US EPA

3860233 

Journal Article 

Reduced mutation rate and increased transformability of transposon-free Acinetobacter baylyi ADP1-ISx 

Suárez, GA; Renda, BA; Dasgupta, A; Barrick, JE 

2017 

Yes 

Applied and Environmental Microbiology
ISSN: 0099-2240
EISSN: 1098-5336 

AMER SOC MICROBIOLOGY 

WASHINGTON 

83 

17 

e01025-17 

English 

28667117 

10.1128/AEM.01025-17 

WOS:000408259000017 

https://search.proquest.com/docview/1957166657?accountid=171501
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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 this 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) 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 compared to 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 uptake DNA from its environment during normal growth. We deleted transposable elements from the ADP1 genome that are capable of copying themselves, inserting into other genes, and thereby inactivating them. 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. 

clean genome; insertion sequence; mutation accumulation; genome engineering; synthetic biology