Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada.
Michael G. DeGroote Institute for Infectious Disease Research, McMaster University, Hamilton, Ontario, Canada.
mBio. 2020 Mar 10;11(2):e00161-20. doi: 10.1128/mBio.00161-20.
Gram-negative bacteria are intrinsically resistant to many antibiotics due to their outer membrane barrier. Although the outer membrane has been studied for decades, there is much to uncover about the biology and permeability of this complex structure. Investigating synthetic genetic interactions can reveal a great deal of information about genetic function and pathway interconnectivity. Here, we performed synthetic genetic arrays (SGAs) in by crossing a subset of gene deletion strains implicated in outer membrane permeability with nonessential gene and small RNA (sRNA) deletion collections. Some 155,400 double-deletion strains were grown on rich microbiological medium with and without subinhibitory concentrations of two antibiotics excluded by the outer membrane, vancomycin and rifampin, to probe both genetic interactions and permeability. The genetic interactions of interest were synthetic sick or lethal (SSL) gene deletions that were detrimental to the cell in combination but had a negligible impact on viability individually. On average, there were ∼30, ∼36, and ∼40 SSL interactions per gene under no-drug, rifampin, and vancomycin conditions, respectively; however, many of these involved frequent interactors. Our data sets have been compiled into an interactive database called the Outer Membrane Interaction (OMI) Explorer, where genetic interactions can be searched, visualized across the genome, compared between conditions, and enriched for gene ontology (GO) terms. A set of SSL interactions revealed connectivity and permeability links between enterobacterial common antigen (ECA) and lipopolysaccharide (LPS) of the outer membrane. This data set provides a novel platform to generate hypotheses about outer membrane biology and permeability. Gram-negative bacteria are a major concern for public health, particularly due to the rise of antibiotic resistance. It is important to understand the biology and permeability of the outer membrane of these bacteria in order to increase the efficacy of antibiotics that have difficulty penetrating this structure. Here, we studied the genetic interactions of a subset of outer membrane-related gene deletions in the model Gram-negative bacterium We systematically combined these mutants with 3,985 nonessential gene and small RNA deletion mutations in the genome. We examined the viability of these double-deletion strains and probed their permeability characteristics using two antibiotics that have difficulty crossing the outer membrane barrier. An understanding of the genetic basis for outer membrane integrity can assist in the development of new antibiotics with favorable permeability properties and the discovery of compounds capable of increasing outer membrane permeability to enhance the activity of existing antibiotics.
革兰氏阴性菌由于其外膜屏障而对许多抗生素具有内在抗性。尽管对外膜的研究已经进行了几十年,但对于这种复杂结构的生物学和通透性仍有很多需要揭示。研究合成遗传相互作用可以揭示大量有关遗传功能和途径相互连接的信息。在这里,我们通过交叉一组与外膜通透性相关的基因缺失菌株与非必需基因和小 RNA(sRNA)缺失文库,在模型革兰氏阴性菌中进行了合成遗传阵列(SGA)。在含有和不含有两种外膜排斥抗生素(万古霉素和利福平)的亚抑菌浓度的丰富微生物培养基中,培养了大约 155400 个双缺失菌株,以探测遗传相互作用和通透性。感兴趣的遗传相互作用是合成的致病或致死(SSL)基因缺失,这些缺失对细胞联合有害,但单独对活力几乎没有影响。平均而言,在无药物、利福平、万古霉素条件下,每个基因分别有∼30、∼36 和∼40 个 SSL 相互作用;然而,其中许多涉及频繁的相互作用者。我们的数据集已经被汇编到一个名为外膜相互作用(OMI)探索者的交互式数据库中,在这个数据库中可以搜索、在基因组上可视化、在不同条件下比较和富集基因本体(GO)术语的遗传相互作用。一组 SSL 相互作用揭示了外膜中的肠杆菌共同抗原(ECA)和脂多糖(LPS)之间的连接和通透性联系。这个数据集为研究外膜生物学和通透性提供了一个新的平台。革兰氏阴性菌对公共卫生构成重大威胁,特别是由于抗生素耐药性的增加。了解这些细菌外膜的生物学和通透性非常重要,这可以提高穿透这种结构困难的抗生素的疗效。在这里,我们研究了模型革兰氏阴性菌中的一组与外膜相关的基因缺失的遗传相互作用。我们系统地将这些突变与基因组中的 3985 个非必需基因和小 RNA 缺失突变相结合。我们检查了这些双缺失菌株的生存能力,并使用两种难以穿过外膜屏障的抗生素探测了它们的通透性特征。对外膜完整性的遗传基础的了解可以帮助开发具有良好通透性的新抗生素,并发现能够增加外膜通透性以增强现有抗生素活性的化合物。