Institute for Integrative Biology of the Cell (I2BC), Université Paris-Saclay, CEA, CNRS, Gif-sur-Yvette, France.
Département Tuberculose & Biologie des Infections, Institut de Pharmacologie et de Biologie Structurale IPBS, UMR5089, Université de Toulouse, CNRS, UPS, Toulouse, France.
PLoS One. 2020 Dec 31;15(12):e0240497. doi: 10.1371/journal.pone.0240497. eCollection 2020.
Corynebacteriales are Actinobacteria that possess an atypical didermic cell envelope. One of the principal features of this cell envelope is the presence of a large complex made up of peptidoglycan, arabinogalactan and mycolic acids. This covalent complex constitutes the backbone of the cell wall and supports an outer membrane, called mycomembrane in reference to the mycolic acids that are its major component. The biosynthesis of the cell envelope of Corynebacteriales has been extensively studied, in particular because it is crucial for the survival of important pathogens such as Mycobacterium tuberculosis and is therefore a key target for anti-tuberculosis drugs. In this study, we explore the biogenesis of the cell envelope of Corynebacterium glutamicum, a non-pathogenic Corynebacteriales, which can tolerate dramatic modifications of its cell envelope as important as the loss of its mycomembrane. For this purpose, we used a genetic approach based on genome-wide transposon mutagenesis. We developed a highly effective immunological test based on the use of anti-cell wall antibodies that allowed us to rapidly identify bacteria exhibiting an altered cell envelope. A very large number (10,073) of insertional mutants were screened by means of this test, and 80 were finally selected, representing 55 different loci. Bioinformatics analyses of these loci showed that approximately 60% corresponded to genes already characterized, 63% of which are known to be directly involved in cell wall processes, and more specifically in the biosynthesis of the mycoloyl-arabinogalactan-peptidoglycan complex. We identified 22 new loci potentially involved in cell envelope biogenesis, 76% of which encode putative cell envelope proteins. A mutant of particular interest was further characterized and revealed a new player in mycolic acid metabolism. Because a large proportion of the genes identified by our study is conserved in Corynebacteriales, the library described here provides a new resource of genes whose characterization could lead to a better understanding of the biosynthesis of the envelope components of these bacteria.
棒杆菌目是一种放线菌，具有非典型的双分层细胞包膜。该细胞包膜的主要特征之一是存在由肽聚糖、阿拉伯半乳聚糖和类脂酸组成的大型复杂结构。这个共价复合物构成了细胞壁的骨干，并支撑着一个外膜，由于其主要成分是类脂酸，所以被称为类脂膜。棒杆菌目的细胞包膜生物合成已经得到了广泛的研究，特别是因为它对重要病原体（如结核分枝杆菌）的生存至关重要，因此是抗结核药物的关键目标。在这项研究中，我们探索了非致病性棒杆菌目的谷氨酸棒杆菌的细胞包膜生物合成，该菌可以容忍其细胞包膜的剧烈改变，例如类脂膜的丧失。为此，我们使用了基于全基因组转座子诱变的遗传方法。我们开发了一种基于使用抗细胞壁抗体的高效免疫测试，使我们能够快速识别具有改变的细胞壁的细菌。通过这种测试筛选了非常大量（10073）的插入突变体，最终选择了 80 个，代表 55 个不同的基因座。对这些基因座的生物信息学分析表明，大约 60%的基因座对应于已经表征的基因，其中 63%的基因已知直接参与细胞壁过程，更具体地说是参与类脂阿拉伯半乳聚糖-肽聚糖复合物的生物合成。我们确定了 22 个新的潜在参与细胞包膜生物合成的基因座，其中 76%的基因编码假定的细胞包膜蛋白。一个特别有趣的突变体进一步被表征，并揭示了一种新的类脂酸代谢参与者。由于我们的研究中确定的基因有很大一部分在棒杆菌目中是保守的，因此所描述的文库提供了一个新的基因资源，其特征的描述可能会导致更好地理解这些细菌包膜成分的生物合成。
