Ulrike Munderloh and team at the University of Minnesota published a new paper on mutational analysis of gene function. 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.
The researchers chose to focus on a method for high-throughput generation of mutants via random mutagenesis as opposed to targeted gene inactivation. In the search for a suitable mutagenesis tool, they 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. They 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.
Researchers describe protocols developed in their 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, researchers describe the discovery and characterization of a putative secreted effector necessary for Ap survival in HL-60 cells.
The paper, Mutational analysis of gene function in the Anaplasmataceae: Challenges and perspectives, is published in Tick and Tick-borne Diseases.