利用Red重组系统对大肠杆菌ClpP基因的敲除

利用含有质粒pKD4 6的菌株BW2 5 113,在阿拉伯糖诱导后 ,表达λ噬菌体的 3个重组蛋白 ,宿主菌就具有了同源重组的能力 .设计的引物 5′端有 5 0bp的拟敲除基因的同源臂 ,3′端为扩增引物 ,以pKD3为模板 ,扩增两侧含FRT位点的氯霉素抗性基因 ,将此线性片段电转入具重组功能的感受态细胞 ,利用氯霉素平板就可以筛选到阳性转化体 .再利用表达Flp重组酶的质粒pCP2 0 ,可将FRT位点之间的氯霉素抗性基因删除 .利用该重组系统 ,构建了ClpP蛋白酶缺失的大肠杆菌工程菌株 ,可望在减少外源蛋白的降解方面发挥一定的作用 .     

志贺菌毒力大质粒大片段敲除方法的建立

目的:构建志贺菌毒力大质粒大片段缺失突变体库。方法:首先利用λ-Red重组系统构建弗氏2a志贺菌301株毒力大质粒特定位点缺失株,再在距离此位点20 kb处缺失另一突变位点,最后根据重组酶识别远端FRT位点的特性,将两个远端FRT位点之间的DNA序列全部缺失。结果:敲除了毒力大质粒24 kb的DNA序列。结论:利用λ-Red重组系统及FLP-FRT位点特异性识别重组系统可以对志贺菌毒力大质粒逐步进行大片段的敲除,构建大质粒大片段缺失突变体库。     

弗氏志贺菌 M90T 株毒力相关膜蛋白复合物组成基因缺失突变体的构建

目的：构建弗氏志贺菌M90T株毒力相关膜蛋白复合物组成蛋白基因的缺失突变株。方法：分别扩增目的基因的上、下游同源臂和卡那霉素抗性基因片段,利用重叠延伸PCR技术将这3个片段融合为打靶片段,电击转化M90T／pKD46感受态细胞,在bRed重组系统的作用下,通过同源重组将目的基因置换为卡那霉素抗性基因,之后在辅助质粒pCP20的作用下切除FRT位点之间的卡那霉素抗性基因,得到基因缺失突变体。结果与结论：分别构建了M90T株毒力相关膜蛋白复合物4个组成蛋白Apy、DnaK、CIpB、YdgA的基因缺失突变体,为进一步研究各基因的功能提供了突变体。     

λRed重组系统用于沙门菌质粒毒力基因spvC敲除株的构建

[目的]利用λRed重组系统敲除沙门菌质粒毒力基因spvC。[方法]首先以质粒p KD4为模板,扩增得到两侧含spvC同源臂、中间为卡那霉素抗性基因的线性DNA片段。再将此线性片段电转入具重组功能的感受态沙门菌菌株,发生重组后,卡那霉素平板筛选阳性转化子。最后利用表达FLP重组酶的质粒p CP20,将FRT位点之间的卡那霉素抗性基因消除,用PCR鉴定。Western Blot检测野生沙门菌和spvC敲除株感染的He La细胞ERK磷酸化水平。[结果]沙门菌质粒毒力基因spvC敲除株构建成功,spvC敲除株感染的He La细胞内ERK磷酸化水平升高。[结论]成功构建沙门菌质粒毒力基因spvC敲除株,验证了spvC基因的功能。     

一种快速、精确构建大肠杆菌组氨酸营养缺陷型的方法*

将表达Red体内重组蛋白的质粒pKD46转化大肠杆菌DH5α,用 5′端与组氨酸基因同源 ,3′端与卡那霉素抗性基因同源的引物获得具有卡那霉素抗性基因的PCR产物 ,然后电击转化DH5α,在λRed重组系统的帮助下 ,通过卡那霉素抗性基因两侧的组氨酸基因序列在体内与大肠杆菌染色体上的组氨酸基因发生同源重组 ,置换了DH5α组氨酸操纵元中的hisDCB基因 ,最后利用卡那霉素抗性基因两端的FRT位点 ,通过FTP位点专一性重组将卡那霉素抗性基因去除 ,最终获得了不具抗性的大肠杆菌组氨酸营养缺陷型菌株。为在大     

利用λRed重组系统敲除鼠伤寒沙门氏菌sopB基因

利用λRed重组系统敲除鼠伤寒沙门氏菌LT2的(Salmonella enterica serovar typhimurium LT2,S.typhimurium LT2)sopB基因。以pKD4质粒为模板,扩增得到中间带有卡那霉素抗性基因且两端各带有59 bp分别与sopB基因上下游序列同源的同源打靶片段,将其转化至表达Red重组酶的S.typhimurium LT2感受态细胞中;在抗生素压力和λRed重组系统帮助下,同源片段和菌体sopB基因发生同源重组,通过卡那霉素筛选得到带有抗性标记的阳性重组菌;转入重组酶表达质粒pCP20以除去抗性标记,得到保留单一FRT位点的突变菌株;利用PCR技术鉴定重组菌,并通过检测沙门氏菌效应蛋白SopB的分泌以及沙门氏菌感染HeLa细胞后pAKT的激活反应来鉴定sopB基因是否被敲除。构建的ΔsopB突变菌株失去了分泌SopB蛋白的能力,且不能够像野生型菌株那样在感染HeLa细胞的过程中激活pAkt。本研究获得了S.typhimurium LT2的sopB基因缺失突变株,为沙门氏菌感染宿主过程中SopB的功能研究提供工具,同时也为进一步探索其他类型细菌的基因敲除提供了线索。     

一种快速、精确构建大肠杆菌组氨酸营养缺陷型的方法

将表达Red体内重组蛋白的质粒pKD46转化大肠杆菌：DH5α,用5′端与组氨酸基因同源,3′端与卡那霉素抗性基因同源的引物获得具有卡那霉素抗性基因的PCR产物,然后电击转化DH5α,在λRed重组系统的帮助下,通过卡那霉素抗性基因两侧的组氨酸基因序列在体内与大肠杆菌染色体上的组氨酸基因发生同源重组,置换了DH5α组氨酸操纵元中的hisDCB基因,最后利用卡那霉素抗性基因两端的FRT位点,通过FTP位点专一性重组将卡那霉素抗性基因去除,最终获得了不具抗性的大肠杆菌组氨酸营养缺陷型菌株。为在大肠杆菌及其他菌株中快速、精确的构建营养缺陷型菌株提供了有益的参考。     

鼠伤寒沙门菌spvBC基因编辑株的构建

利用λRed重组系统和pBAD原核表达载体构建鼠伤寒沙门菌spvBC质粒毒力基因修饰菌株,为深入探究沙门菌毒力基因spv的功能和致病机制及宿主抗感染免疫提供工具菌。以pKD4为模板,PCR扩增含spvBC同源臂的卡那霉素抗性基因以构建同源打靶片段,再将其电转入含有质粒pKD46的鼠伤寒沙门菌中进行同源重组,随后将质粒pCP20电转导入阳性转化子,消除卡那霉素抗性基因,PCR鉴定敲除株的构建。PCR扩增含酶切位点的spvBC基因片段,扩增产物与原核表达载体pBAD/gⅢ分别双酶切后连接构建pBAD-spvBC重组质粒,PCR筛选阳性菌落并测序鉴定。将构建成功的pBAD-spvBC重组质粒电转导入spvBC敲除株中,Western blot测定不同浓度L-阿拉伯糖诱导SpvB和SpvC蛋白表达情况。PCR结果表明鼠伤寒沙门菌spvBC基因敲除成功;PCR及测序结果表明pBAD-spvBC重组质粒构建成功,Western blot结果表明13 mmol/L L-阿拉伯糖可诱导SpvB和SpvC蛋白正常表达。λRed重组系统可用于沙门菌质粒上大片段基因的敲除,pBAD原核表达载体可用于沙门菌质粒上大片段基因的回补,丰富了细菌质粒的基因修饰和编辑策略。     

伤寒沙门菌bcfD基因敲除突变株的构建

目的:构建伤寒沙门菌Ty2菌株菌毛亚单位bcfD基因敲除突变株.方法:利用交错PCR得到bcfD基因缺失且含其两侧翼序列的片段,将该片段与pMD 18-T连接,亚克隆到pYG4,电转入大肠埃希菌S17-1/λpir菌株,阳性菌株与受体菌伤寒沙门氏菌Ty2进行固相杂交后筛选.结果:成功获得敲除bcfD基因序列954bp的敲除突变株.结论:交错PCR有利于细菌基因精确敲除突变株的构建,bcfD基因敲除株的构建将为进一步研究该基因在伤寒沙门菌中的功能奠定了基础.     

利用Red同源重组技术构建产L-苏氨酸的基因工程菌

利用Red重组技术构建不同基因突变的L-苏氨酸工程菌大肠杆菌ITHR,研究单敲除metA、ilvA和双敲除metA、ilvA基因后对L-苏氨酸积累的影响。应用质粒pKD46介导的Red同源重组系统,通过第一次同源重组将拟敲除基因替换为氯霉素抗性基因,再通过重组酶在FRT位点发生第二次同源重组,消除抗性基因,成功敲除了菌株ITHR体内苏氨酸合成的代谢旁路途径中的metA和ilvA基因,构建了三株不同的基因突变株。将携带苏氨酸操纵子的工程质粒pWYE065电转化入敲除不同基因的突变株中,构建基因工程菌。经5 L发酵罐发酵产酸实验,未敲除任何基因的菌株ITHR/pWYE065 L-苏氨酸的产量为5.55±0.51 g/L,metA基因单敲除菌株ITHR△metA/pWYE065 L-苏氨酸产量为9.77±1.83 g/L,ilvA基因单敲除菌株ITHR△ilvA/pWYE065 L-苏氨酸产量为8.65±1.42 g/L,同时敲除ilvA和metA基因的菌株ITHR△metA△ilvA/pWYE065 L-苏氨酸的产量增加到13.6±1.14 g/L。通过敲除L-苏氨酸的旁路代谢途径中的关键酶的基因,可以增强L 苏氨酸积累的效果,为L-苏氨酸工程菌的进一步改造奠定了基础。     

断奶仔猪源大肠杆菌LEE及HPI毒力岛的检测

应用Duplex_PCR方法,对240株断奶仔猪源大肠杆菌分离株的LEE毒力岛的eaeA基因和耶尔森菌强毒力岛核心区的irp2基因进行了检测,并对HPI毒力岛的fyuA基因及其在大肠杆菌染色体中的插入位置进行了分析,以及随机选取部分PCR产物进行了克隆和序列分析。结果表明:其中29株(12.08%)为LEE HPI ,39株(16.25%)为LEE ,11株(4.58%)为HPI ;另外还发现:不同病例来源的分离株之间,两种毒力岛的携带率不同;在断奶仔猪腹泻源分离株中,29株(20.71%)为LEE HPI ,22株(15.71%)为LEE ,9株(6.43%)为HPI ;断奶仔猪水肿病源分离株中,仅5株(6.58%)为LEE ,2株(2.63%)为HPI ,未发现LEE HPI 菌株;断奶仔猪水肿病并发腹泻源分离株中,仅12株(50%)为LEE ,未发现HPI 及LEE HPI 菌株。本实验克隆的eaeA(425bp)与已发表序列完全一致,irp2(280bp)f、yuA(948bp)、asn_tRNA_intB(1391bp)均与已发表的序列高度同源,同源性分别在98.2%、98.3%、95.8%以上;40株LEE HPI 或HPI 分离株中,29株(72.5%)为fyuA ,且其HPI毒力岛位于大肠杆菌染色体asn_tRNA位点。     

禽致病性大肠杆菌（CVCC1565）中耶尔森菌强毒力岛核心区的检测

采用PCR法,检测了大肠杆菌（CVCC1565）中耶尔森菌强毒力岛（high pathogenicity island,HPI）核心区的irp1、irp2、irp3、irp4、irp5及fyuA基因片段,并与小肠结肠炎耶尔森菌毒力岛的类似基因进行同源性比较。结果显示,E.coliCVCC1565菌株irp1、irp2、irp3、irp4、irp5及fyuA基因大小分别为799bp、414bp、798bp、504bp、758bp、948bp,与GenBank中公布的小肠结肠炎耶尔森菌（Yersinia enterocoliticaO：8 WA）HPI的irp1、irp2、irp3、irp4、irp5及fyuA基因同源性分别达到98%、98%、98%、95%、98%、98%。研究结果表明禽致病性大肠杆菌标准株（CVCC1565）携带耶尔森菌强毒力岛基因,这几个毒力岛基因在小肠结肠炎耶尔森菌和禽致病性大肠杆菌之间可能存在水平性转移。     

The Yersinia high-pathogenicity island is present in different members of the family Enterobacteriaceae

A pathogenicity island termed high-pathogenicity island (HPI) is present in pathogenic Yersinia. This 35 to 45 kb island carries genes involved in synthesis, regulation and transport of the siderophore yersiniabactin. Recently, the HPI was also detected in various strains of Escherichia coli. In this study, the distribution of the HPI in the family Enterobacteriaceae was investigated. Among the 67 isolates pertaining to 18 genera and 52 species tested, nine (13.4%) harbored the island. These isolates were three E. coli, one Citrobacter diversus and five Klebsiella of various species (Klebsiella pneumoniae, Klebsiella rhinoscleromatis, Klebsiella ozaenae, Klebsiella planticola, and Klebsiella oxytoca). As in Yersinia sp., all nine isolates synthesized the HPI-encoded iron-repressible proteins HMWP1 and HMWP2. In the K. oxytoca strain, the right-end portion of the HPI was deleted, whereas the entire core region of the island was present in the eight other enterobacteria strains analyzed. In most of these isolates, the HPI was bordered by an asn tRNA locus, as in Yersinia sp. This report thus demonstrates the spread of the HPI among various members of the family Enterobacteriaceae.     

The Yersinia high-pathogenicity island: an iron-uptake island.

Highly pathogenic Yersinia carry a pathogenicity island termed high-pathogenicity island (HPI). The Yersinia HPI comprises genes involved in the synthesis of the siderophore yersiniabactin and can thus be regarded as an iron-uptake island. A unique characteristic of the HPI is its wide distribution among different enterobacteria such as Escherichia coli, Klebsiella, Citrobacter and Salmonella. Other types of iron-uptake systems are also carried by different pathogenicity islands in enterobacteria.     

A novel integrative and conjugative element (ICE) of Escherichia coli: the putative progenitor of the Yersinia high-pathogenicity island

Diversification of bacterial species and pathotypes is largely caused by horizontal transfer of diverse DNA elements such as plasmids, phages and genomic islands (e.g. pathogenicity islands, PAIs). A PAI called high-pathogenicity island (HPI) carrying genes involved in siderophore-mediated iron acquisition (yersiniabactin system) has previously been identified in Yersinia pestis, Y. pseudotuberculosis and Y. enterocolitica IB strains, and has been characterized as an essential virulence factor in these species. Strikingly, an orthologous HPI is a widely distributed virulence determinant among Escherichia coli and other Enterobacteriaceae which cause extraintestinal infections. Here we report on the HPI of E. coli strain ECOR31 which is distinct from all other HPIs described to date because the ECOR31 HPI comprises an additional 35 kb fragment at the right border compared to the HPI of other E. coli and Yersinia species. This part encodes for both a functional mating pair formation system and a DNA-processing region related to plasmid CloDF13 of Enterobacter cloacae. Upon induction of the P4-like integrase, the entire HPI of ECOR31 is precisely excised and circularised. The HPI of ECOR31 presented here resembles integrative and conjugative elements termed ICE. It may represent the progenitor of the HPI found in Y. pestis and E. coli, revealing a missing link in the horizontal transfer of an element that contributes to microbial pathogenicity upon acquisition.     

The Yersinia high-pathogenicity island.

A pathogenicity island present only in highly pathogenic strains of Yersinia (Y. enterocolitica 1B, Y. pseudotuberculosis I and Y. pestis) has been identified on the chromosome of Yersinia spp. and has been designated High-Pathogenicity Island (HPI). The Yersinia HPI carries a cluster of genes involved in the biosynthesis, transport and regulation of the siderophore yersiniabactin. The major function of this island is thus to acquire iron molecules essential for in vivo bacterial growth and dissemination. The presence of an integrase gene and att sites homologous to those of phage P4, together with a G + C content much higher than the chromosomal background, suggests that the HPI is of foreign origin and has been acquired by chromosomal integration of a phage. The HPI can excise from the chromosome of Y. pseudotuberculosis and is found inserted into any of the three copies of the asn tRNA loci present in this species. A unique characteristic of the HPI is its wide distribution in various enterobacteria. Although first identified in Yersinia spp., it has subsequently been detected in other genera such as E. coli, Klebsiella and Citrobacter.     

禽源大肠杆菌的分离及其毒力因子的检测

从临床疑似大肠杆菌感染的病禽组织中分离到69株细菌(其中鹅源29株,鸡源40株);通过常规形态学、培养特性和生化特征的研究,确定为大肠杆菌。PCR检测表明,其中46株(66.7%)为F1 大肠杆菌,10株(14.5%)为F1 HPI 大肠杆菌,2株(2.9%)为HPI 大肠杆菌;通过比较还发现,F1菌毛和HPI在鹅源和鸡源大肠杆菌中以及不同脏器来源的菌株中具有相似的分子流行病学。O抗原鉴定结果表明鹅源大肠杆菌的O抗原型主要有O26、O78、O18、O117,鸡源大肠杆菌的O抗原型主要有O109、O24、O18、O139、O78。药敏试验表明,其中绝大多数菌株对先锋霉素V、呋喃妥因、庆大霉素敏感,对环丙沙星因菌株差异而不同,林可霉素、四环素、多粘菌素多不敏感。     

Presence of virulence and fitness gene modules of enterohemorrhagic Escherichia coli in atypical enteropathogenic Escherichia coli O26

Enterohemorrhagic Escherichia coli (EHEC) strains of serogroup O26 cause hemolytic-uremic syndrome (HUS) whereas atypical enteropathogenic E. coli (aEPEC) O26 typically cause uncomplicated diarrhea but have been also isolated from HUS patients. To gain insight into the virulence of aEPEC O26, we compared the presence of O island (OI) 122, which is associated with enhanced virulence in EHEC strains, among aEPEC O26 and EHEC O26 clinical isolates. We also tested these strains for the high pathogenicity island (HPI) which is a fitness island. All 20 aEPEC O26 and 20 EHEC O26 investigated contained virulence genes located within OI-122 (efa1/lifA, nleB, nleE, ent). In both aEPEC O26 and EHEC O26, OI-122 was linked to the locus for enterocyte effacement, forming a mosaic island which was integrated in pheU. Moreover, strains of these two pathotypes shared a conserved HPI. These data support a close relatedness between aEPEC O26 and EHEC O26 and have evolutionary implications. The presence of OI-122 in aEPEC O26 might contribute to their pathogenic potential.     

The Yersinia high-pathogenicity island is highly predominant in virulence-associated phylogenetic groups of Escherichia coli

The high-pathogenicity island (HPI) present in pathogenic Yersinia and encoding the siderophore yersiniabactin, has recently been identified in the asnT tRNA region of various Escherichia coli pathotypes, especially those responsible for bacteremia and urosepsis. Most E. coli strains causing such extra-intestinal infections belong to phylogenetic groups B2 and D. In this study we investigated (i) the distribution and localization of HPI among the different E. coli phylogenetic groups, using the ECOR reference collection; and (ii) the prevalence of HPI among a set of 124 phylogenetically characterized E. coli strains responsible for neonatal meningitis. Ninety-three percent of the ECOR strains belonging to groups B2 and D harbored HPI. In contrast, the island was present in 32% and 25% of strains belonging to groups A and B1, respectively, which are considered to be non-pathogenic. HPI was found in 100% of the neonatal meningitis strains, 13 of which belonged to groups A and B1, suggesting that HPI might contain virulent factors required for the development of neonatal meningitis. Moreover, we observed for the first time that HPI can be inserted in a site different from the asnT tRNA region.