Analysis of Conjugal Transfer Region of Plasmid R64.

细菌接合是一个复杂的过程，通过细胞间的直接接触将质粒DNA从一个细胞转移到另一个细胞。这一过程通常是由接合质粒介导的。R64是一个122 kb的接合质粒，属于不亲和型I1。通过构建R64的一系列缺失衍生体，将负责接合转移的DNA区域定位在复制区右侧的54 kb区域。为了弄清R64转移区的基因组织，我们测定了它的整个核苷酸序列。R64转移区由54,089个核苷酸组成，虽然在某些区域只有一条链的序列被确定。除了没有开放阅读框架的2.2kb空白区外，在整个转移区序列上至少发现了39个开放阅读框架(ORF)。除nikab基因外，所有基因都位于同一方向。TraABCD基因存在于转运区的一端，TraBC基因似乎是转移性基因表达的正调控因子。菌毛V基因由一个负责细菌毛形成的操纵子组成。PilV基因是该操纵子的最后一个操纵子，受Shufflon控制。R64Shufflon由4个DNA片段组成，两侧各有7个19个碱基的重复序列。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。改组基因被认为通过选择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倒置由RCI基因介导，RCI基因位于重组区附近。三个转移基因，traEFG，似乎构成了RCI的操纵子。编码EDTA抗性核酸酶的nuc基因和编码DNA引物酶的sog基因与TRAH-P共同构成操纵子。该操纵子受traBC基因控制。在R64转移区的另一端存在ORIT操纵子，它构成了转移区、ORIT和nikab基因的起源。

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.