Applied and Environmental Microbiology最新文献

{"title":"Organic particle scavenging by marine bacteria: influences of bacterial nanoscale surface properties.","authors":"Yosuke Yamada, Toshiaki Mochizuki, Nirav Patel, Farooq Azam, Hideki Fukuda, Toshi Nagata, Satoshi Mitarai","doi":"10.1128/aem.01049-25","DOIUrl":"https://doi.org/10.1128/aem.01049-25","url":null,"abstract":"<p><p>Marine bacteria contribute to biogeochemical cycles by scavenging organic nanoparticles such as cell fragments, viruses, excretions from phytoplankton and bacteria, and naturally occurring polymeric substances. Nonetheless, bacterial surface properties influencing this process remain poorly understood. A previous study found that marine bacteria exhibit significant variation in surface roughness, which affects nanoparticle attachment, influencing bacterial survival and marine biogeochemical cycles. However, cell surface characteristics such as Young's modulus and adhesiveness have rarely been measured. This study investigated bacterial nanoscale surface properties and their effects on nanoparticle attachment. Atomic force microscopy was employed to measure these parameters of 559 individual bacterial cells collected from Okinawa coastal waters. These results revealed significant variation in Young's modulus (6-21,000 kPa) and adhesiveness (86-1,200 pN). Subsequent coincubation experiments with polystyrene beads and virus-like particles, as model nanoparticles, demonstrated a significant negative correlation between Young's modulus and the attachment of virus-like particles, whereas no significant relationships were observed for other factors. Our results suggest that measuring bacterial surface properties provides novel insights into their strategies for resource utilization and their contribution to marine biogeochemical cycles.IMPORTANCESurface properties of marine bacteria are believed to influence their ability to acquire nanoparticles for nutrition. However, studies on these properties are limited, and the relationship with nanoparticle attachment remains unclear. This study measured Young's modulus and adhesiveness of marine bacteria, investigating their variability and their influence upon nanoparticle attachment. This work sheds light on biophysical mechanisms driving bacterial nanoparticle utilization, as well as ecological and biogeochemical implications of bacterial surface properties in marine environments.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0104925"},"PeriodicalIF":3.9,"publicationDate":"2025-06-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144301054","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":"Ultrahigh-throughput screening of environmental bacteria for proteolytic activity using droplet-based microfluidics.","authors":"Akihiro Nakamura, Yoshiyuki Suzuki, Nobuyuki Homma, Yosuke Shida, Rikako Sato, Hiroaki Takaku, Wataru Ogasawara","doi":"10.1128/aem.00109-25","DOIUrl":"https://doi.org/10.1128/aem.00109-25","url":null,"abstract":"<p><p>Exploration of diverse microbial sources, particularly environmental bacteria, is needed to identify novel and efficient peptidases/proteases that can be used in a variety of industrial applications. However, conventional function-based screening methods are inefficient and preclude the use of diverse microbial resources. This study illustrates a revolutionary approach to microbial screening using droplet-based microfluidics and fluorescence-activated droplet sorting that targets endopeptidases. Droplet-based microfluidic systems are a powerful tool for culturing microorganisms and for detecting microbial functions inside droplets with ultra-high throughput. However, droplet-based microfluidics for screening the proteolytic activity of environmental bacteria at a large scale remains largely unexplored. Here, we screened approximately 630,000 microorganisms in 6 h and obtained four species with high peptidase activity using droplet-based microfluidics. Furthermore, we isolated an Asp-specific endopeptidase from the isolated bacteria <i>Lysobacter soli</i> and showed that its activity was 2.4-fold higher than that of the related commercially available enzyme. The successful isolation of Asp-specific peptidase with superior activity in a short period of time compared to existing alternatives underscores the efficacy of droplet-based microfluidics for function-based microbial screening.</p><p><strong>Importance: </strong>As global efforts to reduce environmental impact progress, realization of the significance of biomanufacturing bio-based products has risen, increasing the demand for microbial-based manufacturing. Producing diverse bio-based products through biomanufacturing requires isolating suitable host organisms from environmental sources and screening them for essential genetic characteristics. Efficient screening methods based on microbial activity and functionality are thus essential to significantly expand the scope of bio-based products. Here, we demonstrate the development of a highly efficient screening system for functional screening of environmental bacteria using a droplet-based microfluidic device. This platform enables the streamlined isolation of microbial strains and acquisition of genetic resources from the environment and is tailored to specific microbial activities and functions. In this study, we have demonstrated the efficacy of this droplet-based method for functional screening and have shown its potential for scalability to industrial levels for advancing bio-based production.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0010925"},"PeriodicalIF":3.9,"publicationDate":"2025-06-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144282136","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":"Enzymatic property and stabilization mechanism of LysBT1, a novel polyextremotolerant endolysin with a C-terminal S-layer homology domain.","authors":"Yu Li, Ke Luo, Chaofeng Jiang, Yihao Zhang, Yong Yang, Yitong Yao, Huai Li, Fei Gan, Xiao-Feng Tang, Bing Tang","doi":"10.1128/aem.00867-25","DOIUrl":"https://doi.org/10.1128/aem.00867-25","url":null,"abstract":"<p><p>Phage-encoded endolysins are getting increasing attention because of their potential to serve as alternative antimicrobials to combat antibiotic-resistant bacteria. Here, we report a novel endolysin LysBT1, which is encoded by a prophage of thermophilic <i>Brevibacillus thermoruber</i> WF146 and comprises an N-acetylmuramoyl-L-alanine amidase domain and an S-layer homology (SLH) domain not found in known endolysins. LysBT1 is not only extremely thermostable, retaining more than 60% activity after 1 h incubation at 95°C, but also highly stable over a wide pH range of 4.0-11.0. Moreover, the thermostability of LysBT1 could be enhanced by EDTA or reducing agents. Although none of the seven cysteine residues of LysBT1 participate in disulfide bond formation, six of them, including the catalytic Zn²⁺-coordinating Cys156, are involved in stabilizing the enzyme at elevated temperatures. The SLH domain contributes to the thermostability of LysBT1 and mediates cell surface binding of the enzyme to facilitate enzymatic lysis of strain WF146 cells via increasing local enzyme concentration around the substrate. LysBT1 is capable of trimerization, where the SLH domains are predicted to form a three-prong spindle-like trimer similar to that in S-layer proteins. The SLH domain of LysBT1 could bind to cell surfaces of both Gram-positive and Gram-negative bacteria. LysBT1 can lyse not only Gram-positive strain WF146, <i>Geobacillus stearothermophilus</i>, and <i>Bacillus subtilis</i> but also Gram-negative <i>Escherichia coli</i> and <i>Acinetobacter baumannii</i> with the aid of EDTA or citric acid. EDTA also facilitates LysBT1 to lyse <i>Bacillus cereus</i>, probably because EDTA-induced disorganization of the S-layer allows LysBT1 to access and hydrolyze the peptidoglycan.IMPORTANCEThe emergence of antibiotic-resistant bacteria has led to an urgent requirement to develop novel antimicrobials, and endolysins are regarded as ideal alternatives to antibiotics. The thermostability of endolysins plays an important role in the feasibility of enzymatic bacteriolysis. However, reports on thermostable endolysins are limited, and little is known about their stabilization mechanisms. Our results demonstrate that the thermophile-derived prophage endolysin LysBT1 is highly thermostable and functional under polyextreme (multiple forms of stress) conditions, enabling the enzyme to lyse both Gram-positive and Gram-negative bacteria in synergy with outer membrane permeabilizer. Moreover, we found that the unique S-layer homology domain of LysBT1 contributes to the stability, activity, oligomerization, and cell-wall binding ability of the enzyme. This study not only characterizes a novel endolysin but also provides new clues about the stabilization mechanisms of endolysins.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0086725"},"PeriodicalIF":3.9,"publicationDate":"2025-06-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144282233","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":"Rhamnose biosynthesis is not impaired by the deletion of putative <i>rfbC</i> genes, <i>slr0985</i> and <i>slr1933</i>, in <i>Synechocystis</i> sp. PCC 6803.","authors":"João Pissarra, Marina Santos, Sara B Pereira, Catarina C Pacheco, Filipe Pinto, Sónia S Ferreira, Ricardo Monteiro, Cláudia Nunes, Manuel A Coimbra, Didier Cabanes, Rita Mota, Paula Tamagnini","doi":"10.1128/aem.00702-25","DOIUrl":"https://doi.org/10.1128/aem.00702-25","url":null,"abstract":"<p><p>Cyanobacterial extracellular polymeric substances (EPS) mainly composed of heteropolysaccharides can be attached to the cell wall as capsular polysaccharides (CPS) or released to the environment as released polysaccharides (RPS). These polymers have an unusually high diversified monosaccharidic composition, making them attractive for biotechnological/biomedical applications. However, their production is still poorly understood, hindering their optimization for industrial needs. This work aimed to better understand the biosynthesis of the 6-deoxy sugars, fucose and rhamnose, in the model cyanobacterium <i>Synechocystis</i> sp. PCC 6803. To that end, genes encoding proteins putatively involved in the biosynthesis of GDP-L-fucose [<i>sll1213</i> (<i>fucS</i>)] and dTDP-L-rhamnose [<i>slr0985</i> (<i>rfbC1</i>) and <i>slr1933</i> (<i>rfbC2</i>)] were deleted. As previously observed, Δ<i>fucS</i> had significant growth impairment, and its RPS did not contain any fucose or rhamnose. Here, we also showed that both deoxyhexoses' pathways are completely impaired in Δ<i>fucS</i>. In contrast, both Δ<i>rfbC1</i> and Δ<i>rfbC1</i>Δ<i>rfbC2</i>, although producing significantly less RPS and more CPS than the wild type, did not show major differences regarding the RPS monosaccharidic composition. These results strongly suggest that their gene products are not essential for rhamnose biosynthesis. Transcriptional analysis revealed that one of the <i>gmd</i> genes (<i>slr1072</i>) putatively encoding a GDP-mannose 4,6-dehydratase was upregulated in all the knockout strains and that the three EPS-related genes in the same operon as <i>rfbC1</i> (<i>slr0982</i>, <i>slr0983</i>, and <i>slr1610</i>) were upregulated in both Δ<i>rfbC</i> strains. Altogether, our results reveal that rhamnose biosynthesis in <i>Synechocystis</i> depends on FucS but not on the putative RfbC enzymes, underlining the need to further elucidate the mechanisms involved in the biosynthesis of this deoxyhexose.IMPORTANCEThis study contributes to the overall knowledge of deoxyhexoses' biosynthesis in <i>Synechocystis</i> sp. PCC 6803. Here, we demonstrated that the Δ<i>fucS</i> strain not only produces EPS without fucose and rhamnose, but that both pathways are completely impaired. Furthermore, we also showed that the deletion of both putative <i>rfbC</i> genes does not affect rhamnose biosynthesis despite having an impact on carbohydrate production/export, shifting RPS to CPS production. Altogether, our results suggest that the <i>rfbC</i> genes are not correctly annotated and highlight the intricacies and/or potential crosstalk between the two deoxyhexose pathways, yet to be completely unraveled in <i>Synechocystis</i>. The understanding of the cyanobacterial EPS assembly and export is crucial for the optimization of their production and tailoring for industrial/commercial applications.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0070225"},"PeriodicalIF":3.9,"publicationDate":"2025-06-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144282234","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 oligandrin in enhancing post-harvest disease resistance in cherry tomato through salicylic acid and jasmonic acid signaling pathways.","authors":"Jia-Hao Sun, Jia-Hui Xu, Chuanzhi Kang, Lu Cheng, Yu-Tang Gao, Feng-Qiao Hu, Jian Liang, Lan-Ping Guo, Xiao-Kui Ma","doi":"10.1128/aem.00421-25","DOIUrl":"https://doi.org/10.1128/aem.00421-25","url":null,"abstract":"&lt;p&gt;&lt;p&gt;Salicylic acid (SA) and jasmonic acid (JA) pathways are crucial components of plant defense mechanisms; however, their roles in post-harvest preservation remain inadequately explored. Cherry tomatoes are highly susceptible to fungal pathogens, such as &lt;i&gt;Alternaria nees&lt;/i&gt;, after harvest, leading to significant post-harvest losses. This study investigates the effects of oligandrin, an elicitin protein derived from &lt;i&gt;Pythium oligandrum&lt;/i&gt;, on enhancing the resistance of harvested cherry tomatoes to &lt;i&gt;A. nees&lt;/i&gt;. Oligandrin treatment significantly reduced disease symptoms, including black spots and decay, during storage without affecting &lt;i&gt;A. nees&lt;/i&gt; spore germination. Treated samples exhibited enhanced activities of defense-related enzymes, including polyphenol oxidase (PPO), peroxidase (POD), phenylalanine ammonia-lyase (PAL), and catalase (CAT), compared to controls. Key SA-responsive genes, such as &lt;i&gt;NPR1&lt;/i&gt; (Non-expressor of Pathogenesis-Related Genes 1), &lt;i&gt;TGA2&lt;/i&gt; (TGACG-Binding Factor 2), &lt;i&gt;WRKY70&lt;/i&gt;, and &lt;i&gt;PR1&lt;/i&gt; (Pathogenesis-Related Protein 1), were upregulated within 48 h, indicating activation of the SA signaling pathway. Additionally, &lt;i&gt;JAZ1&lt;/i&gt; (Jasmonate ZIM-Domain 1), a negative regulator of JA signaling, was elevated at both 24 and 48 h, while &lt;i&gt;MYC2&lt;/i&gt; (Myelocytomatosis 2), a transcription factor involved in the JA pathway, showed significant upregulation, suggesting activation of the JA signaling pathway. These findings demonstrate that oligandrin enhances post-harvest fungal disease resistance by modulating both SA and JA pathways and sustaining elevated defense enzyme activity, offering a promising strategy for improving post-harvest preservation and quality maintenance of cherry tomatoes.IMPORTANCEThis study provides compelling evidence that oligandrin activates both salicylic acid and jasmonic acid signaling pathways in cherry tomatoes, enhancing resistance to &lt;i&gt;A. nees&lt;/i&gt;. This dual activation not only deepens our understanding of oligandrin's mechanisms but also underscores the potential of oligandrin and its producer &lt;i&gt;P. oligandrum&lt;/i&gt; as a biocontrol agent for post-harvest preservation. By modulating plant immunity and promoting defense enzyme activity, oligandrin strengthens resistance to fungal diseases, offering a promising strategy for maintaining the quality and extending the shelf life of berry-like crops. Given the successful use of &lt;i&gt;P. oligandrum&lt;/i&gt; as a biopesticide in Europe and North America, oligandrin and its producer &lt;i&gt;P. oligandrum&lt;/i&gt; present an environmentally friendly alternative to chemical pesticides for post-harvest disease management. This research lays the groundwork for further studies aimed at optimizing the application of &lt;i&gt;P. oligandrum&lt;/i&gt; in real-world agricultural settings, establishing it as a reliable, sustainable tool for both agricultural and post-harvest management practices. These findings contribute to advancing biocontrol strategies for safer, more sus","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0042125"},"PeriodicalIF":3.9,"publicationDate":"2025-06-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144282135","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":"Influence of the pili of <i>Lacticaseibacillus rhamnosus</i> GG on its encapsulation and survival in mixed protein-starch gels assembled by <i>in situ</i> fermentation.","authors":"Tana Hernandez-Barrueta, Silvia Lorena Amaya-Llano, Nitin Nitin","doi":"10.1128/aem.00248-25","DOIUrl":"https://doi.org/10.1128/aem.00248-25","url":null,"abstract":"<p><p>Preserving the viability of probiotics during storage and gastrointestinal digestion poses a significant challenge in the development of effective probiotic formulations. Thus, this study developed an <i>in situ</i> fermentation approach to encapsulate the probiotic <i>Lacticaseibacillus rhamnosus</i> GG (LGG) in a mixed whey protein/modified starch gel and evaluated (i) the role of the pili on gel formation and on the effectiveness of the gel to maintain cell viability during simulated digestion, and (ii) the storage stability of the encapsulated probiotics. Kinetic data of gels made with the wild-type (WT) or a pilus-depleted mutant (Δ<i>spaCBA</i>) strain showed a rapid <i>in situ</i> gel formation (<30 min) at room temperature after inoculating the polymeric mixture, driven by <i>in situ</i> fermentation and independently of the piliation of the cells. After simulated gastrointestinal digestion, the viability of encapsulated WT cells was ~3 log higher than free WT cells (<i>P</i> value 7 × 10^-4) and ~0.6 times higher than encapsulated Δ<i>spaCBA</i> cells (<i>P</i> value 9 × 10^-3). A higher release of Δ<i>spaCBA</i> vs WT cells from the gels was quantified, and confocal microscopy revealed the aggregation of Δ<i>spaCBA</i> but not WT cells within the gel cavities. These findings suggest the pili-dependent retention of LGG within the gel contributes to its protective effect. Finally, the hydrated gels sustained counts of LGG of 7.76-6.69 log CFU/g (depending on the relative humidity) during 2 months of storage at room temperature. In summary, bacteria-to-matrix interactions might influence the survival of probiotics during delivery, and the protein/starch gels could represent a cost-effective alternative for unrefrigerated storage and delivery of probiotics.</p><p><strong>Importance: </strong>Many probiotic formulations struggle to maintain the viability of microbial cells over time and during their passage through the gastrointestinal tract. This has led to the development of encapsulation strategies for probiotics, most of which are either costly to implement or damage the cells during the encapsulation process. To overcome these limitations, this work developed a rapid fermentation-based approach to encapsulate probiotics in protein/starch gels as a strategy to keep the cells alive during storage and digestion. Moreover, this work explored the role of interactions between bacterial cells and their encapsulation matrix on the formation of the gels and in the protection the gels provided in maintaining the viability of cells during simulated digestion. Developing this <i>in situ</i> fermentation approach for the encapsulation of probiotics and understanding the bacteria-matrix interactions will lead to the development of more effective probiotic products that can be easily deployed in low-resource settings.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0024825"},"PeriodicalIF":3.9,"publicationDate":"2025-06-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144273972","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":"A single-nucleotide variant conditions the ability vs. inability of <i>Propionibacterium freudenreichii</i> to utilize L-lactate.","authors":"Riccardo Cocuzzi, Meral Turgay, Remo S Schmidt, Ueli von Ah, Hans-Peter Bachmann, Laure Weisskopf, Marie-Therese Fröhlich-Wyder","doi":"10.1128/aem.00599-25","DOIUrl":"https://doi.org/10.1128/aem.00599-25","url":null,"abstract":"<p><p><i>Propionibacterium freudenreichii</i> (<i>P. freudenreichii</i>) has various biotechnological applications, notably in the ripening of Swiss-type cheese, where it utilizes the two enantiomers of lactate as the main carbon source, contributing to flavor development and eye formation. Here, we genotypically characterized two <i>P. freudenreichii</i> strains unable to catabolize L-lactate through whole-genome sequencing and variant calling, using <i>P. freudenreichii</i> FAM-14222 as the reference genome, which highlighted a mutation in the <i>lutB</i> gene in both strains. This gene is part of the <i>lutABC</i> operon, which has been previously linked to lactate utilization in other bacterial species. Subsequently, we successfully restored the strains' ability to utilize L-lactate by following an adaptive laboratory evolution approach, which involved repeated subculturing in a medium containing L-lactate as the main carbon source. Sequencing of the <i>lutB</i> gene confirmed that isolates with a restored ability to utilize L-lactate had also reverted the mutation back to wild-type, supporting the involvement of the <i>lutABC</i> operon in L-lactate catabolism in <i>P. freudenreichii</i>. Moreover, the phenotype of the two L-lactate-negative strains was confirmed under cheesemaking conditions, highlighting the potential of the strains as cheese ripening cultures.IMPORTANCELactate catabolism is of paramount importance in <i>Propionibacterium freudenreichii</i>, particularly for its industrial applications, such as Swiss-type cheese ripening. Nevertheless, the genetic background of this metabolic process is not fully understood. In our study, we developed an adaptive laboratory evolution-based approach for the elucidation of L-lactate catabolism, starting from two strains unable to utilize L-lactate. Our results delivered experimental evidence of the role of the <i>lutABC</i> operon in this process, as opposed to the widespread theory of L-lactate dehydrogenase-mediated oxidation. A deeper understanding of this metabolic pathway will be beneficial for a more efficient selection of industrial strains, as well as for metabolic engineering.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0059925"},"PeriodicalIF":3.9,"publicationDate":"2025-06-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144274042","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":"A recombineering-based platform for high-throughput genomic editing in <i>Escherichia coli</i>.","authors":"Zeyu Liang, Chaoyong Huang, Yitian Li, Chao Yang, Ning Wang, Xiaoyan Ma, Yi-Xin Huo","doi":"10.1128/aem.00193-25","DOIUrl":"https://doi.org/10.1128/aem.00193-25","url":null,"abstract":"<p><p>Functional analysis of bacterial genes or genomic fragments <i>in vivo</i> primarily relies on the analysis of knockout strains. Although various methods have successfully generated bacterial knockout mutants, the parallel operation of multiple sites, especially in biofoundries, remains challenging. New technological refinements of existing methods are necessary for high-throughput genomic editing in bacteria. In this study, to modify numerous sites in parallel, we optimized the linear donor DNA by adding modification at the different positions and achieved high-efficiency recombination with chemical transformation. Then, by combining with the CRISPR system, we established a guide sequence-independent and donor DNA-mediated genomic editing (GIDGE) method, enabling efficient and scarless engineering of common <i>E. coli</i> strains as well as wild-type strains such as <i>E. coli</i> MG1655, with particularly marked advantages demonstrated in <i>E. coli</i> Nissle 1917. This method allows for high-throughput genomic engineering in a 96-well format and is useful for sequence deletion with a wide range of lengths, sequence insertion, sequence replacement, and point mutation. As a proof-of-concept study, we constructed 96 single-gene knockout mutants and a genomic large-fragment deletion library in <i>E. coli</i> K-12 MG1655 using the GIDGE method. This high-throughput and easy-to-use method has great potential for automation and can be adapted for use in biofoundries.</p><p><strong>Importance: </strong>With the increasing demand in the microbiology field and the expansion of its application scope, the urgency for genome editing techniques that are not only efficient and versatile but also capable of high-throughput processing and even automation has become increasingly critical. In this study, we enhanced the efficiency of recombination engineering by incorporating modifications and integrated it with the CRISPR system to develop an advanced gene editing method. This method allows for various gene editing events such as insertion, replacement, and long fragment knockout without the need for plasmid construction. It not only demonstrated high efficiency in common <i>E. coli</i> strains but also exhibited marked advantages in the probiotic strain <i>E. coli</i> Nissle 1917. This method is a versatile, efficient approach capable of high-throughput parallel gene editing. Using this method, we successfully constructed a large-scale strain library, significantly accelerating the process of microbial engineering.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0019325"},"PeriodicalIF":3.9,"publicationDate":"2025-06-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144274041","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":"Insights into genetic determinants of volatile fatty acid catabolism in <i>Cupriavidus necator</i> H16.","authors":"Eric C Holmes, Stephanie L Breunig, Christopher W Johnson, Gregg T Beckham, Alissa C Bleem","doi":"10.1128/aem.00515-25","DOIUrl":"https://doi.org/10.1128/aem.00515-25","url":null,"abstract":"<p><p>The soil bacterium <i>Cupriavidus necator</i> H16 is a promising host for upgrading waste-derived volatile fatty acids (VFAs) into renewable biochemicals. While bacterial VFA metabolic pathways are well understood, the <i>C. necator</i> genome encodes multiple enzymes for each catabolic step, and the degree of substrate specificity among these homologs is currently unknown. To gain insight into the catabolism of VFA substrates in <i>C. necator</i>, we performed transcriptomics on cells grown with acetate, propionate, butyrate, valerate, or hexanoate as the sole source of carbon and energy. These data revealed that <i>C. necator</i> upregulates multiple sets of genes putatively involved in substrate activation and <i>β</i>-oxidation in response to VFAs. To better understand this redundancy, we performed biochemical and genetic deletion studies of acyl-CoA synthetase enzymes upregulated during growth on VFA substrates. These results demonstrated the functional redundancy of the <i>C. necator</i> VFA catabolism and led to the identification of a gene cluster, <i>H16_B1332-H16_B1337</i>, that contains several genes that are important for the efficient catabolism of hexanoate. Constitutive expression of a second copy of these hexanoate catabolism genes did not improve growth of <i>C. necator</i> on hexanoate, suggesting that other factors (e.g., redox, transport, or toxicity) may be limiting for growth. Collectively, this work provides new insight into how <i>C. necator</i> uses metabolic regulation to effectively utilize VFA substrates and uncovers the important role of the gene cluster <i>H16_B1332-H16_B1337</i> in the catabolism of hexanoate.</p><p><strong>Importance: </strong>The development of efficient bioprocesses that utilize waste-derived carbon will be important for ensuring the circularity of carbon flows and the sustainability of new biotechnologies. Unfortunately, carbon substrates that can be reliably sourced from waste are often toxic or inefficient growth substrates for industrially relevant bacteria. A more complete understanding of the regulatory and biochemical mechanisms that bacteria use to respond to and catabolize waste-derived carbon resources will enable metabolic engineering strategies to improve bioconversion of these same resources. In this study, we provide new insight into these mechanisms for an emerging and promising host-feedstock pairing: <i>Cupriavidus necator</i> H16 and volatile fatty acids (VFAs). We anticipate that these insights can be leveraged in future work to engineer <i>C. necator</i> to more efficiently convert VFAs into sustainable protein and bioproducts.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0051525"},"PeriodicalIF":3.9,"publicationDate":"2025-06-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144273973","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":"Biodegradation of petroleum tar in contaminated sediments of the Eastern Mediterranean shores and associated microbial dynamics.","authors":"Baraa Al Haj Chehadeh, Farah Ali Ahmad, Darine A Salam","doi":"10.1128/aem.00258-25","DOIUrl":"https://doi.org/10.1128/aem.00258-25","url":null,"abstract":"<p><p>The Eastern Mediterranean coasts were impacted by several oil spills over the past two decades where tar residues were encountered on the shoreline. The majority of research on marine tar residues has focused on tar formation, chemical composition, transport, and fingerprinting to trace petroleum oil spills. Only a few studies have assessed the biodegradation of tar in the marine environment, and no studies have examined the evolution of the associated microbial communities. This research evaluated the biodegradation of tar residues in contaminated beach sediments of the Eastern Mediterranean coast of Lebanon. Biodegradation experiments were conducted in laboratory microcosms over a period of 56 days at 18°C and 28°C. Tar removal throughout the incubation period was monitored by the measurement of residual <i>n</i>-alkanes and polycyclic aromatic hydrocarbons (PAHs) using gas chromatography-mass spectrometry. Additionally, the diversity and evolution of the microbial community structure throughout the tar biodegradation experiments were determined using 16S rRNA gene sequencing. The biodegradation rates of total alkanes and total PAHs were 0.035 day^-1 and 0.023 day^-1, respectively, at 18°C, and increased to 0.110 day^-1 and 0.055 day^-1 at 28°C. Microbial analysis revealed a shift in the microbial community from generalist hydrocarbon degraders at the beginning of the biodegradation process to more specialized groups as the experiments progressed. Key genera involved in tar biodegradation included Erythrobacter, Bacillus, Alcanivorax, Marinobacter, Actinomarinales, and Pseudomonas. These findings highlight the potential of the Eastern Mediterranean coast to naturally degrade tar contamination and enhance our understanding of the microbial dynamics associated with tar biodegradation.</p><p><strong>Importance: </strong>The planned oil and gas extraction activities of the Eastern Mediterranean coasts increase the risk of potential oil spills and threaten the Mediterranean shoreline with devastating impacts. A recent oil spill has resulted in huge amounts of tar residues washing up along the Lebanese southern coastline, affecting Nature Reserve shores known to be a nesting ground for several species of endangered turtles. The majority of research conducted on marine tar residues has studied tar formation, distribution and prevalence, chemical composition and tracing, transport mechanisms, as well as human and ecological effects. The biodegradation of spilled petroleum tar in aquatic media and the associated microbial dynamics are still poorly addressed in the literature. This study contributes to the state of knowledge and current scarce literature on petroleum tar biodegradation in marine environments and provides guidelines to spill responders for an effective bioremediation response plan to address future potential tar contamination.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0025825"},"PeriodicalIF":3.9,"publicationDate":"2025-06-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144274043","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}