Applied and Environmental Microbiology最新文献

{"title":"Rational design and characterization of enhanced alcohol-inducible synthetic promoters in <i>Pichia pastoris</i>.","authors":"Qi Liu, Yun-Hao Li, Liu-Fei Tao, Jia-Yi Yang, Yi-Lun Zhang, Meng-Hao Cai","doi":"10.1128/aem.02191-24","DOIUrl":"10.1128/aem.02191-24","url":null,"abstract":"<p><p>The C1 and C2 alcohols hold great promise as substrates for biomanufacturing due to their low cost and rich resources. <i>Pichia pastoris</i> is considered a preferred host for methanol and ethanol bioconversion due to its natural utilization of methanol and ethanol. However, the scarcity of strong and tightly regulated alcohol-inducible promoters limits its extended use. This study aimed to develop enhanced methanol- and ethanol-inducible promoters capable of improving gene expression in <i>P. pastoris</i>. Rational design strategies were employed to rewire the upstream regulatory sequence of the methanol-inducible P<i>_AOX1</i>, generating several high-strength methanol-inducible promoters with a stringent regulatory pattern. Eleven strong promoters were identified from 36 endogenous ethanol-inducible candidates recognized from transcriptome analysis. Core promoter regions, the crucial element influencing transcriptional strength, were also characterized. Five high-activity core promoters were then combined with four upstream regulatory sequences of high-strength promoters, resulting in four groups of synthetic promoters. Ultimately, the highly active methanol-inducible P<i>_A13</i> and ethanol-inducible P<i>₀₆₈₈</i> and P<i>_synIV-5</i> were selected for the expression of an α-amylase and yielded enzyme activity 1.6, 2.6, and 4.5 times higher as compared to that of P<i>_AOX1</i>. This work expands the genetic toolkit available for <i>P. pastoris</i>, providing more precise and efficient options for regulating gene expression. It benefits the use of <i>P. pastoris</i> as an efficient platform for the C1 and C2 alcohol-based biotransformation in industrial biotechnology.IMPORTANCE<i>P. pastoris</i> represents a preferred microbial host for the bio-utilization of C1 and C2 alcohols that are regarded as renewable carbon sources based on clean energy. However, lack of efficient and regulated expression tools highly limits the C1 and C2 alcohols based bioproduction. By exploring high-strength and strictly regulated alcohol-inducible promoters, this study expands the expression toolkit for <i>P. pastoris</i> on C1 and C2 alcohols. The newly developed methanol-inducible P<i>_A13</i> and ethanol-inducible P<i>_synIV-5</i> demonstrate significantly higher expression levels than the commercial P<i>_AOX1</i> system. The endogenous and synthetic promoter series established in this study provides new construction references and alternative tools for expression control in <i>P. pastoris</i> for C1 and C2 alcohols based biomanufacturing.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0219124"},"PeriodicalIF":3.9,"publicationDate":"2025-01-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11784102/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142851961","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":"Persistence of human Aichi virus infectivity from raw surface water to drinking water.","authors":"Khira Sdiri-Loulizi, Amira Khachou, Stephanie Lemaire, Jean-Baptiste Bour, Siwar Ayouni, Jérôme Kaplon, Nabil Sakly, Mahjoub Aouni, Gael Belliot, Alexis de Rougemont","doi":"10.1128/aem.01189-24","DOIUrl":"10.1128/aem.01189-24","url":null,"abstract":"<p><p>Human Aichi virus 1 (AiV-1) is a water- and food-borne infection-associated picornavirus that causes gastroenteritis in humans. Recent studies on environmental waters showed a high frequency and abundance of AiV-1, suggesting that it might be an appropriate indicator of fecal contamination. We screened 450 surface and drinking water samples from a Tunisian drinking water treatment plant (DWTP) and the Sidi Salem dam for AiV-1 by real time reverse transcriptase PCR (RT-qPCR). The persistence of infectious particles was evaluated using an integrated cell culture approach coupled with quantitative molecular detection (ICC-RT-qPCR). In all, 85 (18.9%) water samples were positive for AiV-1 with viral loads ranging from 0.47 to 11.62 log₁₀ cp/L and a median of 4.97 log₁₀ cp/L, including 30/100 raw, 18/50 decanted, 14/50 flocculated, 9/100 treated, 1/50 tap, and 13/100 surface water samples. Of these, 15 (17.6%) samples contained infectious AiV-1 genotype A particles, including five raw, four decanted, one surface, three flocculated, and two treated water samples. Our data suggest that the persistence of infectious AiV-1 particles in environmental waters might represent a potential threat to public health. This study also indicates that the ICC-RT-qPCR is a practical tool for monitoring human waterborne viral risk in aquatic environments.IMPORTANCEHuman Aichi virus 1 (AiV-1) is a water- and food-borne infection-associated picornavirus that causes gastroenteritis in humans. Its high frequency and abundance in environmental waters would suggest that it might be an appropriate indicator of fecal contamination. The analysis of surface and drinking water samples from a Tunisian drinking water treatment plant (DWTP) and the Sidi Salem dam using an integrated cell culture approach coupled with a quantitative molecular detection (ICC-RT-qPCR) confirmed the persistence of infectious AiV-1 particles in samples at all stages of the treatment process, except in tap water. This suggests that the persistence of AiV-1 infectivity in environmental waters might represent a potential threat to public health. This study also indicates that the ICC-RT-qPCR is a practical tool for monitoring human waterborne viral risk in aquatic environments.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0118924"},"PeriodicalIF":3.9,"publicationDate":"2025-01-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11784453/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142913267","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":"Identification of an antibiotic from an HTS targeting EF-Tu:tRNA interaction: a prospective topical treatment for MRSA skin infections.","authors":"Wlodek Mandecki, Maxim Chudaev, Wenjuan Ye, Amy Q Wang, Kenneth J Wilson, Xin Xu, Jisun Kim, Dane Parker, David Alland, Pradeep Kumar, Barry Li, Jason H Yang, Barry Kreiswirth, Jose R Mediavilla, Juan J Marugan, Mark J Henderson, Emanuel Goldman","doi":"10.1128/aem.02046-24","DOIUrl":"10.1128/aem.02046-24","url":null,"abstract":"<p><p>Because of the urgent need for new antibiotics to treat drug-resistant bacterial pathogens, we employed an assay that rapidly screens large quantities of compounds for their ability to interfere with bacterial protein synthesis, in particular, the delivery of amino acids to the ribosome via tRNA and elongation factor Tu (EF-Tu). We have identified a drug lead, named MGC-10, which kills Gram-positive bacteria, including methicillin-resistant <i>Staphylococcus aureus</i> (MRSA), with a MIC of 6 µM, while being harmless to mammalian cells <i>in vitro</i> in that concentration range. The antibacterial activity of MGC-10 was broad against over 50 strains of antibiotic-resistant samples obtained from hospital infections, where MGC-10 inhibited all tested strains of MRSA. Extensive selection and screening with MGC-10 did not yield any resistant strains, indicating it may have universal antibacterial activity against <i>S. aureus</i>. Pharmacokinetics performed in mice suggested that MGC-10 was too toxic for systemic use; however, it appears to have potential as a topical treatment for difficult-to-treat wounds or skin infections by Gram-positive pathogens such as MRSA. In a mouse skin-infection model with MRSA, MGC-10 performed as well or better than the present topical drug of choice, mupirocin. MGC-10 showed little, if any, accumulation in the livers of topically treated mice. These results bode well for the future use of MGC-10 in clinical application as it could be used to treat a broad range of <i>S. aureus</i> skin infections that are resistant to known antibiotics.IMPORTANCEThere is a critical need for new antibiotics to treat bacterial infections caused by pathogens resistant to many if not all currently available antibiotics. We describe here the identification of a prospective new antibiotic from high-throughput screening of a chemical library. The screening was designed to detect the inhibition of formation of a complex required for bacterial protein synthesis in all bacteria, the \"ternary complex,\" comprised of elongation factor Tu (EF-Tu), aminoacyl-tRNA, and GTP. The inhibitory compound, renamed MGC-10, was effective against all Gram-positive bacteria, including a wide variety of methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) strains. Although apparently too toxic for systemic use, the compound was safe and effective for topical use for treating skin infections in a mouse model. No resistance to the compound has been detected thus far, suggesting the potential to develop this compound for topical use to treat infections, especially those caused by pathogens resistant to existing antibiotics.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0204624"},"PeriodicalIF":3.9,"publicationDate":"2025-01-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11784183/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142875975","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":"Tetracycline induces <i>wsp</i> operon expression to promote biofilm formation in <i>Pseudomonas putida</i>.","authors":"Kexin Mu, Meina He, Haozhe Chen, Tong Liu, Ying Fan, Yongxin Tao, Haoqi Feng, Qiaoyun Huang, Yujie Xiao, Wenli Chen","doi":"10.1128/aem.01071-24","DOIUrl":"10.1128/aem.01071-24","url":null,"abstract":"<p><p>The overuse and wanton discharge of antibiotics produces a threat to bacteria in the environment, which, in turn, stimulates the more rapid emergence of antibiotic-resistant bacteria. <i>Pseudomonas putida</i> actively forms biofilms to protect the population under tetracycline stress, but the molecular mechanism remains unclear. This study found that tetracycline at sub-minimal inhibitory concentrations increased cyclic diguanylate (c-di-GMP), a second messenger that positively regulates biofilm formation. Four c-di-GMP-metabolizing proteins were found to be involved in the tetracycline-mediated biofilm promotion, including DibA, WspR, PP_3242, and PP_3319. Among them, the diguanylate cyclase WspR displayed the most significant effect on c-di-GMP level and biofilm formation. <i>WspR</i> belongs to the <i>wsp</i> operon comprising seven genes (<i>wspA-wspF</i> and <i>wspR</i>). The <i>wsp</i> operon contained six promoters, including one major start promoter (P<i>_wspA</i>) and five internal promoters (P<i>_wspB</i>, P<i>_wspC</i>, P<i>_wspD</i>, P<i>_wspF</i>, and P<i>_wspR</i>), and tetracycline promoted the activity of P<i>_wspA</i>. The stress-response sigma factor RpoS directly bound to P<i>_wspA</i> and positively regulated its activity under tetracycline stress. Moreover, RpoS was required for tetracycline to induce P<i>_wspA</i> activity and promote biofilm formation. Our results enrich the transcriptional regulation of the <i>wsp</i> operon and reveal the mechanism by which tetracycline promotes biofilm formation in <i>P. putida</i>.IMPORTANCEThe overuse and wanton discharge of antibiotics produces a threat to bacteria in the environment, which, in turn, stimulates the more rapid emergence of antibiotic-resistant bacteria. The <i>Pseudomonas putida</i> actively forms biofilm against antibiotic threats, but the mechanism remains unclear. Here, our results showed that tetracycline treatment at sub-minimal inhibitory concentrations could induce the expression of the Wsp system via the sigma factor RpoS in <i>P. putida</i>, resulting in elevated c-di-GMP levels, which leads to increased biofilm formation. The <i>wsp</i> operon contains one major promoter and five internal promoters, and RpoS directly binds to the major promoter to promote its activity.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0107124"},"PeriodicalIF":3.9,"publicationDate":"2025-01-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11784136/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142715079","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":"Evaluating the stability of nursery-established arbuscular mycorrhizal fungal associations in apple rootstocks.","authors":"Huiting Zhang, Wanyan Wang, Loren Honnas, Mark Mazzola, Tracey Somera","doi":"10.1128/aem.01937-24","DOIUrl":"10.1128/aem.01937-24","url":null,"abstract":"<p><p>Arbuscular mycorrhizal fungi (AMF) are promoted as commercial bioinoculants for sustainable agriculture. Little is known, however, about the survival of AMF inoculants in soil and their impacts on native or pre-established AMF communities in root tissue. The current study was designed to assess the stability of pre-existing/nursery-derived AMF in apple rootstocks after being planted into soil containing a known community of AMF with a limited number of species. Root-associated endophytic communities (bacteria and fungi) are known to differ depending on apple rootstock genotype. Thus, an additional aim of this study was to explore the effect of apple rootstock genotype on AMF community structure. A greenhouse experiment was conducted in which a variety of apple rootstock genotypes (G.890, G.935, M.26, and M.7) were inoculated with a commercially available, multi-species AMF consortium. Nursery-derived AMF communities were sequenced, and changes to AMF community structure following cultivation in pasteurized soil (inoculated and non-inoculated) were assessed using a Glomeromycota-specific phylogenetic tree, which included 91 different AMF species from 24 genera. Results show that inoculant colonization potential was limited and that apple rootstocks serve as a significant source of inoculum from the nursery where they are produced. Rootstocks established relationships with introduced AMF in a genotype-specific manner. Regardless of colonization success, however, the inoculant caused alterations to the resident AMF communities of both Geneva and Malling rootstocks, particularly low abundance taxa. In addition, phylogeny-based analysis revealed a unique, well-supported clade of unknown taxonomy, highlighting the importance of using phylogenetic-based classification for accurate characterization of AMF communities.IMPORTANCEUnderstanding the impacts of introduced AMF on residential AMF communities is essential to improving plant productivity in nursery and orchard systems. In general, there is a dearth of data on the interactions of commercial AMF inoculants with pre-established AMF communities living in symbiosis with the host plant. The interplay between apple rootstock genotype and the endophytic root microbiome is also an area where more research is needed. This study demonstrates the potential for nursery-established AMF associations to be maintained when transplanted into the field. In addition to providing insight into rootstock/AMF associations, our study calls attention to the current issues attendant with relying on web-based databases for determining AMF identity. The use of phylogenetic tools represents one possible solution and may be of value to industry practitioners in terms of improving product composition and consistency.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0193724"},"PeriodicalIF":3.9,"publicationDate":"2025-01-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11784189/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142798956","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 <i>Clostridioides difficile</i> cell-free gene expression system for prototyping and gene expression analysis.","authors":"Ji Zeng, Hao Wang, Yuxi Xu, Jianying Han, Yannan Li, Shu'an Wen, Changbu Wu, Dani Li, Zheng Liu, Xiaokang Zhang, Guo-Bao Tian, Min Dong","doi":"10.1128/aem.01566-24","DOIUrl":"10.1128/aem.01566-24","url":null,"abstract":"<p><p><i>Clostridioides difficile</i> is an obligate anaerobic, Gram-positive bacterium that produces toxins. Despite technological progress, conducting gene expression analysis of <i>C. difficile</i> under different conditions continues to be labor-intensive. Therefore, there is a demand for simplified tools to investigate the transcriptional and translational regulation of <i>C. difficile</i>. The cell-free gene expression (CFE) system has demonstrated utility in various applications, including prototyping, protein production, and <i>in vitro</i> screening. In this study, we developed a <i>C. difficile</i> CFE system capable of <i>in vitro</i> transcription and translation (TX-TL) in the presence of oxygen. Through optimization of cell extract preparation and reaction systems, we increased the protein yield significantly. Furthermore, our observations indicated that this system exhibited higher protein yield using linear DNA templates than circular plasmids for <i>in vitro</i> expression. The prototyping capability of the <i>C. difficile</i> CFE system was assessed using a series of synthetic <i>Clostridium</i> promoters, demonstrating a good correlation between <i>in vivo</i> and <i>in vitro</i> expression. Additionally, we tested the expression of <i>tcdB</i> and <i>tcdR</i> from clinically relevant <i>C. difficile</i> strains using the CFE system, confirming higher toxin expression of the hypervirulent strain R20291. We believe that the CFE system can not only serve as a platform for <i>in vitro</i> protein synthesis and genetic part prototyping but also has the potential to be a simplified model for studying metabolic regulations in <i>Clostridioides difficile</i>.IMPORTANCE<i>Clostridioides difficile</i> has been listed as an urgent threat due to its antibiotic resistance, and it is crucial to conduct gene expression analysis to understand gene functionality. However, this task can be challenging, given the need to maintain the bacterium in an anaerobic environment and the inefficiency of introducing genetic material into <i>C. difficile</i> cells. Conversely, the <i>C. difficile</i> cell-free gene expression (CFE) system enables <i>in vitro</i> transcription and translation in the presence of oxygen within just half an hour. Furthermore, the composition of the CFE system is adaptable, permitting the addition or removal of elements, regulatory proteins for example, during the reaction. As a result, this system could potentially offer an efficient and accessible approach to accelerate the study of gene expression and function in <i>Clostridioides difficile</i>.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0156624"},"PeriodicalIF":3.9,"publicationDate":"2025-01-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11784378/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142913753","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":"Genomic and metabolic characterization of <i>Trueperella pyogenes</i> isolated from domestic and wild animals.","authors":"Gabriela Magossi, Katherine E Gzyl, Devin B Holman, T G Nagaraja, Raghavendra Amachawadi, Samat Amat","doi":"10.1128/aem.01725-24","DOIUrl":"10.1128/aem.01725-24","url":null,"abstract":"<p><p><i>Trueperella pyogenes</i> is an important bacterial pathogen implicated in infections such as mastitis, metritis, pneumonia, and liver abscesses in both domestic and wild animals, as well as endocarditis and prosthetic joint infections in humans. Understanding the genomic and metabolic features that enable <i>T. pyogenes</i> to colonize different anatomical sites within a host and its inter-kingdom transmission and survival is important for the effective control of this pathogen. We employed whole-genome sequencing, phenotype microarrays, and antimicrobial susceptibility testing to identify genomic, metabolic and phenotypic features, and antimicrobial resistance (AMR) genes in <i>T. pyogenes</i> recovered from different livestock, companion, and wildlife animals. For comparative genomic analysis, 83 <i>T. pyogenes</i> genomes, including 60 isolated in the current study and 23 publicly available genomes were evaluated. These genomes represented <i>T. pyogenes</i> strains originating from 16 different body sites of 11 different animal hosts (e.g., cattle, swine, ovine, deer, bison, horse, chamois, and cat). Additionally, 49 <i>T. pyogenes</i> isolates (cattle, sheep, deer, swine, and cats) were evaluated for phenotypic AMR using disk diffusion, and for metabolic profiling using the Biology GENIII MicroPlates. The <i>T. pyogenes</i> strains were found not to be host- or body site-specific. The presence of conserved virulence genes (<i>plo</i> and <i>fimA</i>), as well as genotypic and phenotypic AMR may contribute to the ability of <i>T. pyogenes</i> to cause infections in livestock, wildlife, and pets. Most of the tested isolates metabolized diverse carbon sources and chemical compounds, suggesting that this metabolic versatility may enhance the survival, competitiveness, and pathogenic potential of <i>T. pyogenes</i>.IMPORTANCE<i>Trueperella pyogenes</i> is an important animal pathogen with zoonotic potential, posing a significant health concern to both animals and humans due to its ability to cause infections across different animal host species and tissues. Current understanding of this pathogen's adaptability and survival mechanisms is limited. Here, we evaluated the genomic, virulence, metabolic, and antimicrobial resistance (AMR) characteristics of <i>T. pyogenes</i> recovered from 16 different body sites of 11 different animal hosts (livestock, companion, and wild animals). We identified multiple AMR and virulence genes that may enable <i>T. pyogenes</i> for sustained infection and transmission. Additionally, <i>T. pyogenes</i> strains displayed metabolic versatility which could also contribute to its ability to thrive in diverse environments. Understanding the genomic and metabolic, and AMR characteristics that enable <i>T. pyogenes</i> to colonize different anatomical sites within a host and its transmission between different animal species is important for the effective control of this pathogen.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0172524"},"PeriodicalIF":3.9,"publicationDate":"2025-01-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11784230/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142913756","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":"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":"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":"2025-01-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11784079/pdf/","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":"OA","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":"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":"2025-01-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11784247/pdf/","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":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Surviving the storm: exploring the role of natural transformation in nutrition and DNA repair of stressed <i>Deinococcus radiodurans</i>.","authors":"Dhirendra Kumar Sharma, Ishu Soni, Yogendra Singh Rajpurohit","doi":"10.1128/aem.01371-24","DOIUrl":"10.1128/aem.01371-24","url":null,"abstract":"<p><p><i>Deinococcus radiodurans</i>, a natural transformation (NT)-enabled bacterium renowned for its exceptional radiation resistance, employs unique DNA repair and oxidative stress mitigation mechanisms as a strategic response to DNA damage. This study excavates into the intricate roles of NT machinery in the stressed <i>D. radiodurans</i>, focusing on the genes <i>comEA</i>, <i>comEC</i>, <i>endA</i>, <i>pilT</i>, and <i>dprA</i>, which are instrumental in the uptake and processing of extracellular DNA (eDNA). Our data reveal that NT not only supports the nutritional needs of <i>D. radiodurans</i> under stress but also has roles in DNA repair. The study findings establish that NT-specific proteins (ComEA, ComEC, and endonuclease A [EndA]) may contribute to support the nutritional requirements in unstressed and heavily DNA-damaged cells, while DprA contributes differently and in a context-dependent manner to navigating through the DNA damage storm. Thus, this dual functionality of NT-specific genes is proposed to be a contributing factor in the remarkable ability of <i>D. radiodurans</i> to survive and thrive in environments characterized by high levels of DNA-damaging agents.IMPORTANCE<i>Deinococcus radiodurans</i> is a bacterium known for its extraordinary radiation resistance. This study explores the roles of NT machinery in the radiation-resistant bacterium <i>Deinococcus radiodurans</i>, focusing on the genes <i>comEA</i>, <i>comEC</i>, <i>endA</i>, <i>pilT</i>, and <i>dprA</i>. These genes are crucial for the uptake and processing of eDNA and contribute to the bacterium nutritional needs and DNA repair under stress. The findings suggest that the NT-specific proteins ComEA, ComEC, and EndA may help meet the nutritional needs of unstressed and heavily DNA-damaged cells, whereas DprA plays a distinct role that varies, depending on the context in aiding cells to cope with DNA damage. The functionality of NT genes is proposed to enhance <i>D. radiodurans</i> survival in environments with high levels of DNA-damaging agents.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0137124"},"PeriodicalIF":3.9,"publicationDate":"2025-01-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11784314/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142798967","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}