Goncharoff P, Saadi S, Chang C H, Saltman L H, Figurski D H
Department of Microbiology, College of Physicians and Surgeons, Columbia University, New York, New York 10032.
J Bacteriol. 1991 Jun;173(11):3463-77. doi: 10.1128/jb.173.11.3463-3477.1991.
The kil loci (kilA, kilB, kilC, and kilE) of incompatibility group P (IncP), broad-host-range plasmid RK2 were originally detected by their potential lethality to Escherichia coli host cells. Expression of the kil determinants is controlled by different combinations of kor functions (korA, korB, korC, and korE). This system of regulated genes, known as the kil-kor regulon, includes trfA, which encodes the RK2 replication initiator. The functions of the kil loci are unknown, but their coregulation with an essential replication function suggests that they have a role in the maintenance or host range of RK2. In this study, we have determined the nucleotide sequence of a 3-kb segment of RK2 that encodes the entire kilA locus. The region encodes three genes, designated klaA, klaB, and klaC. The phage T7 RNA polymerase-dependent expression system was use to identify three polypeptide products. The estimated masses of klaA and klaB products were in reasonable agreement with the calculated molecular masses of 28,407 and 42,156 Da, respectively. The klaC product is calculated to be 32,380 Da, but the observed polypeptide exhibited an apparent mass of 28 kDa on sodium dodecyl sulfate-polyacrylamide gels. Mutants of klaC were used to confirm that initiation of translation of the observed product occurs at the first ATG in the klaC open reading frame. Hydrophobicity analysis indicated that the KlaA and KlaB polypeptides are likely to be soluble, whereas the KlaC polypeptide was predicted to have four potential membrane-spanning domains. The only recognizable promoter sequences in the kilA region were those of the kilA promoter located upstream of klaA and the promoter for the korA-korB operon located just downstream of a rho-independent terminatorlike sequence following klaC. The transcriptional start sites for these promoters were determined by primer extension. Using isogenic sets of plasmids with nonpolar mutations, we found that klaA, klaB, and klaC are each able to express a host-lethal (Kil+) phenotype in the absence of kor functions. Inactivation of the kilA promoter causes loss of the lethal phenotype, demonstrating that all three genes are expressed from the kilA promoter as a multicistronic operon. We investigated two other phenotypes that have been mapped to the kilA region of RK2 or the closely related IncP plasmids RP1 and RP4: inhibition of conjugal transfer of IncW plasmids (fwB) and resistance to potassium tellurite. The cloned kilA operon was found to express both phenotypes, even in the presence of korA and korB, whose functions are known to regulate the kilA promoter. In addition, mutant and complementation analyses showed that the kilA promoter and the products of all three kla genes are necessary for expression of both phenotypes. Therefore, host lethality, fertility inhibition, and tellurite resistance are all properties of the kilA operon. We discuss the possible role of the kilA operon for RK2.
不相容性P组（IncP）广宿主范围质粒RK2的kil基因座（kilA、kilB、kilC和kilE）最初是因其对大肠杆菌宿主细胞的潜在致死性而被发现的。kil决定簇的表达受kor功能（korA、korB、korC和korE）不同组合的控制。这个被称为kil-kor调控子的调控基因系统包括编码RK2复制起始蛋白的trfA。kil基因座的功能尚不清楚，但它们与基本复制功能的共同调控表明它们在RK2的维持或宿主范围中起作用。在本研究中，我们确定了RK2编码整个kilA基因座的3 kb片段的核苷酸序列。该区域编码三个基因，命名为klaA、klaB和klaC。使用噬菌体T7 RNA聚合酶依赖性表达系统鉴定出三种多肽产物。klaA和klaB产物的估计分子量分别与计算出的28,407 Da和42,156 Da分子量合理一致。klaC产物计算为32,380 Da，但在十二烷基硫酸钠-聚丙烯酰胺凝胶上观察到的多肽表观分子量为28 kDa。klaC突变体用于证实观察到的产物的翻译起始发生在klaC开放阅读框中的第一个ATG处。疏水性分析表明，KlaA和KlaB多肽可能是可溶的，而KlaC多肽预计有四个潜在的跨膜结构域。kilA区域中唯一可识别的启动子序列是位于klaA上游的kilA启动子以及位于klaC之后的类rho非依赖性终止子序列下游的korA-korB操纵子的启动子。这些启动子的转录起始位点通过引物延伸确定。使用具有非极性突变的同基因质粒组，我们发现klaA、klaB和klaC在没有kor功能的情况下各自都能够表达宿主致死（Kil +）表型。kilA启动子的失活导致致死表型丧失，表明所有三个基因作为多顺反子操纵子从kilA启动子表达。我们研究了另外两种已定位到RK2的kilA区域或密切相关的IncP质粒RP1和RP4的表型：对IncW质粒（fwB）接合转移的抑制和对亚碲酸钾的抗性。发现克隆的kilA操纵子即使在存在已知调节kilA启动子功能的korA和korB的情况下也表达这两种表型。此外，突变和互补分析表明，kilA启动子和所有三个kla基因的产物对于这两种表型的表达都是必需的。因此，宿主致死性、育性抑制和亚碲酸盐抗性都是kilA操纵子的特性。我们讨论了kilA操纵子对RK2的可能作用。
