Mutational analysis of gene function in the Anaplasmataceae

Challenges and perspectives

Adela S. Oliva Chávez, Michael J. Herron, Curtis M. Nelson, Roderick F. Felsheim, Jonathan Oliver, Nicole Y. Burkhardt, Timothy J Kurtti, Ulrike G Munderloh

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Pages (from-to)482-494
Number of pages13
JournalTicks and Tick-borne Diseases
Volume10
Issue number2
DOIs
StatePublished - Feb 1 2019

Fingerprint

Anaplasmataceae
Electroporation
electroporation
HL-60 Cells
mutagenesis
Mutagenesis
Microbial Genes
Herbicide Resistance
Anaplasma marginale
Anaplasma phagocytophilum
Spectinomycin
Genes
Transposases
spectinomycin
Granulocyte Precursor Cells
Ixodes
Ixodes scapularis
genes
herbicide resistance
Gene Silencing

Keywords

  • Anaplasma phagocytophilum
  • Effector
  • Selectable markers
  • Transposon mutagenesis

PubMed: MeSH publication types

  • Journal Article
  • Research Support, N.I.H., Extramural

Cite this

Mutational analysis of gene function in the Anaplasmataceae : Challenges and perspectives. / Oliva Chávez, Adela S.; Herron, Michael J.; Nelson, Curtis M.; Felsheim, Roderick F.; Oliver, Jonathan; Burkhardt, Nicole Y.; Kurtti, Timothy J; Munderloh, Ulrike G.

In: Ticks and Tick-borne Diseases, Vol. 10, No. 2, 01.02.2019, p. 482-494.

Research output: Contribution to journalArticle

Oliva Chávez, Adela S. ; Herron, Michael J. ; Nelson, Curtis M. ; Felsheim, Roderick F. ; Oliver, Jonathan ; Burkhardt, Nicole Y. ; Kurtti, Timothy J ; Munderloh, Ulrike G. / Mutational analysis of gene function in the Anaplasmataceae : Challenges and perspectives. In: Ticks and Tick-borne Diseases. 2019 ; Vol. 10, No. 2. pp. 482-494.
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