Natural transformation as a tool in Acinetobacter baylyi: Streamlined engineering and mutational analysis

Stacy R. Bedore, Ellen L. Neidle, Isabel Pardo, Jin Luo, Alyssa C. Baugh, Chantel V. Duscent-Maitland, Melissa P. Tumen-Velasquez, Ville Santala, Suvi Santala

Research output: Chapter in Book/Report/Conference proceedingChapterScientificpeer-review

4 Citations (Scopus)

Abstract

Natural transformation and homologous recombination in a soil bacterium, Acinetobacter baylyi ADP1, occur with exceptionally high efficiency. These genetic features can be harnessed to address a wide variety of fundamental and applied scientific topics. Recent advances in synthetic biology and laboratory evolution have led to renewed appreciation for the use of A. baylyi as a model organism. To complement several review articles that highlight new tool sets, this chapter focuses on simple protocols and examples of transformation assays that facilitate genetic analysis and engineering. Whole genome sequencing often reveals extensive genetic variation among closely related isolates that can confound the association of genotypic and phenotypic changes. In A. baylyi, such associations can be deciphered in unique ways by directly transforming cells with linear DNA fragments. The resulting allelic replacement, which occurs at high frequency, rapidly generates desired mutants via targeted chromosomal editing. Diverse screening and selection methods can be used to test hypotheses and streamline experimental strategies to reveal the significance of specific DNA sequences. Moreover, large procedural variations are well tolerated, and techniques can be readily adapted for new purposes. One goal of highlighting natural transformation methodology in A. baylyi is to expand the community of researchers using this versatile bacterial host.

Original languageEnglish
Title of host publicationGenome Engineering
EditorsVolker Gurtler, Michael Calcutt
PublisherAcademic Press
Chapter8
Pages207-234
ISBN (Electronic)978-0-12-823540-9
DOIs
Publication statusPublished - 2023
Publication typeA3 Book chapter

Publication series

NameMethods in Microbiology
Volume52
ISSN (Print)0580-9517

Funding

I. Pardo wishes to thank the Spanish National Research Council, Reina Sofía Foundation, and Primafrío Foundation for funding under agreement no. 20210510. Research at the University of Georgia U.S., described in this chapter, was funded by grants from the National Science Foundation (MCB2225858) and the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research, Genomic Science Program (DE-SC0022220). S. Santala would like to thank the Novo Nordisk Foundation (grant NNF21OC0067758) and the Academy of Finland (grant no. 334822 and 347204). V. Santala is grateful to the Academy of Finland (no. 310188).

Keywords

  • Acinetobacter baylyi ADP1
  • Allelic replacement
  • Genome editing
  • Natural competence
  • Transformation

Publication forum classification

  • Publication forum level 1

ASJC Scopus subject areas

  • Microbiology
  • Microbiology (medical)

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