Xanthomonas spp. cause severe bacterial diseases. However, effective strategies for prevention and management of these diseases are scarce. Thus, it is necessary to improve the efficiency of control of diseases caused...Xanthomonas spp. cause severe bacterial diseases. However, effective strategies for prevention and management of these diseases are scarce. Thus, it is necessary to improve the efficiency of control of diseases caused by Xanthomonas. In this study, Xanthomonas oryzae pv. oryzae(Xoo), which causes rice bacterial leaf blight, has been studied as a representative. A transposon insertion library of Xoo, comprising approximately 200,000 individual insertion mutants, was generated. Transposon sequencing data indicated that the mariner C9 transposase mapped at 35.7–36.4% of all potential insertion sites, revealing 491 essential genes required for the growth of Xoo in rich media. The results show that, compared to the functions of essential genes of other bacteria, the functions of some essential genes of Xoo are unknown, 25 genes might be dangerous for the Xanthomonas group, and 3 are specific to Xanthomonas. High-priority candidates for developing broad-spectrum, Xanthomonas-specific, and environment-friendly bactericides were identified in this study. In addition, this study revealed the possible targets of dioctyldiethylenetriamine using surface plasmon resonance(SPR) in combination with high performance liquid chromatography–mass spectrometry(HPLC–MS). The study also provided references for the research of some certain bactericides with unknown anti-bacterial mode of action. In conclusion, this study urged a better understanding of Xanthomonas,provided meaningful data for the management of bacterial leaf blight, and disclosed selected targets of a novel bactericide.展开更多
Most in silico evolutionary studies commonly assumed that core genes are essential for cellular function,while accessory genes are dispensable,particularly in nutrient-rich environments.However,this assumption is seld...Most in silico evolutionary studies commonly assumed that core genes are essential for cellular function,while accessory genes are dispensable,particularly in nutrient-rich environments.However,this assumption is seldom tested genetically within the pangenome context.In this study,we conducted a robust pangenomic Tn-seq analysis of fitness genes in a nutrient-rich medium for Sinorhizobium strains with a canonical open pangenome.To evaluate the robustness of fitness category assignment,Tn-seq data for three independent mutant libraries per strain were analyzed by three methods,which indicates that the Hidden Markov Model(HMM)-based method is most robust to variations between mutant libraries and not sensitive to data size,outperforming the Bayesian and Monte Carlo simulation-based methods.Consequently,the HMM method was used to classify the fitness category.Fitness genes,categorized as essential(ES),advantage(GA),and disadvantage(GD)genes for growth,are enriched in core genes,while nonessential genes(NE)are over-represented in accessory genes.Accessory ES/GA genes showed a lower fitness effect than core ES/GA genes.Connectivity degrees in the cofitness network decrease in the order of ES,GD,and GA/NE.In addition to accessory genes,1599 out of 3284 core genes display differential essentiality across test strains.Within the pangenome core,both shared quasi-essential(ES and GA)and strain-dependent fitness genes are enriched in similar functional categories.Our analysis demonstrates a considerable fuzzy essential zone determined by cofitness connectivity degrees in Sinorhizobium pangenome and highlights the power of the cofitness network in understanding the genetic basis of ever-increasing prokaryotic pangenome data.展开更多
The chromosomal position effect can significantly affect the transgene expression,which may provide an efficient strategy for the inauguration of alien genes in new hosts,but has been less explored rationally.The bact...The chromosomal position effect can significantly affect the transgene expression,which may provide an efficient strategy for the inauguration of alien genes in new hosts,but has been less explored rationally.The bacterium Myxococcus xanthus harbors a large circular high-GC genome,and the position effect in this chassis may result in a thousand-fold expression variation of alien natural products.In this study,we conducted transposon insertion at TA sites on the M.xanthus genome,and used enrichment and dilution indexes to respectively appraise high and low expression potentials of alien genes at insertion sites.The enrichment sites are characteristically distributed along the genome,and the dilution sites are overlapped well with the horizontal transfer genes.We experimentally demonstrated the enrichment sites as high expression integration sites(HEISs),and the dilution sites unsuitable for gene integration expression.This work highlights that HEISs are the plug-and-play sites for efficient expression of integrated genes.展开更多
基金This study was supported by the National Natural Science Foundation of China(32272587 and 32202342)the Programs for the Scientific Research Activities of Academic and Technical Leaders of Anhui Province,China(2020D251)+3 种基金the Development Fund for Talent Personnel of Anhui Agricultural University,China(rc342006)the University Synergy Innovation Program of Anhui Province,China(GXXT-2021-059)the Key Project of the Natural Science Foundation of Anhui Provincial Department of Education,China(2023AH040129)Anhui Province Agricultural Eco-Environmental Protection and Quality Safety Industry Technology System,China。
文摘Xanthomonas spp. cause severe bacterial diseases. However, effective strategies for prevention and management of these diseases are scarce. Thus, it is necessary to improve the efficiency of control of diseases caused by Xanthomonas. In this study, Xanthomonas oryzae pv. oryzae(Xoo), which causes rice bacterial leaf blight, has been studied as a representative. A transposon insertion library of Xoo, comprising approximately 200,000 individual insertion mutants, was generated. Transposon sequencing data indicated that the mariner C9 transposase mapped at 35.7–36.4% of all potential insertion sites, revealing 491 essential genes required for the growth of Xoo in rich media. The results show that, compared to the functions of essential genes of other bacteria, the functions of some essential genes of Xoo are unknown, 25 genes might be dangerous for the Xanthomonas group, and 3 are specific to Xanthomonas. High-priority candidates for developing broad-spectrum, Xanthomonas-specific, and environment-friendly bactericides were identified in this study. In addition, this study revealed the possible targets of dioctyldiethylenetriamine using surface plasmon resonance(SPR) in combination with high performance liquid chromatography–mass spectrometry(HPLC–MS). The study also provided references for the research of some certain bactericides with unknown anti-bacterial mode of action. In conclusion, this study urged a better understanding of Xanthomonas,provided meaningful data for the management of bacterial leaf blight, and disclosed selected targets of a novel bactericide.
文摘目的建立转座子突变文库,筛选高毒力肺炎克雷伯菌(hypervirulent Klebsiella pneumoniae,hvKp)的新毒力基因。方法构建转座子突变文库,血清处理,通过转座子测序(transposon sequencing,Tn-seq)比较分析血清处理前后各基因突变株在文库中丰度变化,并对筛选出的基因进行KEGG(kyoto encyclopedia of genes and genomes)注释和富集分析。结果以处理后丰度低于初始丰度20%为界,共筛选出405个基因,其中351个基因在HS11286、NJST258_1、NTUH-K2044和RJF293等4株参考菌株中保守存在,占86.7%,10个基因为NTUH-K2044和RJF293两株高毒力株共有而低毒力株HS11286和NJST258_1缺失;荚膜多糖基因簇中基因如糖基转移酶基因wzy、聚集蛋白基因wzi、荚膜转运蛋白基因wza等突变株在血清处理后不能检出,气杆菌素、沙门氏菌素等铁载体基因簇在各文库中的丰度变化不超过1倍;KEGG注释结果显示,注释最多的基因为参与氨基酸代谢、辅助因子和维生素代谢、碳水化合物代谢等。结论Tn-seq是功能基因筛选的可靠方法,本研究成功筛选出405个hvKp的候选新毒力基因,为后续深入研究hvKp的新毒力基因功能和调控机制提供实验依据。
基金supported by the National Key R&D Program of China(grant number 2022YFA0912100)the National Natural Science Foundation of China(grant number 32070078)to C.F.T.
文摘Most in silico evolutionary studies commonly assumed that core genes are essential for cellular function,while accessory genes are dispensable,particularly in nutrient-rich environments.However,this assumption is seldom tested genetically within the pangenome context.In this study,we conducted a robust pangenomic Tn-seq analysis of fitness genes in a nutrient-rich medium for Sinorhizobium strains with a canonical open pangenome.To evaluate the robustness of fitness category assignment,Tn-seq data for three independent mutant libraries per strain were analyzed by three methods,which indicates that the Hidden Markov Model(HMM)-based method is most robust to variations between mutant libraries and not sensitive to data size,outperforming the Bayesian and Monte Carlo simulation-based methods.Consequently,the HMM method was used to classify the fitness category.Fitness genes,categorized as essential(ES),advantage(GA),and disadvantage(GD)genes for growth,are enriched in core genes,while nonessential genes(NE)are over-represented in accessory genes.Accessory ES/GA genes showed a lower fitness effect than core ES/GA genes.Connectivity degrees in the cofitness network decrease in the order of ES,GD,and GA/NE.In addition to accessory genes,1599 out of 3284 core genes display differential essentiality across test strains.Within the pangenome core,both shared quasi-essential(ES and GA)and strain-dependent fitness genes are enriched in similar functional categories.Our analysis demonstrates a considerable fuzzy essential zone determined by cofitness connectivity degrees in Sinorhizobium pangenome and highlights the power of the cofitness network in understanding the genetic basis of ever-increasing prokaryotic pangenome data.
基金the National Key Research and Development Program of China(2021YFC2101000)the National Natural Science Foundation of China(32301220).
文摘The chromosomal position effect can significantly affect the transgene expression,which may provide an efficient strategy for the inauguration of alien genes in new hosts,but has been less explored rationally.The bacterium Myxococcus xanthus harbors a large circular high-GC genome,and the position effect in this chassis may result in a thousand-fold expression variation of alien natural products.In this study,we conducted transposon insertion at TA sites on the M.xanthus genome,and used enrichment and dilution indexes to respectively appraise high and low expression potentials of alien genes at insertion sites.The enrichment sites are characteristically distributed along the genome,and the dilution sites are overlapped well with the horizontal transfer genes.We experimentally demonstrated the enrichment sites as high expression integration sites(HEISs),and the dilution sites unsuitable for gene integration expression.This work highlights that HEISs are the plug-and-play sites for efficient expression of integrated genes.
