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ABSTRACT The bacteriumAcinetobacter baylyiis a model organism known for its extreme natural competence and metabolic versatility. It is capable of taking up environmental DNA at a high rate across all growth phases. The type strain ADP1 was created by random mutagenesis of a precursor strain, BD4, to prevent it from forming cell chains in culture. ADP1 has since been distributed between research groups over several decades and acquired subsequent mutations during this time. In this study, we compare the genome sequences ofA. baylyiBD4 and its modern descendants to identify and understand the effects of mutations acquired and engineered during its domestication. We demonstrate that the ADP1 variants in use today differ in their competence, growth on different carbon sources, and autoaggregation. In addition, we link the global carbon storage regulator CsrA and a transposon insertion that removes its C-terminal domain specifically to changes in both overall competence and an almost complete loss of competence during the stationary phase. Reconstructing the history of ADP1 and the diversity that has evolved in the variants currently in use improves our understanding of the desirable properties of this experimentally and industrially important bacterium and suggests ways that its reliability can be improved through further genome engineering.IMPORTANCEAcinetobacter baylyiADP1 is a bacterial chassis of interest to microbiologists in academia and industry due to its extreme natural competence and wide metabolic range. Its ability to take up DNA from its environment makes it straightforward to efficiently edit its chromosome. We identify and characterize mutations that have been passed down to modern strains of ADP1 from the initial work in the 1960s, as well as subsequent mutations and genome edits separating strains in use by different research groups today. These mutations, including one in a global regulator (CsrA), have significant phenotypic consequences that have affected the reproducibility and consistency of experiments reported in the literature. We link a mutation in this global regulator to unexpected changes in natural competence. We also show that domesticatedA. baylyistrains have impaired growth on a variety of carbon sources. 

Abstract Adenosine-to-inosine (A-to-I) messenger RNA (mRNA) editing can affect the sequence and function of translated proteins and has been extensively investigated in eukaryotes. However, the prevalence of A-to-I mRNA editing in bacteria, its governing regulatory principles, and its biological significance are poorly understood. Here, we show that A-to-I mRNA editing occurs in hundreds of transcripts across dozens of gammaproteobacterial species, with most edits predicted to recode protein sequences. Furthermore, we reveal conserved regulatory determinants controlling editing across gammaproteobacterial species. Using Acinetobacter baylyi as a model, we show that mutating TadA, the mediating enzyme, reduces editing across all sites. Conversely, overexpressing TadA resulted in the editing of >300 transcripts, attesting to the editing potential of TadA. Notably, we show for the first time, at the protein level, that normal levels of A-to-I mRNA editing lead to wild-type bacteria expressing two protein isoforms from a single gene. Finally, we show that a TadA mutant with deficient editing activity does not grow at high temperatures, suggesting that RNA editing has a functional role in bacteria. Our work reveals that A-to-I mRNA editing in bacteria is widespread and has the potential to reshape the bacterial transcriptome and proteome. 

Abstract Foundational techniques in molecular biology—such as cloning genes, tagging biomolecules for purification or identification, and overexpressing recombinant proteins—rely on introducing non-native or synthetic DNA sequences into organisms. These sequences may be recognized by the transcription and translation machinery in their new context in unintended ways. The cryptic gene expression that sometimes results has been shown to produce genetic instability and mask experimental signals. Computational tools have been developed to predict individual types of gene expression elements, but it can be difficult for researchers to contextualize their collective output. Here, we introduce CryptKeeper, a software pipeline that visualizes predictions of Escherichia coli gene expression signals and estimates the translational burden possible from a DNA sequence. We investigate several published examples where cryptic gene expression in E. coli interfered with experiments. CryptKeeper accurately postdicts unwanted gene expression from both eukaryotic virus infectious clones and individual proteins that led to genetic instability. It also identifies off-target gene expression elements that resulted in truncations that confounded protein purification. Incorporating negative design using CryptKeeper into reverse genetics and synthetic biology workflows can help to mitigate cloning challenges and avoid unexplained failures and complications that arise from unintentional gene expression. 

Abstract Engineered DNA will slow the growth of a host cell if it redirects limiting resources or otherwise interferes with homeostasis. Escape mutants that alleviate this burden can rapidly evolve and take over cell populations, making genetic engineering less reliable and predictable. Synthetic biologists often use genetic parts encoded on plasmids, but their burden is rarely characterized. We measured how 301 BioBrick plasmids affectedEscherichia coligrowth and found that 59 (19.6%) were burdensome, primarily because they depleted the limited gene expression resources of host cells. Overall, no BioBricks reduced the growth rate ofE. coliby >45%, which agreed with a population genetic model that predicts such plasmids should be unclonable. We made this model available online for education (https://barricklab.org/burden-model) and added our burden measurements to the iGEM Registry. Our results establish a fundamental limit on what DNA constructs and genetic modifications can be successfully engineered into cells. 

Naturally competent bacteria can be engineered into platforms for detecting environmental DNA. This capability could be used to monitor the spread of pathogens, invasive species, and resistance genes, among other applications. Here, we create Acinetobacter baylyi ADP1-ISx biosensors that detect specific target DNA sequences through natural transformation. We tested strains with DNA sensors that consisted of either a mutated antibiotic resistance gene (TEM-1 bla or nptII) or a counterselectable gene flanked by sequences from the fungus Pseudogymnoascus destructans, which causes white-nose syndrome in bats. Upon uptake of homologous DNA, recombination restored antibiotic resistance gene function or removed the counterselectable gene, enabling selection of cells that sensed the target DNA. The antibiotic resistance gene and P. destructans biosensors could detect as few as 3,000 or 5,000,000 molecules of their DNA targets, respectively, and their sensitivity was not affected by excess off-target DNA. These results demonstrate how A. baylyi can be reprogrammed into a modular platform for monitoring environmental DNA. 

Organelles and endosymbionts have naturally evolved dramatically reduced genome sizes compared to their free-living ancestors. Synthetic biologists have purposefully engineered streamlined microbial genomes to create more efficient cellular chassis and define the minimal components of cellular life. During natural or engineered genome streamlining, deletion of many non-essential genes in combination often reduces bacterial fitness for idiosyncratic or unknown reasons. We investigated how and to what extent laboratory evolution could overcome these defects in six variants of the transposon-freeAcinetobacter baylyistrain ADP1-ISx that each had a deletion of a different 22- to 42-kilobase region and two strains with larger deletions of 70 and 293 kilobases. We evolved replicate populations of ADP1-ISx and each deletion strain for ~300 generations in a chemically defined minimal medium or a complex medium and sequenced the genomes of endpoint clonal isolates. Fitness increased in all cases that were examined except for two ancestors that each failed to improve in one of the two environments. Mutations affecting nine protein-coding genes and two small RNAs were significantly associated with one of the two environments or with certain deletion ancestors. The global post-transcriptional regulatorsrnd(ribonuclease D),csrA(RNA-binding carbon storage regulator), andhfq(RNA-binding protein and chaperone) were frequently mutated across all strains, though the incidence and effects of these mutations on gene function and bacterial fitness varied with the ancestral deletion and evolution environment. Mutations in this regulatory network likely compensate for how an earlier deletion of a transposon in the ADP1-ISx ancestor of all the deletion strains restoredcsrAfunction. More generally, our results demonstrate that fitness lost during genome streamlining can usually be regained rapidly through laboratory evolution and that recovery tends to occur through a combination of deletion-specific compensation and global regulatory adjustments.