Bacteria can evolve rapidly by acquiring new traits such as virulence,metabolic properties,and most importantly,antimicrobial resistance,through horizontal gene transfer(HGT).Multidrug resistance in bacteria,especiall...Bacteria can evolve rapidly by acquiring new traits such as virulence,metabolic properties,and most importantly,antimicrobial resistance,through horizontal gene transfer(HGT).Multidrug resistance in bacteria,especially in Gram-negative organisms,has become a global public health threat often through the spread of mobile genetic elements.Conjugation represents a major form of HGT and involves the transfer of DNA from a donor bacterium to a recipient by direct contact.Conjugative plasmids,a major vehicle for the dissemination of antimicrobial resistance,are selfish elements capable of mediating their own transmission through conjugation.To spread to and survive in a new bacterial host,conjugative plasmids have evolved mechanisms to circumvent both host defense systems and compete with co-resident plasmids.Such mechanisms have mostly been studied in model plasmids such as the F plasmid,rather than in conjugative plasmids that confer antimicrobial resistance(AMR)in important human pathogens.A better understanding of these mechanisms is crucial for predicting the flow of antimicrobial resistance-conferring conjugative plasmids among bacterial populations and guiding the rational design of strategies to halt the spread of antimicrobial resistance.Here,we review mechanisms employed by conjugative plasmids that promote their transmission and establishment in Gram-negative bacteria,by following the life cycle of conjugative plasmids.展开更多
An imbalance in oral microbial homeostasis is significantly associated with the onset and progression of several systemic diseases.Fusobacterium nucleatum,a ubiquitous periodontitis-causing bacterium in the oral cavit...An imbalance in oral microbial homeostasis is significantly associated with the onset and progression of several systemic diseases.Fusobacterium nucleatum,a ubiquitous periodontitis-causing bacterium in the oral cavity,is frequently detected in focal sites and contributes to the pathogenesis of many extraoral diseases,including cancers,cardiovascular diseases,and adverse pregnancy outcomes(APOs).F.nucleatum is one of the few oral anaerobes that can be cultured purely in vitro and is a‘model species’for studying the impact of oral health on systemic health.The establishment and development of genetic manipulation tools for F.nucleatum and the construction of pathogenic gene-disrupted strains are important strategies for studying the pathogenicity of F.nucleatum.Here,we review the establishment and development of the genetic manipulation systems for F.nucleatum and summarize the characteristics of various genetic manipulation tools,such as suicide plasmid-based systems for gene inactivation,replicable plasmid-based systems controlling gene expression,and transposon-based random mutagenesis systems.Notably,we summarize and analyze their applications in the study of the pathogenic mechanisms of F.nucleatum.We hope to provide reference information and ideas for future research on genetic manipulation tools and the pathogenic mechanisms of F.nucleatum and other Fusobacterium species.展开更多
Saccharopolyspora spinosa is an industrial rare actinomycete capable of producing important environmental-friendly biopesticides, spinosyns. However, exploitation of S. spinosa has been limited due to its genetic inac...Saccharopolyspora spinosa is an industrial rare actinomycete capable of producing important environmental-friendly biopesticides, spinosyns. However, exploitation of S. spinosa has been limited due to its genetic inaccessibility and lack of effective genome engineering tools.In this work, we characterized the activity of an endogenous type I-B CRISPR-Cas system as well as its recognized protospacer adjacent motifs(PAMs) based on bioinformatics analysis combined with a plasmid interference assay in S. spinosa. By delivering editing plasmids containing a designed mini CRISPR array(repeat+self-targeting spacer+repeat) and repair templates, we achieved 100% editing efficiency for gene deletion. Using this tool, the genetic barrier composed of the restriction-modification(RM) systems was systematically disarmed. We showed that by disarming one type I RM system(encoded by A8926_1903/1904/1905) and two type Ⅱ RM systems(encoded by A8926_1725/1726 and A8926_2652/2653) simultaneously, the transformation efficiency of the replicative and integrative plasmids(pSP01 and pSI01) was increased by approximately 3.9-fold and 4.2-fold, respectively. Using the engineered strain with simultaneous knock-out of these three RM genes as the starting strain, we achieved the deletion of 75-kb spinosyns biosynthetic gene cluster(BGC) as well as gene insertion at high efficiency. Collectively, we developed a reliable and highly efficient genome editing tool based on the endogenous type I CRISPR-Cas system combined with the disarmament of the RM systems in S. spinosa. This is the first time to establish an endogenous CRISPR-Cas-based genome editing tool in the non-model industrial actinomycetes.展开更多
基金the Wellcome Trust,BBSRC,and the National Natural Science Foundation of China(81802065,102908/Z/13/Z).
文摘Bacteria can evolve rapidly by acquiring new traits such as virulence,metabolic properties,and most importantly,antimicrobial resistance,through horizontal gene transfer(HGT).Multidrug resistance in bacteria,especially in Gram-negative organisms,has become a global public health threat often through the spread of mobile genetic elements.Conjugation represents a major form of HGT and involves the transfer of DNA from a donor bacterium to a recipient by direct contact.Conjugative plasmids,a major vehicle for the dissemination of antimicrobial resistance,are selfish elements capable of mediating their own transmission through conjugation.To spread to and survive in a new bacterial host,conjugative plasmids have evolved mechanisms to circumvent both host defense systems and compete with co-resident plasmids.Such mechanisms have mostly been studied in model plasmids such as the F plasmid,rather than in conjugative plasmids that confer antimicrobial resistance(AMR)in important human pathogens.A better understanding of these mechanisms is crucial for predicting the flow of antimicrobial resistance-conferring conjugative plasmids among bacterial populations and guiding the rational design of strategies to halt the spread of antimicrobial resistance.Here,we review mechanisms employed by conjugative plasmids that promote their transmission and establishment in Gram-negative bacteria,by following the life cycle of conjugative plasmids.
基金foundation support of the National Natural Science Foundation of China(82270980,82071122)the National Young Scientist Support Foundation(2019),the Major Innovation Projects in Shandong Province(2021SFGC0502)+2 种基金the Oral Microbiome Innovation Team of Shandong Province(2020KJK001)Shandong Province Key Research and Development Program(2021ZDSYS18)Intramural Joint Program Fund of State Key Laboratory of Microbial Technology(SKLMTIJP-2024-08)。
文摘An imbalance in oral microbial homeostasis is significantly associated with the onset and progression of several systemic diseases.Fusobacterium nucleatum,a ubiquitous periodontitis-causing bacterium in the oral cavity,is frequently detected in focal sites and contributes to the pathogenesis of many extraoral diseases,including cancers,cardiovascular diseases,and adverse pregnancy outcomes(APOs).F.nucleatum is one of the few oral anaerobes that can be cultured purely in vitro and is a‘model species’for studying the impact of oral health on systemic health.The establishment and development of genetic manipulation tools for F.nucleatum and the construction of pathogenic gene-disrupted strains are important strategies for studying the pathogenicity of F.nucleatum.Here,we review the establishment and development of the genetic manipulation systems for F.nucleatum and summarize the characteristics of various genetic manipulation tools,such as suicide plasmid-based systems for gene inactivation,replicable plasmid-based systems controlling gene expression,and transposon-based random mutagenesis systems.Notably,we summarize and analyze their applications in the study of the pathogenic mechanisms of F.nucleatum.We hope to provide reference information and ideas for future research on genetic manipulation tools and the pathogenic mechanisms of F.nucleatum and other Fusobacterium species.
基金supported by the National Key Research and Development Program of China(2019YFA0905400)the National Natural Science Foundation of China(32270095)the Open Funding Project(MMLKF23-08)of State Key Laboratory of Microbial Metabolism.
文摘Saccharopolyspora spinosa is an industrial rare actinomycete capable of producing important environmental-friendly biopesticides, spinosyns. However, exploitation of S. spinosa has been limited due to its genetic inaccessibility and lack of effective genome engineering tools.In this work, we characterized the activity of an endogenous type I-B CRISPR-Cas system as well as its recognized protospacer adjacent motifs(PAMs) based on bioinformatics analysis combined with a plasmid interference assay in S. spinosa. By delivering editing plasmids containing a designed mini CRISPR array(repeat+self-targeting spacer+repeat) and repair templates, we achieved 100% editing efficiency for gene deletion. Using this tool, the genetic barrier composed of the restriction-modification(RM) systems was systematically disarmed. We showed that by disarming one type I RM system(encoded by A8926_1903/1904/1905) and two type Ⅱ RM systems(encoded by A8926_1725/1726 and A8926_2652/2653) simultaneously, the transformation efficiency of the replicative and integrative plasmids(pSP01 and pSI01) was increased by approximately 3.9-fold and 4.2-fold, respectively. Using the engineered strain with simultaneous knock-out of these three RM genes as the starting strain, we achieved the deletion of 75-kb spinosyns biosynthetic gene cluster(BGC) as well as gene insertion at high efficiency. Collectively, we developed a reliable and highly efficient genome editing tool based on the endogenous type I CRISPR-Cas system combined with the disarmament of the RM systems in S. spinosa. This is the first time to establish an endogenous CRISPR-Cas-based genome editing tool in the non-model industrial actinomycetes.
