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Genetic and molecular analysis of bacteriocins of enterococcus clinical isolates-a bacterial colonization factor-

肠球菌临床分离株细菌素的遗传和分子分析-细菌定植因子-

基本信息

批准号：
19390123
负责人：
IKE Yasuyoshi
金额：
$ 11.98万
依托单位：
Gunma University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2007
资助国家：
日本
起止时间：
2007 至 2009
项目状态：
已结题

项目摘要

1. The conjugative plasmid pYI14 (61kbp) was isolated from Enterococcus faecalis YI714, a clinical isolate. pYI14 conferred a pheromone response on its host and encoded bacteriocin 41 (bac41). Bacteriocin 41 (Bac41) only showed activity against E.faecalis. Genetic analysis showed that the determinant was located in a 6.6-kbp region Six open reading frames (ORFs) were identified in this region and designated ORF7 (bacL_1) ORF8 (bacL_2), ORF9, ORF10, ORF11 (bacA), and ORF12 (bacI). They were aligned in this order and oriented in the same direction. ORFs bacL_1, bacL_2, bacA, and bacI were essential for expression of the bacteriocin in E.faecalis. Extracellular complementation of bacteriocin expression was possible for bacL_1 and -L_2 and bacA mutants. bacL_1 and -L_2 and bacA encoded bacteriocin component L and activator component A, respectively. The products of these genes are secreted into the culture medium and extracellularly complement bacteriocin expression. bacI encoded immunity, … More providing the host with resistance to its own bacteriocin activity.2. The bacteriocin 51 (Bac51) was encoded on the mobilizable plasmid pHY (6,037bp) isolated from vancomycin resistant E.faecium V38. Bacteriocin 51 was active against E.faecium, E.hirae, and E.duraus. The sequence analysis of pHY showed that plasmid pHY encoded nine ORFs from ORF1 to ORF9 in this order. ORF3, ORF4 and ORF5 showed homology to mobC, mobA, and mobB, respectively. ORF7 and ORF8 showed homology to repA and repB, respectively. ORF1, ORF2, ORF6 and ORF9 had no homology to the reported genes. The Tn5 insertion mutants in ORF1 did not show both bacteriocin and immunity activities, implying that ORF1 and ORF2 were the bacteriocin determinant, and were designated as bacA and bacB, respectively. Detailed DNA sequence analysis of bacA and bacB was performed. bacA encoded a 144-amino-acid protein. The ATG start codon was preceded by a potential ribosome binding site 8 bp upstream. The deduced bacA protein had a span of hydrophobic residues typical of the signal sequence at its amino terminus and a potential signal peptidase processing site corresponding to the VEA sequence was located at position 37 to 39 amino acid. The predicted BacA mature protein consisted of 105 amino acids. There was a potential promoter sequence upstream of the start codon. bacB encoded a 55 amino acid protein. The ATG start codon was preceded by a potential ribosome binding site 12 bp upstream. There was no obvious promoter sequence upstream of the ribosome binding site. A putative transcription terminator signal was identified downstream of bacB. There was no obvious promoter or terminator sequence between bacA and bacB. The transcript of bacA and bacB were analyzed by Northern hybridization. The results of Northern analysis of bacA and bacB with bacA-RNA probe showed about 700 nucleotides which corresponded to the nucleotide size of both bacA and bacB, indicating that a transcription initiated from the promoter upstream bacA, continued through bacB, and terminated at the terminator downstream of bacB. The transcription start site was determined at the nucleotide T located at six nucleotides downstream from -10 promoter sequence by Rapid Amplification of cDNA Ends (RACE) method. These results indicated that the bacA and bacB composed of operon structure, and bacA was bacteriocin structure gene and bacB was the immunity gene. Less

1.从粪肠球菌YI714临床分离株中分离到接合质粒pYI14(61kbp)。PYI14对寄主产生信息素反应，编码细菌素41(Bac41)。细菌素41(Bac41)只对粪肠球菌有抗菌活性。基因分析表明，该决定簇位于6.6kbP区域，共鉴定出6个开放阅读框，分别命名为ORF7(BaCl1)、ORF8(BaCl2)、ORF9、ORF10、ORF11(BaA)和ORF12(BaCI)。它们按此顺序排列，并朝向相同的方向。BaCl1、BaCl2、BaCa和BaCi是细菌素在粪肠球菌中表达所必需的ORF。BaCl1、-L 2和bacA突变体细菌素表达的胞外互补是可能的。BaCl1、-L 2和BaCa分别编码细菌素组分L和激活组分A。这些基因的产物被分泌到培养基中，并在细胞外补充细菌素的表达。BACI编码免疫，…更多地为寄主提供对自身细菌素活性的抗性。细菌素51(baciocin 51，Bac51)编码于从耐万古霉素的屎埃希菌V38中分离到的可动员质粒PHY(6037bp)上。细菌素51对粪肠球菌、平肠球菌和杜老肠杆菌有较强的抗菌活性。序列分析表明，质粒PHY依次编码ORF1~ORF9的9个ORF。ORF3、ORF4和ORF5分别与mobC、MOBA和Mobb同源。ORF7和ORF8分别与REPA和RepB同源。ORF1、ORF2、ORF6和ORF9与已报道的基因没有同源性。ORF1中的Tn5插入突变体没有细菌素和免疫活性，表明ORF1和ORF2是细菌素决定簇，分别命名为BACA和BACB。对BAKA和BACB进行了详细的DNA序列分析。BACA编码一个144个氨基酸的蛋白质。在ATG起始密码子之前，上游有一个潜在的核糖体结合位点8bp。推导出的BacA蛋白在其氨基末端有一段典型的信号序列的疏水残基，与VEA序列相对应的潜在信号肽加工位点位于第37-39位氨基酸。预测的BAKA成熟蛋白由105个氨基酸组成。在起始密码子的上游有一个潜在的启动子序列。BACB编码一个55个氨基酸的蛋白质。在ATG起始密码子之前有一个潜在的核糖体结合位点，其上游为12个碱基。核糖体结合位点上游没有明显的启动子序列。在BACB下游发现了一个可能的转录终止信号。BACA和BACB之间没有明显的启动子和终止子序列。用Northern杂交技术分析BACA和BACB的转录本。用BACA-RNA探针对BAKA和BACB进行Northern分析，结果显示约有700个核苷酸，与BACA和BACB的核苷酸大小一致，表明转录起始于BACA上游的启动子，继续穿过BACB，终止于BACB下游的终止子。采用快速扩增-10启动子末端(RACE)的方法，确定转录起始点位于-10启动子下游6个核苷酸的T。这些结果表明，BACA和BACB由操纵子结构组成，BACA是细菌素结构基因，BACB是免疫基因。较少

项目成果

期刊论文数量（0）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Nationwide epidemiological study revealed the dissemination of meticillin-resistant Staphylococcus aureus carrying a specific set of virulence-associated genes in Japanese hospitals.

全国流行病学研究表明，携带一组特定毒力相关基因的耐甲氧西林金黄色葡萄球菌在日本医院中传播。

DOI：
发表时间：
2009
期刊：
Journal of Medical Microbiology 58
影响因子：
0
作者：
Ohkura;T.;K. Yamada;A. Okamoto;H. Baba;Y. Ike;Y. Arakawa;T. Hasegawa;M. Ohta.
通讯作者：
M. Ohta.

Vancomycin-Resistant Enterococci(VRE)in Japan

日本耐万古霉素肠球菌（VRE）