Analysis of Conjugal Transfer Region of Plasmid R64.

细菌接合是一个复杂的过程，其中质粒DNA通过直接细胞与细胞接触从一个细胞转移到另一个细胞。这一过程通常由接合质粒介导。R64是一个122 kb的接合质粒，属于不亲和组I1。通过构建一系列质粒R64的缺失衍生物，将负责接合转移的DNA区域定位在复制区右侧的54 kb区域内。为了阐明R64转移区的基因结构，我们测定了其全核苷酸序列。R64转移区由54，089 bp组成，尽管在某些区域中仅确定了一条链的序列。在转移区的整个序列上发现至少39个开放阅读框（ORF），除了没有ORF的2.2-kb缺口区。除nikAB基因外，所有基因都位于同一方向。traABCD基因存在于转移区的一端，traBC基因似乎起着转移基因表达的正调节剂的作用。pilI-V基因由负责薄菌毛形成的操纵子组成。pilV基因是该操纵子的最后一个，受shufflon基因控制。R64 shufflon由四个DNA片段组成，它们被七个19-bp重复序列侧翼和分隔。任何两个反向重复序列之间的位点特异性重组导致复杂的DNA重排。人们认为，shufflon通过选择pilV基因的七个C末端之一来决定R64液体交配中的受体特异性。DNA倒位由位于shufflon区附近的rci基因介导。三个转移基因，traEFG，似乎构成了一个操纵子与rci。编码EDTA抗性核酸酶的nuc基因和编码DNA引发酶的sog基因与traH-P一起形成操纵子。在R64转移区的另一端，存在oriT操纵子，其构成转移、oriT和nikAB基因的起点。

Bacterial conjugation is a complex process whereby plasmid DNA is transferred from one cell to another by direct cell-to-cell contact. This process is usually mediated by conjugative plasmids. R64 is a 122-kb conjugative plasmid belonging to the incompatibility group I1.By constructing a series of deletion-derivatives of plasmid R64, a DNA region responsible for the conjugal transfer was located within a 54-kb region to the right of replication region. To clarify the gene organization of R64 transfer region, we determined its entire nucleotide sequence. R64 transfer region was shown to consist of 54, 089 bp although sequences of only one strand were determined in some regions. At least 39 open reading frames (ORFs) are found over the entire sequence of the transfer region except for 2.2-kb gap region without ORF. All genes except nikAB genes are situated in the same deirection. traABCD genes are present at one end of the transfer region and traBC genes appear to function as positive regulators of transfer gene expression. pilI-V genes consist of an operon responsible for thin pilus formation. pilV gene, the last one of this operon, is under the control of shufflon. R64 shufflon consists of four DNA segments, which are flanked and separated by seven 19-bp repeat sequences. Site-specific recombination between any two inverted repeats results in a complex DNA rearrangement. The shufflon is thought to determine the recipient specificity in R64 liquid mating, by selecting one of the seven C-termini of the pilV gene. DNA inversions are mediated by rci gene lacated adjacent to shufflon region. Three transfer genes, traEFG, seem to constitute an operon with rci. nuc gene encoding an EDTA-resistant nuclease and sog gene encoding DNA primase make an operon together with traH-P. This operon is under control of traBC gene. At the other end of R64 transfer region, oriT operon is present, which constitutes an origin of transfer, oriT, and nikAB genes.