氦氧饱和高气压暴露对铜绿假单胞菌PAO1基因表达的诱导调节

研究氦氧饱和高气压暴露对铜绿假单胞菌基因表达谱的系统影响,对急性毒力基因表达的调节。用全基因组DNA芯片分析技术比较菌株暴露前后基因表达谱差异;RT-PCR方法验证部分差异表达基因;用分光光度法在细胞水平验证弹性蛋白酶含量;小鼠染毒法观察暴露组细菌毒力在整体动物水平的变化。基因表达谱分析结果表明,铜绿假单胞菌暴露12 h差异表达基因达243个、72 h差异表达基因为1 168个。72 h差异表达基因中与细菌应激响应、蛋白折叠、转录调节、菌毛和鞭毛合成、毒力因子调节与合成、细菌外膜蛋白和抗原合成的基因大量上调;部分基因的RT-PCR验证结果与芯片结果一致;细胞水平验证结果显示暴露72 h细菌毒力表型增强。因此,氦氧饱和高气压暴露对铜绿假单胞菌基因表达谱有明显影响,对急性侵袭性感染毒力因子基因表达水平有正向诱导调节作用。     

基于生物信息学的致病菌特有基因预测及分析

目的:利用生物信息学方法对致病菌特有基因进行大规模预测,同时探讨致病菌特有基因与致病菌毒力之间的关系。方法:构建致病性细菌蛋白质序列数据库和非致病性细菌蛋白质序列数据库,利用同源性比对的方法(BlastP工具)对致病菌特有基因进行预测;同时从文献中提取与致病菌毒力紧密相关的毒力因子,构建具有代表性的毒力因子分析库,对预测的致病菌特有基因进行比较分析。结果:在致病菌780310个基因中,预测了致病菌特有基因79166个,约占致病菌总基因的10.15%;预测的致病菌特有基因包含了构建的毒力因子分析库中的大部分毒力基因。结论:预测的致病菌特有基因与致病菌毒力紧密相关,大大减少了进一步在致病菌基因组中鉴定毒力基因时整个基因组的数据量。     

CodY在单核细胞增生李斯特菌运动和毒力方面的作用

目的:探究全局性转录调控因子CodY在单核细胞增生李斯特菌(Listeria monocytogenes,Lm)鞭毛运动和细菌毒力方面的作用。方法:通过同源重组的方法敲除Lm染色体上CodY的编码基因codY并成功构建缺失菌株的回复菌株;利用平板泳动法观测鞭毛运动的变化,RT-qPCR检测与鞭毛运动相关基因的转录表达;比较野生型菌株EGDe与CodY缺失菌株对细菌溶血活性、棉铃虫幼虫的半致死剂量和主要的毒力因子LLO和毒力基因调控蛋白PrfA转录表达的影响。结果:同野生型菌株相比,CodY缺失菌株鞭毛运动和相关基因,以及主要的毒力因子LLO和PrfA的转录表达显著降低(P≤0.01),溶血活性显著降低(P≤0.01),对棉铃虫幼虫的半致死剂量上升了5.8倍。结论:CodY在Lm鞭毛运动和细菌毒力调控方面具有重要作用。     

CRISPR/Cas系统与志贺菌毒力和耐药的关系及插入序列IS600对cse2表达水平的影响北大核心CSCD

【目的】了解志贺菌中成簇的规律间隔短回文重复序列(Clustered regularly interspaced short palindromic repeats,CRISPR)的分布及其与毒力和耐药的关系,并分析志贺菌中插入序列IS600对CRISPR相关蛋白基因cse2 m RNA表达水平的影响。【方法】利用课题组前期设计的引物PCR扩增志贺菌的3个CRISPR位点、CRISPR相关蛋白基因cse2、耐药基因和毒力基因;改良Kirby-Bauer(K-B)纸片法进行药敏试验;台盼蓝计数试验检测细菌毒力;Real-time PCR检测志贺菌中cse2基因m RNA表达水平。分别分析志贺菌中CRISPR/Cas系统与耐药基因、耐药表型、毒力基因、毒力表型的关系;了解IS600对CRISPR相关蛋白基因cse2 m RNA表达水平的影响。【结果】志贺菌中CRISPR1位点阴性细菌的毒力强;插入序列IS600使cse2 m RNA表达水平降低。【结论】志贺菌中存在CRISPR1、2、3位点;CRISPR1位点与毒力有关;插入序列IS600对cse2 m RNA表达水平有影响。     

产单核细菌李氏菌毒力因子的分子遗传学研究进展

产单核细菌李氏菌（ＬＭ）是重要的食品卫生污染菌，其毒力由多基因决定。本文就其已发现的产毒基因们点、基因克隆、重组和表达、毒力 调控以及基因产物的功能作一简介 。     

Tn5转座突变技术在革兰氏阴性细菌分子遗传研究中的应用

随着广宿主载体系统的发展,Tn5及其衍生载体已经广泛应用于革兰氏阴性细菌的分子遗传学研究。主要综述了Tn5转座突变技术在生防细菌生防机理研究、细菌必需基因的鉴定、病原细菌毒力相关基因研究、代谢调控基因研究和菌株的遗传改良方面的应用研究进展。     

rmlB基因在单核细胞增生李斯特菌耐药性、生物被膜形成和毒力方面的作用

【目的】探究单核细胞增生李斯特菌(Listeria monocytogenes,Lm)rmlB基因在细菌耐药、生物被膜形成和毒力方面的作用。【方法】通过同源重组的方法敲除Lm染色体上的rmlB基因,比较野生株与rmlB缺失株在耐药性方面的差异;利用微孔板法观测rmlB缺失菌株生物被膜形成能力的变化;利用RT-PCR检测缺失菌株中主要毒力基因转录表达,并观察rmlB缺失对细菌溶血活性的影响。【结果】同野生菌株相比,rmlB缺失菌株对头孢菌素和杆菌肽等作用位点在细菌细胞壁和细胞膜的敏感性显著增加(P≤0.01),生物被膜形成能力显著降低(P≤0.01),细菌主要毒力基因hly的转录表达及溶血活性也发生显著降低(P≤0.01)。【结论】rmlB基因在Lm生物被膜形成和耐受作用位点位于细胞壁和细胞膜的抗生素及细菌毒力方面具有重要作用。     

基因芯片技术在检测肠道致病菌方面的应用

基因芯片技术具有高通量、自动化、快速检测等特点,因此被广泛地应用于各种研究领域,如细菌分子流行病学、细菌基因鉴定、致病分子机理、基因突变及多态性分析、表达谱分析、DNA测序和药物筛选等。现介绍基因芯片检测肠道致病菌方面的国外研究进展,基因芯片应用于检测肠道致病菌的3个方面:结合多重PCR对致病菌的毒力因子或者特异性基因进行鉴定;直接检测细菌的DNA或者RNA;以致病细菌核糖体RNA作为检测的靶基因同时检测多种肠道致病菌。由于其检测的高效率,该技术要优于其他分子生物学检测方法。基因芯片技术在肠道致病菌检测中有着巨大的应用价值,具有广阔的应用前景。     

细菌非编码小RNA的筛选及鉴定方法

细菌非编码小RNA（smallnon．codingRNAs,sRNAs）是一类长度为50～500nt、不编码蛋白质的功能RNA,在应对胁迫、毒力产生和新陈代谢等生命过程中起重要的调控作用。其主要通过碱基配对与靶mRNA发生作用,导致mRNA翻译和稳定性改变,从而在转录后水平调节基因的表达,最终影响细菌各种生命活动。近年来,利用生物信息学和分子生物学技术,已在细菌中筛选并鉴定得到了几百个sRNA。该文对细菌sRNA的筛选和鉴定方法作一简要综述。     

差减杂交技术在细菌学研究中的应用

差减杂交技术是一种用于寻找基因组之间差异的有效的方法。其通过去除被比较的两组基因组之间的共有序列,富集差异序列的方法来达到寻找差异基因的目的。其操作简便,价格低廉,且避免了已知细菌基因组信息匮乏对其应用的限制,从而在细菌学方面得到了广泛的应用,包括鉴定可能与毒力有关的致病基因岛/遗传岛、查找细菌的致弱机理、鉴定可移动的遗传成分、寻找毒力基因、寻找与导致宿主差异有关的基因、检测基因表达上的变化等多个方面。从而为病原的鉴定,疾病的诊断,预防,细菌的致病机理等方面的研究奠定了坚实的基础。     

Involvement of Streptococcus gordonii beta-glucoside metabolism systems in adhesion, biofilm formation, and in vivo gene expression

Streptococcus gordonii genes involved in beta-glucoside metabolism are induced in vivo on infected heart valves during experimental endocarditis and in vitro during biofilm formation on saliva-coated hydroxyapatite (sHA). To determine the roles of beta-glucoside metabolism systems in biofilm formation, the loci of these induced genes were analyzed. To confirm the function of genes in each locus, strains were constructed with gene inactivation, deletion, and/or reporter gene fusions. Four novel systems responsible for beta-glucoside metabolism were identified, including three phosphoenolpyruvate-dependent phosphotransferase systems (PTS) and a binding protein-dependent sugar uptake system for metabolizing multiple sugars, including beta-glucosides. Utilization of arbutin and esculin, aryl-beta-glucosides, was defective in some mutants. Esculin and oligochitosaccharides induced genes in one of the three beta-glucoside metabolism PTS and in four other genetic loci. Mutation of genes in any of the four systems affected in vitro adhesion to sHA, biofilm formation on plastic surfaces, and/or growth rate in liquid medium. Therefore, genes associated with beta-glucoside metabolism may regulate S. gordonii in vitro adhesion, biofilm formation, growth, and in vivo colonization.     

Directed formation of chromosomal deletions in Salmonella typhimurium : targeting of specific genes induced during infection

In vivo expression technology (IVET) has resulted in the isolation of more than 100 Salmonella typhimurium genes that are induced during infection. Many of these in vivo induced (ivi) genes, as well as other virulence genes, are clustered in regions of the chromosome that are specific for Salmonella and are not present in Escherichia coli (e.g., pathogenicity islands). It would be desirable to be able to delete such putative virulence regions of the chromosome, and if the deletion removes genes that play a role in pathogenesis subsequent efforts can then be focused on individual genes that reside within that region. We therefore have developed a strategy for constructing chromosomal deletions which are not limited in size, have defined endpoints with a selectable marker at the joint point, and are not dependent on prior knowledge of sequences contained within the deleted region. Such deletion strategies can be applied to almost any bacterium with homologous recombination and to plasmid-based mutational systems where homologous recombination is not desired or feasible.     

Strategies for isolation of in vivo expressed genes from bacteria

The discovery and characterization of genes specifically induced in vivo upon infection and/or at a specific stage of the infection will be the next phase in studying bacterial virulence at the molecular level. Genes isolated are most likely to encode virulence-associated factors or products essential for survival, bacterial cell division and multiplication in situ. Identification of these genes is expected to provide new means to prevent infection, new targets for, antimicrobial therapy, as well as new insights into the infection process. Analysis of genes and their sequences initially discovered as in vivo induced may now be revealed by functional and comparative genomics. The new field of virulence genomics and their clustering as pathogenicity islands makes feasible their in-depth analysis. Application of new technologies such as in vivo expression technologies, signature-tagged mutagenesis, differential fluorescence induction, differential display using polymerase chain reaction coupled to bacterial genomics is expected to provide a strong basis for studying in vivo induced genes, and a better understanding of bacterial pathogenicity in vivo. This review presents technologies for characterization of genes expressed in vivo.     

致病菌体内诱导基因的功能分析—体内表达技术的应用

病原菌体内诱导的基因在致病过程中起重要作用,体内表达技术是一类很有前景的研究体内诱导基因的技术,本介绍了体内表达技术的基本原理,类型以及在致病菌体内诱导基因方面的研究进展和应用前景。     

Directed formation of chromosomal deletions in Salmonella typhimurium : targeting of specific genes induced during infection

In vivo expression technology (IVET) has resulted in the isolation of more than 100 Salmonella typhimurium genes that are induced during infection. Many of these in vivo induced (ivi) genes, as well as other virulence genes, are clustered in regions of the chromosome that are specific for Salmonella and are not present in Escherichia coli (e.g., pathogenicity islands). It would be desirable to be able to delete such putative virulence regions of the chromosome, and if the deletion removes genes that play a role in pathogenesis subsequent efforts can then be focused on individual genes that reside within that region. We therefore have developed a strategy for constructing chromosomal deletions which are not limited in size, have defined endpoints with a selectable marker at the joint point, and are not dependent on prior knowledge of sequences contained within the deleted region. Such deletion strategies can be applied to almost any bacterium with homologous recombination and to plasmid-based mutational systems where homologous recombination is not desired or feasible. Received: 6 October 1997 / Accepted: 30 December 1997     

Identification and analysis of bacterial virulence genes in vivo

Signature-tagged mutagenesis is a mutation-based screening method for the identification of virulence genes of microbial pathogens. Genes isolated by this approach fall into three classes: those with known biochemical function, those of suspected function and some whose functions cannot be predicted from database searches. A variety of in vitro and in vivo methods are available to elucidate the function of genes of the second and third classes. We describe the use of some of these approaches to study the function of the Salmonella pathogenicity island 2 type III secretion system of Salmonella typhimurium. This virulence determinant is required for intracellular survival. Secretion by this system is induced by an acidic pH, and its function may be to alter trafficking of the Salmonella-containing vacuole. Use of a temperature-sensitive non-replicating plasmid and competitive index tests with other genes show that in vivo phenotypes do not always correspond to those predicted from in vitro studies.     

A subset of Actinobacillus pleuropneumoniae in vivo induced promoters respond to branched-chain amino acid limitation

Actinobacillus pleuropneumoniae is the causative agent of a necrotizing hemorrhagic pleuropneumonia in swine. In this study, we investigate the possibility that the limitation of branched-chain amino acids is a stimulus that A. pleuropneumoniae will encounter during infection and will respond to by up-regulation of genes involved in branched-chain amino acid biosynthesis and virulence. Actinobacillus pleuropneumoniae genetic loci that are specifically induced during infection were screened in vitro for expression in response to limitation of branched-chain amino acids. Of 32 in vivo induced promoter clones screened in vitro, eight were induced on chemically defined medium without isoleucine, leucine and valine as compared to complete chemically defined medium. We identify the genomic context of each clone and discuss its relevance to branched-chain amino acid limitation and virulence. We conclude that limitation of branched-chain amino acids is a cue for expression of a subset in vivo induced genes, including not only genes involved in the biosynthesis of branched-chain amino acids, but also other genes that are induced during infection of the natural host. These results suggest that limitation of branched-chain amino acids may be one of an array of environmental cues responsible for the induction of virulence-associated genes in A. pleuropneumoniae.     

A comparison study of Agrobacterium-mediated transformation methods for root-specific promoter analysis in soybean

Key message

Both in vitro and in vivo hairy root transformation systems could not replace whole plant transformation for promoter analysis of root-specific and low-P induced genes in soybean.

Abstract

An efficient genetic transformation system is crucial for promoter analysis in plants. Agrobacterium-mediated transformation is the most popular method to produce transgenic hairy roots or plants. In the present study, first, we compared the two different Agrobacterium rhizogenes-mediated hairy root transformation methods using either constitutive CaMV35S or the promoters of root-preferential genes, GmEXPB2 and GmPAP21, in soybean, and found the efficiency of in vitro hairy root transformation was significantly higher than that of in vivo transformation. We compared Agrobacterium rhizogenes-mediated hairy root and Agrobacterium tumefaciens-mediated whole plant transformation systems. The results showed that low-phosphorous (P) inducible GmEXPB2 and GmPAP21 promoters could not induce the increased expression of the GUS reporter gene under low P stress in both in vivo and in vitro transgenic hairy roots. Conversely, GUS activity of GmPAP21 promoter was significantly higher at low P than high P in whole plant transformation. Therefore, both in vitro and in vivo hairy root transformation systems could not replace whole plant transformation for promoter analysis of root-specific and low-P induced genes in soybean.