Department of Microbiology and Immunology, Dalhousie University, Halifax, NS, Canada.
Can J Microbiol. 2011 Nov;57(11):943-52. doi: 10.1139/w11-086. Epub 2011 Oct 27.
HtpB, the chaperonin of the intracellular bacterial pathogen Legionella pneumophila , displays several virulence-related functions in vitro. To confirm HtpB's role in vivo, host infections with an htpB deletion mutant would be required. However, we previously reported that the htpAB operon (encoding co-chaperonin and chaperonin) is essential. We attempted here to delete htpAB in a L. pneumophila strain carrying the groE operon (encoding the Escherichia coli co-chaperonin and chaperonin). The groE operon was inserted into the chromosome of L. pneumophila Lp02, and then allelic replacement of htpAB with a gentamicin resistance cassette was attempted. Although numerous potential postallelic replacement transformants showed a correct selection phenotype, we still detected htpAB by PCR and full-size HtpB by immunoblot. Southern blot and PCR analysis indicated that the gentamicin resistance cassette had apparently integrated in a duplicated htpAB region. However, we showed by Southern blot that strain Lp02, and the Lp02 derivative carrying the groE operon, have only one copy of htpAB. These results confirmed that the htpAB operon cannot be deleted, not even in the presence of the groE operon, and suggested that attempts to delete htpAB under strong phenotypic selection result in aberrant genetic recombinations that could involve duplication of the htpAB locus.
细胞内细菌病原体军团菌的伴侣蛋白 HtpB 在体外表现出几种与毒力相关的功能。为了确认 HtpB 在体内的作用，需要进行宿主感染缺失突变体的实验。然而，我们之前曾报道过 htpAB 操纵子（编码共伴侣蛋白和伴侣蛋白）是必需的。我们试图在携带 groE 操纵子（编码大肠杆菌共伴侣蛋白和伴侣蛋白）的军团菌菌株中删除 htpAB。groE 操纵子被插入到军团菌 Lp02 的染色体中，然后尝试用庆大霉素抗性盒替代 htpAB。尽管许多潜在的等位基因替换转化子表现出正确的选择表型，但我们仍然通过 PCR 和免疫印迹检测到 htpAB。Southern blot 和 PCR 分析表明，庆大霉素抗性盒显然整合到 htpAB 区域的重复序列中。然而，我们通过 Southern blot 表明，菌株 Lp02 和携带 groE 操纵子的 Lp02 衍生物只有一个 htpAB 拷贝。这些结果证实 htpAB 操纵子不能被删除，即使存在 groE 操纵子也是如此，并表明在强表型选择下尝试删除 htpAB 会导致涉及 htpAB 基因座重复的异常遗传重组。
