Horizontal 'gene drives' harness indigenous bacteria for bioremediation.

Engineering bacteria to clean-up oil spills is rapidly advancing but faces regulatory hurdles and environmental concerns. Here, we develop a new technology to harness indigenous soil microbial communities for bioremediation by flooding local populations with catabolic genes for petroleum hydrocarbon degradation. Overexpressing three enzymes (almA, xylE, p450cam) in Escherichia coli led to degradation of 60-99% of target hydrocarbon substrates. Mating experiments, fluorescence microscopy and TEM revealed indigenous bacteria could obtain these vectors from E. coli through several mechanisms of horizontal gene transfer (HGT), including conjugation and cytoplasmic exchange through nanotubes. Inoculating petroleum-polluted sediments with E. coli carrying the vector pSF-OXB15-p450camfusion showed that the E. coli cells died after five days but a variety of bacteria received and carried the vector for over 60 days after inoculation. Within 60 days, the total petroleum hydrocarbon content of the polluted soil was reduced by 46%. Pilot experiments show that vectors only persist in indigenous populations when under selection pressure, disappearing when this carbon source is removed. This approach to remediation could prime indigenous bacteria for degrading pollutants while providing minimal ecosystem disturbance.

A High-Throughput Fluorescence-Based Assay for Rapid Identification of Petroleum-Degrading Bacteria.

Over the past 100 years, oil spills and long-term waste deposition from oil refineries have significantly polluted the environment. These contaminants have widespread negative effects on human health and ecosystem functioning. Natural attenuation of long chain and polyaromatic hydrocarbons is slow and often incomplete. Bioaugmentation of polluted soils with indigenous bacteria that naturally consume petroleum hydrocarbons could speed up this process. However, the characterization of bacterial crude oil degradation efficiency - which often relies upon expensive, highly specialized gas-chromatography mass spectrometry analyses - can present a substantial bottleneck in developing and implementing these bioremediation strategies. Here, we develop a low-cost, rapid, high-throughput fluorescence-based assay for identifying wild-type bacteria that degrade crude oil using the dye Nile Red. We show that Nile Red fluoresces when in contact with crude oil and developed a robust linear model to calculate crude oil content in liquid cell cultures based on fluorescence intensity (FI). To test whether this assay could identify bacteria with enhanced metabolic capacities to break down crude oil, we screened bacteria isolated from a former Shell Oil refinery in Bay Point, CA, and identified one strain (Cupriavidus sp. OPK) with superior crude oil depletion efficiencies (up to 83%) in only 3 days. We further illustrate that this assay can be combined with fluorescence microscopy to study how bacteria interact with crude oil and the strategies they use to degrade this complex substance. We show for the first time that bacteria use three key strategies for degrading crude oil: biofilm formation, direct adherence to oil droplets, and vesicle encapsulation of oil. We propose that the quantitative and qualitative data from this assay can be used to develop new bioremediation strategies based on bioaugmentation and/or biomimetic materials that imitate the natural ability of bacteria to degrade crude oil.

Targeted and Untargeted Metabolic Profiling of Wild Grassland Plants identifies Antibiotic and Anthelmintic Compounds Targeting Pathogen Physiology, Metabolism and Reproduction.

Plants traditionally used by farmers to manage livestock ailments could reduce reliance on synthetic antibiotics and anthelmintics but in many cases their chemical composition is unknown. As a case study, we analyzed the metabolite profiles of 17 plant species and 45 biomass samples from agricultural grasslands in England using targeted and untargeted metabolite profiling by liquid-chromatography mass spectrometry. We identified a range of plant secondary metabolites, including 32 compounds with known antimicrobial/anthelmintic properties which varied considerably across the different plant samples. These compounds have been shown previously to target multiple aspects of pathogen physiology and metabolism in vitro and in vivo, including inhibition of quorum sensing in bacteria and egg viability in nematodes. The most abundant bioactive compounds were benzoic acid, myricetin, p-coumaric acid, rhamnetin, and rosmarinic acid. Four wild plants (Filipendula ulmaria (L.) Maxim., Prunella vulgaris L., Centuarea nigra L., and Rhinanthus minor L.) and two forage legumes (Medicago sativa L., Trifolium hybridium L.) contained high levels of these compounds. Forage samples from native high-diversity grasslands had a greater abundance of medicinal compounds than samples from agriculturally improved grasslands. Incorporating plants with antibiotic/anthelmintic compounds into livestock feeds may reduce global drug-resistance and preserve the efficacy of last-resort drugs.

Engineering Mycorrhizal Symbioses to Alter Plant Metabolism and Improve Crop Health.

Creating sustainable bioeconomies for the 21st century relies on optimizing the use of biological resources to improve agricultural productivity and create new products. Arbuscular mycorrhizae (phylum Glomeromycota) form symbiotic relationships with over 80% of vascular plants. In return for carbon, these fungi improve plant health and tolerance to environmental stress. This symbiosis is over 400 million years old and there are currently over 200 known arbuscular mycorrhizae, with dozens of new species described annually. Metagenomic sequencing of native soil communities, from species-rich meadows to mangroves, suggests biologically diverse habitats support a variety of mycorrhizal species with potential agricultural, medical, and biotechnological applications. This review looks at the effect of mycorrhizae on plant metabolism and how we can harness this symbiosis to improve crop health. I will first describe the mechanisms that underlie this symbiosis and what physiological, metabolic, and environmental factors trigger these plant-fungal relationships. These include mycorrhizal manipulation of host genetic expression, host mitochondrial and plastid proliferation, and increased production of terpenoids and jasmonic acid by the host plant. I will then discuss the effects of mycorrhizae on plant root and foliar secondary metabolism. I subsequently outline how mycorrhizae induce three key benefits in crops: defense against pathogen and herbivore attack, drought resistance, and heavy metal tolerance. I conclude with an overview of current efforts to harness mycorrhizal diversity to improve crop health through customized inoculum. I argue future research should embrace synthetic biology to create mycorrhizal chasses with improved symbiotic abilities and potentially novel functions to improve plant health. As the effects of climate change and anthropogenic disturbance increase, the global diversity of arbuscular mycorrhizal fungi should be monitored and protected to ensure this important agricultural and biotechnological resource for the future.