Genome Instability Mediates the Loss of Key Traits by Acinetobacter baylyi ADP1 during Laboratory Evolution

Abstract

Acinetobacter baylyi ADP1 has the potential to be a versatile bacterial host for synthetic biology because it is naturally transformable. To examine the genetic reliability of this desirable trait and to understand the potential stability of other engineered capabilities, we propagated ADP1 for 1,000 generations of growth in rich nutrient broth and analyzed the genetic changes that evolved by whole-genome sequencing. Substantially reduced transformability and increased cellular aggregation evolved during the experiment. New insertions of IS1236 transposable elements and IS1236-mediated deletions led to these phenotypes in most cases and were common overall among the selected mutations. We also observed a 49-kb deletion of a prophage region that removed an integration site, which has been used for genome engineering, from every evolved genome. The comparatively low rates of these three classes of mutations in lineages that were propagated with reduced selection for 7,500 generations indicate that they increase ADP1 fitness under common laboratory growth conditions. Our results suggest that eliminating transposable elements and other genetic failure modes that affect key organismal traits is essential for improving the reliability of metabolic engineering and genome editing in undomesticated microbial hosts, such as Acinetobacter baylyi ADP1.T he large-scale engineering of genome sequences is a growing area of synthetic biology and bioengineering with many applications (1, 2). Projects such as the development of reduced bacterial genomes (3), modifying the genetic code (4), and optimizing metabolic pathways (5) require whole-scale genetic rewriting of genomes in order to accommodate progressively more ambitious goals. The process of making such modifications is a resourceintensive undertaking with significant technical, human-resource, and time costs. Acinetobacter baylyi ADP1 has been proposed as a model organism for metabolic engineering, synthetic biology, and genome evolution studies because it is naturally transformable (6-8). During normal growth in the laboratory, ADP1 expresses competence machinery that enables it to efficiently import extracellular DNA without any sequence constraints (9, 10). By designing DNA constructs with homology to the ADP1 genome, targeted deletions or insertions of new genes can be made with high efficiency without the need for artificial transformation techniques or the expression of heterologous genes, as is typically required when engineering the Escherichia coli genome (5, 11). Thus, ADP1 could be a useful chassis organism for studies that require large-scale or combinatorial genome modifications.One known challenge associated with ADP1, however, is that the competence phenotype is unstable when it is cultured in the laboratory (12). In general, genetic reliability is a key trait sought after in chassis organisms; one does not want to design a DNA sequence only to see it be rapidly deleted or mutated when placed in a living organism. In this vein, efforts to engineer E. coli v...

Emergence of a Competence-Reducing Filamentous Phage from the Genome of Acinetobacter baylyi ADP1

“…We and others have found that mutants with reduced transformability arise and take over populations of ADP1 during long-term evolution experiments (14,15). This result was thought to occur due to a strong selective pressure to lose competence, either a direct cost for producing the DNA uptake apparatus or a deleterious effect from active DNA uptake.…”

“…Strains in this study were derived from an A. baylyi ADP1 evolution experiment (15). Growth conditions were incubation at 30°C with orbital shaking at 140 rpm over a 1-inch diameter in LB (10 g/liter tryptone, 5 g/liter yeast extract, and 10 g/liter NaCl).…”

“…Measurements of transformation frequency were conducted as previously described (15). Briefly, we combined 1 ml fresh LB, 100 ng AB-KAN genomic DNA, and 70 l preconditioned culture in a sterile test tube, incubated the resulting culture for 18 to 24 h under standard growth conditions, and then plated dilutions in sterile saline solution onto selective (kanamycin [Kan]) and nonselective (no antibiotic) agar plates.…”

“…Then, they were preconditioned by transferring 10 l of this culture into 10 ml of LB in a sterile 50-ml Erlenmeyer flask and growth for 24 Ϯ 1 h. ADP1 clones from evolved populations were isolated by plating portions of preconditioned populations diluted in sterile saline solution on LB agar (1.6% [wt/vol]) plates, growing the plates overnight at 30°C, and then picking single colonies. ADP1 knockout strains were obtained courtesy of the collection maintained at Genoscope (Évry, France) (16), except that the pilB knockout was constructed through chromosomal replacement of this gene with a Kan r -tdk cassette using methods described elsewhere (15). Phage isolation.…”

“…Genome sequencing of A. baylyi ADP1 clones was carried out as previously described (15). Briefly, genomic DNA was isolated from strains of interest and sheared to an average size of ϳ550 bp using a Covaris S2 ultrasonicator.…”