Applied and Environmental Microbiology最新文献

{"title":"Robust measurement of microbial reduction of graphene oxide nanoparticles using image analysis.","authors":"Danielle T Bennett, Anne S Meyer","doi":"10.1128/aem.00360-25","DOIUrl":"https://doi.org/10.1128/aem.00360-25","url":null,"abstract":"<p><p><i>Shewanella oneidensis</i> (<i>S. oneidensis</i>) has the capacity to reduce electron acceptors within a medium and is thus used frequently in microbial fuel generation, pollutant breakdown, and nanoparticle fabrication. Microbial fuel setups, however, often require costly or labor-intensive components, thus making optimization of their performance onerous. For rapid optimization of setup conditions, a model reduction assay can be employed to allow simultaneous, large-scale experiments at lower cost and effort. Since <i>S. oneidensis</i> uses different extracellular electron transfer pathways depending on the electron acceptor, it is essential to use a reduction assay that mirrors the pathways employed in the microbial fuel system. For microbial fuel setups that use nanoparticles to stimulate electron transfer, reduction of graphene oxide provides a more accurate model than other commonly used assays as it is a bulk material that forms flocculates in solutions with a large ionic component. However, graphene oxide flocculates can interfere with traditional absorbance-based measurement techniques. This study introduces a novel image analysis method for quantifying graphene oxide reduction, showing improved performance and statistical accuracy over traditional methods. A comparative analysis shows that the image analysis method produces smaller errors between replicates and reveals more statistically significant differences between samples than traditional plate reader measurements under conditions causing graphene oxide flocculation. Image analysis can also detect reduction activity at earlier time points due to its use of larger solution volumes, enhancing color detection. These improvements in accuracy make image analysis a promising method for optimizing microbial fuel cells that use nanoparticles or bulk substrates.IMPORTANCE<i>Shewanella oneidensis</i> (<i>S. oneidensis</i>) is widely used in reduction processes such as microbial fuel generation due to its capacity to reduce electron acceptors. Often, these setups are labor-intensive to operate and require days to produce results, so use of a model assay would reduce the time and expenses needed for optimization. Our research developed a novel digital analysis method for analysis of graphene oxide flocculates that may be utilized as a model assay for reduction platforms featuring nanoparticles. Use of this model reduction assay will enable rapid optimization and drive improvements in the microbial fuel generation sector.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0036025"},"PeriodicalIF":3.9,"publicationDate":"2025-03-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143717932","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":"The evaluation of biogenic silica in brackish and freshwater strains reveals links between phylogeny and silica accumulation in picocyanobacteria.","authors":"Anabella Aguilera, Daniel Lundin, Evangelia Charalampous, Yelena Churakova, Christian Tellgren-Roth, Sylwia Śliwińska-Wilczewska, Daniel J Conley, Hanna Farnelid, Jarone Pinhassi","doi":"10.1128/aem.02527-24","DOIUrl":"https://doi.org/10.1128/aem.02527-24","url":null,"abstract":"<p><p>Through biosilicification, organisms incorporate dissolved silica (dSi) and deposit it as biogenic silica (bSi), driving the silicon (Si) cycle in aquatic systems. While Si accumulation in marine picocyanobacteria has been recently observed, its mechanisms and ecological implications remain unclear. This study investigates biosilicification in marine and brackish picocyanobacteria of the <i>Synechococcus</i> clade and two model freshwater coccoid cyanobacteria. Brackish strains showed significantly higher Si quotas when supplemented with external dSi (100 µM) compared to controls (up to 60.0 ± 7.3 amol Si.cell-1 versus 9.2 to 16.3 ± 2.9 amol Si.cell-1). Conversely, freshwater strains displayed no significant differences in Si quotas between dSi-enriched treatments and controls, emphasizing that not all phytoplanktons without an obligate Si requirement accumulate this element. The Si-accumulating marine and brackish picocyanobacteria clustered within the <i>Synechococcus</i> clade, whereas their freshwater counterparts formed a distinct sister group, suggesting a link between phylogeny and silicification. Rapid culture growth caused increased pH and led to dSi precipitation, influencing apparent dSi uptake; this was mitigated by pH control through bubbling. This phenomenon has significant implications for natural systems affected by phytoplankton blooms. In such environments, pH-induced silicon precipitation may reduce dSi availability impacting Si-dependent populations like diatoms. Our findings suggest brackish picocyanobacteria could significantly influence the Si cycle through at least two mechanisms: cellular Si accumulation and biologically induced changes in dSi concentrations.IMPORTANCEThis work provides the first evidence of biogenic silica accumulation in brackish picocyanobacteria and uncovers a link between phylogeny and biosilicification patterns. Our findings demonstrate that picocyanobacterial growth induces pH-dependent silica precipitation, which could lead to overestimations of cellular Si quotas by up to 85%. This process may drive substantial silica precipitation in highly productive freshwater and coastal marine systems, with potential effects on silica cycling and the population dynamics of Si-dependent phytoplankton. The extent of biosilicification in modern picocyanobacteria offers insights into the rock record, shedding light on the evolutionary and ecological dynamics that influence sedimentary processes and the preservation of biosilicification signatures in geological formations. Overall, this research adds to the significant impact that microorganisms lacking an obligate silica requirement may have on silica dynamics.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0252724"},"PeriodicalIF":3.9,"publicationDate":"2025-03-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143717933","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":"Growth-coupled continuous directed evolution by MutaT7 enables efficient and automated enzyme engineering.","authors":"Yijie Deng, Kai Etheridge, Xinping Ran, Hannah E Maurais, Rahul Sarpeshkar","doi":"10.1128/aem.02491-24","DOIUrl":"https://doi.org/10.1128/aem.02491-24","url":null,"abstract":"<p><p>Traditional directed evolution is limited by labor-intensive iterative steps and low-throughput selection and screening. To address these challenges, we developed a growth-coupled continuous directed evolution (GCCDE) approach, enabling automated and efficient enzyme engineering. By linking enzyme activity to bacterial growth and utilizing the MutaT7 system, GCCDE combines <i>in vivo</i> mutagenesis and high-throughput selection of superior enzyme variants in a single process. To validate this approach, we evolved the thermostable enzyme CelB from <i>Pyrococcus furiosus</i> to enhance its β-galactosidase activity at lower temperatures while maintaining thermal stability. CelB activity was coupled to the growth of <i>E. coli</i>, allowing variants with improved activity to utilize lactose more efficiently and promote faster growth in a minimal medium. Using a continuous culture system, we achieved automated high-throughput mutagenesis and simultaneous real-time selection of over 10⁹ variants per culture. Integrating <i>in vitro</i> and <i>in vivo</i> mutagenesis further increased genetic diversity, yielding CelB variants with significantly enhanced low-temperature activity compared to the wild type while preserving thermostability. DNA sequencing identified key mutations likely responsible for improved substrate binding and catalytic turnover. This GCCDE approach is broadly applicable for optimizing diverse enzymes, demonstrating the potential of automated continuous evolution for industrial and research applications.</p><p><strong>Importance: </strong>Enzyme engineering aims to develop enzymes with improved or novel traits, but traditional methods are slow and require repetitive manual steps. This study presents a faster, automated protein engineering approach. We utilized an <i>in vivo</i> mutagenesis technique, MutaT7 tools, to induce mutations in living bacteria and established a direct link between enzyme activity and bacterial growth. A continuous culture setup enables automated mutagenesis and growth-coupled selection of better-performing variants in real time. Bacteria with improved enzymes grew faster, selecting superior variants without manual intervention. Using this method, we engineered CelB with better performance at lower temperatures while maintaining thermal stability. By combining high-throughput mutagenesis and selection in a single process, this system bypasses iterative cycles of error-prone PCR, transformation, and screening. Our approach is adaptable to various enzymes, providing a faster and more efficient solution for enzyme engineering.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0249124"},"PeriodicalIF":3.9,"publicationDate":"2025-03-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143717931","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":"The GCN4-Swi6B module mediates low nitrogen-induced cell wall remodeling in <i>Ganoderma lucidum</i>.","authors":"Lingyan Shi, Lingshuai Wang, Rui Liu, Jing Zhu, Liang Shi, Ang Ren, Huhui Chen, Mingwen Zhao","doi":"10.1128/aem.00164-25","DOIUrl":"https://doi.org/10.1128/aem.00164-25","url":null,"abstract":"<p><p>In natural habitats, microorganisms encounter various unfavorable environmental stresses, including nitrogen deficiency. As the outermost barrier, the cell wall plays a crucial role in the interaction between the cell and the external environment. However, the effect of low nitrogen on cell wall thickness, especially the underlying molecular mechanism, is unclear. Here, we found that compared with those under normal nitrogen conditions, both the cell wall thickness and polysaccharide content of <i>Ganoderma lucidum</i> are increased under low nitrogen conditions. Furthermore, the abundance of SWI6B, a transcription factor that participates in cell wall remodeling, is also increased in low-nitrogen environments. The thickness and polysaccharide content of the cell wall increased in <i>SWI6B</i>-overexpression strains (<i>SWI6B-OEs</i>) but decreased in <i>SWI6-</i>knockdown strains (<i>swi6-kds</i>). Moreover, although the cell wall thickness of all the genotypes increased under nitrogen-limited conditions, the percentage of upregulated <i>swi6-kds</i> was significantly lower than that of the WT, and the percentage of increased <i>SWI6B-OEs</i> was the highest. Moreover, GCN4, a key transcription factor of the low-nitrogen signaling pathway, was found to directly bind to the promoter of <i>SWI6</i>. The transcriptional and translational levels of <i>SWI6B</i> were reduced in <i>GCN4</i>-knockdown strains (<i>gcn4-kds</i>), indicating a positive regulation of <i>SWI6B</i> by GCN4. Consistently, the cell wall thickness of <i>gcn4-kds</i> was also lower than that of the wild type. Taken together, our results revealed that the GCN4-Swi6B module regulates cell wall remodeling in <i>G. lucidum</i> under nitrogen deficiency conditions.</p><p><strong>Importance: </strong>To survive in stressful environments, fungi initiate cell wall remodeling pathways to adaptively modify the cell wall composition and structure. Here, we found that nitrogen deficiency upregulated the cell wall polysaccharide content and cell wall thickness through the GCN4-SWI6B signaling pathway. Our findings provide valuable insights into the environmental adaptation of fungal cell walls, contributing to a deeper understanding of fungal responses to environmental stress.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0016425"},"PeriodicalIF":3.9,"publicationDate":"2025-03-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143717634","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":"Inducible promoters of bacterial microcompartments improve the CRISPR/Cas9 tools for efficient metabolic engineering of <i>Clostridium ljungdahlii</i>.","authors":"Jun-Zhe Zhang, Yu-Zhen Li, Zhi-Ning Xi, Yue Zhang, Zi-Yong Liu, Xiao-Qing Ma, Fu-Li Li","doi":"10.1128/aem.02183-24","DOIUrl":"https://doi.org/10.1128/aem.02183-24","url":null,"abstract":"<p><p><i>Clostridium ljungdahlii</i>, as a model acetogen strain, represents a novel platform for biotechnological production for CO₂ fixation. The genome of <i>C. ljungdahlii</i> harbors two gene loci associated with glycyl radical enzyme-associated microcompartments (GRMs), which are predicted to play essential roles in choline and 1,2-propanediol (1,2-PD) metabolism. This study validated the functions of these GRM loci and identified two inducible promoters, of which P_choline1 was induced by choline, while P_1,2-PD was induced by 1,2-PD. Subsequently, the highly expressed P_1,2-PD and tightly controlled P_choline1 were applied to improve CRISPR/Cas9 gene editing tools. Specifically, P_1,2-PD was used to develop a highly efficient gene knockout tool based on an all-in-one plasmid, achieving 100% deletion efficiency for multiple genes, including <i>pyrE</i>, <i>pduS</i>, <i>aor2</i>, and <i>eutT</i>. On the other hand, the <i>cas9</i> gene was integrated downstream of P_choline1 into the genome. The integrated <i>cas9</i> efficiently mediated gene editing in <i>C. ljungdahlii</i> by introducing plasmids containing a gRNA cassette along with the relevant homology arms. This was exemplified by the construction of the <i>Δbdh::pdc</i> strain, where the 2,3-butanediol dehydrogenase gene was replaced with a pyruvate decarboxylase gene from <i>Zymomonas mobilis</i> and the 3-HB Syn KI strain, in which an artificial 3-hydroxybutyric acid synthesis pathway was inserted into the genome. This study highlights the effectiveness and convenience of the inducible CRISPR/Cas9 gene editing systems, thereby enriching the CRISPR/Cas toolkit in acetogens.</p><p><strong>Importance: </strong>A CRISPR/Cas9 genetic tool controlled by a constitutive promoter has been developed for precise gene deletion in <i>Clostridium ljungdahlii</i>. However, its efficiency was hindered by the toxicity resulting from the constitutive expression of cas9 and the large plasmids, leading to a low overall success rate. Inducible promoters, which allow for the transcription of target genes to be switched on and off in the presence or absence of inducers, have a broad range of applications. In this study, we identify two inducible promoters and apply them to enhance the CRISPR/Cas9 tools. The improved CRISPR/Cas9 tools facilitate gene editing with high efficiency, potentially playing significant roles in advancing genetic research and metabolic engineering of <i>C. ljungdahlii</i>.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0218324"},"PeriodicalIF":3.9,"publicationDate":"2025-03-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143708256","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":"pH-dependent genotypic and phenotypic variability in <i>Oleidesulfovibrio alaskensis</i> G20.","authors":"Priya Saxena, Dipayan Samanta, Payal Thakur, Kian Mau Goh, Mahadevan Subramaniam, Brent M Peyton, Matthew Fields, Rajesh K Sani","doi":"10.1128/aem.02565-24","DOIUrl":"https://doi.org/10.1128/aem.02565-24","url":null,"abstract":"<p><p>Sulfate-reducing bacteria (SRB) exhibit versatile metabolic adaptability with significant flexibility influenced by pH fluctuations, which play a critical role in biogeochemical cycles. In this study, we used a model SRB, <i>Oleidesulfovibrio alaskensis</i> G20, to determine the temporal effects of pH variations (pH 6, 7, and 8) on both growth dynamics and metabolic gene expressions. The specific growth rate at pH 6 (0.014 h^-1) closely matched that at pH 7 (0.016 h^-1), while pH 8 exhibited a lower growth rate (0.010 h^-1). Lactate consumption peaked at pH 7 (0.35 mM lactate.h^-1) and declined at pH 8 (0.09 mM lactate.h^-1). Significant hydrogen production was evident under both acidic and alkaline conditions. Gene expression studies revealed that ATPases function as proton pumps, while hydrogenases mediate reversible proton-to-hydrogen conversion. Sulfate and energy metabolism act as electron acceptors and donors, while amino acid synthesis regulates basic and acidic amino acids to mitigate pH stress. Downregulation of <i>FtsZ</i> at pH 6 suggests impaired division, correlating with slightly longer lengths (~2 µm), while upregulation of divisome proteins at pH 8 suggests efficient division processes, aligning with shorter lengths (~1.8 µm). This study will facilitate the employment of <i>O. alaskensis</i> G20 in extreme pH environments, enhancing its effectiveness in optimizing bioremediation and anaerobic digestion processes.</p><p><strong>Importance: </strong>Sulfate-reducing bacteria (SRB) play essential roles in global sulfur and carbon cycling and are critical for bioremediation and anaerobic digestion processes. However, detailed studies on the genotypic and phenotypic responses of SRB under varying pH conditions are limited. This study addresses this gap by examining the pH-dependent genetic and metabolic adaptations of <i>Oleidesulfovibrio alaskensis</i> G20, revealing key mechanisms regulating hydrogenase and ATPase activities, cell division, and extracellular polymeric substance formation. These findings provide new insights into how SRB maintains pH homeostasis, showcasing their ability to survive and function in both acidic and alkaline environments. Furthermore, this study reveals critical genetic and phenotypic characteristics that will directly aid to engineer industrial effluent management systems, bioremediation, and dissolved heavy metal recovery. By elucidating the dynamic response of <i>O. alaskensis</i> G20 to varied pH environments, the research provides a foundation for enhancing the resilience and performance of SRB-based systems, paving the way for improved environmental and industrial applications.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0256524"},"PeriodicalIF":3.9,"publicationDate":"2025-03-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143708259","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":"Synergistic effect of transporter and pathway engineering on the key performance indicators of erythritol synthesis by the yeast <i>Yarrowia lipolytica</i>.","authors":"Shuo Xu, Qian Li, Ye Li, Yue Zhang, Qing Li, Liyun Ji, Hairong Cheng","doi":"10.1128/aem.00061-25","DOIUrl":"https://doi.org/10.1128/aem.00061-25","url":null,"abstract":"<p><p>Erythritol, a food additive, is produced on an industrial scale using the yeast <i>Yarrowia lipolytica</i>. Nevertheless, the key performance indicators (KPIs) have been found to be unsatisfactory, resulting in elevated erythritol production cost. This study demonstrated that the KPIs (titer, productivity, and yield) of erythritol can be improved by the synergistic application of transporter and pathway engineering strategies in the producing strain. The engineered <i>Y. lipolytica</i> strain Ylxs48 exhibits a glucose consumption rate of 310 g/L of glucose within 46 h during batch culture in 3, 100, and 200 L bioreactors as compared to above 72 h for the parental strain Ylxs01. The erythritol yield achieved ranges from 0.69 to 0.74 g/g depending on the culture conditions as compared to 0.55-0.57 g/g for the parental strain Ylxs01. The productivity surpasses 4.60 g/(L·h), representing a 1.91-fold improvement over the parental strain Ylxs01 in 3, 100, or 200 L bioreactors. Under fed-batch conditions in a 200 L bioreactor, an erythritol titer of 355.81 g/L was achieved, marking the highest titer ever reported. This increased erythritol titer enabled crystallization at 4°C directly from the clear supernatant, eliminating the requirement for evaporation or concentration steps. A comprehensive techno-economic analysis of the entire process conclusively demonstrated that implementing the industrial process based on the engineered strain Ylxs48 led to a significant 23% reduction in production cost. This approach holds the potential to substantially reduce erythritol costs and provides novel insights for engineering other industrial strains.</p><p><strong>Importance: </strong>The expansion of the erythritol market attracted excessive capital injection, resulting in overcapacity, operational difficulties, and even bankruptcy of erythritol manufacturers. Technology upgrades in the industry are imminent. However, the production technology of existing enterprises is seriously homogenized, and there is a lack of competitive core-producing strains. In this study, the industrial erythritol-producing strain <i>Y. lipolytica</i> CGMCC7326 was genetically modified by integrating substrate transport and pathway modification, which improved the conversion of glucose and significantly improved KPIs, thereby reducing the erythritol production cost.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0006125"},"PeriodicalIF":3.9,"publicationDate":"2025-03-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143707894","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":"Comparative analyses of the biological characteristics, fluconazole resistance, and heat adaptation mechanisms of <i>Candida auris</i> and members of the <i>Candida haemulonii</i> complex.","authors":"Wei Xiao, Hao Zhou, Jian Huang, Caiyan Xin, Jinping Zhang, Huan Wen, Zhangyong Song","doi":"10.1128/aem.02406-24","DOIUrl":"https://doi.org/10.1128/aem.02406-24","url":null,"abstract":"<p><p><i>Candida auris</i> and the emerging fungal pathogens comprising the <i>Candida haemulonii</i> complex (<i>C. haemulonii</i> and <i>C. duobushaemulonii</i>) are phylogenetically closely related. Notably, each can cause serious nosocomial infection and acquire multidrug resistance. We isolated various strains of <i>C. auris</i> and <i>C. haemulonii</i> complex from patient specimens. The results of growth curve analysis and the spot assay showed that <i>C. auris</i> was the most tolerant to high temperatures, while differences were found among the five strains of the <i>C. haemulonii</i> complex. We selected a representative strain from each of the three types of fungi that exhibit differences in heat resistance for further research. In addition, three strains were resistant to fluconazole, whereas sensitivity to common antifungal agents differed, as determined by the micro liquid-based dilution method. Moreover, <i>C. auris</i> was more virulent in <i>Galleria mellonella</i> than members of the <i>C. haemulonii</i> complex. In addition, there were notable differences in biofilm formation, hydrophobicity, adhesion, and enzyme activity among the three <i>Candida</i> strains. The results of transcriptomics, real-time quantitative PCR, and biochemical analyses showed that <i>C. auris</i> was most tolerant to heat stress due to the expression of genes in regulating pyruvate consumption and the accumulation of intracellular pyruvate. These results provide valuable references for further studies of the biological characteristics, pathogenesis, and treatment of <i>Candida</i> infection.</p><p><strong>Importance: </strong><i>Candida auris</i> and the <i>Candida haemulonii</i> complex are multidrug-resistant fungi that have emerged in recent years, posing a significant threat to human health. The biological characteristics of two strains of the <i>Candida haemulonii</i> complex and one strain of <i>C. auris</i> isolated from clinical patient samples were analyzed. Our primary focus was to compare the heat resistance between <i>C. auris</i> and the <i>C. haemulonii</i> complex, with a particular emphasis on understanding the differences in the heat resistance mechanisms. The main distinction between environmental and pathogenic fungi is that the latter can survive at human body temperature. Despite their close phylogenetic relationship, the <i>C. haemulonii</i> complex and <i>C. auris</i> exhibit significant differences in heat resistance. Studying these heat resistance mechanisms may aid in our understanding of the evolutionary process of environmental fungi transforming into pathogenic fungi.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0240624"},"PeriodicalIF":3.9,"publicationDate":"2025-03-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143708253","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":"Role of the TPR family protein VPA1365 in regulating type III secretion system 2 and virulence in <i>Vibrio parahaemolyticus</i>.","authors":"Wenliang Yin, Mengyan Wan, Youkun Zhang, Hongmei Meng, Zhiming Pan, Xinan Jiao, Dan Gu","doi":"10.1128/aem.02201-24","DOIUrl":"https://doi.org/10.1128/aem.02201-24","url":null,"abstract":"<p><p><i>Vibrio parahaemolyticus</i> is a notable seafood-borne pathogen capable of colonizing the intestines of hosts and inducing acute gastroenteritis. The intestinal colonization and enterotoxicity of <i>V. parahaemolyticus</i> are highly reliant on the type III secretion system 2 (T3SS2), encoded within the pathogenicity island (Vp-PAI). The expression of Vp-PAI is strictly regulated by bile acid signals and transcriptional regulators VtrA/VtrB. In this study, we identified a tetratricopeptide repeat (TPR) family protein named VPA1365, which regulates the expression of T3SS2 and is indispensable for the intestinal colonization of <i>V. parahaemolyticus</i>. The expression and secretion of the T3SS2-dependent protein VopD2 were significantly reduced in Δ<i>vpa1365</i> compared to that of the wild type (WT), suggesting that VPA1365 positively regulates the function of T3SS2. Further research indicated that VPA1365 directly binds to the promoters of <i>vtrA</i>, thereby increasing the expression levels of T3SS2-associated genes. Additionally, the deletion of <i>vpa1365</i> markedly reduced the cytotoxicity, adhesion ability, biofilm formation, and hemolytic activity of <i>V. parahaemolyticus</i>. VPA1365 was found to control the expression levels of these virulence-associated genes by binding to the promoters of <i>scrG</i>, <i>pilA</i>, and <i>mshA</i>. In a zebrafish infection model, the Δ<i>vpa1365</i> infected groups demonstrated a higher survival rate compared to the zebrafish infected with WT. In conclusion, this study identified a TPR family protein VPA1365, which regulates the expression levels of T3SS2 and virulence-associated genes in <i>V. parahaemolyticus</i>, further broadening our understanding of its virulence factors.</p><p><strong>Importance: </strong>The type III secretion system 2 (T3SS2) is of crucial significance for the pathogenicity of <i>Vibrio parahaemolyticus</i>; nevertheless, the biological functions of many genes within the T3SS2 gene cluster and the transcriptional regulatory network of T3SS2 remain ambiguous. In this study, we identified VPA1365, a tetratricopeptide repeat family regulator encoded in the T3SS2 gene cluster, which differs from other known T3SS2 regulatory factors, such as OmpR, ToxR, or LysR family proteins. VPA1365 not only positively regulated the expression and secretion of T3SS2-related proteins but also enhanced the virulence in infant rabbits and zebrafish. Moreover, we identified several novel functions of VPA1365, such as its contribution to hemolytic activity, biofilm formation, cytotoxicity, and adhesion ability, uncovering its global physiological role in <i>V. parahaemolyticus</i>. The putative VPA1365-binding site was predicted and identified through the MEME-Suite tool and electrophoretic mobility shift analysis. Collectively, these results broaden our understanding of the regulatory pathways of T3SS2 and virulence.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0220124"},"PeriodicalIF":3.9,"publicationDate":"2025-03-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143699472","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":"Lipid hydrogen isotope compositions primarily reflect growth water in the model archaeon <i>Sulfolobus acidocaldarius</i>.","authors":"Carolynn M Harris, Sebastian Kopf, Jeemin H Rhim, Alec Cobban, Felix J Elling, Xiahong Feng, Jamie McFarlin, Yuki Weber, Yujiao Zhang, Alice Zhou, Harpreet Batther, Ann Pearson, William D Leavitt","doi":"10.1128/aem.01983-24","DOIUrl":"https://doi.org/10.1128/aem.01983-24","url":null,"abstract":"<p><p>The stable hydrogen isotope composition (δ²H) of lipid biomarkers can track environmental processes and remain stable over geologically relevant time scales, enabling studies of past climate, hydrology, and ecology. Most research has focused on lipids from the domain Eukarya (e.g., plant waxes, long-chain alkanes), and the potential of prokaryotic lipid biomarkers from the domain Archaea to offer unique insights into environments not captured by eukaryotic lipids remains unclear. Here, we investigate the H-isotope composition of biphytanes in <i>Sulfolobus acidocaldarius</i>, a model thermoacidophile and obligate heterotroph. We conducted a series of experiments that varied temperature, pH, shaking rate, electron acceptor availability, or electron donor flux. From these experiments, we quantified the lipid/water H-isotope fractionation (²ε_L/W) values for core biphytane chains derived from tetraether lipids. The ²ε_L/W values are consistently negative (-230‰ to -180‰) and are relatively invariant across all experiments despite a 20-fold change in doubling times and a twofold change in lipid cyclization. The magnitude and relative invariance of ²ε_L/W values are consistent with studies on other heterotrophic archaea and suggest archaeal lipids may be faithful recorders of the δ²H composition of growth water. Our study highlights the potential of archaeal lipid δ²H values as a hydrological proxy, offering new insights into environments where traditional proxies, such as plant-derived lipids, are not available, including extreme environments and extraterrestrial settings.IMPORTANCEReconstructing past climates is crucial for understanding Earth's environmental history and its responses to changing conditions. This study examines <i>Sulfolobus acidocaldarius</i>, a thermoacidophilic archaeon that thrives in extreme environments like hot springs. These microorganisms incorporate hydrogen water in the growth environment into membrane lipids, creating hydrogen isotope signatures that can reflect hydroclimate conditions. Our findings show that these hydrogen isotope ratios remain consistent even under varying temperatures, pH, oxygen levels, and electron donor fluxes, indicating a stable fractionation between lipids and water. This invariance suggests that <i>S. acidocaldarius</i> lipids could serve as a robust proxy for reconstructing ancient water H-isotope values, especially in extreme environments where traditional proxies, such as plant waxes, are absent. This research has broader implications for planetary-scale reconstructions, including potential applications in studying past climates on other planets, such as Mars, where similar microorganisms may have existed in hydrothermal conditions.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0198324"},"PeriodicalIF":3.9,"publicationDate":"2025-03-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143699458","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}