Nataraj Vijayashankar, Pang Poh-choo, Haslam Stuart M, Veerapen Natacha, Minnikin David E, Dell Anne, Besra Gurdyal S, Bhatt Apoorva
School of Biosciences and Institute of Microbiology and Infection, University of Birmingham, Edgbaston, Birmingham, B15 2TT, United Kingdom.
Department of Life Sciences, Imperial College London, London, SW7 2AZ, United Kingdom.
PLoS One. 2015 Apr 20;10(3):e0122804. doi: 10.1371/journal.pone.0122804. eCollection 2015.
Lipooligosaccharides are glycolipids found in the cell wall of many mycobacterial species including the opportunistic pathogen Mycobacterium kansasii. The genome of M. kansasii ATCC12478 contains a cluster with genes orthologous to Mycobacterium marinum LOS biosynthesis genes. To initiate a genetic dissection of this cluster and demonstrate its role in LOS biosynthesis in M. kansasii, we chose MKAN27435, a gene encoding a putative glycosyltransferase. Using Specialized Transduction, a phage-based gene knockout tool previously used to generate null mutants in other mycobacteria, we generated a MKAN27435 null mutant. The mutant strain was found to be defective in the biosynthesis of higher LOS subspecies, viz LOS-IV, LOS-V, LOS-VI and LOS-VII. Additionally, a range of low abundance species were detected in the mutant strain and mass spectroscopic analysis indicated that these were shunt products generated from LOS-III by the addition of up to six molecules of a pentose.
脂寡糖是一种糖脂，存在于包括机会致病菌堪萨斯分枝杆菌在内的许多分枝杆菌属物种的细胞壁中。堪萨斯分枝杆菌ATCC12478的基因组包含一个与海分枝杆菌脂寡糖生物合成基因直系同源的基因簇。为了对该基因簇进行遗传学剖析，并证明其在堪萨斯分枝杆菌脂寡糖生物合成中的作用，我们选择了MKAN27435，一个编码假定糖基转移酶的基因。利用专门转导（一种以前用于在其他分枝杆菌中产生缺失突变体的基于噬菌体的基因敲除工具），我们构建了MKAN27435缺失突变体。发现该突变菌株在较高分子量脂寡糖亚种（即脂寡糖-IV、脂寡糖-V、脂寡糖-VI和脂寡糖-VII）的生物合成中存在缺陷。此外，在突变菌株中检测到一系列低丰度物种，质谱分析表明这些是由脂寡糖-III通过添加多达六个戊糖分子产生的分流产物。
