Laboratory evolution and natural transformation in Acinetobacter baylyi ADP1

The bacterium Acinetobacter baylyi ADP1 has been proposed as a next-generation chassis for genome engineering due to its high natural transformation rates and metabolic proficiency. Development of ADP1 for biotechnological use however is inhibited by the evolutionary instability of the competence, the large homology requirement for the efficient transformation of DNA into the ADP1 genome, and incomplete knowledge regarding the composition and regulation of the protein complex involved in DNA uptake.

Chapters 1 and 2 describe an investigation of the evolutionary stability of competence. We conducted a 1000 generation evolution experiment with ADP1 and characterized the genetic basis of competence loss. We found that mutations caused by the mobile genetic element IS1236 mediated loss of were a driving force for genetic instability in the strain. We also found that a previously uncharacterized filamentous phage (CRAϕ) emerged from the ADP1 genome. CRAϕ appears to infect via the ADP1 competence machinery – providing a potential explanation for the fitness benefit of competence loss.

Chapter 3 describes studies aimed at improving transformation rates and better characterizing the ADP1 transformation system. We examined how perturbing factors that affect the fate of internalized DNA effects transformation rates at different homology lengths. We found that some factors, such as exonuclease activity and recombinase activity, limited transformation rates at certain homology lengths and DNA concentrations. Other factors, such as adding random DNA to the ends of fragments to buffer against exonucleases and altering the abundance of a single-stranded DNA protecting protein, did not.

Chapter 4 describes an effort to better characterize the machinery required for competence in ADP1. We coupled transformation-mediated selection of an ADP1 transposon library with Tn-seq to identify genes required for competence. Through this method, we were able to positively identify two additional competence genes (pilR and ACIAD3188) and other candidates.

Together, the work presented in this dissertation provides insights into the evolutionary stability and molecular workings of ADP1 competence. These insights represent progress toward further improving ADP1 for use as a versatile genome and metabolic engineering platform.