Applied and Environmental Microbiology最新文献

{"title":"Genetic and phenotypic stability of <i>Lacticaseibacillus paracasei</i> DG (DSM 34154) over 10 years of industrial production.","authors":"Laura Brunelli, Susanna Perotti, Giorgio Gargari, Valerio De Vitis, Giacomo Mantegazza, Roberto Ferrari, Mario Minuzzo, Elena Pierallini, Giovanni Ricci, Walter Fiore, Simone Guglielmetti","doi":"10.1128/aem.02394-24","DOIUrl":"https://doi.org/10.1128/aem.02394-24","url":null,"abstract":"<p><p>The commercialization of a probiotic microorganism requires genetic and phenotypic consistency across production lots. However, the large-scale industrial production of probiotic microbial biomasses over years poses a risk of genetic drift, potentially affecting the probiotic's ability to confer health benefits. This study assessed the stability of <i>Lacticaseibacillus paracasei</i> DG (DSM 34154), a commercial probiotic. Seven isolates from different commercial lots over 10 years, one 8-year-old laboratory subculture, one isolate from human feces, and the DG strain deposited at DSMZ were analyzed. Shotgun and PacBio sequencing were combined to obtain the complete genome of the 10 isolates. Comparative analysis showed that the seven commercial isolates had identical genomes, differing from the DSMZ isolate by one synonymous transition and one non-synonymous transversion. The laboratory subculture strain had two additional mutations. Phenotypic analyses, including antibiotic resistance, carbohydrate fermentation profile, survival to simulated gastrointestinal transit, immunomodulatory capacity, and radical scavenging ability, found no significant differences among isolates. Overall, this study demonstrates the substantial equivalence of <i>L. paracasei</i> DG over 10 years of industrial production, indicating that the current industrial practices help prevent genome alterations that could compromise probiotic performance. Similar studies should be part of continuous monitoring and quality control measures for the probiotic products on the market.IMPORTANCEThe genetic and functional stability of probiotic strains during years of industrial production is essential but has not been clearly demonstrated for many strains. This study shows that careful industrial practices can maintain the genetic integrity and functionality of probiotics. Using advanced genome sequencing and detailed laboratory tests, we confirmed that the probiotic <i>Lacticaseibacillus paracasei</i> DG (DSM 34154) has remained stable over a decade of production, consistently delivering its health-promoting properties. These findings support the quality and reliability of probiotic products, fostering consumer trust and highlighting the importance of continuous monitoring in probiotic manufacturing to sustain quality assurance.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0239424"},"PeriodicalIF":3.9,"publicationDate":"2025-04-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143967290","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":"Highly efficient CRISPR-Cas9 base editing in <i>Bifidobacterium</i> with bypass of restriction modification systems.","authors":"Hung-Chun Lin, Wan-Chi Hsiao, Ya-Chen Hsu, Meng-Chieh Lin, Cheng-Chih Hsu, Mingzi M Zhang","doi":"10.1128/aem.01985-24","DOIUrl":"10.1128/aem.01985-24","url":null,"abstract":"<p><p>Intestinal microbiota members of the <i>Bifidobacterium</i> genus are increasingly explored as probiotics and therapeutics. However, the paucity of genetic tools and the widespread restriction modification (RM) systems in <i>Bifidobacterium</i> limit our ability to genetically manipulate these bacteria. Here we established a CRISPR-Cas9 cytosine base editor system (cBEST) for portable genome editing in bifidobacteria. Harboring different promoters characterized in this study, these cBEST plasmids showed a range of editing efficiencies in different strains and genomic contexts, highlighting the importance of fine-tuning base editor and sgRNA expression. Additionally, we showed that disruption or bypass of RM systems dramatically improved editing efficiencies in otherwise hard-to-edit genomic loci and <i>Bifidobacterium</i> strains. Notably, we demonstrated the use of RM-disrupted <i>Bifidobacterium longum</i> strains for simultaneous assembly, amplification, and methylation of the all-in-one editing plasmids, greatly streamlining the workflow for high-efficiency base editing. Last but not least, we showed the portability of cBESTs using the same editing construct to disrupt a conserved metabolic gene in multiple <i>Bifidobacterium</i> species. Looking ahead, the ability to efficiently edit and engineer bifidobacterial genomes will give rise to new opportunities for research and applications toward improving human health.IMPORTANCEThe ability to genetically manipulate specific genes and biological pathways in <i>Bifidobacterium</i> is essential to unlocking their probiotic and therapeutic potential in human health applications. The DNA double-strand break-free CRISPR-Cas9 cytosine base editor system established in this work allows portable and efficient base editing in <i>Bifidobacterium</i> spp. We further showed that bypass of restriction modification systems significantly improved base editing efficiency, especially for hard-to-edit genomic loci and strains. This expanded <i>Bifidobacterium</i> genome editing toolbox should facilitate mechanistic investigations into the roles of <i>Bifidobacterium</i> in host physiology and disease.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0198524"},"PeriodicalIF":3.9,"publicationDate":"2025-04-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12016496/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143584329","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A call for the United States to continue investing in science.","authors":"Ira Blader, Felicia Goodrum, Michael J Imperiale, Arturo Casadevall, Cesar A Arias, Andreas Baumler, Carey-Ann D Burnham, Christina A Cuomo, Corrella S Detweiler, Graeme N Forrest, Jack A Gilbert, Susan Lovett, Stanley Maloy, Alexander McAdam, Irene Newton, Gemma Reguera, George A O'Toole, Patrick D Schloss, Ashley Shade, Marvin Whiteley","doi":"10.1128/aem.00467-25","DOIUrl":"10.1128/aem.00467-25","url":null,"abstract":"","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0046725"},"PeriodicalIF":3.9,"publicationDate":"2025-04-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12016512/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143514443","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":"Effect of beaver dam analogs (BDAs) on waterborne protozoal pathogens <i>Giardia duodenalis</i> and <i>Cryptosporidium parvum</i>.","authors":"Ariel I Loredo, Andrea Packham, Nick Graham, Skylar Johnson, Stephanie Elliott, Brian H Bird, Carrie Monohan, Sarah Yarnell, Woutrina A Smith","doi":"10.1128/aem.01569-24","DOIUrl":"10.1128/aem.01569-24","url":null,"abstract":"<p><p>Installing beaver dam analogs (BDAs) in freshwater ecosystems is a process-based restoration technique mimicking natural beaver dams to increase stream channel complexity, floodplain connectivity, and hydrological residence time. BDAs have been applied in recent years by resource managers to improve riparian habitats with beneficial effects on ecosystem services, including improved water quality. While BDAs have been shown to reduce suspended sediments and nutrients in surface waters, research is needed to evaluate BDA effects on waterborne fecal pathogens such as <i>Giardia duodenalis</i> (syn. <i>G. lamblia</i>, <i>G. intestinalis</i>) and <i>Cryptosporidium parvum</i>. At a California riparian field site, this study used an <i>in situ</i> release trial of inactivated protozoal (oo)cysts upstream of BDAs to compare the concentrations and loads detected above and below the BDAs. The median percent decrease in load-based recovery rates was 78% for <i>G. duodenalis</i> and 80% for <i>C. parvum</i> at sites with BDAs. Mixed-effect linear regression analyses showed a significant reduction (by 81%) of the <i>G. duodenalis</i> adjusted cyst concentrations in downstream surface waters at sites with BDAs compared to similar sites without BDAs. While a reduction of <i>C. parvum</i> oocysts was noted, it was not statistically significant. The improved surface water quality downstream of BDAs could be because the BDA structures promote hyporheic exchange and act as a passive filter to remove pathogenic protozoa from streams. If BDAs are used to aid the restoration of riparian meadow ecosystems to slow water and raise the water table, they may also help to promote cleaner and safer waters for communities downstream.</p><p><strong>Importance: </strong>Beaver dam analogs (BDAs) are a cost-effective, low-environmental impact technique for stream and riparian meadow restoration that provide a variety of beneficial ecosystem services; however, their impact on waterborne fecal protozoal pathogens has not been evaluated. This study used an <i>in situ</i> protozoal release trial to quantify the effect of BDAs on the load of <i>Giardia duodenalis</i> and <i>Cryptosporidium parvum</i> in streams in California. Results showed that <i>G. duodenalis</i> concentrations below BDAs were significantly reduced by 81%. The median percent decrease in load-based recovery rates below a BDA for <i>G. duodenalis</i> and <i>C. parvum</i> was 78% and 80%, respectively. This finding indicates that BDAs may promote passive filtration of waterborne pathogens, thereby improving water quality in downstream reaches and adding to the beneficial outcomes associated with using BDAs in stream and meadow restoration efforts. The potential benefit to resource managers and communities is immense.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0156924"},"PeriodicalIF":3.9,"publicationDate":"2025-04-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12016503/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143690506","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 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":"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":"2025-04-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12016540/pdf/","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":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"No-tillage systems promote bacterial photosynthetic gene expression in low carbon, semi-arid surface soils.","authors":"Mark D McDonald, Katie L Lewis, Terry J Gentry","doi":"10.1128/aem.00184-25","DOIUrl":"10.1128/aem.00184-25","url":null,"abstract":"<p><p>Managing soils in semi-arid agricultural croplands generally focuses on reducing wind erosion, increasing fertility, and storing carbon. Thus, converting conventionally tilled systems to no-tillage and cover-cropped systems are often the first steps towards a conservation management approach across the growing area of semi-arid croplands. From a soil biological perspective, introducing cover crops to semi-arid soils has been shown to alter microbial community structure, which may lead to changes in the biogeochemical pathways expressed in these soils. In this study, we examined the impact of single-species wheat cover cropping and no-tillage on microbial gene expression after 4 and 5 years of implementation. We sequenced the metatranscriptomes of three production systems with varying levels of conservation management: conventional tillage winter fallow, no-tillage winter fallow, and no-tillage with a winter wheat cover crop. Removing tillage was the biggest factor altering microbial gene expression in this study, specifically resulting in upregulation of several photosystem-associated functions. These functions were taxonomically linked to organisms that make up the early stages of biological soil crusts, which may introduce additional benefits to these semi-arid agricultural systems beyond a reduction in wind erosion. Implementing a cover crop did not clearly alter gene expression beyond the effect of tillage removal; however, it did indicate a potential to reduce fungal disease incidence in 1 year of the study. These alterations of microbial activities and selection for potentially beneficial functions should be considered and further studied to aid in maintaining sustainable croplands for our changing climate.IMPORTANCEEliminating tillage from semi-arid agricultural soils has the potential to significantly alter the activities of the soil bacterial community compared with conventionally tilled soils. A major driver of this change was the activities of biological soil crust forming organisms that can provide several environmental benefits to the soil ecosystem beyond the typically associated benefits of conservation management. Furthermore, this study revealed that the implementation of a cover crop regime on no-tillage soils does not confer a major change in the function of the organisms present. Overall, the study reported here reveals that soil management practices aimed at reducing wind erosion and improving sustainability will positively impact the function of the microbial community and suggests that future investigations into the consequences of these functional changes may provide valuable services to these agricultural ecosystems.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0018425"},"PeriodicalIF":3.9,"publicationDate":"2025-04-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12016546/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143584330","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":"Comparative proteogenomics reveals ecological and evolutionary insights into the organohalide-respiring <i>Dehalobacter restrictus</i> strain T.","authors":"Xiaocui Li, Xiuying Li, Huijuan Jin, Jingjing Wang, Lian Yu, Jun Yan, Yi Yang","doi":"10.1128/aem.01719-24","DOIUrl":"10.1128/aem.01719-24","url":null,"abstract":"<p><p>1,1,1-Trichloroethane (1,1,1-TCA) and chloroform (CF) are persistent groundwater contaminants because of their widespread industrial use as organic solvents and improper disposal in the past. Obligate organohalide-respiring bacteria (OHRB), such as <i>Dehalobacter</i> (<i>Dhb</i>), play crucial roles in biotransforming and detoxifying natural or anthropogenic halogenated organics including 1,1,1-TCA and CF through reductive dechlorination. Despite their significance, only five <i>Dhb</i> strains have been associated with the reductive dechlorination of 1,1,1-TCA or CF. Therefore, cultivating and characterizing novel <i>Dhb</i> strains from various environments of different origins worldwide is crucial for understanding the ecology and evolution of <i>Dhb</i> and the associated reductive dehalogenase (RDase) genes. This study reports the enrichment and investigation of a novel <i>Dhb</i> population capable of reducing 1,1,1-TCA to 1,1-dichloroethane, CF to dichloromethane, 1,1,2-TCA to vinyl chloride/1,2-dichloroethane, and 1,2,4-trichlorobenzene to 1,2-dichlorobenzene. The capability for dechlorinating both aliphatic and aromatic compounds was observed for the first time in the sediment sourced from the Xi River situated in the North China Plain. Comparative genomic analysis of <i>Dhb</i> strains revealed genome contraction might have resulted in the loss of various gene family members, contributing to the syntrophy interactions (e.g., cobalamin, hydrogen, and acetate) of <i>Dhb</i> with other anaerobes (e.g., fermenters and acetogens). Proteogenomic and phylogenetic analysis confirmed the highly expressed 1,1,1-TCA/CF-dechlorinating RDase, designated as TcaA, shared 94.7-96.7% amino acid sequence similarities with RDases, such as ThmA, CfrA, and TmrA. This study expands knowledge on <i>Dhb</i> biogeography and evolution while providing insights into potential syntrophy interactions supporting organohalide respiration by <i>Dhb</i>. The findings have implications for developing the novel biotechnologies for the remediation of halogenated alkane-contaminated sites.IMPORTANCEOrganohalide-respiring bacteria (OHRB) are essential for breaking down harmful pollutants in the environment. This study investigates a newly discovered OHRB capable of degrading multiple contaminants, including persistent 1,1,1-trichloroethane and chloroform. By understanding its unique abilities and interactions with other microbes, we gain valuable insights into how these bacteria evolve and function, enabling the development of improved bioremediation strategies to clean up polluted sites.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0171924"},"PeriodicalIF":3.9,"publicationDate":"2025-04-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12016555/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143623261","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":"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":"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":"2025-04-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12016504/pdf/","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":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Immobilization of alginate C-5 epimerases using <i>Bacillus subtilis</i> spore display.","authors":"Jan Benedict Spannenkrebs, Agnes Beenfeldt Petersen, Finn Lillelund Aachmann, Johannes Kabisch","doi":"10.1128/aem.00298-25","DOIUrl":"10.1128/aem.00298-25","url":null,"abstract":"<p><p>Alginates are the most abundant polysaccharides found in brown seaweed, composed of (1→4)-linked β-D-mannuronate (M) and its C-5 epimer, α-L-guluronate (G). The G-blocks of alginate possess viscosifying and gelling properties, making alginates valuable industrial polysaccharides. Alginate epimerases are enzymes epimerizing M to G, enhancing the usability and value of alginate. The three alginate epimerases AlgE1, AlgE4, and AlgE6 were immobilized using <i>Bacillus subtilis</i> spores displaying the epimerases fused to the spore crust protein CotY. To our knowledge, this is the first display of immobilized alginate-modifying enzymes. Activity assays of the four AlgE4-displaying spore strains showed that AlgE4 produced MG-blocks from polyM alginate. AlgE4 was tested linked by its N- and C-termini. Two linkers with different flexibility were tested, both containing a TEV protease cleavage site. Immobilizing alginate epimerases on <i>B. subtilis</i> spores resulted in a recyclable system that is easy to isolate and reuse, thus opening possibilities for industrial application. Recyclability was demonstrated by performing five consecutive reactions with the same batch of AlgE4 spores, with the spores retaining 24% of the starting activity after four rounds of reuse. TEV cleavage of spore-displayed enzyme was optimized using spores displaying a green fluorescent protein, and these optimized conditions were used to cleave AlgE4 off the spores. The cleavage of four AlgE4-displaying spores was successful, but cleavage efficiency varied depending on which terminus of AlgE4 was fused to CotY.</p><p><strong>Importance: </strong>Seaweed is a scalable resource that requires no fresh water, fertilizer, or arable land, making it an important biomass for bioeconomies. Alginates are a major component of brown seaweed and are widely used in food, feed, technical, and pharmacological industries. To tailor the functional properties of alginates, alginate epimerases have shown to be promising for postharvest valorization of alginate. This study investigates an efficient and easy method to produce immobilized alginate epimerases, thus opening new industrial use cases. In this study, the alginate epimerases are immobilized on the surface of <i>Bacillus subtilis</i> spores. The bacterium forms spores in reaction to nutrient starvation, which are highly resistant to external influences and can be repurposed as a stable protein display platform for numerous applications due to its ease of genomic manipulation and cultivation.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0029825"},"PeriodicalIF":3.9,"publicationDate":"2025-04-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12016494/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143771097","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":"Expanding the potential soil carbon sink: unraveling carbon sequestration accessory genes in vermicompost phages.","authors":"Shujian Yuan, Yunling Wu, Jose Luis Balcazar, Danrui Wang, Dong Zhu, Mao Ye, Mingming Sun, Feng Hu","doi":"10.1128/aem.00296-25","DOIUrl":"10.1128/aem.00296-25","url":null,"abstract":"<p><p>The compost microbiome is important in regulating soil carbon sequestration. However, there is limited information concerning phage communities and phage-encoded auxiliary metabolic genes (AMGs) in compost-applied soils. We combined metagenomics and meta-viromes to explore the potential role of bacterial and phage communities in carbon sequestration in the compost microbiome. The experiment comprised swine manure compost (SW) and vermicompost (VE) applied to the soil along with a control treatment (CK). The bacterial community richness decreased after swine manure application and increased after vermicomposting compared to the control treatment. The phage community in the vermicompost-applied soil was dominated (63.1%) by temperate phages. In comparison, the communities of the swine manure compost-applied soil (92.7%) and control treatments (75.4%) were dominated by virulent phages. Phage-encoded carbon sequestration AMGs were detected in all three treatments, with significant enrichment in the vermicompost-applied soil. The average carbon sequestration potential (the coverage ratio of phage AMGs:total genes) of phage AMGs (<i>aceF</i>, <i>GT</i>11, and <i>GT</i>6) in the vermicompost-applied soil (65.18%) was greater than in the swine manure-applied (0) and control soils (50.21%). The results highlight the role of phage-encoded AMGs in improving soil carbon sequestration in vermicompost-applied soil. The findings provide new avenues for increasing soil carbon sequestration.IMPORTANCEThe phage-bacteria interactions have a significant impact on the global carbon cycle. Soil microbial carbon sequestration is a process in combination withcarbon sequestration genes and growth activity. This is the first study aimed at understanding the carbon sequestration potential of phage communities in vermicompost. The results of this study provide variations in carbon sequestration genes in vermicompost microbial communities, and some novel phage auxiliary metabolic genes were revealed to assist bacterial communities to increase soil carbon sequestration potential. Our results highlight the importance of phages in soil carbon sequestration from the perspective of phage-bacterial community interactions.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0029625"},"PeriodicalIF":3.9,"publicationDate":"2025-04-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12016548/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143623263","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}