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. 2017 Aug 17;83(17):e01025-17.
doi: 10.1128/AEM.01025-17. Print 2017 Sep 1.

Reduced Mutation Rate and Increased Transformability of Transposon-Free Acinetobacter Baylyi ADP1-ISx

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Reduced Mutation Rate and Increased Transformability of Transposon-Free Acinetobacter Baylyi ADP1-ISx

Gabriel A Suárez et al. Appl Environ Microbiol. .
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Abstract

The genomes of most bacteria contain mobile DNA elements that can contribute to undesirable genetic instability in engineered cells. In particular, transposable insertion sequence (IS) elements can rapidly inactivate genes that are important for a designed function. We deleted all six copies of IS1236 from the genome of the naturally transformable bacterium Acinetobacter baylyi ADP1. The natural competence of ADP1 made it possible to rapidly repair deleterious point mutations that arose during strain construction. In the resulting ADP1-ISx strain, the rates of mutations inactivating a reporter gene were reduced by 7- to 21-fold. This reduction was higher than expected from the incidence of new IS1236 insertions found during a 300-day mutation accumulation experiment with wild-type ADP1 that was used to estimate spontaneous mutation rates in the strain. The extra improvement appears to be due in part to eliminating large deletions caused by IS1236 activity, as the point mutation rate was unchanged in ADP1-ISx. Deletion of an error-prone polymerase (dinP) and a DNA damage response regulator (umuDAb [the umuD gene of A. baylyi]) from the ADP1-ISx genome did not further reduce mutation rates. Surprisingly, ADP1-ISx exhibited increased transformability. This improvement may be due to less autolysis and aggregation of the engineered cells than of the wild type. Thus, deleting IS elements from the ADP1 genome led to a greater than expected increase in evolutionary reliability and unexpectedly enhanced other key strain properties, as has been observed for other clean-genome bacterial strains. ADP1-ISx is an improved chassis for metabolic engineering and other applications.IMPORTANCEAcinetobacter baylyi ADP1 has been proposed as a next-generation bacterial host for synthetic biology and genome engineering due to its ability to efficiently take up DNA from its environment during normal growth. We deleted transposable elements that are capable of copying themselves, inserting into other genes, and thereby inactivating them from the ADP1 genome. The resulting "clean-genome" ADP1-ISx strain exhibited larger reductions in the rates of inactivating mutations than expected from spontaneous mutation rates measured via whole-genome sequencing of lineages evolved under relaxed selection. Surprisingly, we also found that IS element activity reduces transformability and is a major cause of cell aggregation and death in wild-type ADP1 grown under normal laboratory conditions. More generally, our results demonstrate that domesticating a bacterial genome by removing mobile DNA elements that have accumulated during evolution in the wild can have unanticipated benefits.

Keywords: clean genome; genome engineering; insertion sequence; mutation accumulation; synthetic biology.

Figures

FIG 1
FIG 1
Mutations observed in A. baylyi ADP1 by whole-genome sequencing of 17 strains that each evolved separately for 7,500 generations under relaxed selection in the MA experiment. (A) Mutations observed in each clone from the MA experiment divided into major categories. Overall, 26% of the mutations were directly related to IS1236 element activity. The precise location and nature of each mutation is reported in Table S1 in the supplemental material. (B) Summed observations of each base pair substitution across all sequenced clones from the MA experiment.
FIG 2
FIG 2
Construction of A. baylyi ADP1-ISx. (A) Five unmarked genomic deletions were made to remove all six IS1236 elements, designated by numbers 1 to 6, found in the wild-type ADP1 chromosome (shown in red) to create the ADP1-ISx strain. Sites 1 to 4 (shown in blue) were used for transformation and mutation assays. IS1236 elements number 2 and number 3 form a composite transposon (Tn5613), and element number 6 is inactive (IS1236φ). (B) Example of qPCR data used to monitor IS element deletion steps. The six IS copies per genome found in wild-type ADP1 register above a given fluorescence threshold (ΔRn value) during early PCR cycles when amplifying a 119-bp fragment located within the IS1236 transposase gene. The sequential removal of IS elements in the deletion strains progressively increases the number of cycles necessary to reach this threshold from the same input quantity of genomic DNA. One example of a rejected candidate strain that accumulated a new IS element insertion elsewhere in the genome such that its IS copy number did not decrease after the deletion of IS number 4 is shown (dotted line).
FIG 3
FIG 3
Repair of unplanned cyoB and rpoD mutations sustained during ADP1-ISx strain construction. Growth curves in LB broth (A) and MS defined medium (B) for wild-type ADP1, ADP1-ISx with the cyoB and rpoD mutations, and ADP1-ISx with these genes repaired to the wild-type sequence (as verified by whole-genome sequencing) were recorded by monitoring the OD600. The error bars represent standard deviations of three biological replicates.
FIG 4
FIG 4
Reduced rates of inactivating mutations in ADP1-ISx. (A) Rates of mutations leading to loss of function of a counterselectable marker gene (tdk) were determined using fluctuation tests that selected for resistance to azidothymidine (AZTr). The error bars are 95% confidence intervals. The marker was placed at three different sites in the A. baylyi genome that differed in how near they were to IS1236 elements in the wild-type ADP1 genome (Fig. 2A). (B) Mutation rates were measured for reversion of a stop codon in the leader region of an antibiotic marker gene that restored spectinomycin resistance (Specr). This assay is expected to primarily reflect the point mutation rate. The error bars are 95% confidence intervals. (C) Spectrum of inactivating mutations determined by using PCR to amplify a fragment containing the tdk gene from the genome of one AZTr mutant from each of the 24 biological replicates of the fluctuation tests in panel A. IS1236 mutations and point mutations were inferred from an expected size change or no size change in this fragment, respectively. When no PCR product resulted and the function of the adjacent aph marker gene that was inserted alongside the tdk gene was also lost (yielding a Kans versus a Kanr phenotype), then the inactivating mutation was inferred to be a large deletion. If there was no PCR product and the mutant remained Kanr, then it was likely a large deletion that overlapped the tdk gene but not the aph gene. However, since the PCR could have failed to amplify a band for other reasons, we conservatively classified this as an “ambiguous” result. This logic for classifying mutations is illustrated in the schematic below the legend and further explained in the text (see “Altered mutational spectrum in ADP1-ISx”). As expected, no IS1236 insertions were found in ADP1-ISx. The proportion of large deletions among the inactivating mutations was also greatly reduced at site 1 and site 2.
FIG 5
FIG 5
ADP1-ISx exhibits increased transformability and reduced autolysis. (A) Transformation frequencies measured as transformants per CFU under normal growth conditions in LB medium for PCR products containing the tdk-Kanr cassette (into site 2) or a spectinomycin resistance marker (into site 4). (B) CFU measured by plating dilutions of cultures on LB agar after saturating growth. (C) Reduced settling behavior of ADP1-ISx compared to ADP1 in saturated cultures left at room temperature for 48 h after growth. (D) Accumulation of extracellular DNA over time in the supernatants of MS cultures grown under standard conditions. (E) Fraction of cells staining as dead in LB cultures after 24 h of growth. The error bars are 95% confidence intervals.
FIG 6
FIG 6
Inactivating mutation rates are unchanged in ADP1-ISx after deletion of error-prone polymerase DinP and DNA damage response regulator UmuDAb. The effects of deleting each gene on the rates of mutations inactivating the tdk counterselectable marker gene inserted into the genome at site 2 were measured for each strain. Mutation rates in ADP1-ISx and the umuDAb deletion strain were also determined in the presence of a subinhibitory concentration of nalidixic acid, which induces double-strand breaks in DNA. The error bars are 95% confidence intervals.

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