Biology subjects

Gifford, I.

Publications and source records attributed to Gifford, I..

7 recordsLinked to original sources

bioRxiv2026

Dual-Chassis Strategy for Bridging Adaptive Evolution and Rational Design for Synthetic Biology

Genome streamlining and pathway refactoring are powerful strategies for constructing controllable microbial chassis for both fundamental studies and applications. While rational design benefits from reduced genetic complexity, adaptive laboratory evolution (ALE) thrives on metabolic redundancy, creating a mismatch between optimal hosts for design and evolution. Here, we introduce a dual chassis framework (DUET) in which rational pathway construction and adaptive evolution are first carried out in an evolution-competent host, and the resulting optimized designs are subsequently transferred into a genetically stable chassis for deployment. Using the naturally evolvable bacterium Acinetobacter baylyi ADP1 and its genome-stabilized derivative (ISx), we applied this framework to the {beta}-ketoadipate pathway, a central hub for aromatic compound catabolism. We first streamlined the native network by deleting individual pathway branches and then engineered a minimal synthetic route that merges protocatechuate and catechol metabolism. Subsequent ALE enabled efficient growth through this synthetic pathway, and reverse-engineering identified key adaptive mutations underlying functional recovery. Both the synthetic pathway and the mutations were transferred unchanged into ISx, where robust growth was maintained without further adaptation. These results demonstrate that DUET enables portable, host-independent deployment of rational metabolic streamlining combined with evolution, providing a generalizable strategy for building reduced yet robust microbial platforms. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/729570v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@19ba68forg.highwire.dtl.DTLVardef@1d40eb4org.highwire.dtl.DTLVardef@1115597org.highwire.dtl.DTLVardef@10a50f4_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2025

Engineered Acinetobacter baylyi ADP1-ISx cells are sensitive DNA biosensors for antibiotic resistance genes and a fungal pathogen of bats

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.

synthetic biology↗

bioRxiv2024

Virulence-linked adhesin drives mutualist colonization of the bee gut via biofilm formation

Bacterial biofilms are stable multicellular structures that can enable long term host association. Yet, the role of biofilms in supporting gut mutualism is still not fully understood. Here, we investigate Snodgrassella alvi, a beneficial bacterial symbiont of honey bees, and find that biofilm formation is required for its colonization of the bee gut. We constructed fifteen S. alvi mutants containing knockouts of genes known to promote colonization with putative roles in biofilm formation. Genes required for colonization included staA and staB, encoding trimeric autotransporter adhesins (TAAs) and mltA, encoding a lytic transglycosylase. Intriguingly, TAAs are considered virulence factors in pathogens but support mutualism by the symbiont S. alvi. In vitro, biofilm formation was reduced in {Delta}staB cells and abolished in the other two mutants. Loss of staA also reduced auto-aggregation and cell-cell connections. Based on structural predictions, StaA/B are massive (>300 nm) TAAs with many repeats in their stalk regions. Further, we find that StaA/B are conserved across Snodgrassella species, suggesting that StaA/B-dependent colonization is characteristic of this symbiont lineage. Finally, staA deletion increases sensitivity to bactericidal antimicrobials, suggesting that the biofilm indirectly buffers against antibiotic stress. In all, the inability of two biofilm-deficient strains ({Delta}staA and {Delta}mltA) to effectively mono-colonize bees indicates that S. alvi biofilm formation is required for colonization of the bee gut. We envision the bee gut system as a genetically tractable model for studying the physical basis of biofilm-mutualist-gut interactions.

microbiology↗

bioRxiv2024

Evolution recovers the fitness of Acinetobacter baylyi strains with large deletions through mutations in deletion-specific targets and global post-transcriptional regulators

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-free Acinetobacter baylyi strain 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 regulators rnd (ribonuclease D), csrA (RNA-binding carbon storage regulator), and hfq (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 restored csrA function. 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. Author SummaryGenome streamlining reduces the complexity of organisms by eliminating large, non-essential portions of their genomes. This process occurs naturally in endosymbiont lineages and can be engineered to create microbial chassis that operate more efficiently and predictably. However, genome reduction often compromises the fitness of an organism when genes and combinations of genes are deleted that, while not essential, are advantageous. In this study, we used laboratory evolution to improve the fitness of a collection of Acinetobacter baylyi strains with large engineered deletions. In most cases, we found that spontaneous mutations could recover fitness lost due to deletions spanning many genes in these strains. These beneficial mutations were sometimes general, occurring in multiple strains and environments regardless of what genes were deleted, or specific, observed solely or more often in one environment or in strains with certain deletions. Mutations affecting proteins and small RNAs involved in post-transcriptional regulation of gene expression were especially common. Thus, recovering fitness often involves a combination of mutations that adjust global regulatory networks and compensate for lost gene functions. More broadly, our findings validate using laboratory evolution as a strategy for improving the fitness of reduced-genome strains created for biotechnology applications.

genetics↗

bioRxiv2022

Gut microbe Lactiplantiballus plantarum undergoes different evolutionary trajectories between insects and mammals

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 as Drosophila 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. Here, 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. While the symbiosis between Drosophila and L. plantarum is mainly determined by host diet, with the host influencing the fitness of its gut microbes only over short timescales, bacterial evolution within the mammalian host follows a divergent evolutionary path. Here, 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.

microbiology↗

bioRxiv2020

Rates of gene conversions between Escherichia coli ribosomal operons

Due to their universal presence and high sequence conservation, rRNA sequences are used widely in phylogenetics for inferring evolutionary relationships between microbes and in metagenomics for analyzing the composition of microbial communities. Most microbial genomes encode multiple copies of ribosomal RNA (rRNA) genes to supply cells with sufficient capacity for protein synthesis. These copies typically undergo concerted evolution that keeps their sequences identical, or nearly so, due to gene conversion, a type of intragenomic recombination that changes one copy of a homologous sequence to exactly match another. Widely varying rates of rRNA gene conversion have previously been estimated by comparative genomics methods and using genetic reporter assays. To more directly measure rates of rRNA intragenomic recombination, we sequenced the seven Escherichia coli rRNA operons in 15 lineages of cells that were evolved for ~13,750 generations with frequent single-cell bottlenecks that reduce the effects of selection. We identified 34 gene conversion events and estimate an overall rate of intragenomic recombination events between rRNA copies of 3.2 x 10-4 per generation or 5.3 x 10-5 per potential donor sequence. This rate varied only slightly from random expectations between different portions of the rRNA genes and between rRNA operons located at different locations in the genome. This accurate estimate of the rate of rRNA gene conversions fills a gap in our quantitative understanding of how ribosomal sequences and other multicopy elements diversify and homogenize during microbial genome evolution.

genetics↗

bioRxiv2018

Stable Genetic Transformation and Heterologous Expression in the Nitrogen-fixing Plant Endosymbiont Frankia alni ACN14a

Genus Frankia is comprised primarily of nitrogen-fixing actinobacteria that form root nodule symbioses with a group of hosts known as the actinorhizal plants. These plants are evolutionarily closely related to the legumes, which are nodulated by the rhizobia. Both host groups utilize homologs of nodulation genes for root-nodule symbiosis, derived from common plant ancestors. However the corresponding endosymbionts, Frankia and the rhizobia, are distantly related groups of bacteria, leading to questions of their symbiotic mechanisms and evolutionary history. To date, a stable system of genetic transformation has been lacking in Frankia. Here, we report the successful electrotransformation of Frankia alni ACN14a, by means of replicating plasmids expressing chloramphenicol-resistance for selection, and the use of GFP as a marker of gene expression. We have identified type IV methyl-directed restriction systems, highly-expressed in a range of actinobacteria, as a likely barrier to Frankia transformation and circumvented this barrier by using unmethylated plasmids, which allowed the transformation of F. alni as well as the maintenance of the plasmid. During nitrogen limitation, Frankia differentiates into two cell types: the vegetative hyphae and nitrogen-fixing vesicles. When the plasmid transformation system was used with expression of egfp under the control of the nif gene cluster promoter, it was possible to demonstrate by fluorescence imaging the expression of nitrogen fixation in vesicles but not hyphae in nitrogen-limited culture.\n\nImportanceTo date, the study of Frankia-actinorhizal symbioses has been complicated by the lack of genetic tools for manipulation of Frankia, especially stable genetic transformation. The transformation system reported here, particularly coupled with marker genes, can be used to differentiate patterns of gene expression between Frankia hyphae and vesicles in symbiosis or in free-living conditions. This will enable deeper comparisons between Frankia-actinorhizal symbioses and rhizobia-legume symbioses in terms of molecular signaling and metabolic exchange that will broaden understanding of the evolution of these symbioses and potentially make possible their application in agriculture. The development of transformation methods will allow further down-stream applications including gene knock-outs and complementation that will, in turn, open up a much broader range of experiments into Frankia and its symbioses.

microbiology↗