Analyses of Clustered Inversion Region in Incl Plasmids.

Shufflon，或集群倒置区，是在IncI1质粒R64中发现的一种新型DNA重排。R64的洗牌子由4个DNA片段组成，分别为A、B、C和D，它们的DNA长度和序列不同。这四个DNA片段被7个19bp的重复序列所分隔。任何两个反向重复序列之间的位点特异性重组都会导致复杂的DNA重排。对开放阅读框的分析表明，沙弗伦是一种生物开关，用于选择基因的7个c末端pilV中的一个。通过构建质粒R64的一系列缺失衍生物，在复制区右侧54kb的DNA区域内定位了一个负责共轭转移的区域。我们确定了一个负责薄菌形成的19kb DNA区域的核苷酸序列，发现在相同的方向上存在18个开放阅读框（orf）。pliv基因是18个orf中的最后一个。通过在所有18个orf中引入缺失和插入突变，我们证明了18个orf中的大多数是形成薄菌毛和进行配偶转移所必需的。为了揭示DNA重排在shufflon区域的作用，我们构建了固定pilv基因c端的R64衍生物。将携带R64衍生质粒的供体细胞与大肠杆菌K-12、B和C、鼠伤寒沙门菌LT2和福氏志贺氏菌等受体细菌进行配对。结果表明，受体菌株与供体中pilv基因的可变c端结合后，液体配种的传递频率发生了显著变化，表明沙洗子决定了质粒R64中液体配种的受体特异性。我们还对R64转移区另一端的oriT区进行了测序，发现了两个开放阅读帧，分别命名为nikA和nikB。NikA和nikB对于R64的转移是不可或缺的，并被证明作为一种特异性的镍酶一起起作用。少

Shufflon, or clustered inversion region, is a novel type of DNA rearrangement found in the IncI1 plasmid R64. The shufflon of R64 consists of four DNA segments, designated A, B, C and D, which differ in DNA length and sequence. The four DNA segments are flanked and separated by seven 19-bp repeat sequences. Site-specific recombination between any two inverted repeats results in a complex DNA rearrangement. An analysis of open reading frames revealed that the shufflon is a biological switch to select one of the seven C-termini of a gene, pilV.By constructing a series of deletion-derivatives of plasmid R64, a region responsible for the conjugal transfer was located within a 54-kb DNA region to the right of replication region. We determined the nucleotide sequence of a 19-kb DNA region responsible for the formation of thin pilus, and found that 18 open reading frames (ORFs) are present in the same orientation. The pliv gene is the last one of the 18 ORFs. By introducing deletion and inser … More tion mutations into all the 18 ORFs, we demonstrated that most of the 18 ORFs are required for the formation of thin pilus and for the conjugal transfer. To reveal the role of the DNA rearrangement in the shufflon region, we constructed R64 derivatives where the C-termini of the pilv gene are fixed. Donor cells carrying R64 derivative plasmids with the seven fixed pilv C-termini were mated with one of several recipient bacterial strains including E. Coli K-12, B and C, Salmonella typhimurium LT2 and Shigella flexneri. The results indicated that the transfer frequencies of liquid mating dramatically change according to the combination of recipient strains and variable C-terminiof the pilv gene in the donors, suggesting that the shufflon determines the recipient specificity in liquid mating in plasmid R64. We also sequenced the oriT region located at the other end of transfer region of R64 and found two open reading frames, designated nikA and nikB. NikA and nikB were found indispensable for the transfer of R64 and were demonstrated to function together as an oriT-specific nickase. Less