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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. 

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. 

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. 

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. 

Abstract BackgroundAnimals form complex symbiotic associations with their gut microbes, whose evolution is determined by an intricate network of host and environmental factors. In many insects, such asDrosophila melanogaster, the microbiome is flexible, environmentally determined, and less diverse than in mammals. In contrast, mammals maintain complex multispecies consortia that are able to colonize and persist in the gastrointestinal tract. Understanding the evolutionary and ecological dynamics of gut microbes in different hosts is challenging. This requires disentangling the ecological factors of selection, determining the timescales over which evolution occurs, and elucidating the architecture of such evolutionary patterns. ResultsWe employ experimental evolution to track the pace of the evolution of a common gut commensal,Lactiplantibacillus plantarum, within invertebrate (Drosophila melanogaster) and vertebrate (Mus musculus) hosts and their respective diets. We show that inDrosophila, the nutritional environment dictates microbial evolution, while the host benefitsL. plantarumgrowth only over short ecological timescales. By contrast, in a mammalian animal model,L. plantarumevolution results to be divergent between the host intestine and its diet, both phenotypically (i.e., host-evolved populations show higher adaptation to the host intestinal environment) and genomically. Here, both the emergence of hypermutators and the high persistence of mutated genes within the host’s environment strongly differed from the low variation observed in the host’s nutritional environment alone. ConclusionsOur results demonstrate thatL. plantarumevolution diverges between insects and mammals. While the symbiosis betweenDrosophilaandL. plantarumis mainly determined by the host diet, in mammals, the host and its intrinsic factors play a critical role in selection and influence both the phenotypic and genomic evolution of its gut microbes, as well as the outcome of their symbiosis.