Analyses of Clustered Inversion Region in Incl Plasmids.

Shufflon，或簇状反转区，是在IncI1质粒R64中发现的一种新型DNA重排。R64的洗牌子由四个DNA片段组成，分别命名为A、B、C和D，它们的DNA长度和序列不同。这四个DNA片段位于两侧，由7个19个碱基的重复序列分隔。任何两个反向重复序列之间的特定位点重组会导致复杂的DNA重排。对开放阅读框的分析表明，改组是选择基因7个C末端之一的生物开关，通过构建一系列R64的缺失衍生品，负责接合转移的区域位于复制区右侧的54kb DNA区域内。我们测定了负责形成细菌毛的19kb DNA区域的核苷酸序列，并发现18个开放阅读框架(ORF)存在于同一方向。Pliv基因是18个ORF中的最后一个。通过引入删除和插入…在所有18个ORF中都有更多的基因突变，我们证明了18个ORF中的大多数都是形成细菌毛和配偶转移所必需的。为了揭示DNA重排在改组区域中的作用，我们构建了PilV基因C末端固定的R64衍生物。携带带有7个固定PilV C末端的R64衍生质粒的供体细胞与几个受体菌株之一交配，包括E.Coli K-12、B和C、鼠伤寒沙门氏菌LT2和福氏志贺氏菌。结果表明，在供体中，液体交配的转移频率因受体菌株的组合和PilV基因的可变C末端而发生显著变化，提示在R64中，改组决定了液体交配的受体专一性。我们还对位于R64转移区另一端的ORIT区进行了测序，发现了两个开放阅读框，命名为Nika和nikB。Nika和nikB被发现对于R64的转移是必不可少的，并且被证明作为ORIT特有的镍酶一起发挥作用。较少

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