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首页> 外文期刊>Journal of bacteriology >Structure, expression, and regulation of the kilC operon of promiscuous IncP alpha plasmids.
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Structure, expression, and regulation of the kilC operon of promiscuous IncP alpha plasmids.

机译：混杂IncPα质粒的kilC操纵子的结构，表达和调控。

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The kil-kor regulon was first identified on the broad-host-range IncP alpha plasmid RK2 by the presence of multiple kil loci (kilA, kilB, kilC, and recently kilE) that are lethal to Escherichia coli host cells in the absence of regulation by kor functions in various combinations. Whereas the kilB operon is required for mating-pair formation during conjugation, the functions encoded by the other kil loci are not known. They are not essential for replication or conjugal transfer, but their coregulation with replication and transfer genes indicates that they are likely to be important for RK2. In this report, we describe molecular and genetic studies on kilC. We determined the nucleotide sequence of the kilC region, which is located between the origin of vegetative replication (oriV) and transposon Tn1 on RK2. Primer extension analysis identified the transcriptional start site and showed that a sequence corresponding to a strong sigma 70 promoter is functional. The abundance of RNA initiated from the kilC promoter is reduced in the presence of korA and korC, as predicted from genetic analysis of kilC regulation. The first gene of the kilC operon (klcA) is sufficient to express the host-lethal phenotype of the kilC determinant in the absence of korA and korC. By comparing RK2 to the related IncP alpha plasmids pUZ8 and R995, we determined that the Tn1 transposon in RK2 interrupts a gene (klcB) immediately downstream of klcA. Thus, the kilC determinant is normally part of an autoregulated operon of three genes: klcA, klcB, and korC. klcA is predicted to encode a 15,856-Da polypeptide that is related to the ArdB antirestriction protein of the IncN plasmid pKM101, suggesting a role for klcA in the broad host ranges of IncP alpha plasmids. The predicted product of the uninterrupted klcB gene is a polypeptide of 51,133 Da that contains a segment with significant similarity to the RK2 regulatory proteins KorA and TrbA. Located 145 bp upstream of the kilC promoter is a 10th copy of the 17-bp oriV iteron sequence in inverted orientation relative to that of the other nine iterons of oriV. Iteron 10 is identical to the "orphan" iteron 1, and both have identical 6-bp flanking sequences that make them likely to be strong binding sites for the TrfA replication initiator protein. The locations and relative orientation of orphan iterons 10 and 1 raise the possibility that these iterons promote the formation of a DNA loop via protein-protein interactions by bound TrfA and lead us to propose that they demarcate the functional origin of replication. This analysis of the kilC region and our previous studies on the other kil loci of RK2 have revealed that the region between oriV and the korABF operon in wild-type IncP alpha plasmids is saturated by the kilC, kilE, and kilA loci arranged in four kor-regulated operons encoding a total of 12 genes.
机译：通过在不存在调控的情况下对大肠杆菌宿主细胞具有致死性的多个kil位点（kilA，kilB，kilC和最近的kilE）的存在，首先在宽宿主范围的IncP alpha质粒RK2上鉴定了kil-kor regulon。通过kor函数的各种组合。尽管在偶联过程中形成配对对需要kilB操纵子，但其他kil位点编码的功能尚不清楚。它们对于复制或结合转移不是必需的，但是它们与复制和转移基因的共调节表明它们可能对RK2很重要。在本报告中，我们描述了关于kilC的分子和遗传研究。我们确定了kilC区的核苷酸序列，该区位于营养复制（oriV）和RK2上的转座子Tn1之间。引物延伸分析鉴定了转录起始位点，并表明对应于强σ70启动子的序列具有功能。如对kilC调控的遗传分析所预测的，在korA和korC存在的情况下，从kilC启动子启动的RNA的丰度降低。在不存在korA和korC的情况下，kilC操纵子的第一个基因（klcA）足以表达kilC决定簇的宿主致死表型。通过将RK2与相关的IncPα质粒pUZ8和R995进行比较，我们确定RK2中的Tn1转座子中断了klcA下游的一个基因（klcB）。因此，kilC决定簇通常是三个基因：klcA，klcB和korC的自调控操纵子的一部分。预测klcA编码与IncN质粒pKM101的ArdB抗限制性蛋白有关的15,856-Da多肽，这表明klcA在IncPα质粒的广泛宿主范围中的作用。不间断的klcB基因的预测产物是51,133 Da的多肽，其包含与RK2调节蛋白KorA和TrbA显着相似的区段。位于kilC启动子上游145 bp处的是相对于oriV的其他9个iteron反向排列的17 bp oriV iteron序列的第十个拷贝。 Iteron 10与“ orphan” iteron 1相同，并且都具有相同的6 bp侧翼序列，这使它们很可能成为TrfA复制起始蛋白的强结合位点。孤儿迭代子10和1的位置和相对方向，增加了这些迭代子通过结合的TrfA通过蛋白质-蛋白质相互作用促进DNA环形成的可能性，并导致我们提出它们界定复制的功能起源的可能性。对kilC区域的分析以及我们先前对RK2其他kil基因座的研究表明，野生型IncP alpha质粒中oriV和korABF操纵子之间的区域被四个kor中的kilC，kilE和kilA基因座饱和。 -调控的操纵子编码总共12个基因。

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