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

1. 结合质粒pYI14 （61kbp）从粪肠球菌YI714临床分离株中分离得到。pYI14向宿主传递信息素反应并编码细菌素41 （bac41）。细菌素41 （Bac41）仅对粪肠杆菌有活性。遗传分析表明，该区域共鉴定出6个开放阅读框（orf），分别为ORF7 （bacL_1）、ORF8 （bacL_2）、ORF9、ORF10、ORF11 （bacA）和ORF12 （bacI）。它们按这个顺序排列，朝向相同的方向。orf bacL_1、bacL_2、bacA和bacI在粪肠杆菌中表达细菌素是必需的。bacL_1、-L_2和bacA突变体的细菌素表达可以在细胞外互补。bacL_1、-L_2和bacA分别编码细菌素成分L和激活剂成分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…更多的是为宿主提供抵抗自身细菌素活性的能力。细菌素51 （Bac51）编码在耐万古霉素大肠杆菌V38的可移动质粒pHY （6037bp）上。细菌素51对粪肠杆菌、hirae肠杆菌和duraus肠杆菌均有抑制作用。pHY的序列分析显示，质粒pHY编码ORF1到ORF9的9个orf。ORF3、ORF4、ORF5分别与mobC、mobA、mobB具有同源性。ORF7和ORF8分别与repA和repB具有同源性。ORF1、ORF2、ORF6和ORF9与报道的基因无同源性。ORF1的Tn5插入突变体没有同时表现出细菌素和免疫活性，这意味着ORF1和ORF2是细菌素的决定因素，分别被命名为bacA和bacB。对bacA和bacB进行详细的DNA序列分析。双歧杆菌编码144个氨基酸的蛋白质。ATG起始密码子在上游8bp处有一个潜在的核糖体结合位点。推导出的bacA蛋白在其氨基端具有典型信号序列的疏水残基，并且与VEA序列对应的潜在信号肽酶加工位点位于第37至39个氨基酸位置。预测的BacA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55个氨基酸的蛋白质。ATG起始密码子在上游12bp处有一个潜在的核糖体结合位点。核糖体结合位点上游没有明显的启动子序列。在bacB的下游发现了一个假定的转录终止信号。bacA和bacB之间没有明显的启动子和终止子序列。用Northern杂交分析了bacA和bacB的转录本。利用bacA- rna探针对bacA和bacB进行Northern分析，结果显示大约有700个核苷酸与bacA和bacB的核苷酸大小相对应，表明转录从bacA上游启动子开始，继续通过bacB，并在bacB下游终止子终止。利用快速扩增cDNA Ends （RACE）方法确定转录起始位点在-10启动子序列下游6个核苷酸的核苷酸T上。这些结果表明，bacA和bacB由操纵子结构组成，bacA为细菌素结构基因，bacB为免疫基因。少

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