摘要

突变分析是鉴定微生物基因功能的有效方法。直到最近，缺乏有效的无形体科植物工具产生可重复的结果已经成为功能基因组学的障碍，因为替代系统，例如大肠杆菌中的异位基因表达和分析，可能无法提供准确的答案。我们选择通过随机诱变而不是靶向基因失活来专注于高通量产生突变体的方法。在我们寻找合适的诱变工具时，我们考虑了Himar1转座酶系统的属性，即随机插入AT二核苷酸位点，其在无形体科中是丰富的，并且缺乏对特定宿主因子的要求。我们选择了Anaplasma marginale tr启动子和临床上无关的抗生素壮观霉素进行选择，并且另外使用除草剂抗性基因成功实施了非抗生素选择。这些构建体在从人早幼粒细胞HL-60细胞或肩胛骨蜱细胞收获的无形体嗜吞噬细胞中起相当好的作用。我们描述了在我们实验室开发的协议，并讨论了它们成功的可能性。使Anaplasmataceae电穿孔能力的原因尚不清楚，操纵电穿孔条件并未改善突变效率。需要协同努力来解决专性细胞内细菌固有的剩余问题。最后，使用这种方法，我们描述了在HL-60细胞中Ap存活所必需的推定的分泌效应子的发现和表征。

Mutational analysis is an efficient approach to identifying microbial gene function. Until recently, lack of an effective tool for Anaplasmataceae yielding reproducible results has created an obstacle to functional genomics, because surrogate systems, e.g., ectopic gene expression and analysis in E. coli, may not provide accurate answers. We chose to focus on a method for high-throughput generation of mutants via random mutagenesis as opposed to targeted gene inactivation. In our search for a suitable mutagenesis tool, we considered attributes of the Himar1 transposase system, i.e., random insertion into AT dinucleotide sites, which are abundant in Anaplasmataceae, and lack of requirement for specific host factors. We chose the Anaplasma marginale tr promoter, and the clinically irrelevant antibiotic spectinomycin for selection, and in addition successfully implemented non-antibiotic selection using an herbicide resistance gene. These constructs function reasonably well in Anaplasma phagocytophilum harvested from human promyelocyte HL-60 cells or Ixodes scapularis tick cells. We describe protocols developed in our laboratory, and discuss what likely makes them successful. What makes Anaplasmataceae electroporation competent is unknown and manipulating electroporation conditions has not improved mutational efficiency. A concerted effort is needed to resolve remaining problems that are inherent to the obligate intracellular bacteria. Finally, using this approach, we describe the discovery and characterization of a putative secreted effector necessary for Ap survival in HL-60 cells.

https://www.ncbi.nlm.nih.gov/pubmed/30466964