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Engineered plasmids have been workhorses of recombinant DNA technology for nearly half a century. Plasmids are used to clone DNA sequences encoding new genetic parts and to reprogram cells by combining these parts in new ways. Historically, many genetic parts on plasmids were copied and reused without routinely checking their DNA sequences. With the widespread use of high-throughput DNA sequencing technologies, we now know that plasmids often contain variants of common genetic parts that differ slightly from their canonical sequences. Because the exact provenance of a genetic part on a particular plasmid is usually unknown, it is difficult to determine whether these differences arose due to mutations during plasmid construction and propagation or due to intentional editing by researchers. In either case, it is important to understand how the sequence changes alter the properties of the genetic part. We analyzed the sequences of over 50,000 engineered plasmids using depositor metadata and a metric inspired by the natural language processing field. We detected 217 uncatalogued genetic part variants that were especially widespread or were likely the result of convergent evolution or engineering. Several of these uncatalogued variants are known mutants of plasmid origins of replication or antibiotic resistance genes that are missing from current annotation databases. However, most are uncharacterized, and 3/5 of the plasmids we analyzed contained at least one of the uncatalogued variants. Our results include a list of genetic parts to prioritize for refining engineered plasmid annotation pipelines, highlight widespread variants of parts that warrant further investigation to see whether they have altered characteristics, and suggest cases where unintentional evolution of plasmid parts may be affecting the reliability and reproducibility of science.

 

Parasites, including pathogens, can adapt to better exploit their hosts on many scales, ranging from within an infection of a single individual to series of infections spanning multiple host species. However, little is known about how the genomes of parasites in natural communities evolve when they face diverse hosts. We investigated howBartonellabacteria that circulate in rodent communities in the dunes of the Negev Desert in Israel adapt to different species of rodent hosts. We propagated 15Bartonellapopulations through infections of either a single host species (Gerbillus andersoniorGerbillus pyramidum) or alternating between the two. After 20 rodent passages, strains withde novomutations replaced the ancestor in most populations. Mutations in two mononucleotide simple sequence repeats (SSRs) that caused frameshifts in the same adhesin gene dominated the evolutionary dynamics. They appeared exclusively in populations that encounteredG.andersoniand altered the dynamics of infections of this host. Similar SSRs in other genes are conserved and exhibit ON/OFF variation inBartonellaisolates from the Negev Desert dunes. Our results suggest that SSR-based contingency loci could be important not only for rapidly and reversibly generating antigenic variation to escape immune responses but that they may also mediate the evolution of host specificity. 

The phenomenon of de novo gene birth—the emergence of genes from non-genic sequences—has received considerable attention due to the widespread occurrence of genes that are unique to particular species or genomes. Most instances of de novo gene birth have been recognized through comparative analyses of genome sequences in eukaryotes, despite the abundance of novel, lineage-specific genes in bacteria and the relative ease with which bacteria can be studied in an experimental context. Here, we explore the genetic record of the Escherichia coli long-term evolution experiment (LTEE) for changes indicative of “proto-genic” phases of new gene birth in which non-genic sequences evolve stable transcription and/or translation. Over the time span of the LTEE, non-genic regions are frequently transcribed, translated and differentially expressed, with levels of transcription across low-expressed regions increasing in later generations of the experiment. Proto-genes formed downstream of new mutations result either from insertion element activity or chromosomal translocations that fused preexisting regulatory sequences to regions that were not expressed in the LTEE ancestor. Additionally, we identified instances of proto-gene emergence in which a previously unexpressed sequence was transcribed after formation of an upstream promoter, although such cases were rare compared to those caused by recruitment of preexisting promoters. Tracing the origin of the causative mutations, we discovered that most occurred early in the history of the LTEE, often within the first 20,000 generations, and became fixed soon after emergence. Our findings show that proto-genes emerge frequently within evolving populations, can persist stably, and can serve as potential substrates for new gene formation. 

Animals 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.

We 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.

Our 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.

 

Evolutionary innovations generate phenotypic and species diversity. Elucidating the genomic processes underlying such innovations is central to understanding biodiversity. In this study, we addressed the genomic basis of evolutionary novelties in the glassy-winged sharpshooter (Homalodisca vitripennis, GWSS), an agricultural pest. Prominent evolutionary innovations in leafhoppers include brochosomes, proteinaceous structures that are excreted and used to coat the body, and obligate symbiotic associations with two bacterial types that reside within cytoplasm of distinctive cell types. Using PacBio long-read sequencing and Dovetail Omni-C technology, we generated a chromosome-level genome assembly for the GWSS and then validated the assembly using flow cytometry and karyotyping. Additional transcriptomic and proteomic data were used to identify novel genes that underlie brochosome production. We found that brochosome-associated genes include novel gene families that have diversified through tandem duplications. We also identified the locations of genes involved in interactions with bacterial symbionts. Ancestors of the GWSS acquired bacterial genes through horizontal gene transfer (HGT), and these genes appear to contribute to symbiont support. Using a phylogenomics approach, we inferred HGT sources and timing. We found that some HGT events date to the common ancestor of the hemipteran suborder Auchenorrhyncha, representing some of the oldest known examples of HGT in animals. Overall, we show that evolutionary novelties in leafhoppers are generated by the combination of acquiring novel genes, produced both de novo and through tandem duplication, acquiring new symbiotic associations that enable use of novel diets and niches, and recruiting foreign genes to support symbionts and enhance herbivory.

 

ABSTRACT Iron acquisition is essential for almost all living organisms. In certain environments, ferrous iron is the most prevalent form of this element. Feo is the most widespread system for ferrous iron uptake in bacteria and is critical for virulence in some species. The canonical architecture of Feo consists of a large transmembrane nucleoside triphosphatase (NTPase) protein, FeoB, and two accessory cytoplasmic proteins, FeoA and FeoC. The role of the latter components and the mechanism by which Feo orchestrates iron transport are unclear. In this study, we conducted a comparative analysis of Feo protein sequences to gain insight into the evolutionary history of this transporter. We identified instances of how horizontal gene transfer contributed to the evolution of Feo. Also, we found that FeoC, while absent in most lineages, is largely present in the Gammaproteobacteria group, although its sequence is poorly conserved. We propose that FeoC, which may couple FeoB NTPase activity with pore opening, was an ancestral element that has been dispensed with through mutations in FeoA and FeoB in some lineages. We provide experimental evidence supporting this hypothesis by isolating and characterizing FeoC-independent mutants of the Vibrio cholerae Feo system. Also, we confirmed that the closely related species Shewanella oneidensis does not require FeoC; thus, Vibrio FeoC sequences may resemble transitional forms on an evolutionary pathway toward FeoC-independent transporters. Finally, by combining data from our bioinformatic analyses with this experimental evidence, we propose an evolutionary model for the Feo system in bacteria. IMPORTANCE Feo, a ferrous iron transport system composed of three proteins (FeoA, -B, and -C), is the most prevalent bacterial iron transporter. It plays an important role in iron acquisition in low-oxygen environments and some host-pathogen interactions. The large transmembrane protein FeoB provides the channel for the transport of iron into the bacterial cell, but the functions of the two small, required accessory proteins FeoA and FeoC are not well understood. Analysis of the evolution of this transporter shows that FeoC is poorly conserved and has been lost from many bacterial lineages. Experimental evidence indicates that FeoC may have different functions in different species that retain this protein, and the loss of FeoC is promoted by mutations in FeoA or by the fusion of FeoA and FeoB. 

Abstract Insertion sequences (IS) are ubiquitous bacterial mobile genetic elements, and the mutations they cause can be deleterious, neutral, or beneficial. The long-term dynamics of IS elements and their effects on bacteria are poorly understood, including whether they are primarily genomic parasites or important drivers of adaptation by natural selection. Here, we investigate the dynamics of IS elements and their contribution to genomic evolution and fitness during a long-term experiment with Escherichia coli . IS elements account for ~35% of the mutations that reached high frequency through 50,000 generations in those populations that retained the ancestral point-mutation rate. In mutator populations, IS-mediated mutations are only half as frequent in absolute numbers. In one population, an exceptionally high ~8-fold increase in IS 150 copy number is associated with the beneficial effects of early insertion mutations; however, this expansion later slowed down owing to reduced IS 150 activity. This population also achieves the lowest fitness, suggesting that some avenues for further adaptation are precluded by the IS 150 -mediated mutations. More generally, across all populations, we find that higher IS activity becomes detrimental to adaptation over evolutionary time. Therefore, IS-mediated mutations can both promote and constrain evolvability.