring its critical role in neurotropism. Furthermore, we show that the absence of Hly III results in enhanced environmental resilience, as indicated by increased desiccation resistance and hydrophilicity. This metabolic trade-off between virulence and environmental adaptability reveals a novel aspect of <i>C. sakazakii</i>'s pathogenesis and survival strategies. These insights open new avenues for developing targeted interventions to prevent neonatal meningitis and enhance food safety measures against this opportunistic pathogen.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0033925"},"PeriodicalIF":3.9,"publicationDate":"2025-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143555574","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":"Microbial magnetite oxidation via MtoAB porin-multiheme cytochrome complex in <i>Sideroxydans lithotrophicus</i> ES-1.","authors":"Jessica L Keffer, Nanqing Zhou, Danielle D Rushworth, Yanbao Yu, Clara S Chan","doi":"10.1128/aem.01865-24","DOIUrl":"https://doi.org/10.1128/aem.01865-24","url":null,"abstract":"<p><p>Most of Earth's iron is mineral-bound, but it is unclear how and to what extent iron-oxidizing microbes can use solid minerals as electron donors. A prime candidate for studying mineral-oxidizing growth and pathways is <i>Sideroxydans lithotrophicus</i> ES-1, a robust, facultative iron oxidizer with multiple possible iron oxidation mechanisms. These include Cyc2 and Mto pathways plus other multiheme cytochromes and cupredoxins, and so we posit that the mechanisms may correspond to different Fe(II) sources. Here, <i>S. lithotrophicus</i> ES-1 was grown on dissolved Fe(II)-citrate and magnetite. <i>S. lithotrophicus</i> ES-1 oxidized all dissolved Fe²⁺ released from magnetite and continued to build biomass when only solid Fe(II) remained, suggesting it can utilize magnetite as a solid electron donor. Quantitative proteomic analyses of <i>S. lithotrophicus</i> ES-1 grown on these substrates revealed global proteome remodeling in response to electron donor and growth state and uncovered potential proteins and metabolic pathways involved in the oxidation of solid magnetite. While the Cyc2 iron oxidases were highly expressed on both dissolved and solid substrates, MtoA was only detected during growth on solid magnetite, suggesting this protein helps catalyze oxidation of solid minerals in <i>S. lithotrophicus</i> ES-1. A set of cupredoxin domain-containing proteins were also specifically expressed during solid iron oxidation. This work demonstrated that the iron oxidizer <i>S. lithotrophicus</i> ES-1 utilized additional extracellular electron transfer pathways when growing on solid mineral electron donors compared to dissolved Fe(II).</p><p><strong>Importance: </strong>Mineral-bound iron could be a vast source of energy to iron-oxidizing bacteria, but there is limited physiological evidence of this metabolism, and it has been unknown whether the mechanisms of solid and dissolved Fe(II) oxidation are distinct. In iron-reducing bacteria, multiheme cytochromes can facilitate iron mineral reduction, and here, we link a multiheme cytochrome-based pathway to mineral oxidation, expanding the known functionality of multiheme cytochromes. Given the growing recognition of microbial oxidation of minerals and cathodes, increasing our understanding of these mechanisms will allow us to recognize and trace the activities of mineral-oxidizing microbes. This work shows how solid iron minerals can promote microbial growth, which, if widespread, could be a major agent of geologic weathering and mineral-fueled nutrient cycling in sediments, aquifers, and rock-hosted environments.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0186524"},"PeriodicalIF":3.9,"publicationDate":"2025-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143555578","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":"Computer-directed rational engineering of dioxygenase TcsAB for triclosan biodegradation under cold conditions.","authors":"Yiran Yin, Xinjie Yu, Zongxin Tao, Christopher E French, Zhenmei Lu","doi":"10.1128/aem.00346-25","DOIUrl":"https://doi.org/10.1128/aem.00346-25","url":null,"abstract":"<p><p>The dioxygenase TcsAB is a specific dioxygenase involved in the initial biodegradation of the broad-spectrum antibacterial agent triclosan (TCS). However, it exhibits significantly reduced activity under cold conditions. In this study, a computer-directed approach combining loop engineering and N-terminal truncation was utilized to decrease the thermostability of TcsAB, thereby enhancing its catalytic activity in cold environments. The iterative mutant TcsAB (TcsA^{Y277P/F279P/S311W/A313W}TcsB^{N-terminal truncation}) exhibited a 2.54-fold greater catalytic efficiency than the wild type at 15°C. Molecular dynamics simulations showed that the mutations introduced in the substrate-binding pocket increased its flexibility, leading to enhanced catalytic activity through binding in a more advantageous conformation. This modified dioxygenase was employed as a biological component, and <i>Pseudomonas knackmussii</i> B13 was used as a chassis cell to construct an engineered strain for the efficient degradation of TCS at low temperatures. The objective was to enhance the capacity of TCS bioremediation in natural environments. Insights gained from this study may inform the rational redesign of enzymes related to the robustness of biodegradation of emerging contaminants.IMPORTANCEThe presence of TCS in surface water and wastewater poses a significant risk to aquatic organisms and human health due to its high resistance to degradation. The biodegradation of TCS pollution in the environment through the metabolic processes of microorganisms represents a significant and effective remediation strategy. The dioxygenase TcsAB is the only specific enzyme that has been identified as responsible for the initial biodegradation of TCS. Nevertheless, the enzyme activity responsible for the degradation of TCS was markedly diminished at low temperatures. The actual ambient temperature is frequently lower than the optimum temperature for enzyme reaction, and maintaining the 30°C reaction condition results in high costs and energy consumption for TCS removal. Accordingly, the rational engineering of dioxygenase TcsAB for low-temperature activity will facilitate more efficient and realistic removal of TCS in an aqueous environment.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0034625"},"PeriodicalIF":3.9,"publicationDate":"2025-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143555573","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":"Expression pattern, subcellular localization of <i>Aspergillus oryzae</i> ergosterol synthases, and their effects on ergosterol and fatty acid metabolism.","authors":"Xueqin Tian, Kunhai Qin, Yunhong Deng, Pinghong Xue, Chaozheng Huang, Shaofang Liu, Zhihong Hu","doi":"10.1128/aem.02273-24","DOIUrl":"https://doi.org/10.1128/aem.02273-24","url":null,"abstract":"<p><p>Ergosterol is an important component of fungal cell membranes and participates in the regulation of its fluidity, permeability, and material transport. In our study, we found that the ergosterol biosynthesis pathway in <i>Aspergillus oryzae</i> is more complex than that in <i>Saccharomyces cerevisiae</i>, involving 49 enzymes and 25 reactions. In this study, we systematically analyzed the expression pattern and subcellular localization of <i>A. oryzae</i> ergosterol synthases and their effects on ergosterol and fatty acid metabolism. The results showed that ergosterol synthase genes were distributed across the eight chromosomes of the <i>A. oryzae</i> genome and were differentially expressed at different growth times. Subcellular localization analysis revealed that the mevalonate biosynthesis-related enzymes were localized in the cytoplasm, mitochondria, and peroxisomes; farnesyl pyrophosphate biosynthesis-related enzymes were mainly localized in the cytoplasm, peroxisomes, and mitochondria; while several ergosterol biosynthesis-related enzymes were localized in the endoplasmic reticulum and lipid droplets. Overexpression (OE) of these enzymes affected both ergosterol and fatty acid contents in <i>A. oryzae</i>. The ergosterol content was the highest in the <i>AoHmgB</i>-OE strain and the lowest in the <i>AoHmgA</i>-OE strain, while the fatty acid content was the highest in the <i>AoErg26B</i>-OE strain and the lowest in the <i>AoErg26A</i>-OE strain. Moreover, the ergosterol content was significantly increased in the <i>AoHmgB</i>/<i>AoErg7B</i>-, <i>AoHmgB</i>/<i>AoErg9</i>-, and <i>AoErg1</i>/<i>AoErg7B</i>-co-OE strains compared to the single gene OE strains. Our study results may offer a scientific foundation for genetic engineering research focusing on lipid metabolism in <i>A. oryzae</i> and related fungi, thereby facilitating the creation of fungal strains with enhanced ergosterol production.IMPORTANCEErgosterol, an important fungal cell membrane component, participates in the regulation of membrane fluidity, permeability, and material transport. Previous studies have demonstrated that the ergosterol biosynthesis pathway in <i>Aspergillus oryzae</i> exhibits greater complexity compared to that in <i>Saccharomyces cerevisiae</i>; nonetheless, research on the ergosterol biosynthesis pathway in <i>A. oryzae</i> remains limited. In this study, we determined the expression pattern and subcellular localization of ergosterol biosynthesis-related enzymes in <i>A. oryzae</i>. Additionally, we assessed the effects of the overexpression (OE) of ergosterol biosynthesis-related genes on ergosterol and fatty acid contents in <i>A. oryzae</i>. Therefore, our study may provide a scientific basis for genetic engineering research on lipid metabolism in <i>A. oryzae</i> and other fungal species.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0227324"},"PeriodicalIF":3.9,"publicationDate":"2025-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143539805","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":"Transcriptional regulation of two redundant 3-bromo-4-hydroxybenzoate catabolic operons via two different regulatory modes in <i>Pigmentiphaga kullae</i> strain H8.","authors":"Zhuang Ke, Ke Yang, Zonghui Zhang, Ru Guo, Yuan Gao, Minjian Lan, Jiandong Jiang, Kai Chen","doi":"10.1128/aem.02403-24","DOIUrl":"https://doi.org/10.1128/aem.02403-24","url":null,"abstract":"&lt;p&gt;&lt;p&gt;Gene redundancy endows bacteria with enhanced adaptability to complex and fluctuating environments but results in genetic costs. Transcriptional regulation is considered an effective strategy for harmonizing adaptive benefits with physiological burdens. In our previous study, two redundant gene clusters (phbh1pcaApcaBorf404bhbR1 and phbh2pcaB2pcaA2bhbR2) involved in 3-bromo-4-hydroxybenzoate (3-Br-4-HB) catabolism were identified in &lt;i&gt;Pigmentiphaga kullae&lt;/i&gt; strain H8. The LysR-type transcription regulator BhbR1, encoded by the bhbR1 gene, activated phbh1pcaApcaBorf404 transcription. Through DNase I footprinting assays, the presence of the inducer 3-Br-4-HB was found to shorten the BhbR1-bound region in the promoter, uncovering the protected -35 box, thereby activating transcription. The MarR-family transcription factor (MFTF) BhbR2, encoded by the bhbR2 gene, was different from typical inhibitive MFTFs and activated phbh2pcaB2pcaA2 transcription. BhbR2 was found to bind a 17-bp imperfect palindromic sequence (TTGATT-N5-AATCAA) in the target promoter. Intriguingly, the presence of 3-Br-4-HB neither dissociated BhbR2 from the promoter nor modified its binding site, indicating a novel regulatory mode. Despite a coincident trend in activating their respective operons in response to different concentrations of 3-Br-4-HB, BhbR1 and BhbR2 both showed a significant attenuation of the activation effect at high concentrations (&gt;480 μM), highlighting the requisite co-existence of redundant 3-Br-4-HB catabolic operons and their regulatory genes. This study presents two distinct transcriptional regulation mechanisms of these two redundant 3-Br-4-HB catabolic operons in strain H8, expanding our understanding of the diversity of transcriptional regulation for enhancing adaptation.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Importance: &lt;/strong&gt;In bacteria, catabolic genes for pollutant degradation often possess functionally redundant duplicates, providing a genetic basis for rapid adaptation to complex polluted environments. Synergic regulation plays an important role in balancing the physiological burden of extra genetic material with the adaptive benefits conferred by genetic redundancy. Although the co-existence of two redundant 3-bromo-4-hydroxybenzoate (3-Br-4-HB)-catabolic operons has been proven to enhance the metabolic robustness and adaptability of the host strain &lt;i&gt;Pigmentiphaga kullae&lt;/i&gt; H8, how these two inducible catabolic operons are regulated remains unclear. This study identified two regulators, the LysR-type transcription regulator BhbR1 and the MarR-family transcription factor BhbR2, which activated transcription of the two 3-Br-4-HB-catabolic operons using different modes, and also revealed interactions of these two regulators with their effectors and target promoters. These findings not only clarify two distinct transcriptional strategies employed by redundant catabolic operons but also enhance our understanding of the significance of regulatory diversity for","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0240324"},"PeriodicalIF":3.9,"publicationDate":"2025-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143539839","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":"ChIP-exo and CRISPRi/a illuminate the role of Pdr1 and Yap1 in acetic acid tolerance in <i>Saccharomyces cerevisiae</i>.","authors":"Ibai Lenitz, Christoph Börlin, Luca Torello Pianale, Darshan Balachandran, Jens Nielsen, Florian David, Verena Siewers, Yvonne Nygård","doi":"10.1128/aem.01824-24","DOIUrl":"https://doi.org/10.1128/aem.01824-24","url":null,"abstract":"<p><p>Budding yeast <i>Saccharomyces cerevisiae</i> has great potential as a host organism for various biorefinery applications. Nevertheless, the utilization of renewable plant biomass as feedstock for yeast in industrial applications remains a bottleneck, largely due to the presence of inhibitory substances such as acetic acid that are released in the biomass pretreatment processes. Exposure to acetic acid leads to different cellular stress mechanisms, several of which are directed by transcription factors. In this work, the role of the transcription factors Pdr1 and Yap1 in acetic acid tolerance was investigated using ChIP-exo and CRISPR interference/activation (CRISPRi/a). Pdr1 is the main regulator of the pleiotropic drug response, whereas Yap1 governs the oxidative stress response. CRISPRa targeting <i>YAP1</i> for overexpression conferred a higher specific growth rate of <i>S. cerevisiae</i>, whereas CRISPRi-based downregulation of <i>PDR1</i> proved to be beneficial for growth in medium containing acetic acid. ChIP-exo experiments showed increased binding of Pdr1 or Yap1 to their target promoters in the presence of acetic acid, and a large number of promoters were bound by either transcription factor. Promoters of genes involved in amino acid synthesis or encoding ABC transporters had the highest level of binding enrichment in the presence of acetic acid. The results highlight the potential for developing more acetic acid-tolerant yeast by altering the expression of transcription factor-encoding genes and demonstrate how expression can be fine-tuned by CRISPRi/a.IMPORTANCEBiotechnological conversion of plant biomass into a variety of commodity chemicals and specialty molecules is an important step towards a bioeconomy. This study highlights the importance of two transcription factors, Pdr1 and Yap1, in the tolerance of <i>Saccharomyces cerevisiae</i> to acetic acid, a common inhibitor in bioprocesses using lignocellulosic biomass. CRISPR interference/activation and ChIP-exo were used to manipulate the expression and binding of these transcription factors in response to acetic acid stress. The study provides new insights into adaptation to acetic acid and suggests ways to improve yeast performance in industrial applications.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0182424"},"PeriodicalIF":3.9,"publicationDate":"2025-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143539780","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}