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

1.从临床分离的粪肠球菌YI 714中分离到接合质粒pYI 14（61 kbp）。pYI 14能使宿主产生信息素反应，并编码细菌素41（bac 41）。细菌素41（Bac 41）仅对粪肠球菌表现出活性。在该区域内鉴定出6个开放阅读框（ORF），分别命名为ORF 7（bacL_1）、ORF 8（bacL_2）、ORF 9、ORF 10、ORF 11（巴卡A）和ORF 12（bacI）。它们按这个顺序排列，朝向同一个方向。ORF bacL_1、bacL_2、巴卡A和bacI是粪肠球菌中细菌素表达所必需的。bacL_1和-L_2以及巴卡A突变体的细菌素表达可能存在胞外互补。bacL_1、-L_2和巴卡A分别编码细菌素组分L和激活剂组分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表达。bacI编码免疫， ...更多信息 为宿主提供对其自身细菌素活性的抗性。细菌素51（Bac 51）编码在从万古霉素抗性屎肠球菌V38分离的可移动质粒pHY（6，037 bp）上。细菌素51对屎肠球菌、肠球菌和硬肠球菌有活性。序列分析表明，pHY质粒共编码9个ORF，从ORF 1到ORF 9。ORF 3、ORF 4和ORF 5分别与mobC、mobA和mobB具有同源性。ORF 7和ORF 8分别与repA和repB具有同源性。ORF 1、ORF 2、ORF 6和ORF 9与已报道的基因没有同源性。ORF 1和ORF 2的Tn 5插入突变体均不具有细菌素和免疫活性，表明ORF 1和ORF 2是细菌素决定簇，分别命名为巴卡A和bacB。对巴卡A和巴卡B进行了详细的DNA序列分析。巴卡编码一个144个氨基酸的蛋白质。ATG起始密码子之前是上游8bp的潜在核糖体结合位点。推导的巴卡蛋白在其氨基末端具有典型的信号序列的疏水残基跨度，并且对应于VEA序列的潜在信号肽酶加工位点位于第37至39位氨基酸处。预测的巴卡成熟蛋白由105个氨基酸组成。在起始密码子的上游存在一个潜在的启动子序列。bacB编码一个55个氨基酸的蛋白质。ATG起始密码子之前是一个潜在的核糖体结合位点上游12 bp。核糖体结合位点上游无明显的启动子序列。在bacB下游鉴定了推定的转录终止子信号。在巴卡A和bacB之间没有明显的启动子或终止子序列。北方杂交分析巴卡和bacB的转录本。用bacA-RNA探针对巴卡和bacB的北方分析结果显示，约700个核苷酸，与巴卡和bacB的核苷酸大小相对应，表明转录起始于巴卡上游的启动子，继续通过bacB，并终止于bacB下游的终止子。通过cDNA末端快速扩增（RACE）方法，在位于-10启动子序列下游6个核苷酸的核苷酸T处确定转录起始位点。这些结果表明，巴卡A和bacB由操纵子结构组成，其中巴卡A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