Chahat Upreti, Pranav Kumar, Lisa M Durso, Kelli L Palmer
The horizontal transfer of antibiotic resistance genes among bacteria is a pressing global issue. The bacterial defense system clustered regularly interspaced short palindromic repeats (CRISPR)-Cas acts as a barrier to the spread of antibiotic resistance plasmids, and CRISPR-Cas-based antimicrobials can be effective to selectively deplete antibiotic-resistant bacteria. While significant surveillance efforts monitor the spread of antibiotic-resistant bacteria in the clinical context, a major, often overlooked aspect of the issue is resistance emergence in agriculture. Farm animals are commonly treated with antibiotics, and antibiotic resistance in agriculture is on the rise. Yet, CRISPR-Cas efficacy has not been investigated in this setting. Here, we evaluate the prevalence of CRISPR-Cas in agricultural Enterococcus faecalis strains and its antiplasmid efficacy in an agricultural niche: manure. Analyzing 1,986 E. faecalis genomes from human and animal hosts, we show that the prevalence of CRISPR-Cas subtypes is similar between clinical and agricultural E. faecalis strains. Using plasmid conjugation assays, we found that CRISPR-Cas is a significant barrier against resistance plasmid transfer in manure. Finally, we used a CRISPR-based antimicrobial approach to cure resistant E. faecalis of erythromycin resistance, but this was limited by delivery efficiency of the CRISPR antimicrobial in manure. However, immunization of bacteria against resistance gene acquisition in manure was highly effective. Together, our results show that E. faecalis CRISPR-Cas is prevalent and effective in an agricultural setting and has the potential to be utilized for depleting antibiotic-resistant populations. Our work has broad implications for tackling antibiotic resistance in the increasingly relevant agricultural setting, in line with a One Health approach.IMPORTANCEAntibiotic resistance is a growing global health crisis in human and veterinary medicine. Previous work has shown technologies based on CRISPR-Cas-a bacterial defense system-to be effective in tackling antibiotic resistance. Here we test if CRISPR-Cas is present and effective in agricultural niches, specifically in the ubiquitously present bacterium, Enterococcus faecalis. We show that CRISPR-Cas is both prevalent and functional in manure and has the potential to be used to specifically kill bacteria carrying antibiotic resistance genes. This study demonstrates the utility of CRISPR-Cas-based strategies for control of antibiotic resistance in agricultural settings.
{"title":"CRISPR-Cas inhibits plasmid transfer and immunizes bacteria against antibiotic resistance acquisition in manure.","authors":"Chahat Upreti, Pranav Kumar, Lisa M Durso, Kelli L Palmer","doi":"10.1128/aem.00876-24","DOIUrl":"10.1128/aem.00876-24","url":null,"abstract":"<p><p>The horizontal transfer of antibiotic resistance genes among bacteria is a pressing global issue. The bacterial defense system clustered regularly interspaced short palindromic repeats (CRISPR)-Cas acts as a barrier to the spread of antibiotic resistance plasmids, and CRISPR-Cas-based antimicrobials can be effective to selectively deplete antibiotic-resistant bacteria. While significant surveillance efforts monitor the spread of antibiotic-resistant bacteria in the clinical context, a major, often overlooked aspect of the issue is resistance emergence in agriculture. Farm animals are commonly treated with antibiotics, and antibiotic resistance in agriculture is on the rise. Yet, CRISPR-Cas efficacy has not been investigated in this setting. Here, we evaluate the prevalence of CRISPR-Cas in agricultural <i>Enterococcus faecalis</i> strains and its antiplasmid efficacy in an agricultural niche: manure. Analyzing 1,986 <i>E. faecalis</i> genomes from human and animal hosts, we show that the prevalence of CRISPR-Cas subtypes is similar between clinical and agricultural <i>E. faecalis</i> strains. Using plasmid conjugation assays, we found that CRISPR-Cas is a significant barrier against resistance plasmid transfer in manure. Finally, we used a CRISPR-based antimicrobial approach to cure resistant <i>E. faecalis</i> of erythromycin resistance, but this was limited by delivery efficiency of the CRISPR antimicrobial in manure. However, immunization of bacteria against resistance gene acquisition in manure was highly effective. Together, our results show that <i>E. faecalis</i> CRISPR-Cas is prevalent and effective in an agricultural setting and has the potential to be utilized for depleting antibiotic-resistant populations. Our work has broad implications for tackling antibiotic resistance in the increasingly relevant agricultural setting, in line with a One Health approach.IMPORTANCEAntibiotic resistance is a growing global health crisis in human and veterinary medicine. Previous work has shown technologies based on CRISPR-Cas-a bacterial defense system-to be effective in tackling antibiotic resistance. Here we test if CRISPR-Cas is present and effective in agricultural niches, specifically in the ubiquitously present bacterium, <i>Enterococcus faecalis</i>. We show that CRISPR-Cas is both prevalent and functional in manure and has the potential to be used to specifically kill bacteria carrying antibiotic resistance genes. This study demonstrates the utility of CRISPR-Cas-based strategies for control of antibiotic resistance in agricultural settings.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":null,"pages":null},"PeriodicalIF":3.9,"publicationDate":"2024-08-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141999238","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}
The characterization of surface microbiota living in biofilms within livestock buildings has been relatively unexplored, despite its potential impact on animal health. To enhance our understanding of these microbial communities, we characterized 11 spore-forming strains isolated from two commercial broiler chicken farms. Sequencing of the strains revealed them to belong to three species Bacillus velezensis, Bacillus subtilis, and Bacillus licheniformis. Genomic analysis revealed the presence of antimicrobial resistance genes and genes associated with antimicrobial secretion specific to each species. We conducted a comprehensive characterization of the biofilm formed by these strains under various conditions, and we revealed significant structural heterogeneity across the different strains. A macro-colony interaction model was employed to assess the compatibility of these strains to coexist in mixed biofilms. We identified highly competitive B. velezensis strains, which cannot coexist with other Bacillus spp. Using confocal laser scanning microscopy along with a specific dye for extracellular DNA, we uncovered the importance of extracellular DNA for the formation of B. licheniformis biofilms. Altogether, the results highlight the heterogeneity in both genome and biofilm structure among Bacillus spp. isolated from biofilms present within livestock buildings.IMPORTANCELittle is known about the microbial communities that develop on farms in direct contact with animals. Nonpathogenic strains of Bacillus velezensis, Bacillus subtilis, and Bacillus licheniformis were found in biofilm samples collected from surfaces in contact with animals. Significant genetic and phenotypic diversity was described among these Bacillus strains. The strains do not possess mobile antibiotic resistance genes in their genomes and have a strong capacity to form structured biofilms. Among these species, B. velezensis was noted for its high competitiveness compared with the other Bacillus spp. Additionally, the importance of extracellular DNA in the formation of B. licheniformis biofilms was observed. These findings provide insights for the management of these surface microbiota that can influence animal health, such as the use of competitive strains to minimize the establishment of undesirable bacteria or enzymes capable of specifically deconstructing biofilms.
尽管生活在畜舍生物膜中的表面微生物群对动物健康有潜在影响,但对其特征的研究相对较少。为了加深我们对这些微生物群落的了解,我们对从两个商业肉鸡养殖场分离出的 11 株孢子形成菌株进行了鉴定。这些菌株的测序结果表明,它们分别属于韦氏芽孢杆菌、枯草芽孢杆菌和地衣芽孢杆菌三个物种。基因组分析表明,每个菌种都存在抗菌药耐药性基因和与抗菌药分泌相关的基因。我们对这些菌株在各种条件下形成的生物膜进行了全面鉴定,发现不同菌株的生物膜在结构上存在显著的异质性。我们采用了大菌落相互作用模型来评估这些菌株在混合生物膜中共存的兼容性。利用激光共聚焦扫描显微镜和细胞外 DNA 的特异性染料,我们发现了细胞外 DNA 对地衣芽孢杆菌生物膜形成的重要性。总之,这些结果凸显了从畜牧建筑内的生物膜中分离出的芽孢杆菌属在基因组和生物膜结构上的异质性。从与动物接触的表面采集的生物膜样本中发现了非致病性的韦氏芽孢杆菌、枯草芽孢杆菌和地衣芽孢杆菌。在这些芽孢杆菌菌株中发现了显著的遗传和表型多样性。这些菌株的基因组中没有可移动的抗生素耐药性基因,并且具有形成结构化生物膜的强大能力。地衣芽孢杆菌生物膜的形成过程中,细胞外 DNA 起着重要作用。这些发现为管理这些可能影响动物健康的表面微生物群提供了启示,例如使用具有竞争力的菌株来尽量减少不良细菌的建立,或使用能够专门分解生物膜的酶。
{"title":"Insights into the genomic and phenotypic characteristics of <i>Bacillus</i> spp. strains isolated from biofilms in broiler farms.","authors":"Virgile Guéneau, Guillermo Jiménez, Mathieu Castex, Romain Briandet","doi":"10.1128/aem.00663-24","DOIUrl":"https://doi.org/10.1128/aem.00663-24","url":null,"abstract":"<p><p>The characterization of surface microbiota living in biofilms within livestock buildings has been relatively unexplored, despite its potential impact on animal health. To enhance our understanding of these microbial communities, we characterized 11 spore-forming strains isolated from two commercial broiler chicken farms. Sequencing of the strains revealed them to belong to three species <i>Bacillus velezensis</i>, <i>Bacillus subtilis</i>, and <i>Bacillus licheniformis</i>. Genomic analysis revealed the presence of antimicrobial resistance genes and genes associated with antimicrobial secretion specific to each species. We conducted a comprehensive characterization of the biofilm formed by these strains under various conditions, and we revealed significant structural heterogeneity across the different strains. A macro-colony interaction model was employed to assess the compatibility of these strains to coexist in mixed biofilms. We identified highly competitive <i>B. velezensis</i> strains, which cannot coexist with other <i>Bacillus</i> spp. Using confocal laser scanning microscopy along with a specific dye for extracellular DNA, we uncovered the importance of extracellular DNA for the formation of <i>B. licheniformis</i> biofilms. Altogether, the results highlight the heterogeneity in both genome and biofilm structure among <i>Bacillus</i> spp. isolated from biofilms present within livestock buildings.IMPORTANCELittle is known about the microbial communities that develop on farms in direct contact with animals. Nonpathogenic strains of <i>Bacillus velezensis</i>, <i>Bacillus subtilis</i>, and <i>Bacillus licheniformis</i> were found in biofilm samples collected from surfaces in contact with animals. Significant genetic and phenotypic diversity was described among these <i>Bacillus</i> strains. The strains do not possess mobile antibiotic resistance genes in their genomes and have a strong capacity to form structured biofilms. Among these species, <i>B. velezensis</i> was noted for its high competitiveness compared with the other <i>Bacillus</i> spp. Additionally, the importance of extracellular DNA in the formation of <i>B. licheniformis</i> biofilms was observed. These findings provide insights for the management of these surface microbiota that can influence animal health, such as the use of competitive strains to minimize the establishment of undesirable bacteria or enzymes capable of specifically deconstructing biofilms.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":null,"pages":null},"PeriodicalIF":3.9,"publicationDate":"2024-08-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141999251","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}
Rekha Rana, Praveen Kumar Nayak, Vishnu Narayanan Madhavan, Ramesh V Sonti, Hitendra K Patel, Prabhu B Patil
Xanthomonas species are major pathogens of plants and have been studied extensively. There is increasing recognition of the importance of non-pathogenic species within the same genus. With this came the need to understand the genomic and functional diversity of non-pathogenic Xanthomonas (NPX) at the species and strain level. This study reports isolation and investigation into the genomic diversity and variation in NPX isolates, chiefly Xanthomonas indica, a newly discovered NPX species from rice. The study establishes the relationship of X. indica strains within clade I of Xanthomonads with another NPX species, X. sontii, also associated with rice seeds. Identification of highly diverse strains, open-pan genome, and systematic hyper-variation at the lipopolysaccharide biosynthetic locus when compared to pathogenic Xanthomonas indicates the acquisition of new functions for adaptation. Furthermore, comparative genomics studies established the absence of major virulence genes such as type III secretion system and effectors, which are present in the pathogens, and the presence of a known bacterial-killing type IV secretion system (X-T4SS). The diverse non-pathogenic strains of X. indica and X. sontii were found to protect rice from bacterial leaf blight pathogen, X. oryzae pv. oryzae (Xoo). The absence of phenotype of an X-T4SS mutant suggests redundancy in the genetic basis of the mechanisms involved in the bioprotection function, which may include multiple genetic loci, such as putative bacteriocin-encoding gene clusters and involvement of other factors such as nutrient and niche competition apart from induction of innate immunity through shared microbial-associated molecular patterns. The rice-NPX community and its pathogenic counterpart can be a promising model for understanding plant-microbe-microbiome interaction studies.IMPORTANCEThe Xanthomonas group of bacteria is known for its characteristic lifestyle as a phytopathogen. However, the discovery of non-pathogenic Xanthomonas (NPX) species is a major shift in understanding this group of bacteria. Multi-strain, in-depth genomic, evolutionary and functional studies on each of these NPX species are still lacking. This study on diverse non-pathogenic strains provides novel insights into genome diversity, dynamics, and evolutionary trends of NPX species from rice microbiome apart from its relationship with other relatives that form a sub-clade. Interestingly, we also uncovered that NPX species protect rice from pathogenic Xanthomonas species. The plant protection property shows their importance as a part of a healthy plant microbiome. Furthermore, finding an open pan-genome and large-scale variation at lipopolysaccharide biosynthetic locus indicates a significant role of the NPX community in host adaptation. The findings and high-quality genomic resources of NPX species and the strains will allow furth
{"title":"Comparative genomics-based insights into <i>Xanthomonas indica</i>, a non-pathogenic species of healthy rice microbiome with bioprotection function.","authors":"Rekha Rana, Praveen Kumar Nayak, Vishnu Narayanan Madhavan, Ramesh V Sonti, Hitendra K Patel, Prabhu B Patil","doi":"10.1128/aem.00848-24","DOIUrl":"https://doi.org/10.1128/aem.00848-24","url":null,"abstract":"<p><p><i>Xanthomonas</i> species are major pathogens of plants and have been studied extensively. There is increasing recognition of the importance of non-pathogenic species within the same genus. With this came the need to understand the genomic and functional diversity of non-pathogenic <i>Xanthomonas</i> (NPX) at the species and strain level. This study reports isolation and investigation into the genomic diversity and variation in NPX isolates, chiefly <i>Xanthomonas indica</i>, a newly discovered NPX species from rice. The study establishes the relationship of <i>X. indica</i> strains within clade I of Xanthomonads with another NPX species, <i>X. sontii</i>, also associated with rice seeds. Identification of highly diverse strains, open-pan genome, and systematic hyper-variation at the lipopolysaccharide biosynthetic locus when compared to pathogenic <i>Xanthomonas</i> indicates the acquisition of new functions for adaptation. Furthermore, comparative genomics studies established the absence of major virulence genes such as type III secretion system and effectors, which are present in the pathogens, and the presence of a known bacterial-killing type IV secretion system (X-T4SS). The diverse non-pathogenic strains of <i>X. indica</i> and <i>X. sontii</i> were found to protect rice from bacterial leaf blight pathogen, <i>X. oryzae</i> pv. <i>oryzae</i> (Xoo). The absence of phenotype of an X-T4SS mutant suggests redundancy in the genetic basis of the mechanisms involved in the bioprotection function, which may include multiple genetic loci, such as putative bacteriocin-encoding gene clusters and involvement of other factors such as nutrient and niche competition apart from induction of innate immunity through shared microbial-associated molecular patterns. The rice-NPX community and its pathogenic counterpart can be a promising model for understanding plant-microbe-microbiome interaction studies.IMPORTANCEThe <i>Xanthomonas</i> group of bacteria is known for its characteristic lifestyle as a phytopathogen. However, the discovery of non-pathogenic <i>Xanthomonas</i> (NPX) species is a major shift in understanding this group of bacteria. Multi-strain, in-depth genomic, evolutionary and functional studies on each of these NPX species are still lacking. This study on diverse non-pathogenic strains provides novel insights into genome diversity, dynamics, and evolutionary trends of NPX species from rice microbiome apart from its relationship with other relatives that form a sub-clade. Interestingly, we also uncovered that NPX species protect rice from pathogenic <i>Xanthomonas</i> species. The plant protection property shows their importance as a part of a healthy plant microbiome. Furthermore, finding an open pan-genome and large-scale variation at lipopolysaccharide biosynthetic locus indicates a significant role of the NPX community in host adaptation. The findings and high-quality genomic resources of NPX species and the strains will allow furth","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":null,"pages":null},"PeriodicalIF":3.9,"publicationDate":"2024-08-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141999237","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}
Xiufang Gong, Yao Zhou, Qijian Qin, Bin Wang, Linqi Wang, Cheng Jin, Wenxia Fang
Phosphoglucose isomerase (PGI) links glycolysis, the pentose phosphate pathway (PPP), and the synthesis of cell wall precursors in fungi by facilitating the reversible conversion between glucose-6-phosphate (Glc6p) and fructose-6-phosphate (Fru6P). In a previous study, we established the essential role of PGI in cell wall biosynthesis in the opportunistic human fungal pathogen Aspergillus fumigatus, highlighting its potential as a therapeutic target. In this study, we conducted transcriptomic analysis and discovered that the Δpgi mutant exhibited enhanced glycolysis, reduced PPP, and an upregulation of cell wall precursor biosynthesis pathways. Phenotypic analysis revealed defective protein N-glycosylation in the mutant, notably the absence of glycosylated virulence factors DPP V and catalase 1. Interestingly, the cell wall defects in the mutant were not accompanied by activation of the MpkA-dependent cell wall integrity (CWI) signaling pathway. Instead, nitrate assimilation was activated in the Δpgi mutant, stimulating glutamine synthesis and providing amino donors for chitin precursor biosynthesis. Blocking the nitrate assimilation pathway severely impaired the growth of the Δpgi mutant, highlighting the crucial role of nitrate assimilation in rescuing cell wall defects. This study unveils the connection between nitrogen assimilation and cell wall compensation in A. fumigatus.IMPORTANCEAspergillus fumigatus is a common and serious human fungal pathogen that causes a variety of diseases. Given the limited availability of antifungal drugs and increasing drug resistance, it is imperative to understand the fungus' survival mechanisms for effective control of fungal infections. Our previous study highlighted the essential role of A. fumigatus PGI in maintaining cell wall integrity, phosphate sugar homeostasis, and virulence. The present study further illuminates the involvement of PGI in protein N-glycosylation. Furthermore, this research reveals that the nitrogen assimilation pathway, rather than the canonical MpkA-dependent CWI pathway, compensates for cell wall deficiencies in the mutant. These findings offer valuable insights into a novel adaptation mechanism of A. fumigatus to address cell wall defects, which could hold promise for the treatment of infections.
{"title":"Nitrate assimilation compensates for cell wall biosynthesis in the absence of <i>Aspergillus fumigatus</i> phosphoglucose isomerase.","authors":"Xiufang Gong, Yao Zhou, Qijian Qin, Bin Wang, Linqi Wang, Cheng Jin, Wenxia Fang","doi":"10.1128/aem.01138-24","DOIUrl":"https://doi.org/10.1128/aem.01138-24","url":null,"abstract":"<p><p>Phosphoglucose isomerase (PGI) links glycolysis, the pentose phosphate pathway (PPP), and the synthesis of cell wall precursors in fungi by facilitating the reversible conversion between glucose-6-phosphate (Glc6p) and fructose-6-phosphate (Fru6P). In a previous study, we established the essential role of PGI in cell wall biosynthesis in the opportunistic human fungal pathogen <i>Aspergillus fumigatus</i>, highlighting its potential as a therapeutic target. In this study, we conducted transcriptomic analysis and discovered that the Δ<i>pgi</i> mutant exhibited enhanced glycolysis, reduced PPP, and an upregulation of cell wall precursor biosynthesis pathways. Phenotypic analysis revealed defective protein <i>N</i>-glycosylation in the mutant, notably the absence of glycosylated virulence factors DPP V and catalase 1. Interestingly, the cell wall defects in the mutant were not accompanied by activation of the MpkA-dependent cell wall integrity (CWI) signaling pathway. Instead, nitrate assimilation was activated in the Δ<i>pgi</i> mutant, stimulating glutamine synthesis and providing amino donors for chitin precursor biosynthesis. Blocking the nitrate assimilation pathway severely impaired the growth of the Δ<i>pgi</i> mutant, highlighting the crucial role of nitrate assimilation in rescuing cell wall defects. This study unveils the connection between nitrogen assimilation and cell wall compensation in <i>A. fumigatus</i>.IMPORTANCE<i>Aspergillus fumigatus</i> is a common and serious human fungal pathogen that causes a variety of diseases. Given the limited availability of antifungal drugs and increasing drug resistance, it is imperative to understand the fungus' survival mechanisms for effective control of fungal infections. Our previous study highlighted the essential role of <i>A. fumigatus</i> PGI in maintaining cell wall integrity, phosphate sugar homeostasis, and virulence. The present study further illuminates the involvement of PGI in protein <i>N</i>-glycosylation. Furthermore, this research reveals that the nitrogen assimilation pathway, rather than the canonical MpkA-dependent CWI pathway, compensates for cell wall deficiencies in the mutant. These findings offer valuable insights into a novel adaptation mechanism of <i>A. fumigatus</i> to address cell wall defects, which could hold promise for the treatment of infections.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":null,"pages":null},"PeriodicalIF":3.9,"publicationDate":"2024-08-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141999253","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}
Stéphane Perchat, Alicia Nevers, Markus Kranzler, Monika Ehling-Schulz, Didier Lereclus, Michel Gohar
The Bacillus cereus group includes closely related spore-forming Gram-positive bacteria. In this group, plasmids play a crucial role in species differentiation and are essential for pathogenesis and adaptation to ecological niches. The B. cereus emetic strains are characterized by the presence of the pCER270 megaplasmid, which encodes the non-ribosomal peptide synthetase for the production of cereulide, the emetic toxin. This plasmid carries several genes that may be involved in the sporulation process. Furthermore, a transcriptomic analysis has revealed that pCER270 influences the expression of chromosome genes, particularly under sporulation conditions. In this study, we investigated the role of pCER270 on spore properties in different species of the B. cereus group. We showed that pCER270 plays a role in spore wet heat resistance and germination, with varying degrees of impact depending on the genetic background. In addition, pCER270 ensures that sporulation occurs at the appropriate time by delaying the expression of sporulation genes. This regulation of sporulation timing is controlled by the pCER270-borne Rap-Phr system, which likely regulates the phosphorylation state of Spo0A. Acquisition of the pCER270 plasmid by new strains could give them an advantage in adapting to new environments and lead to the emergence of new pathogenic strains.
Importance: The acquisition of new mobile genetic elements, such as plasmids, is essential for the pathogenesis and adaptation of bacteria belonging to the Bacillus cereus group. This can confer new phenotypic traits and beneficial functions that enable bacteria to adapt to changing environments and colonize new ecological niches. Emetic B. cereus strains cause food poisoning linked to the production of cereulide, the emetic toxin whose synthesis is due to the presence of plasmid pCER270. In the environment, cereulide provides a competitive advantage in producing bacteria against various competitors or predators. This study demonstrates that pCER270 also regulates the sporulation process, resulting in spores with improved heat resistance and germination capacity. The transfer of plasmid pCER270 among different strains of the B. cereus group may enhance their adaptation to new environments. This raises the question of the emergence of new pathogenic strains, which could pose a serious threat to human health.
{"title":"The megaplasmid pCER270 of <i>Bacillus cereus</i> emetic strain affects the timing of the sporulation process, spore resistance properties, and germination.","authors":"Stéphane Perchat, Alicia Nevers, Markus Kranzler, Monika Ehling-Schulz, Didier Lereclus, Michel Gohar","doi":"10.1128/aem.01029-24","DOIUrl":"https://doi.org/10.1128/aem.01029-24","url":null,"abstract":"<p><p>The <i>Bacillus cereus</i> group includes closely related spore-forming Gram-positive bacteria. In this group, plasmids play a crucial role in species differentiation and are essential for pathogenesis and adaptation to ecological niches. The <i>B. cereus</i> emetic strains are characterized by the presence of the pCER270 megaplasmid, which encodes the non-ribosomal peptide synthetase for the production of cereulide, the emetic toxin. This plasmid carries several genes that may be involved in the sporulation process. Furthermore, a transcriptomic analysis has revealed that pCER270 influences the expression of chromosome genes, particularly under sporulation conditions. In this study, we investigated the role of pCER270 on spore properties in different species of the <i>B. cereus</i> group. We showed that pCER270 plays a role in spore wet heat resistance and germination, with varying degrees of impact depending on the genetic background. In addition, pCER270 ensures that sporulation occurs at the appropriate time by delaying the expression of sporulation genes. This regulation of sporulation timing is controlled by the pCER270-borne Rap-Phr system, which likely regulates the phosphorylation state of Spo0A. Acquisition of the pCER270 plasmid by new strains could give them an advantage in adapting to new environments and lead to the emergence of new pathogenic strains.</p><p><strong>Importance: </strong>The acquisition of new mobile genetic elements, such as plasmids, is essential for the pathogenesis and adaptation of bacteria belonging to the <i>Bacillus cereus</i> group. This can confer new phenotypic traits and beneficial functions that enable bacteria to adapt to changing environments and colonize new ecological niches. Emetic <i>B. cereus</i> strains cause food poisoning linked to the production of cereulide, the emetic toxin whose synthesis is due to the presence of plasmid pCER270. In the environment, cereulide provides a competitive advantage in producing bacteria against various competitors or predators. This study demonstrates that pCER270 also regulates the sporulation process, resulting in spores with improved heat resistance and germination capacity. The transfer of plasmid pCER270 among different strains of the <i>B. cereus</i> group may enhance their adaptation to new environments. This raises the question of the emergence of new pathogenic strains, which could pose a serious threat to human health.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":null,"pages":null},"PeriodicalIF":3.9,"publicationDate":"2024-08-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141999254","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}
Giulia Longhi, Gabriele Andrea Lugli, Chiara Tarracchini, Federico Fontana, Massimiliano Giovanni Bianchi, Elisa Carli, Ovidio Bussolati, Douwe van Sinderen, Francesca Turroni, Marco Ventura
The microbial ecology of raw milk cheeses is determined by bacteria originating from milk and milk-producing animals. Recently, it has been shown that members of the Bifidobacterium mongoliense species may become transmitted along the Parmigiano Reggiano cheese production chain and ultimately may colonize the consumer intestine. However, there is a lack of knowledge regarding the molecular mechanisms that mediate the interaction between B. mongoliense and the human gut. Based on 128 raw milk cheeses collected from different Italian regions, we isolated and characterized 10 B. mongoliense strains. Comparative genomics allowed us to unveil the presence of enzymes required for the degradation of sialylated host-glycans in B. mongoliense, corroborating the appreciable growth on de Man-Rogosa-Sharpe (MRS) medium supplemented with 3'-sialyllactose (3'-SL) or 6'-sialyllactose (6'-SL). The B. mongoliense BMONG18 was chosen, due to its superior ability to utilize 3'-SL and mucin as representative strain, to investigate its behavior when co-inoculated with other bifidobacterial species. Conversely, members of other bifidobacterial species did not appear to benefit from the presence of BMONG18, highlighting a competitive scenario for nutrient acquisition. Transcriptomic data of BMONG18 reveal no significant differences in gene expression when cultivated in a gut simulating medium (GSM), regardless of whether cheese was included or not. Furthermore, BMONG18 was shown to exhibit high adhesion capabilities to HT29-MTX human cells, in line with its colonization ability of a human host.IMPORTANCEFermented foods are nourishments produced through controlled microbial growth that play an essential role in worldwide human nutrition. Research interest in fermented foods has increased since the 80s, driven by growing awareness of their potential health benefits beyond mere nutritional content. Bifidobacterium mongoliense, previously identified throughout the production process of Parmigiano Reggiano cheese, was found to be capable of establishing itself in the intestines of its consumers. Our study underscores molecular mechanisms through which this bifidobacterial species, derived from food, interacts with the host and other gut microbiota members.
{"title":"From raw milk cheese to the gut: investigating the colonization strategies of <i>Bifidobacterium mongoliense</i>.","authors":"Giulia Longhi, Gabriele Andrea Lugli, Chiara Tarracchini, Federico Fontana, Massimiliano Giovanni Bianchi, Elisa Carli, Ovidio Bussolati, Douwe van Sinderen, Francesca Turroni, Marco Ventura","doi":"10.1128/aem.01244-24","DOIUrl":"https://doi.org/10.1128/aem.01244-24","url":null,"abstract":"<p><p>The microbial ecology of raw milk cheeses is determined by bacteria originating from milk and milk-producing animals. Recently, it has been shown that members of the <i>Bifidobacterium mongoliense</i> species may become transmitted along the Parmigiano Reggiano cheese production chain and ultimately may colonize the consumer intestine. However, there is a lack of knowledge regarding the molecular mechanisms that mediate the interaction between <i>B. mongoliense</i> and the human gut. Based on 128 raw milk cheeses collected from different Italian regions, we isolated and characterized 10 <i>B. mongoliense</i> strains. Comparative genomics allowed us to unveil the presence of enzymes required for the degradation of sialylated host-glycans in <i>B. mongoliense</i>, corroborating the appreciable growth on de Man-Rogosa-Sharpe (MRS) medium supplemented with 3'-sialyllactose (3'-SL) or 6'-sialyllactose (6'-SL). The <i>B. mongoliense</i> BMONG18 was chosen, due to its superior ability to utilize 3'-SL and mucin as representative strain, to investigate its behavior when co-inoculated with other bifidobacterial species. Conversely, members of other bifidobacterial species did not appear to benefit from the presence of BMONG18, highlighting a competitive scenario for nutrient acquisition. Transcriptomic data of BMONG18 reveal no significant differences in gene expression when cultivated in a gut simulating medium (GSM), regardless of whether cheese was included or not. Furthermore, BMONG18 was shown to exhibit high adhesion capabilities to HT29-MTX human cells, in line with its colonization ability of a human host.IMPORTANCEFermented foods are nourishments produced through controlled microbial growth that play an essential role in worldwide human nutrition. Research interest in fermented foods has increased since the 80s, driven by growing awareness of their potential health benefits beyond mere nutritional content. <i>Bifidobacterium mongoliense</i>, previously identified throughout the production process of Parmigiano Reggiano cheese, was found to be capable of establishing itself in the intestines of its consumers. Our study underscores molecular mechanisms through which this bifidobacterial species, derived from food, interacts with the host and other gut microbiota members.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":null,"pages":null},"PeriodicalIF":3.9,"publicationDate":"2024-08-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141987259","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}
Seamounts are widespread underwater topographic features in the ocean that exert an influential role in shaping the microbial biogeographic distribution. Nevertheless, research on the differences in microbial biogeographic distribution between seamount and non-seamount upper water column is still lacking, particularly in the Indian Ocean where studies are limited. In the present study, a total of 45 seawater samples were collected from the water column (5-200 m) of seamounts (HS) and non-seamounts (E87 transect) regions in the Eastern Indian Ocean (EIO) for the analysis of microbial biogeographic patterns and community assembly processes. The results indicated that bacterial community diversity did not differ significantly between the HS and E87 transect regions; however, the community composition was significantly different. Additionally, bacterial community diversity, composition, as well as structure were more affected by depth than by region. Community diversity tended to increase with depth in E87 transect region, while it tended to decrease in HS region. A distance decay analysis also demonstrated that bacterial communities were more influenced by environmental and depth distances than geographic distances. In the assembly of bacterial communities on HS and E87 transect regions, as well as at different depths, stochastic processes, particularly dispersal limitation, were found to be predominant. These findings enhance our comprehension of bacterial community characteristics in the upper seawater of seamounts and non-seamounts regions in the EIO and offer insights into the assembly processes shaping microbial communities at varying depths.
Importance: By comparing the bacterial diversity, composition, and structure in the upper seawater of seamount and non-seamount areas, we provide valuable insights into the influential role of seamounts in shaping microbial biogeography. The finding that the depth had a more significant impact on bacterial community characteristics than region underscores the importance of considering vertical stratification when examining microbial distributions. Moreover, the dominance of stochastic processes, particularly dispersal limitation, in governing community assembly across both seamount and non-seamount areas offers critical implications for the mechanisms underlying microbial biogeographic patterns in these dynamic ocean environments. This study expands the current knowledge and lays the groundwork for further investigations into the complex interactions between oceanographic features, environmental gradients, and microbial community dynamics in the Indian Ocean.
{"title":"Biogeographic patterns and community assembly mechanisms of bacterial community in the upper seawater of seamounts and non-seamounts in the Eastern Indian Ocean.","authors":"Yaqian Jiao, Shanshan Yang, Wenya Bao","doi":"10.1128/aem.01424-24","DOIUrl":"10.1128/aem.01424-24","url":null,"abstract":"<p><p>Seamounts are widespread underwater topographic features in the ocean that exert an influential role in shaping the microbial biogeographic distribution. Nevertheless, research on the differences in microbial biogeographic distribution between seamount and non-seamount upper water column is still lacking, particularly in the Indian Ocean where studies are limited. In the present study, a total of 45 seawater samples were collected from the water column (5-200 m) of seamounts (HS) and non-seamounts (E87 transect) regions in the Eastern Indian Ocean (EIO) for the analysis of microbial biogeographic patterns and community assembly processes. The results indicated that bacterial community diversity did not differ significantly between the HS and E87 transect regions; however, the community composition was significantly different. Additionally, bacterial community diversity, composition, as well as structure were more affected by depth than by region. Community diversity tended to increase with depth in E87 transect region, while it tended to decrease in HS region. A distance decay analysis also demonstrated that bacterial communities were more influenced by environmental and depth distances than geographic distances. In the assembly of bacterial communities on HS and E87 transect regions, as well as at different depths, stochastic processes, particularly dispersal limitation, were found to be predominant. These findings enhance our comprehension of bacterial community characteristics in the upper seawater of seamounts and non-seamounts regions in the EIO and offer insights into the assembly processes shaping microbial communities at varying depths.</p><p><strong>Importance: </strong>By comparing the bacterial diversity, composition, and structure in the upper seawater of seamount and non-seamount areas, we provide valuable insights into the influential role of seamounts in shaping microbial biogeography. The finding that the depth had a more significant impact on bacterial community characteristics than region underscores the importance of considering vertical stratification when examining microbial distributions. Moreover, the dominance of stochastic processes, particularly dispersal limitation, in governing community assembly across both seamount and non-seamount areas offers critical implications for the mechanisms underlying microbial biogeographic patterns in these dynamic ocean environments. This study expands the current knowledge and lays the groundwork for further investigations into the complex interactions between oceanographic features, environmental gradients, and microbial community dynamics in the Indian Ocean.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":null,"pages":null},"PeriodicalIF":3.9,"publicationDate":"2024-08-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141987258","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}
The cell size of phytoplankton is an important defining functional trait that can serve as a driver and sentinel of phytoplankton community structure and function. However, the study of the assembly patterns and drivers of phytoplankton metacommunities with different cell sizes has not been widely carried out. In this study, we systematically investigated the biodiversity patterns, drivers, and assembly processes of the three phytoplankton cell sizes (micro: 20-200 μm; nano: 2-20 μm; pico: 0.2-2 μm) in the Za'gya Zangbo River from the source to the estuary using 18S rDNA amplicon sequencing. The results demonstrated that the alpha diversity and co-occurrence network complexity for all three sizes of phytoplankton increased to a peak downstream of the glacier sources and then decreased to the estuary. The nanophytoplankton subcommunity consistently had the highest alpha diversity and co-occurrence network complexity. On the other hand, total beta diversity followed a unimodal trend of decreasing and then increasing from source to estuary, and was dominated by species replacement components. In addition, deterministic processes driven mainly by physiochemical indices (PCIs) and biogenic elements (BGEs) dominated the assembly of micro- and nanophytoplankton subcommunities, whereas stochastic processes driven by geographical factors (GGFs) dominated the assembly of picophytoplankton subcommunities. The results explained the contradictions in previous studies of phytoplankton community assembly processes in highland aquatic ecosystems, elucidating the different contributions of deterministic and stochastic processes, and the complexity of compositional mechanisms in shaping the assembly of micro-, nano-, and picophytoplankton in this highland glacial river.
Importance: The cell size of phytoplankton is a key life-history trait and key determinant, and phytoplankton of different cell sizes are differentially affected by ecological processes. However, the study of the assembly patterns and drivers of phytoplankton metacommunities with different cell sizes has not been widely carried out. We provide an in-depth analysis of phytoplankton community diversity across three cell sizes in the glacier-fed river, describing how the pattern of phytoplankton communities differs across cell sizes in response to geochemical gradients. The results show that the smaller phytoplankton (picophytoplankton) are relatively more influenced by dispersal-based stochastic processes, whereas larger ones (microphytoplankton and nanophytoplankton) are more structured by selection-based deterministic processes.
{"title":"The enhanced neutral process with decreasing cell size: a study on phytoplankton metacommunities from the glacier-fed river of Qinghai-Xizang Plateau.","authors":"Zhihua Wu, Xiong Xiong, Guoxiang Liu, Huan Zhu","doi":"10.1128/aem.00457-24","DOIUrl":"https://doi.org/10.1128/aem.00457-24","url":null,"abstract":"<p><p>The cell size of phytoplankton is an important defining functional trait that can serve as a driver and sentinel of phytoplankton community structure and function. However, the study of the assembly patterns and drivers of phytoplankton metacommunities with different cell sizes has not been widely carried out. In this study, we systematically investigated the biodiversity patterns, drivers, and assembly processes of the three phytoplankton cell sizes (micro: 20-200 μm; nano: 2-20 μm; pico: 0.2-2 μm) in the Za'gya Zangbo River from the source to the estuary using 18S rDNA amplicon sequencing. The results demonstrated that the alpha diversity and co-occurrence network complexity for all three sizes of phytoplankton increased to a peak downstream of the glacier sources and then decreased to the estuary. The nanophytoplankton subcommunity consistently had the highest alpha diversity and co-occurrence network complexity. On the other hand, total beta diversity followed a unimodal trend of decreasing and then increasing from source to estuary, and was dominated by species replacement components. In addition, deterministic processes driven mainly by physiochemical indices (PCIs) and biogenic elements (BGEs) dominated the assembly of micro- and nanophytoplankton subcommunities, whereas stochastic processes driven by geographical factors (GGFs) dominated the assembly of picophytoplankton subcommunities. The results explained the contradictions in previous studies of phytoplankton community assembly processes in highland aquatic ecosystems, elucidating the different contributions of deterministic and stochastic processes, and the complexity of compositional mechanisms in shaping the assembly of micro-, nano-, and picophytoplankton in this highland glacial river.</p><p><strong>Importance: </strong>The cell size of phytoplankton is a key life-history trait and key determinant, and phytoplankton of different cell sizes are differentially affected by ecological processes. However, the study of the assembly patterns and drivers of phytoplankton metacommunities with different cell sizes has not been widely carried out. We provide an in-depth analysis of phytoplankton community diversity across three cell sizes in the glacier-fed river, describing how the pattern of phytoplankton communities differs across cell sizes in response to geochemical gradients. The results show that the smaller phytoplankton (picophytoplankton) are relatively more influenced by dispersal-based stochastic processes, whereas larger ones (microphytoplankton and nanophytoplankton) are more structured by selection-based deterministic processes.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":null,"pages":null},"PeriodicalIF":3.9,"publicationDate":"2024-08-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141987260","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}
Morgane Wuckelt, Audrey Laurent, Clémence Mouville, Julie Meyer, Anne Jamet, Hervé Lecuyer, Xavier Nassif, Emmanuelle Bille, Vladimir Pelicic, Mathieu Coureuil
The efficient natural transformation of Neisseria meningitidis allows the rapid construction of bacterial mutants in which the genes of interest are interrupted or replaced by antibiotic-resistance cassettes. However, this proved to be a double-edged sword, i.e., although facilitating the genetic characterization of this important human pathogen, it has limited the development of strategies for constructing markerless mutants without antibiotic-resistance markers. In addition, efficient tools for complementation or labeling are also lacking in N. meningitidis. In this study, we significantly expand the meningococcal genetic toolbox by developing new and efficient tools for the construction of markerless mutants (using a dual counterselection strategy), genetic complementation (using integrative vectors), and cell labeling (using a self-labeling protein tag). This expanded toolbox paves the way for more in-depth genetic characterization of N. meningitidis and might also be useful in other Neisseria species.IMPORTANCENeisseria meningitidis and Neisseria gonorrhoeae are two important human pathogens. Research focusing on these bacteria requires genetic engineering, which is facilitated by their natural ability to undergo transformation. However, the ease of mutant engineering has led the Neisseria community to neglect the development of more sophisticated tools for gene editing, particularly for N. meningitidis. In this study, we have significantly expanded the meningococcal genetic toolbox by developing novel and efficient tools for markerless mutant construction, genetic complementation, and cell tagging. This expanded toolbox paves the way for more in-depth genetic characterization of N. meningitidis and might also be useful in other Neisseria species.
{"title":"Expanding the genetic toolbox for <i>Neisseria meningitidis</i> with efficient tools for unmarked gene editing, complementation, and labeling.","authors":"Morgane Wuckelt, Audrey Laurent, Clémence Mouville, Julie Meyer, Anne Jamet, Hervé Lecuyer, Xavier Nassif, Emmanuelle Bille, Vladimir Pelicic, Mathieu Coureuil","doi":"10.1128/aem.00880-24","DOIUrl":"https://doi.org/10.1128/aem.00880-24","url":null,"abstract":"<p><p>The efficient natural transformation of <i>Neisseria meningitidis</i> allows the rapid construction of bacterial mutants in which the genes of interest are interrupted or replaced by antibiotic-resistance cassettes. However, this proved to be a double-edged sword, i.e., although facilitating the genetic characterization of this important human pathogen, it has limited the development of strategies for constructing markerless mutants without antibiotic-resistance markers. In addition, efficient tools for complementation or labeling are also lacking in <i>N. meningitidis</i>. In this study, we significantly expand the meningococcal genetic toolbox by developing new and efficient tools for the construction of markerless mutants (using a dual counterselection strategy), genetic complementation (using integrative vectors), and cell labeling (using a self-labeling protein tag). This expanded toolbox paves the way for more in-depth genetic characterization of <i>N. meningitidis</i> and might also be useful in other <i>Neisseria</i> species.IMPORTANCE<i>Neisseria meningitidis</i> and <i>Neisseria gonorrhoeae</i> are two important human pathogens. Research focusing on these bacteria requires genetic engineering, which is facilitated by their natural ability to undergo transformation. However, the ease of mutant engineering has led the <i>Neisseria</i> community to neglect the development of more sophisticated tools for gene editing, particularly for <i>N. meningitidis</i>. In this study, we have significantly expanded the meningococcal genetic toolbox by developing novel and efficient tools for markerless mutant construction, genetic complementation, and cell tagging. This expanded toolbox paves the way for more in-depth genetic characterization of <i>N. meningitidis</i> and might also be useful in other <i>Neisseria</i> species.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":null,"pages":null},"PeriodicalIF":3.9,"publicationDate":"2024-08-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141974927","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}
The ability to genetically manipulate bacteria is a staple of modern molecular microbiology. Since the 2000s, marker-less mutants of Streptococcus pneumoniae (Spn) have been made by allelic exchange predominantly using the kanR-rpsL cassette known as "Janus." The conventional Janus protocol involves two transformation steps using multiple PCR-assembled products containing the Janus cassette and the target gene's flanking DNA. We present an innovative strategy to achieve marker-less allelic replacement through a single transformation step. Our strategy involves integrating an additional copy of the target's downstream region before the Janus cassette, leading to a modified genetic arrangement. This single modification reduced the number of required PCR fragments from five to four, lowered the number of assembly reactions from two to one, and simplified the transformation process to a single step. To validate the efficacy of our approach, we implemented this strategy to delete in Spn serotype 4 strain TIGR4 the virulence gene pspA, the entire capsular polysaccharide synthesis locus cps4, and to introduce a single-nucleotide replacement into the chromosome. Notably, beyond streamlining the procedure, our method markedly reduced false positives typically encountered during negative selection with streptomycin when employing the traditional Janus protocol. Furthermore, and as consequence of reducing the amount of exogenous DNA required for construct synthesis, we show that our new method is amendable to the use of commercially available synthetic DNA for construct creation, further reducing the work needed to obtain a mutant. Our streamlined strategy, termed easyJanus, substantially expedites the genetic manipulation of Spn facilitating future research endeavors.
Importance: We introduce a new strategy aimed at streamlining the process for marker-less allelic replacement in Streptococcus pneumoniae, a Gram-positive bacterium and leading cause of pneumonia, meningitis, and ear infections. Our approach involves a modified genetic arrangement of the Janus cassette to facilitate self-excision during the segregation step. Since this new method reduces the amount of exogenous DNA required, it is highly amendable to the use of synthetic DNA for construction of the mutagenic construct. Our streamlined strategy, called easyJanus, offers significant time and cost savings while concurrently enhancing the efficiency of obtaining marker-less allelic replacement in S. pneumoniae.
对细菌进行遗传操作的能力是现代分子微生物学的主要特征。自 2000 年代以来,肺炎链球菌(Spn)的无标记突变体主要是通过使用被称为 "Janus "的 kanR-rpsL 盒进行等位基因交换制成的。传统的 Janus 方案涉及两个转化步骤,使用含有 Janus 盒和目标基因侧翼 DNA 的多个 PCR 组装产物。我们提出了一种创新策略,通过单一转化步骤实现无标记等位基因替换。我们的策略是在 Janus 基因盒之前整合目标基因下游区域的额外拷贝,从而改变基因排列。这种单一的修改将所需的 PCR 片段数量从五个减少到四个,将组装反应的数量从两个减少到一个,并将转化过程简化为单一步骤。为了验证我们的方法是否有效,我们采用这一策略删除了 Spn 血清型 4 菌株 TIGR4 的毒力基因 pspA 和整个荚膜多糖合成基因座 cps4,并在染色体中引入了单核苷酸替换。值得注意的是,除了简化程序外,我们的方法还显著减少了在使用链霉素进行阴性选择时,采用传统 Janus 方案通常会遇到的假阳性。此外,由于减少了构建体合成所需的外源 DNA 数量,我们证明我们的新方法适用于使用市售合成 DNA 创建构建体,从而进一步减少了获得突变体所需的工作。我们的简化策略被称为 easyJanus,它大大加快了 Spn 的遗传操作,促进了未来的研究工作:我们介绍了一种旨在简化肺炎链球菌无标记等位基因置换过程的新策略,肺炎链球菌是一种革兰氏阳性细菌,也是肺炎、脑膜炎和中耳炎的主要致病菌。我们的方法涉及改进 Janus 盒的基因排列,以促进分离步骤中的自切割。由于这种新方法减少了所需的外源 DNA 数量,因此非常适合使用合成 DNA 来构建诱变构建体。我们的简化策略被称为 easyJanus,它大大节省了时间和成本,同时提高了肺炎双球菌无标记等位基因置换的效率。
{"title":"Streamlining marker-less allelic replacement in <i>Streptococcus pneumoniae</i> through a single transformation step strategy: easyJanus.","authors":"Vipin Chembilikandy, Adonis D'Mello, Hervé Tettelin, Eriel Martínez, Carlos J Orihuela","doi":"10.1128/aem.01010-24","DOIUrl":"https://doi.org/10.1128/aem.01010-24","url":null,"abstract":"<p><p>The ability to genetically manipulate bacteria is a staple of modern molecular microbiology. Since the 2000s, marker-less mutants of <i>Streptococcus pneumoniae</i> (<i>Spn</i>) have been made by allelic exchange predominantly using the <i>kan<sup>R</sup>-rpsL</i> cassette known as \"Janus.\" The conventional Janus protocol involves two transformation steps using multiple PCR-assembled products containing the Janus cassette and the target gene's flanking DNA. We present an innovative strategy to achieve marker-less allelic replacement through a single transformation step. Our strategy involves integrating an additional copy of the target's downstream region before the Janus cassette, leading to a modified genetic arrangement. This single modification reduced the number of required PCR fragments from five to four, lowered the number of assembly reactions from two to one, and simplified the transformation process to a single step. To validate the efficacy of our approach, we implemented this strategy to delete in <i>Spn</i> serotype 4 strain TIGR4 the virulence gene <i>pspA,</i> the entire capsular polysaccharide synthesis locus <i>cps4</i>, and to introduce a single-nucleotide replacement into the chromosome. Notably, beyond streamlining the procedure, our method markedly reduced false positives typically encountered during negative selection with streptomycin when employing the traditional Janus protocol. Furthermore, and as consequence of reducing the amount of exogenous DNA required for construct synthesis, we show that our new method is amendable to the use of commercially available synthetic DNA for construct creation, further reducing the work needed to obtain a mutant. Our streamlined strategy, termed easyJanus, substantially expedites the genetic manipulation of <i>Spn</i> facilitating future research endeavors.</p><p><strong>Importance: </strong>We introduce a new strategy aimed at streamlining the process for marker-less allelic replacement in <i>Streptococcus pneumoniae</i>, a Gram-positive bacterium and leading cause of pneumonia, meningitis, and ear infections. Our approach involves a modified genetic arrangement of the Janus cassette to facilitate self-excision during the segregation step. Since this new method reduces the amount of exogenous DNA required, it is highly amendable to the use of synthetic DNA for construction of the mutagenic construct. Our streamlined strategy, called easyJanus, offers significant time and cost savings while concurrently enhancing the efficiency of obtaining marker-less allelic replacement in <i>S. pneumoniae</i>.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":null,"pages":null},"PeriodicalIF":3.9,"publicationDate":"2024-08-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141974937","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}