Root-Secreted Coumarins and the Microbiota Interact to Improve Iron Nutrition in Arabidopsis.

Plants benefit from associations with a diverse community of root-colonizing microbes. Deciphering the mechanisms underpinning these beneficial services are of interest for improving plant productivity. We report a plant-beneficial interaction between Arabidopsis thaliana and the root microbiota under iron deprivation that is dependent on the secretion of plant-derived coumarins. Disrupting this pathway alters the microbiota and impairs plant growth in iron-limiting soil. Furthermore, the microbiota improves iron-limiting plant performance via a mechanism dependent on plant iron import and secretion of the coumarin fraxetin. This beneficial trait is strain specific yet functionally redundant across phylogenetic lineages of the microbiota. Transcriptomic and elemental analyses revealed that this interaction between commensals and coumarins promotes growth by relieving iron starvation. These results show that coumarins improve plant performance by eliciting microbe-assisted iron nutrition. We propose that the bacterial root microbiota, stimulated by secreted coumarins, is an integral mediator of plant adaptation to iron-limiting soils.

Plant-derived coumarins shape the composition of an Arabidopsis synthetic root microbiome.

The factors that contribute to the composition of the root microbiome and, in turn, affect plant fitness are not well understood. Recent work has highlighted a major contribution of the soil inoculum in determining the composition of the root microbiome. However, plants are known to conditionally exude a diverse array of unique secondary metabolites, that vary among species and environmental conditions and can interact with the surrounding biota. Here, we explore the role of specialized metabolites in dictating which bacteria reside in the rhizosphere. We employed a reduced synthetic community (SynCom) of Arabidopsis thaliana root-isolated bacteria to detect community shifts that occur in the absence of the secreted small-molecule phytoalexins, flavonoids, and coumarins. We find that lack of coumarin biosynthesis in f6'h1 mutant plant lines causes a shift in the root microbial community specifically under iron deficiency. We demonstrate a potential role for iron-mobilizing coumarins in sculpting the A. thaliana root bacterial community by inhibiting the proliferation of a relatively abundant Pseudomonas species via a redox-mediated mechanism. This work establishes a systematic approach enabling elucidation of specific mechanisms by which plant-derived molecules mediate microbial community composition. Our findings expand on the function of conditionally exuded specialized metabolites and suggest avenues to effectively engineer the rhizosphere with the aim of improving crop growth in iron-limited alkaline soils, which make up a third of the world's arable soils.

Nutrient-responsive regulation determines biodiversity in a colicin-mediated bacterial community.

BACKGROUND: Antagonistic interactions mediated by antibiotics are strong drivers of bacterial community dynamics which shape biodiversity. Colicin production by Escherichia coli is such an interaction that governs intraspecific competition and is involved in promoting biodiversity. It is unknown how environmental cues affect regulation of the colicin operon and thus influence antibiotic-mediated community dynamics. RESULTS: Here, we investigate the community dynamics of colicin-producing, -sensitive, and -resistant/non-producer E. coli strains that colonize a microfabricated spatially-structured habitat. Nutrients are found to strongly influence community dynamics: when growing on amino acids and peptides, colicin-mediated competition is intense and the three strains do not coexist unless spatially separated at large scales (millimeters). Surprisingly, when growing on sugars, colicin-mediated competition is minimal and the three strains coexist at the micrometer scale. Carbon storage regulator A (CsrA) is found to play a key role in translating the type of nutrients into the observed community dynamics by controlling colicin release. We demonstrate that by mitigating lysis, CsrA shapes the community dynamics and determines whether the three strains coexist. Indeed, a mutant producer that is unable to suppress colicin release, causes the collapse of biodiversity in media that would otherwise support co-localized growth of the three strains. CONCLUSIONS: Our results show how the environmental regulation of an antagonistic trait shapes community dynamics. We demonstrate that nutrient-responsive regulation of colicin release by CsrA, determines whether colicin producer, resistant non-producer, and sensitive strains coexist at small spatial scales, or whether the sensitive strain is eradicated. This study highlights how molecular-level regulatory mechanisms that govern interference competition give rise to community-level biodiversity patterns.

Transient gene expression in tobacco using Gibson assembly and the Gene Gun.

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately using complex regulatory strategies including differential promoters and/or signal sequences. Metabolic engineers and synthetic biologists interested in targeting enzymes to a particular organelle are faced with a challenge: For a protein that is to be localized to more than one organelle, the engineer must clone the same gene multiple times. This work presents a solution to this strategy: harnessing alternative splicing of mRNA. This technology takes advantage of established chloroplast and peroxisome targeting sequences and combines them into a single mRNA that is alternatively spliced. Some splice variants are sent to the chloroplast, some to the peroxisome, and some to the cytosol. Here the system is designed for multiple-organelle targeting with alternative splicing. In this work, GFP was expected to be expressed in the chloroplast, cytosol, and peroxisome by a series of rationally designed 5' mRNA tags. These tags have the potential to reduce the amount of cloning required when heterologous genes need to be expressed in multiple subcellular organelles. The constructs were designed in previous work(11), and were cloned using Gibson assembly, a ligation independent cloning method that does not require restriction enzymes. The resultant plasmids were introduced into Nicotiana benthamiana epidermal leaf cells with a modified Gene Gun protocol. Finally, transformed leaves were observed with confocal microscopy.

Targeting a heterologous protein to multiple plant organelles via rationally designed 5' mRNA tags.

BACKGROUND: Plant bioengineers require simple genetic devices for predictable localization of heterologous proteins to multiple subcellular compartments. RESULTS: We designed novel hybrid signal sequences for multiple-compartment localization and characterize their function when fused to GFP in Nicotiana benthamiana leaf tissue. TriTag-1 and TriTag-2 use alternative splicing to generate differentially localized GFP isoforms, localizing it to the chloroplasts, peroxisomes and cytosol. TriTag-1 shows a bias for targeting the chloroplast envelope while TriTag-2 preferentially targets the peroxisomes. TriTag-3 embeds a conserved peroxisomal targeting signal within a chloroplast transit peptide, directing GFP to the chloroplasts and peroxisomes. CONCLUSIONS: Our novel signal sequences can reduce the number of cloning steps and the amount of genetic material required to target a heterologous protein to multiple locations in plant cells. This work harnesses alternative splicing and signal embedding for engineering plants to express multi-functional proteins from single genetic constructs.

Expression of the sub-pathways of the Chloroflexus aurantiacus 3-hydroxypropionate carbon fixation bicycle in E. coli: Toward horizontal transfer of autotrophic growth.

The 3-hydroxypropionate (3-HPA) bicycle is unique among CO2-fixing systems in that none of its enzymes appear to be affected by oxygen. Moreover, the bicycle includes a number of enzymes that produce novel intermediates of biotechnological interest, and the CO2-fixing steps in this pathway are relatively rapid. We expressed portions of the 3-HPA bicycle in a heterologous organism, E. coli K12. We subdivided the 3-HPA bicycle into four sub-pathways: (1) synthesis of propionyl-CoA from acetyl-CoA, (2) synthesis of succinate from propionyl-CoA, (3) glyoxylate production and regeneration of acetyl-CoA, and (4) assimilation of glyoxylate and propionyl-CoA to form pyruvate and regenerate acetyl-CoA. We expressed the novel enzymes of the 3-HPA bicycle in operon form and used phenotypic tests for activity. Sub-pathway 1 activated a propionate-specific biosensor. Sub-pathway 2, found in non-CO2-fixing bacteria, was reassembled in E. coli using genes from diverse sources. Sub-pathway 3, operating in reverse, generated succinyl-CoA sufficient to rescue a sucAD(-) double mutant of its diaminopimelic acid (DAP) auxotrophy. Sub-pathway 4 was able to reduce the toxicity of propionate and allow propionate to contribute to cell biomass in a prpC(-)(2 methylcitrate synthase) mutant strain. These results indicate that all of the sub-pathways of the 3-HPA bicycle can function to some extent in vivo in a heterologous organism, as indicated by growth tests. Overexpression of certain enzymes was deleterious to cell growth, and, in particular, expression of MMC-CoA lyase caused a mucoid phenotype. These results have implications for metabolic engineering and for bacterial evolution through horizontal gene transfer.

Incorporation of non-natural amino acids improves cell permeability and potency of specific inhibitors of proteasome trypsin-like sites.

Proteasomes degrade the majority of proteins in mammalian cells by a concerted action of three distinct pairs of active sites. The chymotrypsin-like sites are targets of antimyeloma agents bortezomib and carfilzomib. Inhibitors of the trypsin-like site sensitize multiple myeloma cells to these agents. Here we describe systematic effort to develop inhibitors with improved potency and cell permeability, yielding azido-Phe-Leu-Leu-4-aminomethyl-Phe-methyl vinyl sulfone (4a, LU-102), and a fluorescent activity-based probe for this site. X-ray structures of 4a and related inhibitors complexed with yeast proteasomes revealed the structural basis for specificity. Nontoxic to myeloma cells when used as a single agent, 4a sensitized them to bortezomib and carfilzomib. This sensitizing effect was much stronger than the synergistic effects of histone acetylase inhibitors or additive effects of doxorubicin and dexamethasone, raising the possibility that combinations of inhibitors of the trypsin-like site with bortezomib or carfilzomib would have stronger antineoplastic activity than combinations currently used clinically.

A toolkit to enable hydrocarbon conversion in aqueous environments.

This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli and presents a proof of principle of its applicability. The toolkit consists of multiple standard interchangeable parts (BioBricks)(9) addressing the conversion of alkanes, regulation of gene expression and survival in toxic hydrocarbon-rich environments. A three-step pathway for alkane degradation was implemented in E. coli to enable the conversion of medium- and long-chain alkanes to their respective alkanols, alkanals and ultimately alkanoic-acids. The latter were metabolized via the native ß-oxidation pathway. To facilitate the oxidation of medium-chain alkanes (C5-C13) and cycloalkanes (C5-C8), four genes (alkB2, rubA3, rubA4and rubB) of the alkane hydroxylase system from Gordonia sp. TF6(8,21) were transformed into E. coli. For the conversion of long-chain alkanes (C15-C36), theladA gene from Geobacillus thermodenitrificans was implemented. For the required further steps of the degradation process, ADH and ALDH (originating from G. thermodenitrificans) were introduced(10,11). The activity was measured by resting cell assays. For each oxidative step, enzyme activity was observed. To optimize the process efficiency, the expression was only induced under low glucose conditions: a substrate-regulated promoter, pCaiF, was used. pCaiF is present in E. coli K12 and regulates the expression of the genes involved in the degradation of non-glucose carbon sources. The last part of the toolkit - targeting survival - was implemented using solvent tolerance genes, PhPFDα and ß, both from Pyrococcus horikoshii OT3. Organic solvents can induce cell stress and decreased survivability by negatively affecting protein folding. As chaperones, PhPFDα and ß improve the protein folding process e.g. under the presence of alkanes. The expression of these genes led to an improved hydrocarbon tolerance shown by an increased growth rate (up to 50%) in the presences of 10% n-hexane in the culture medium were observed. Summarizing, the results indicate that the toolkit enables E. coli to convert and tolerate hydrocarbons in aqueous environments. As such, it represents an initial step towards a sustainable solution for oil-remediation using a synthetic biology approach.

Nature of pharmacophore influences active site specificity of proteasome inhibitors.

Proteasomes degrade most proteins in mammalian cells and are established targets of anti-cancer drugs. The majority of proteasome inhibitors are composed of short peptides with an electrophilic functionality (pharmacophore) at the C terminus. All eukaryotic proteasomes have three types of active sites as follows: chymotrypsin-like, trypsin-like, and caspase-like. It is widely believed that active site specificity of inhibitors is determined primarily by the peptide sequence and not the pharmacophore. Here, we report that active site specificity of inhibitors can also be tuned by the chemical nature of the pharmacophore. Specifically, replacement of the epoxyketone by vinyl sulfone moieties further improves the selectivity of ß5-specific inhibitors NC-005, YU-101, and PR-171 (carfilzomib). This increase in specificity is likely the basis of the decreased cytotoxicity of vinyl sulfone-based inhibitors to HeLa cells as compared with that of epoxyketone-based inhibitors.