Applied and Environmental Microbiology最新文献

{"title":"Molecular piracy in deep-sea hydrothermal vent: phage-plasmid interactions revealed by phage-FISH in <i>Marinitoga piezophila</i>.","authors":"Min Jin, Ouafae Rouxel, Nadège Quintin, Claire Geslin","doi":"10.1128/aem.02306-24","DOIUrl":"10.1128/aem.02306-24","url":null,"abstract":"<p><p>Prokaryotes and mobile genetic elements (MGEs, such as viruses and plasmids) interact extensively, leading to horizontal gene transfer (HGT) and consequent microbial evolution and diversity. However, our knowledge of the interactions between MGEs in deep-sea hydrothermal ecosystems is limited. In this study, we adapted a phage-fluorescence <i>in situ</i> hybridization (phage-FISH) approach to visualize and quantify the dynamics of phage-plasmid interactions in an anaerobic, thermophilic deep-sea bacterium, <i>Marinitoga piezophila</i>. Notably, our results revealed that plasmid signals were detected in viral particles released from lysed cells, indicating that mitomycin C not only induced plasmid replication but also its packaging into phage particles. Further analysis of the DNA content in purified virions showed that the phage capsids incorporated plasmid DNA even without induction, and the majority of capsids (up to 70%) preferentially packaged plasmid DNA rather than viral DNA after induction. Therefore, this study provided direct evidence of molecular piracy in the deep-sea hydrothermal ecosystem, highlighting the important roles of selfish MGEs in virus-host interactions and HGT in extreme marine environments.</p><p><strong>Importance: </strong>Deep-sea hydrothermal vents are hotspots for microbes. Several studies revealed that virus-mediated horizontal gene transfer (HGT) in deep-sea hydrothermal vent ecosystems may be crucial to the survival and stability of prokaryotes in these extreme environments. However, little is known about the interaction between viruses and other mobile genetic elements (MGEs, such as plasmids), and how their interactions influence virus-mediated HGT in these ecosystems. In this study, we adapted a phage-fluorescence <i>in situ</i> hybridization approach to directly monitor the dynamics of phage-plasmid-host interactions at the single-cell level in the <i>Marinitoga piezophila</i> model. Interestingly, our results indicate that plasmid DNA could not only be induced by mitomycin C to a great extent but also hijacked viral assembly machinery to facilitate its propagation and spread. Therefore, the data presented here imply that the interaction between the viruses and other MGEs could play profound roles in virus-host interaction and virus-mediated HGT in the deep-sea hydrothermal ecosystem.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0230624"},"PeriodicalIF":3.9,"publicationDate":"2025-03-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11921389/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143514466","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":"Eco-microbiology: discovering biochemical enhancers of PET biodegradation by <i>Piscinibacter sakaiensis</i>.","authors":"Felipe-Andrés Piedra, Miguel A Salazar, Sara Abouelniaj, Raayed Rahman, Justin C Clark, Yimo Han, Zhao Wang, Anthony Maresso","doi":"10.1128/aem.02118-24","DOIUrl":"10.1128/aem.02118-24","url":null,"abstract":"<p><p>The scale of plastic pollution boggles the mind. Nearly 400 megatons of virgin plastics are produced annually, with an environmental release rate of 80%, and plastic waste, including microplastics and nanoplastics, is associated with a plethora of problems. The naturally evolved abilities of plastic-degrading microbes offer a starting point for generating sustainable and eco-centric solutions to plastic pollution-a field of endeavor we term eco-microbiology. Here, we developed an iterative discovery procedure coupling faster polyethylene terephthalate (PET)-dependent bioactivity screens with longer-term PET biodegradation assays to find biochemical boosters of PET consumption by the bacterium <i>Piscinibacter sakaiensis</i>. We discovered multiple hits supporting the enhancement of PET biodegradation, with a 0.39% dilution of growth medium #802, a rich medium similar to Luria-Bertani broth, on average more than doubling the rate of PET biodegradation both alone and in combination with 0.125% ethylene glycol. In addition, we identified other chemical species (sodium phosphate, L-serine, GABA) worth further exploring, especially in combination with growth medium #802, for enhanced PET biodegradation by <i>P. sakaiensis</i>. This work represents an important step toward the creation of a low-cost PET fermentation process needed to help solve PET plastic pollution.</p><p><strong>Importance: </strong>Plastic pollution is an urgent issue. Adding to the well-known problems of bulk plastic litter, shed microplastics and nanoplastics are globally distributed, found in diverse organisms including human foodstuffs and tissues, and increasingly associated with chronic disease. Solutions are needed and the microbial world offers abundant help via naturally evolved consumers of plastic waste. We are working to accelerate polyethylene terephthalate (PET) plastic biodegradation by <i>Piscinibacter sakaiensis</i>, a recently described bacterium that evolved to slowly but completely consume PET, one of the most common types of plastic pollution. We used a combination of PET-dependent bioactivity screens and biodegradation tests to find stimulators of PET biodegradation. Out of hundreds, we found a small number of biochemical conditions that more than double the PET biodegradation rate. Our work provides a foundation for further studies to realize a fermentation process needed to help solve PET plastic pollution.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0211824"},"PeriodicalIF":3.9,"publicationDate":"2025-03-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11921318/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143482016","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":"Insights into the mechanism of substrate specificity in a novel PL15_3 subfamily oligo-alginate lyase VBAly15A.","authors":"Yongqi Tang, Ziyan Song, Xiaodong Xu, Yingjie Li, Lushan Wang","doi":"10.1128/aem.02351-24","DOIUrl":"10.1128/aem.02351-24","url":null,"abstract":"<p><p>Alginate is a major component of brown algae cell walls and can be degraded via β-elimination by alginate lyases. These enzymes are classified into polysaccharide lyases and oligo-alginate lyases (Oals), with Oals mainly represented by the PL15 and PL17 families. Unlike PL17 Oals, which are widely present in alginate-degrading microorganisms, PL15 enzymes are only identified in a limited number of microorganisms, and their biochemical characteristics remain poorly understood. In this research, a novel PL15 alginate lyase, VBAly15A, from the marine bacterium, <i>Vibrio</i> sp. B1Z05, was identified and characterized. It belongs to a new PL15_3 subfamily and exhibits high activity toward polyM substrates. VBAly15A is thermostable in medium temperatures, tolerant to alkaline up to 11.0, and polyM-specific Oal, and it can first degrade alginate polymers into disaccharides and subsequently catalyze disaccharides into monomers via an exolytic mode. Site-directed mutagenesis showed that Arg¹¹⁴, Tyr⁴⁷⁰, and Arg¹¹⁰ in the active groove are essential for the stable binding of the substrate. In addition, the amino acid His²²⁶ in VBAly15A, previously suggested to act as a catalytic base, is not essential for catalysis, whereas Tyr²⁸⁰, previously proposed to act as a catalytic acid, is required for enzyme activity. Structural bioinformatic and biochemical analyses revealed that His²²⁶ functions as a catalytic base, specifically abstracting protons from G-type substrates, while Tyr²⁸⁰ acts as both a catalytic acid and a base. This catalytic mechanism is likely conserved in PL15 family alginate lyases.IMPORTANCEAlginate, as a renewable resource for sustainability, has great application prospects. In addition to polysaccharide lyases, Oals are critical for the full degradation of alginate, a key prerequisite for biorefinery. So far, most identified and well-characterized Oals belong to the PL17 family. However, the catalytic mechanism of PL15 Oals is limited, and even the catalytic base and acid are not fully elucidated. The significance of this study lies in discovering and characterizing a novel Oal VBAly15A that divides into a new PL15 subfamily, PL15_3. Not only are key amino acid residues involved in enzyme activity identified, but residues acting as the catalytic base and acid are also demonstrated. The distance of the catalytic residues His and Tyr to the C5 proton of the sugar ring determines the substrate specificity. Therefore, this work provides new insights into the mechanism of substrate specificity in alginate lyases.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0235124"},"PeriodicalIF":3.9,"publicationDate":"2025-03-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11921355/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143514460","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":"Generation of novel prebiotic oligosaccharide pools from fiber drives biological insight in bacterial glycan metabolism.","authors":"Chad Masarweh, Maria Maldonado-Gomez, Bruna Paviani, Mrittika Bhattacharya, Cheng-Yu Weng, Christopher Suarez, Shawn Ehlers-Cheang, Aaron Stacy, Juan Castillo, Nithya Krishnakumar, Karen A Kalanetra, Daniela Barile, J Bruce German, Carlito B Lebrilla, David A Mills","doi":"10.1128/aem.02077-24","DOIUrl":"10.1128/aem.02077-24","url":null,"abstract":"&lt;p&gt;&lt;p&gt;Prebiotic oligosaccharides are dietary supplements that modulate the intestinal gut microbiome by selectively nourishing subsets of the microbial community with a goal to enhance host health. To date, the diversity of polysaccharide compositions in the fiber consumed by humans is not well represented by the limited scope of oligosaccharide compositions present in current commercial prebiotics. Recently, our UC Davis group developed a novel method to generate oligosaccharides from any polysaccharide fiber, termed &lt;u&gt;F&lt;/u&gt;enton's &lt;u&gt;I&lt;/u&gt;nitiation &lt;u&gt;T&lt;/u&gt;oward &lt;u&gt;D&lt;/u&gt;efined &lt;u&gt;O&lt;/u&gt;ligosaccharide &lt;u&gt;G&lt;/u&gt;roups (FITDOG). Using this method, sugar beet pulp (SBP) was transformed into sugar beet oligosaccharides (SBOs) composed of arabinose- and galactose-containing oligosaccharides. Fecal fermentations of SBO and SBP produced similar shifts in donor-specific bacterial communities and acid metabolite profiles with a general enrichment of &lt;i&gt;Bacteroides&lt;/i&gt; and &lt;i&gt;Bifidobacterium&lt;/i&gt;. However, &lt;i&gt;in vitro&lt;/i&gt; tests revealed more &lt;i&gt;Bifidobacterium&lt;/i&gt; strains could consume SBO than sugar beet arabinan, and specific strains showed differential consumption of arabinofuranooligosaccharides or galactooligosaccharide (GOS) portions of the SBO pool. Genomic and glycomic comparisons suggest that previously characterized, arabinan-specific, extracellular arabinofuranosidases from &lt;i&gt;Bifidobacterium&lt;/i&gt; are not necessary to metabolize the arabino-oligosaccharides within SBO. Synbiotic application of SBO with an SBO-consuming strain &lt;i&gt;Bifidobacterium longum&lt;/i&gt; subsp. &lt;i&gt;longum&lt;/i&gt; SC596 in serial fecal enrichments resulted in enhanced persistence among 9 of 10 donor feces. This work demonstrates a novel workflow whereby FITDOG creates novel oligosaccharide pools that can provide insight into how compositional differences in fiber drive differential gut fermentation behaviors as well as their downstream health impacts. Moreover, these oligosaccharides may be useful in new prebiotic and synbiotic applications.&lt;b&gt;IMPORTANCE&lt;/b&gt;Prebiotics seek to selectively alter the host microbiome composition or function, resulting in a concurrent health benefit to the host. However, commercial prebiotics represent a small fraction of the diversity of food polysaccharide compositions. In this work a novel method, &lt;u&gt;F&lt;/u&gt;enton's &lt;u&gt;I&lt;/u&gt;nitiation &lt;u&gt;T&lt;/u&gt;oward &lt;u&gt;D&lt;/u&gt;efined &lt;u&gt;O&lt;/u&gt;ligosaccharide &lt;u&gt;G&lt;/u&gt;roups (FITDOG) was used to generate an oligosaccharide pool from sugar beet pulp (SBP). Sugar beet oligosaccharides (SBOs) resulted in similar changes to SBP in fecal enrichments; however, SBO could be consumed by more beneficial bifidobacterial strains than the cognate polysaccharide. These results demonstrate how the details of glycan structure have a profound influence on how gut bacteria metabolize food carbohydrates. The implications of this work are relevant to understanding how different dietary sources influence the human microbiome and extend to developing nove","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0207724"},"PeriodicalIF":3.9,"publicationDate":"2025-03-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11921329/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143254354","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":"Fungicide use intensity influences the soil microbiome and links to fungal disease suppressiveness in amenity turfgrass.","authors":"Ming-Yi Chou, Apoorva Tarihalkar Patil, Daowen Huo, Qiwei Lei, Jenny Kao-Kniffin, Paul Koch","doi":"10.1128/aem.01771-24","DOIUrl":"10.1128/aem.01771-24","url":null,"abstract":"<p><p>Disease-suppressive soils have been documented in many economically important crops, but not in turfgrass, one of the most intensively managed plant systems in the United States. Dollar spot, caused by the fungus <i>Clarireedia jacksonii</i>, is the most economically important disease of managed turfgrass and has historically been controlled through the intensive use of fungicides. However, previous anecdotal observations of lower dollar spot severity on golf courses with less intensive fungicide histories suggest that intensive fungicide usage may suppress microbial antagonism of pathogen activity. This study explored the suppressive activity of transplanted microbiomes against dollar spot from seven locations in the Midwestern U.S. and seven locations in the Northeastern U.S. with varying fungicide use histories. Creeping bentgrass was established in pots containing homogenized sterile potting mix and field soil and inoculated with <i>C. jacksonii</i> upon maturity. Bacterial and fungal communities of root-associated soil and phyllosphere were profiled with short-amplicon sequencing to investigate the microbial community associated with disease suppression. The results showed that plants grown in the transplanted soil microbiome collected from sites with lower fungicide intensities exhibited reduced disease severity. Plant growth-promoting and pathogen-antagonistic microbes may be responsible for disease suppression, but further validation is required. Additional least squares regression analysis of the fungicides used at each location suggested that contact fungicides such as chlorothalonil and fluazinam had a greater influence on the microbiome disease suppressiveness than penetrant fungicides. Potential organisms antagonistic to <i>Clarireedia</i> were identified in the subsequent amplicon sequencing analysis, but further characterization and validation are required.</p><p><strong>Importance: </strong>Given the current reliance on fungicides for plant disease control, this research provides new insights into the potential non-target effects of repeated fungicide usage on disease-suppressive soils. It also indicates that intensive fungicide usage can decrease the activity of beneficial soil microbes and lead to a more disease conducive microbial environment in turfgrass. The results from this study can be used to identify more sustainable disease management strategies for a variety of economically important and intensively managed pathosystems. Understanding the factors that facilitate disease-suppressive soils will contribute to more sustainable plant protection practices.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0177124"},"PeriodicalIF":3.9,"publicationDate":"2025-03-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11921360/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143466710","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":"The impact of wild-boar-derived microbiota transplantation on piglet microbiota, metabolite profile, and gut proinflammatory cytokine production differs from sow-derived microbiota.","authors":"Rajibur Rahman, Janelle M Fouhse, Tingting Ju, Yi Fan, Tulika Bhardwaj, Ryan K Brook, Roman Nosach, John Harding, Benjamin P Willing","doi":"10.1128/aem.02265-24","DOIUrl":"10.1128/aem.02265-24","url":null,"abstract":"<p><p>Colonization of co-evolved, species-specific microbes in early life plays a crucial role in gastrointestinal development and immune function. This study hypothesized that modern pig production practices have resulted in the loss of co-evolved species and critical symbiotic host-microbe interactions. To test this, we reintroduced microbes from wild boars (WB) into conventional piglets to explore their colonization dynamics and effects on gut microbial communities, metabolite profiles, and immune responses. At postnatal day (PND) 21, 48 piglets were assigned to four treatment groups: (i) WB-derived mixed microbial community (MMC), (ii) sow-derived MMC, (iii) a combination of WB and sow MMC (Mix), or (iv) Control (PBS). Post-transplantation analyses at PND 48 revealed distinct microbial communities in WB-inoculated piglets compared with Controls, with trends toward differentiation from Sow but not Mix groups. WB-derived microbes were more successful in colonizing piglets, particularly in the Mix group, where they competed with Sow-derived microbes. WB group cecal digesta enriched with <i>Lactobacillus helveticus</i>, <i>Lactobacillus mucosae</i>, and <i>Lactobacillus pontis</i>. Cecal metabolite analysis showed that WB piglets were enriched in histamine, acetyl-ornithine, ornithine, citrulline, and other metabolites, with higher histamine levels linked to <i>Lactobacillus</i> abundance. WB piglets exhibited lower cecal IL-1β and IL-6 levels compared with Control and Sow groups, whereas the Mix group showed reduced IFN-γ, IL-2, and IL-6 compared with the Sow group. No differences in weight gain, fecal scores, or plasma cytokines were observed, indicating no adverse effects. These findings support that missing WB microbes effectively colonize domestic piglets and may positively impact metabolite production and immune responses.IMPORTANCEThis study addresses the growing concern over losing co-evolved, species-specific microbes in modern agricultural practices, particularly in pig production. The implementation of strict biosecurity measures and widespread antibiotic use in conventional farming systems may disrupt crucial host-microbe interactions that are essential for gastrointestinal development and immune function. Our research demonstrates that by reintroducing wild boar-derived microbes into domestic piglets, these microbes can successfully colonize the gut, influence microbial community composition, and alter metabolite profiles and immune responses without causing adverse effects. These findings also suggest that these native microbes can fill an intestinal niche, positively impacting immune activation. This research lays the groundwork for future strategies to enhance livestock health and performance by restoring natural microbial populations that produce immune-modulating metabolites.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0226524"},"PeriodicalIF":3.9,"publicationDate":"2025-03-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11921332/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143188121","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":"Bumble bee gut microbial community structure differs between species and commercial suppliers, but metabolic potential remains largely consistent.","authors":"Michelle Z Hotchkiss, Alexandre J Poulain, Jessica R K Forrest","doi":"10.1128/aem.02036-24","DOIUrl":"10.1128/aem.02036-24","url":null,"abstract":"<p><p>Bumble bees are key pollinators for natural and agricultural plant communities. Their health and performance are supported by a core gut microbiota composed of a few bacterial taxa. However, the taxonomic composition and community structure of bumble bee gut microbiotas can vary with bee species, environment, and origin (i.e., whether colonies come from the wild or a commercial rearing facility), and it is unclear whether metabolic capabilities therefore vary as well. Here we used metagenomic sequencing to examine gut microbiota community composition, structure, and metabolic potential across bumble bees from two different commercial <i>Bombus impatiens</i> suppliers, wild <i>B. impatiens</i>, and three other wild bumble bee species sampled from sites within the native range of all four species. We found that the community structure of gut microbiotas varied between bumble bee species, between populations from different origins within species, and between commercial suppliers. Notably, we found that <i>Apibacter</i> is consistently present in some wild bumble bee species-suggesting it may be a previously unrecognized core phylotype of bumble bees-and that commercial <i>B. impatiens</i> colonies can lack core phylotypes consistently found in wild populations. However, despite variation in community structure, the high-level metabolic potential of gut microbiotas was largely consistent across all hosts, including for metabolic capabilities related to host performance, though metabolic activity remains to be investigated.IMPORTANCEOur study is the first to compare genome-level taxonomic structure and metabolic potential of whole bumble bee gut microbiotas between commercial suppliers and between commercial and wild populations. In addition, we profiled the full gut microbiotas of three wild bumble bee species for the first time. Overall, our results provide new insight into bumble bee gut microbiota community structure and function and will help researchers evaluate how well studies conducted in one bumble bee population will translate to other populations and species. Research on taxonomic and metabolic variation in bumble bee gut microbiotas across species and origins is of increasing relevance as we continue to discover new ways that social bee gut microbiotas influence host health, and as some bumble bee species decline in range and abundance.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0203624"},"PeriodicalIF":3.9,"publicationDate":"2025-03-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11921327/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143254330","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":"TK2268 encodes the major aminotransferase involved in the conversion from oxaloacetic acid to aspartic acid in <i>Thermococcus kodakarensis</i>.","authors":"Yu Su, Yuta Michimori, Yuto Fukuyama, Shigeru Shimamura, Takuro Nunoura, Haruyuki Atomi","doi":"10.1128/aem.02017-24","DOIUrl":"10.1128/aem.02017-24","url":null,"abstract":"<p><p>Amino acid metabolism in archaea in many cases differs from those reported in bacteria and eukaryotes. The hyperthermophilic archaeon <i>Thermococcus kodakarensis</i> possesses an incomplete tricarboxylic cycle, and the biosynthesis pathway of aspartate is unknown. Here, four Class I aminotransferases in <i>T. kodakarensis</i> encoded by TK0186, TK0548, TK1094, and TK2268 were examined to identify the enzyme(s) responsible for the conversion of oxaloacetate to aspartate. Among the four proteins, the TK2268 protein (TK2268p) was the only protein to recognize oxaloacetate as the amino acceptor. With oxaloacetate, TK2268p only recognized glutamate as the amino donor. The protein also catalyzed the reverse reaction, the transamination between aspartate and 2-oxoglutarate. Substrate inhibition was observed in the presence of high concentrations of oxaloacetate or 2-oxoglutarate. Aminotransferase activity between oxaloacetate and glutamate was observed in cell extracts of the <i>T. kodakarensis</i> host strain KU216. Among the individual gene disruption strains of the four aminotransferases, a significant decrease in activity was only observed in the ΔTK2268 strain. <i>T. kodakarensis</i> KU216 does not display growth in synthetic amino acid medium when aspartate/asparagine are absent. Growth was restored upon the addition of both oxaloacetate and glutamate. Although this restoration in growth was maintained in ΔTK0186, ΔTK0548, and ΔTK1094, growth was not observed in the ΔTK2268 strain. Our results suggest that TK2268p is the predominant aminotransferase responsible for the conversion of oxaloacetate to aspartate. The growth experiments and tracer-based metabolomics using ¹³C₃-pyruvate indicated that pyruvate is a precursor of aspartate and that this conversion is dependent on TK2268p.</p><p><strong>Importance: </strong>Based on genome sequence, the hyperthermophilic archaeon <i>Thermococcus kodakarensis</i> possesses an incomplete tricarboxylic cycle, raising questions on how this organism carries out the biosynthesis of aspartate and glutamate. The results of this study clarify two main points related to aspartate biosynthesis. We show that aspartate can be produced from oxaloacetate and identify TK2268p as the aminotransferase responsible for this reaction. The other point demonstrated in this study is that pyruvate can act as the precursor for oxaloacetate synthesis. Together with previous results, we can propose some of the roles of the individual aminotransferases in <i>T. kodakarensis</i>. TK0548p and TK0186p are involved in amino acid catabolism, with the latter along with TK1094p involved in the conversion of glyoxylate to glycine. TK2268p is responsible for the biosynthesis of aspartate from oxaloacetate.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0201724"},"PeriodicalIF":3.9,"publicationDate":"2025-03-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11921379/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143482026","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":"Antimicrobial peptide DiPGLa-H exhibits the most outstanding anti-infective activity among the PGLa variants based on a systematic comparison.","authors":"Liangjun Zheng, Muhammad Zafir, Ziqian Zhang, Yadong Ma, Fengyi Yang, Xiaokun Wang, Xuemei Xue, Chen Wang, Ping Li, Pilong Liu, Fatma A El-Gohary, Xin Zhao, Huping Xue","doi":"10.1128/aem.02062-24","DOIUrl":"10.1128/aem.02062-24","url":null,"abstract":"<p><p>The escalating threat of antibiotic-resistant bacteria has heightened global interest in antimicrobial peptides as promising candidates due to their potent broad-spectrum activity and low likelihood of resistance development. Despite this potential, these peptides face challenges, including modest bactericidal efficacy, insufficient safety assessment, and expensive production. In this study, we systematically evaluated a panel of nine AMP variants of PGLa, a natural AMP derived from <i>Xenopus laevis</i>. All peptides retained α-helical structures and exhibited high biocompatibility, with hemolytic concentrations above 128 µg/mL and macrophage survival rates over 80%. Among them, a tandem-repeat variant DiPGLa-H demonstrated the most potent antimicrobial activity, with a therapeutic index of 35.94, against key pathogens such as <i>Escherichia coli, Staphylococcus aureus,</i> and <i>Acinetobacter baumannii</i>. A DAMP4-DiPGLa-H fusion protein was engineered to mitigate potential host toxicity, and we achieved high-purity biosynthesis of DiPGLa-H by employing a combination of acid cleavage and non-chromatographic purification, with yields reaching 21.2 mg/mL. The biosynthesized DiPGLa-H exhibited robust stability across a wide pH range and high temperatures, effectively disrupting biofilms formed by multiple pathogenic species. Mechanistically, DiPGLa-H disrupts both the inner and outer bacterial membranes, causing cell shrinkage, vesiculation, and intracellular leakage. <i>In vivo</i>, DiPGLa-H significantly improved survival rates in mice with induced peritoneal inflammation by 31%-38% while reducing bacterial burdens in key organs by 100-fold to 1,000-fold. These findings unearthed DiPGLa-H as a highly promising AMP. Moreover, the successful development of a cost-effective, high-purity biosynthesis method for DiPGLa-H, utilizing DAMP4 fusion technology, enables its low-cost application in combating multidrug-resistant pathogens.</p><p><strong>Importance: </strong>AMPs are innate defense molecules in animals, plants, and microorganisms. Notably, one-third of these peptides in databases originate from amphibians. We discovered that naturally weak AMPs from this source can be enhanced through artificial design. Specifically, variant DiPGLa-H showed superior germicidal efficacy and cell selectivity both <i>in vivo</i> and <i>in vitro</i> and can be biosynthesized and purified by combining DAMP4 fusion protein strategy and a simple non-chromatographic method that facilitates large-scale production. Our focus is on understanding the structure-activity relationships of PGLa. Furthermore, the development of a non-chromatographic purification technique for AMPs offers a viable pathway for the large-scale production of these essential compounds.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0206224"},"PeriodicalIF":3.9,"publicationDate":"2025-03-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11921344/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143188105","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":"Polysulfides promote protein disulfide bond formation in microorganisms growing under anaerobic conditions.","authors":"Yuping Xin, Qingda Wang, Jianming Yang, Xiaohua Wu, Yongzhen Xia, Luying Xun, Huaiwei Liu","doi":"10.1128/aem.01926-24","DOIUrl":"10.1128/aem.01926-24","url":null,"abstract":"<p><p>Polysulfides commonly occur in anaerobic, microbial active environments, where they play key roles in sulfur cycling and redox transformations. Anaerobic survival of microorganisms requires the formation of protein disulfide bond (DSB). The relationship between polysulfides and anaerobic DSB formation has not been studied so far. Herein, we discovered that polysulfides can efficiently mediate protein DSB formation of microorganisms under anaerobic conditions. We used polysulfides to treat proteins, including roGFP2, Trx1, and DsbA, under anaerobic conditions and found that all three proteins formed intramolecular DSB <i>in vitro</i>. Under anaerobic conditions, <i>Escherichia coli</i> Δ<i>dsbB</i> displayed reduced growth and decreased intracellular protein DSB levels, but polysulfide treatment restored both growth and DSB content. Similarly, polysulfide treatment of <i>E. coli</i> Δ<i>dsbA</i> promoted periplasmic roGFP2 DSB formation and recovered growth under anaerobic conditions. Furthermore, treating <i>Schizosaccharomyces pombe</i> and <i>Cupriavidus pinatubonensis</i> JMP134 with polysulfides increased their intracellular protein DSB content. Collectively, these findings demonstrate that polysulfides can promote DSB formation independently of known enzymatic DSB-mediated systems and the presence of oxygen, thereby benefiting the survival of microorganisms in anaerobic habitats.IMPORTANCEHow polysulfides enhance the adaption of microorganisms to anaerobic environments remains unclear. Our study reveals that polysulfides efficiently facilitate protein DSB formation under anaerobic conditions. Polysulfides contain zero-valent sulfur atoms (S⁰), which can be transferred to the thiol group of cysteine residue. This S⁰ atom then accepts two electrons from two cysteine residues and is reduced to H₂S, leaving the two cysteines linked by a disulfide bond. Anaerobic growth of microorganisms benefits from the formation of DSB. These findings pave the way for a deeper understanding of the intricate relationship between polysulfides and microorganisms in various environmental contexts.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0192624"},"PeriodicalIF":3.9,"publicationDate":"2025-03-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11921322/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143363610","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}