Insulin sensitization by small molecules enhancing GLUT4 translocation.

Insulin resistance (IR) is the root cause of type II diabetes, yet no safe treatment is available to address it. Using a high throughput compatible assay that measures real-time translocation of the glucose transporter glucose transporter 4 (GLUT4), we identified small molecules that potentiate insulin action. In vivo, these insulin sensitizers improve insulin-stimulated GLUT4 translocation, glucose tolerance, and glucose uptake in a model of IR. Using proteomic and CRISPR-based approaches, we identified the targets of those compounds as Unc119 proteins and solved the structure of Unc119 bound to the insulin sensitizer. This study identifies compounds that have the potential to be developed into diabetes treatment and establishes Unc119 proteins as targets for improving insulin sensitivity.

Diversity and distribution of microbial communities in floral nectar of two night-blooming plants of the Sonoran Desert.

Nectar-inhabiting microbes are increasingly appreciated as important components of plant-pollinator interactions. We quantified the incidence, abundance, diversity, and composition of bacterial and fungal communities in floral nectar of two night-blooming plants of the Sonoran Desert over the course of a flowering season: Datura wrightii (Solanaceae), which is pollinated by hawkmoths, and Agave palmeri (Agavaceae), which is pollinated by bats but visited by hawkmoths that forage for nectar. We examined the relevance of growing environment (greenhouse vs. field), time (before and after anthesis), season (from early to late in the flowering season), and flower visitors (excluded via mesh sleeves or allowed to visit flowers naturally) in shaping microbial assemblages in nectar. We isolated and identified bacteria and fungi from >300 nectar samples to estimate richness and taxonomic composition. Our results show that microbes were common in D. wrightii and A. palmeri nectar in the greenhouse but more so in field environments, both before and especially after anthesis. Bacteria were isolated more frequently than fungi. The abundance of microbes in nectar of D. wrightii peaked near the middle of the flowering season. Microbes generally were more abundant as time for floral visitation increased. The composition of bacterial and especially fungal communities differed significantly between nectars of D. wrightii and A. palmeri, opening the door to future studies examining their functional roles in shaping nectar chemistry, attractiveness, and pollinator specialization.

Multiple phenotypic changes associated with large-scale horizontal gene transfer.

Horizontal gene transfer often leads to phenotypic changes within recipient organisms independent of any immediate evolutionary benefits. While secondary phenotypic effects of horizontal transfer (i.e., changes in growth rates) have been demonstrated and studied across a variety of systems using relatively small plasmids and phage, little is known about the magnitude or number of such costs after the transfer of larger regions. Here we describe numerous phenotypic changes that occur after a large-scale horizontal transfer event (∼1 Mb megaplasmid) within Pseudomonas stutzeri including sensitization to various stresses as well as changes in bacterial behavior. These results highlight the power of horizontal transfer to shift pleiotropic relationships and cellular networks within bacterial genomes. They also provide an important context for how secondary effects of transfer can bias evolutionary trajectories and interactions between species. Lastly, these results and system provide a foundation to investigate evolutionary consequences in real time as newly acquired regions are ameliorated and integrated into new genomic contexts.

Bigger is not always better: transmission and fitness burden of ∼1MB Pseudomonas syringae megaplasmid pMPPla107.

BACKGROUND: Horizontal gene transfer (HGT) is a widespread process that enables the acquisition of genes and metabolic pathways in single evolutionary steps. Previous reports have described fitness costs of HGT, but have largely focused on the acquisition of relatively small plasmids. We have previously shown that a Pseudomonas syringae pv. lachrymans strain recently acquired a cryptic megaplasmid, pMPPla107. This extrachromosomal element contributes hundreds of new genes to P. syringae and increases total genomic content by approximately 18%. However, this early work did not directly explore transmissibility, stability, or fitness costs associated with acquisition of pMPPla107. RESULTS: Here, we show that pMPPla107 is self-transmissible across a variety of diverse pseudomonad strains, on both solid agar and within shaking liquid cultures, with conjugation dependent on a type IV secretion system. To the best of our knowledge, this is the largest self-transmissible megaplasmid known outside of Sinorhizobium. This megaplasmid can be lost from all novel hosts although the rate of loss depends on medium type and genomic background. However, in contrast, pMPPla107 is faithfully maintained within the original parent strain (Pla107) even under direct negative selection during laboratory assays. These results suggest that Pla107 specific stabilizing mutations have occurred either on this strain's chromosome or within the megaplasmid. Lastly, we demonstrate that acquisition of pMPPla107 by strains other than Pla107 imparts severe (20%) fitness costs under competitive conditions in vitro. CONCLUSIONS: We show that pMPPla107 is capable of transmitting and maintaining itself across multiple Pseudomonas species, rendering it one of the largest conjugative elements discovered to date. The relative stability of pMPPla107, coupled with extensive fitness costs, makes it a tractable model system for investigating evolutionary and genetic mechanisms of megaplasmid maintenance and a unique testing ground to explore evolutionary dynamics after HGT of large secondary elements.