Pérez-Mendoza Daniel, de la Cruz Fernando
Departamento de Biología Molecular (Universidad de Cantabria) and Instituto de Biomedicina y Biotecnología de Cantabria (CSIC-UC-IDICAN), Santander, Spain.
BMC Genomics. 2009 Feb 9;10:71. doi: 10.1186/1471-2164-10-71.
How does the recipient cell contribute to bacterial conjugation? To answer this question we systematically analyzed the individual contribution of each Escherichia coli gene in matings using plasmid R388 as a conjugative plasmid. We used an automated conjugation assay and two sets of E. coli mutant collections: the Keio collection (3,908 E. coli single-gene deletion mutants) and a collection of 20,000 random mini-Tn10::Km insertion mutants in E. coli strain DH5alpha. The combined use of both collections assured that we screened > 99% of the E. coli non-essential genes in our survey.
Results indicate that no non-essential recipient E. coli genes exist that play an essential role in conjugation. Mutations in the lipopolysaccharide (LPS) synthesis pathway had a modest effect on R388 plasmid transfer (6 - 32% of wild type). The same mutations showed a drastic inhibition effect on F-plasmid transfer, but only in liquid matings, suggesting that previously isolated conjugation-defective mutants do in fact impair mating pair formation in liquid mating, but not conjugative DNA processing or transport per se.
We conclude from our genome-wide screen that recipient bacterial cells cannot avoid being used as recipients in bacterial conjugation. This is relevant as an indication of the problems in curbing the dissemination of antibiotic resistance and suggests that conjugation acts as a pure drilling machine, with little regard to the constitution of the recipient cell.
受体细胞如何促进细菌接合作用?为了回答这个问题,我们使用质粒R388作为接合质粒,系统地分析了大肠杆菌在交配过程中每个基因的个体贡献。我们采用了自动接合检测方法以及两组大肠杆菌突变体文库:Keio文库(3908个大肠杆菌单基因缺失突变体)和大肠杆菌DH5α菌株中20000个随机mini-Tn10::Km插入突变体文库。这两个文库的联合使用确保了我们在研究中筛选了超过99%的大肠杆菌非必需基因。
结果表明,不存在对接合作用起关键作用的非必需受体大肠杆菌基因。脂多糖(LPS)合成途径中的突变对R388质粒转移有适度影响(为野生型的6 - 32%)。相同的突变对F质粒转移显示出强烈的抑制作用,但仅在液体交配中如此,这表明先前分离出的接合缺陷突变体实际上确实损害了液体交配中的交配配对形成,但对接合DNA的加工或转运本身没有影响。
我们从全基因组筛选中得出结论,受体细菌细胞无法避免在细菌接合中被用作受体。这对于遏制抗生素耐药性传播方面的问题具有重要意义,并表明接合作用就像一台纯粹的钻孔机,几乎不考虑受体细胞的组成。