Applied and Environmental Microbiology最新文献

{"title":"Nitrous oxide is the main product during nitrate reduction by a novel lithoautotrophic iron(II)-oxidizing culture from an organic-rich paddy soil.","authors":"Hanna Grimm, Jennifer Lorenz, Daniel Straub, Prachi Joshi, Jeremiah Shuster, Christiane Zarfl, E Marie Muehe, Andreas Kappler","doi":"10.1128/aem.01262-24","DOIUrl":"10.1128/aem.01262-24","url":null,"abstract":"<p><p>Microbial nitrate reduction coupled to iron(II) oxidation (NRFeOx) occurs in paddy soils due to high levels of dissolved iron(II) and regular application of nitrogen fertilizer. However, to date, there is no lithoautotrophic NRFeOx isolate or enrichment culture available from this soil environment. Thus, resulting impacts on greenhouse gas emissions during nitrate reduction (i.e., nitrous oxide [N₂O]) and on toxic metalloid (i.e., arsenic) mobility can hardly be investigated. We enriched a lithoautotrophic NRFeOx culture, culture HP (Huilongpu paddy, named after its origin), from a paddy soil (Huilongpu Town, China), which was dominated by <i>Gallionella</i> (71%). The culture reduced 0.45 to 0.63 mM nitrate and oxidized 1.76 to 2.31 mM iron(II) within 4 days leading to N₂O as the main N-product (62%-88% N₂O-N of total reduced NO₃^--N). Nitrite was present as an intermediate at a maximum of 0.16 ± 0.1 mM. Cells were associated with, but mostly not encrusted by, poorly crystalline iron(III) minerals (ferrihydrite). Culture HP performed best below an iron(II) threshold of 2.5-3.5 mM and in a pH range of 6.50-7.05. In the presence of 100 µM arsenite, only 0%-18% of iron(II) was oxidized. Due to low iron(II) oxidation, arsenite was not immobilized. However, the proportion of N₂O-N of total reduced NO₃^--N decreased from 77% to 30%. Our results indicate that lithoautotrophic NRFeOx occurs even in organic-rich paddy soils, resulting in denitrification and subsequent N₂O emissions. The obtained novel enrichment culture allows us to study the impact of lithoautotrophic NRFeOx on arsenic mobility and N₂O emissions in paddy soils.IMPORTANCEPaddy soils are naturally rich in iron(II) and regularly experience nitrogen inputs due to fertilization. Nitrogen fertilization increases nitrous oxide emissions as it is an intermediate product during nitrate reduction. Microorganisms can live using nitrate and iron(II) as electron acceptor and donor, respectively, but mostly require an organic co-substrate. By contrast, microorganisms that only rely on nitrate, iron(II), and CO₂ could inhabit carbon-limited ecological niches. So far, no isolate or consortium of lithoautotrophic iron(II)-oxidizing, nitrate-reducing microorganisms has been obtained from paddy soil. Here, we describe a lithoautotrophic enrichment culture, dominated by a typical iron(II)-oxidizer (<i>Gallionella</i>), that oxidized iron(II) and reduced nitrate to nitrous oxide, negatively impacting greenhouse gas dynamics. High arsenic concentrations were toxic to the culture but decreased the proportion of nitrous oxide of the total reduced nitrate. Our results suggest that autotrophic nitrate reduction coupled with iron(II) oxidation is a relevant, previously overlooked process in paddy soils.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0126224"},"PeriodicalIF":3.9,"publicationDate":"2024-12-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142783916","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":"Evaluation of aqueous chlorine and peracetic acid sanitizers to inactivate protozoa and bacteria of concern in agricultural water.","authors":"Kyle J McCaughan, Zoe Scott, Channah Rock, Kalmia E Kniel","doi":"10.1128/aem.01653-24","DOIUrl":"https://doi.org/10.1128/aem.01653-24","url":null,"abstract":"<p><p>Agricultural water is a potential source of microbial contamination whereby <i>Escherichia coli, Salmonella, Cryptosporidium,</i> and <i>Cyclospora cayetenensis</i> can enter the food supply. To reduce this risk, effective sanitization of agricultural water may be critical to food safety. As such, it is important to investigate the effects of aqueous peracetic acid (PAA) and chlorine (Cl) on bacteria and protozoa at different treatment times and temperatures in agricultural water with respect to key water characteristics. Multiple concentrations of each sanitizer, ranging from 3 to 200 ppm, were prepared in recently collected agricultural water, the solution was brought to the desired temperature, and the target organisms were added and left for the desired contact time (5 or 10 minutes) when sodium metabisulfite was added to neutralize the sanitizers. Bacterial samples were enumerated on MacConkey or XLT4 agar. Samples with protozoa were added to mammalian cell culture (HCT-8 cells for <i>Cryptosporidium parvum</i> and MDBK cells for <i>Eimeria tenella</i>). After 48 hours, the infected cells were collected, DNA extracted and infectivity assessed by quantitative PCR (qPCR). Low and high concentrations of sanitizer were effective at eliminating bacteria with Cl being significantly (<i>P</i> < 0.05) more effective. The greatest reductions in <i>E. coli</i> and <i>Salmonella</i> (3.48 log and 2.5 log cfu/mL, respectively) were observed after 10 minutes of exposure to 10 ppm Cl. Concentrations of sanitizer 50 ppm and lower resulted in insignificant (<i>P</i> > 0.05) reductions in parasite infectivity of less than 1 log for both organisms. A 200 ppm PAA treatment reduced infectious oocyst populations by 3.8 log for <i>C. parvum</i> and 2.6 log for <i>E. tenella</i>, with Cl being significantly (<i>P</i> < 0.05) less effective against these organisms.</p><p><strong>Importance: </strong>This research is critical to inform decisions regarding the application and use of sanitizers in pre-harvest agricultural water settings to enhance food safety. Understanding the effectiveness of chlorine (Cl) and peracetic acid (PAA) on bacteria and protozoa will allow for the more efficient and practical use of these sanitizers, thus improving agricultural practices in ways that are beneficial to both growers and consumers.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0165324"},"PeriodicalIF":3.9,"publicationDate":"2024-12-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142783912","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Transcriptional delineation of polysaccharide utilization loci in the human gut commensal <i>Segatella copri</i> DSM18205 and co-culture with exemplar <i>Bacteroides</i> species on dietary plant glycans.","authors":"Deepesh Panwar, Jonathon Briggs, Alexander S C Fraser, William A Stewart, Harry Brumer","doi":"10.1128/aem.01759-24","DOIUrl":"https://doi.org/10.1128/aem.01759-24","url":null,"abstract":"<p><p>There is growing interest in members of the genus <i>Segatella</i> (family <i>Prevotellaceae</i>) as members of a well-balanced human gut microbiota (HGM). <i>Segatella</i> are particularly associated with the consumption of a diet rich in plant polysaccharides comprising dietary fiber. However, understanding of the molecular basis of complex carbohydrate utilization in <i>Segatella</i> species is currently incomplete. Here, we used RNA sequencing (RNA-seq) of the type strain <i>Segatella copri</i> DSM 18205 (previously <i>Prevotella copri</i> CB7) to define precisely individual polysaccharide utilization loci (PULs) and associated carbohydrate-active enzymes (CAZymes) that are implicated in the catabolism of common fruit, vegetable, and grain polysaccharides (<i>viz</i>. mixed-linkage β-glucans, xyloglucans, xylans, pectins, and inulin). Although many commonalities were observed, several of these systems exhibited significant compositional and organizational differences <i>vis-à-vis</i> homologs in the better-studied <i>Bacteroides</i> (sister family <i>Bacteroidaceae</i>), which predominate in post-industrial HGM. Growth on β-mannans, β(1, 3)-galactans, and microbial β(1, 3)-glucans was not observed, due to an apparent lack of cognate PULs. Most notably, <i>S. copri</i> is unable to grow on starch, due to an incomplete starch utilization system (Sus). Subsequent transcriptional profiling of bellwether Ton-B-dependent transporter-encoding genes revealed that PUL upregulation is rapid and general upon transfer from glucose to plant polysaccharides, reflective of de-repression enabling substrate sensing. Distinct from previous observations of <i>Bacteroides</i> species, we were unable to observe clearly delineated substrate prioritization on a polysaccharide mixture designed to mimic <i>in vitro</i> diverse plant cell wall digesta. Finally, co-culture experiments generally indicated stable co-existence and lack of exclusive competition between <i>S. copri</i> and representative HGM <i>Bacteroides</i> species (<i>Bacteroides thetaiotaomicron</i> and <i>Bacteroides ovatus</i>) on individual polysaccharides, except in cases where corresponding PULs were obviously lacking.</p><p><strong>Importance: </strong>There is currently a great level of interest in improving the composition and function of the human gut microbiota (HGM) to improve health. The bacterium <i>Segatella copri</i> is prevalent in people who eat plant-rich diets and is therefore associated with a healthy lifestyle. On one hand, our study reveals the specific molecular systems that enable <i>S. copri</i> to proliferate on individual plant polysaccharides. On the other, a growing body of data suggests that the inability of <i>S. copri</i> to grow on starch and animal glycans, which dominate in post-industrial diets, as well as host mucin, contributes strongly to its displacement from the HGM by <i>Bacteroides</i> species, in the absence of direct antagonism.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0175924"},"PeriodicalIF":3.9,"publicationDate":"2024-12-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142783494","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":"Structural and mechanism-based engineering of sulfotransferase CHST15 for the efficient synthesis of chondroitin sulfate E.","authors":"Zhonghua Wang, Wei Song, Wanqing Wei, Hejia Qi, Weiwei Meng, Jia Liu, Xiaomin Li, Cong Gao, Liming Liu, Guipeng Hu, Yiwen Zhou, Jing Wu","doi":"10.1128/aem.01573-24","DOIUrl":"https://doi.org/10.1128/aem.01573-24","url":null,"abstract":"<p><p>Natural chondroitin sulfate (CS), extracted from animal cartilage, is widely used in the pharmaceuticals and foods. However, contamination with animal-derived heteropolysaccharides presents significant challenges, including potential immune responses. To address this, we developed a green and efficient method for synthesizing chondroitin sulfate E (CSE) via enzymatic synthesis, identifying <i>Ec</i>CHST15, a sulfotransferase that catalyzes the conversion of chondroitin sulfate A (CSA) to CSE. We investigated the novel catalytic mechanism of CHST15 through quantum mechanical (QM) calculations and experimental validation, confirming its alignment with the SN2 reaction mechanism. Subsequently, we enhanced the catalytic efficiency of CHST15 using protein engineering, improving the catalytic efficiency from 18.1% in the wild type (WT) to 62.5% in the M7 mutant-a 3.5-fold increase. Finally, we constructed a six-enzyme cascade whole-cell catalyst, achieving a 72.2% conversion of 15 g/L CSA to produce CSE within 24 h. These findings offer a promising strategy for the industrial production of CSE.IMPORTANCECurrent methods for obtaining chondroitin sulfate (CS) primarily rely on tissue extraction and chemical synthesis. However, these approaches are hindered by contamination risks from animal-derived heteropolysaccharides and the technical challenges inherent in complex chemical synthesis, limiting the scalability of industrial CS production. To address this, we developed a green and efficient enzymatic biosynthesis method for chondroitin sulfate E (CSE). By identifying and engineering the sulfotransferase CHST15 from <i>Erpetoichthys calabaricus</i>, we created a mutant (<i>Ec</i>CHST15^M7) with a 3.5-fold increase in catalytic efficiency toward chondroitin sulfate A (CSA) compared to the wild-type enzyme. Additionally, we constructed a six-enzyme cascade whole-cell biocatalyst, achieving a 72.2% conversion rate from CSA to CSE. This study opens new avenues for the industrial-scale production of CSE through sustainable enzymatic processes.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0157324"},"PeriodicalIF":3.9,"publicationDate":"2024-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142765697","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":"<i>Wolbachia</i> strain <i>w</i>MelM disrupts egg retention by <i>Aedes aegypti</i> females prevented from ovipositing.","authors":"Perran A Ross, Ella Yeatman, Mel S Berran, Xinyue Gu, Ary A Hoffmann, Belinda van Heerwaarden","doi":"10.1128/aem.01491-24","DOIUrl":"https://doi.org/10.1128/aem.01491-24","url":null,"abstract":"<p><p><i>Aedes aegypti</i> mosquitoes are well adapted to dry climates and can retain their eggs for extended periods in the absence of suitable habitat. <i>Wolbachia</i> strains transferred from other insects to mosquitoes can be released to combat dengue transmission by blocking virus replication and spreading through populations, but host fitness costs imposed by <i>Wolbachia</i>, particularly under some environments, can impede spread. We, therefore, assessed the impact of two <i>Wolbachia</i> strains being released for dengue control (<i>w</i>AlbB and <i>w</i>MelM) on fecundity and egg viability following extended egg retention (up to 24 days) under laboratory conditions. Egg viability following retention decreased to a greater extent in females carrying <i>w</i>MelM compared to uninfected or <i>w</i>AlbB females. Fertility fully recovered in uninfected females following a second blood meal after laying retained eggs, while <i>w</i>MelM females experienced only partial recovery. Effects of <i>w</i>MelM on egg retention were similar regardless of whether females were crossed to uninfected or <i>w</i>MelM males, suggesting that fitness costs were triggered by <i>Wolbachia</i> presence in females. The fecundity and hatch proportions of eggs of <i>w</i>MelM females declined with age, regardless of whether females used stored sperm or were recently inseminated. Costs of some <i>Wolbachia</i> strains during egg retention may affect the invasion and persistence of <i>Wolbachia</i> in release sites where larval habitats are scarce and/or intermittent.IMPORTANCE<i>Wolbachia</i> mosquito releases are expanding around the world with substantial impacts on dengue transmission. Releases have succeeded in many locations, but the establishment of <i>Wolbachia</i> has been challenging in some environments, and the factors contributing to this outcome remain unresolved. Here, we explore the effects of <i>Wolbachia</i> on a novel trait, egg retention, which is likely to be important for the persistence of mosquito populations in locations with intermittent rainfall. We find substantial impacts of the <i>Wolbachia</i> strain <i>w</i>MelM on the quality of retained eggs but not the <i>w</i>AlbB strain. This cost is driven by the <i>Wolbachia</i> infection status of the female and can partially recover following a second blood meal. The results of our study may help to explain the difficulty in establishing <i>Wolbachia</i> strains at some field release sites and emphasize the need to characterize <i>Wolbachia</i> phenotypes across a variety of traits and strains.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0149124"},"PeriodicalIF":3.9,"publicationDate":"2024-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142765706","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":"Ferruginous hemeprotein HhuH facilitates the cadmium adsorption and chromium reduction in <i>Stenotrophomonas</i> sp. SY1.","authors":"Zijie Zhou, Hongbo Yu, Jiahui Liu, Lin Zhu, Gejiao Wang, Kaixiang Shi","doi":"10.1128/aem.02097-24","DOIUrl":"https://doi.org/10.1128/aem.02097-24","url":null,"abstract":"<p><p>Cadmium (Cd) and chromium (Cr) are frequently encountered toxicants, while iron (Fe) plays a crucial role in bacterial survival under conditions of heavy metal stress. However, intracellular Fe ion depletion by heavy metals leads to a state of Fe starvation. Therefore, it is imperative to investigate the mechanism through which bacteria maintain a balance between heavy metal detoxification and Fe homeostasis. This study demonstrates Cd(II) immobilization and Cr(VI) reduction abilities of <i>Stenotrophomonas</i> sp. SY1, while proteomics reveals the upregulation of heme metabolism in response to Cd(II) and Cr(VI) exposure. The expression of the heme-uptake system in <i>Escherichia coli</i> can enhance Cd(II) immobilization and facilitate Cr(VI) reduction. The ferruginous hemeprotein HhuH exhibits the ability to chelate Cd(II) and reduce Cr(VI). The presence of Cd(II) and Cr(VI) in strain SY1 initially led to Fe starvation. Subsequently, the hemeprotein HhuH facilitated Cd(II) adsorption and Cr(VI) reduction, thereby restoring normal cellular Fe homeostasis. Our findings explain the hemeprotein-mediated mechanism for Cd(II) adsorption and Cr(VI) reduction, providing further insights into the correlation between heavy metal and Fe metabolism.IMPORTANCEIron (Fe) is an indispensable trace element for many organisms, and virtually, all bacteria require Fe as a cofactor in enzymes to facilitate redox reactions involved in fundamental cellular processes during periods of heavy metal stress. Understanding bacterial response to Fe in heavy metal contamination is essential. Therefore, our study elucidates Cd(II) adsorption and Cr(VI) reduction processes mediated by the Fe-bearing hemeprotein HhuH. It is a unique trifunctional protein capable of chelating Cd(II) and reducing Cr(VI), demonstrating significant potential in the environmental remediation of heavy metals.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0209724"},"PeriodicalIF":3.9,"publicationDate":"2024-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142765710","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":"<i>Wohlfahrtiimonas chitiniclastica</i>: current insights and complementary review from Chinese cases.","authors":"Qin Yuan, Cheng Peng, Xin-Lin Sun, Zi-Chun Nie, Yi-Wen Zhang, Ying-Ye Miao","doi":"10.1128/aem.00965-24","DOIUrl":"https://doi.org/10.1128/aem.00965-24","url":null,"abstract":"<p><p><i>Wohlfahrtiimonas chitiniclastica</i> is an emerging zoonotic pathogen associated with bacteremia, myiasis, and soft tissue infections. It is insufficiently identified and underestimated due to reasons, such as shortcomings of the traditional identification techniques and language barriers in local case reports from different regions. In this review, we summarize the currently available literature. In particular, we added previously overlooked cases from Chinese and other medical communities. The clinical characteristics, identification, and treatment of <i>W. chitiniclastica</i> are discussed. This work provides a complete review of the previous work including cases from human, animal, and other sources.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0096524"},"PeriodicalIF":3.9,"publicationDate":"2024-11-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142724991","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":"Correction for Tothero et al., \"<i>Leptothrix ochracea</i> genomes reveal potential for mixotrophic growth on Fe(II) and organic carbon\".","authors":"Gracee K Tothero, Rene L Hoover, Ibrahim F Farag, Daniel I Kaplan, Pamela Weisenhorn, David Emerson, Clara S Chan","doi":"10.1128/aem.02044-24","DOIUrl":"https://doi.org/10.1128/aem.02044-24","url":null,"abstract":"","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0204424"},"PeriodicalIF":3.9,"publicationDate":"2024-11-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142724994","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":"Recombinant <i>Saccharomyces cerevisiae</i> EBY100/pYD1-FaeG: a candidate for an oral subunit vaccine against F4+ ETEC infection.","authors":"Dayue Hu, Xiangmin Li, Xiaochao Duan, Liuyue Yang, Baizhi Luo, Linkang Wang, Zihui Hu, Yang Zhou, Ping Qian","doi":"10.1128/aem.01817-24","DOIUrl":"https://doi.org/10.1128/aem.01817-24","url":null,"abstract":"<p><p>Diarrheal diseases attributable to multidrug-resistant F4+ enterotoxigenic <i>Escherichia coli</i> (ETEC) are escalating in severity, posing significant risks to the health and safety of both humans and animals. This study used <i>Saccharomyces cerevisiae</i> EBY100 to display the FaeG subunit of F4 colonizing factor as an oral vaccine against F4+ ETEC infection. Mice were orally immunized twice with 10⁸ CFU of EBY100/pYD1-FaeG, followed by a challenge with F4+ ETEC EC6 on day 7 post-immunization. The results showed that the recombinant strain EBY100/pYD1-FaeG orally enhanced the growth of the small intestine villi, significantly boosted the expression of tight junction proteins (<i>ZO-1</i>, <i>Occludin</i>, <i>MUC2</i>, and <i>Claudin</i>) (<i>P</i> < 0.05), and modulated the gut microbiota composition. Additionally, immunization with EBY100/pYD1-FaeG also upregulated the levels of IL-2, IL-4, and IFN-γ in the intestines of mice (<i>P</i> < 0.01), while serum IgG and fecal sIgA titer significantly increased (<i>P</i> < 0.05). These immune responses enhanced the capacity to fight against ETEC, leading to an increased survival rate of mice and relieved damage to tissues and organs of mice infection. In summary, the study suggested that the recombinant <i>Saccharomyces cerevisiae</i> EBY100/pYD1-FaeG could effectively stimulate the immune response and generate specific antibodies against F4+ ETEC, showing its potential to serve as a subunit oral vaccine candidate for preventing F4+ ETEC infection.IMPORTANCEThe multidrug-resistant F4+ enterotoxigenic <i>Escherichia coli</i> (ETEC) strains are the primary clinical pathogens responsible for post-weaning diarrhea in piglets, resulting in substantial economic losses in the pig farming industry. In the study, we developed an oral vaccine candidate, <i>Saccharomyces cerevisiae</i> EBY100/pYD1-FaeG, to prevent diarrhea caused by multidrug-resistant F4+ ETEC. Oral administration of EBY100/pYD1-FaeG significantly enhanced immune responses, improved intestinal health, and provided protection against F4+ ETEC infection in mice. This approach offers a potential application prospect for preventing F4+ ETEC infections that lead to post-weaning diarrhea in clinical settings and provides a promising solution for addressing the growing threat of antibiotic resistance in bacterial pathogens.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0181724"},"PeriodicalIF":3.9,"publicationDate":"2024-11-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142724996","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":"Papain expression in the <i>Escherichia coli</i> cytoplasm by T7-promoter engineering and co-expression with human protein disulfide isomerase (PDI) and thiol peroxidase (GPx7) genes.","authors":"Md Anarul Hoque, Richard A Gross, Mattheos A G Koffas","doi":"10.1128/aem.02119-24","DOIUrl":"https://doi.org/10.1128/aem.02119-24","url":null,"abstract":"&lt;p&gt;&lt;p&gt;Difficulties exist in obtaining full-length, correctly folded, and soluble papain or papain-like proteases that necessitate the exploration of alternative strategies. This study describes the development of an &lt;i&gt;Escherichia coli&lt;/i&gt; strain capable of producing soluble papain without the need for complex and time-consuming &lt;i&gt;in vitro&lt;/i&gt; refolding steps. To enhance the production of soluble papain, engineered T7 promoters and a recombinant papain translationally fused with varying tags were constructed. The tags investigated include the maltose-binding protein, small ubiquitin modifier protein, and glutathione transferase. An &lt;i&gt;E. coli&lt;/i&gt; SHuffle strain was engineered to accumulate hydrogen peroxide (H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;) by disruption of the redox pathway. This was accomplished by co-expression of the fusion constructs with two human endoplasmic reticulum-resident proteins, thiol peroxidase glutathione peroxidase-7 (GPx7), and protein disulfide isomerase (PDI). The oxidizing capacity of H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt; was used to improve disulfide bond formation in papain. The GPx7-PDI fusion dyad played a significant role in consuming harmful H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt; generated by the SHuffle cells. This consumption of H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt; helped provide the necessary oxidizing conditions for the efficient production of soluble papain. In shake-flask experiments, the recombinant strain produced ~110 mg/L of papain. Moreover, in batch fermentation, the volumetric yield reached ~349 mg/L. This work provides insights into recombinant papain microbial production that can lead to an industrial viable production strain.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Importance: &lt;/strong&gt;Papain, a cysteine-like protease, has extensive applications across various industries including food, chemical, pharmaceutical, drug, and polymer. However, the traditional isolation of papain from &lt;i&gt;Carica papaya&lt;/i&gt; plants results in a complex mixture of proteases. Such protease mixtures result in an inability to understand which component enzyme contributed to substrate conversions. Concentrations of constituent enzymes likely differ based on the ripeness of the papaya fruit. Also, constituent enzymes from papaya differ in optimal activity as a function of temperature and pH. Thus, by using papain-like enzymes from papaya fruit, valuable information on component enzyme activity and specificity is lost. Numerous methods have been reported to purify papain and papain-like enzymes from the crude mixture. Often, methods involve at least three steps including column chromatography to separate five cysteine proteases. Such procedures represent tedious processes to manufacture the pure enzymes in &lt;i&gt;Carica papaya&lt;/i&gt; extracts. The numerous uses of papain for industrial processes, as well as the probability that certain components of papain crude mixtures will be preferred for specific applications, necessitate alternative methods such as recombinant expression from microbial production ","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0211924"},"PeriodicalIF":3.9,"publicationDate":"2024-11-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142715077","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}