RESUMO
Rare earth elements (REE) are essential ingredients of sustainable energy technologies, but separation of individual REE is one of the hardest problems in chemistry today. Biosorption, where molecules adsorb to the surface of biological materials, offers a sustainable alternative to environmentally harmful solvent extractions currently used for separation of rare earth elements (REE). The REE-biosorption capability of some microorganisms allows for REE separations that, under specialized conditions, are already competitive with solvent extractions, suggesting that genetic engineering could allow it to leapfrog existing technologies. To identify targets for genomic improvement we screened 3,373 mutants from the whole genome knockout collection of the known REE-biosorbing microorganism Shewanella oneidensis MR-1. We found 130 genes that increased biosorption of the middle REE europium, and 112 that reduced it. We verified biosorption changes from the screen for a mixed solution of three REE (La, Eu, Yb) using Inductively Coupled Plasma Mass Spectrometry (ICP-MS) in solution conditions with a range of ionic strengths and REE concentrations. We identified 18 gene ontologies and 13 gene operons that make up key systems that affect biosorption. We found, among other things, that disruptions of a key regulatory component of the arc system (hptA), which regulates cellular response to anoxic environments and polysaccharide biosynthesis related genes (wbpQ, wbnJ, SO_3183) consistently increase biosorption across all our solution conditions. Our largest total biosorption change comes from our SO_4685, a capsular polysaccharide (CPS) synthesis gene, disruption of which results in an up to 79% increase in biosorption; and nusA, a transcriptional termination/anti-termination protein, disruption of which results in an up to 35% decrease in biosorption. Knockouts of glnA, pyrD, and SO_3183 produce small but significant increases (≈ 1%) in relative biosorption affinity for ytterbium over lanthanum in multiple solution conditions tested, while many other genes we explored have more complex binding affinity changes. Modeling suggests that while these changes to lanthanide biosorption selectivity are small, they could already reduce the length of repeated enrichment process by up to 27%. This broad exploratory study begins to elucidate how genetics affect REE-biosorption by S. oneidensis, suggests new areas of investigation for better mechanistic understanding of the membrane chemistry involved in REE binding, and offer potential targets for improving biosorption and separation of REE by genetic engineering.
Assuntos
Genômica , Shewanella , Shewanella/genética , Európio , SolventesRESUMO
By the end of the century, tens of gigatonnes of CO2 will need to be removed from the atmosphere every year to maintain global temperatures. Natural weathering of ultramafic rocks and subsequent mineralization reactions can convert CO2 into ultra-stable carbonates. Although this will draw down all excess CO2, it will take thousands of years. CO2 mineralization could be accelerated by weathering ultramafic rocks with biodegradable lixiviants. We show that if these lixiviants come from cellulosic biomass, this demand could monopolize the world's biomass supply. We demonstrate that electromicrobial production technologies (EMP) that combine renewable electricity and microbial metabolism could produce lixiviants for as little as $200 to $400 per tonne at solar electricity prices achievable within the decade. We demonstrate that EMP could make enough lixiviants to sequester a tonne of CO2 for less than $100. This work highlights the potential of this approach and the need for extensive R&D.
RESUMO
Bioleaching of rare earth elements (REEs), using microorganisms such as Gluconobacter oxydans, offers a sustainable alternative to environmentally harmful thermochemical extraction, but is currently not very efficient. Here, we generate a whole-genome knockout collection of single-gene transposon disruption mutants for G. oxydans B58, to identify genes affecting the efficacy of REE bioleaching. We find 304 genes whose disruption alters the production of acidic biolixiviant. Disruption of genes underlying synthesis of the cofactor pyrroloquinoline quinone (PQQ) and the PQQ-dependent membrane-bound glucose dehydrogenase nearly eliminates bioleaching. Disruption of phosphate-specific transport system genes enhances bioleaching by up to 18%. Our results provide a comprehensive roadmap for engineering the genome of G. oxydans to further increase its bioleaching efficiency.
Assuntos
Proteínas de Bactérias/genética , Técnicas de Inativação de Genes/métodos , Genoma Bacteriano/genética , Gluconobacter oxydans/genética , Glucose Desidrogenase/genética , Cofator PQQ/genética , Proteínas de Bactérias/metabolismo , Engenharia Genética/métodos , Gluconobacter oxydans/metabolismo , Glucose Desidrogenase/metabolismo , Microbiologia Industrial/métodos , Metais Terras Raras/metabolismo , Cofator PQQ/metabolismo , Reprodutibilidade dos TestesRESUMO
Under nutrient-limiting conditions, plants will enter into symbiosis with arbuscular mycorrhizal (AM) fungi for the enhancement of mineral nutrient acquisition from the surrounding soil. AM fungi live in close, intracellular association with plant roots where they transfer phosphate and nitrogen to the plant in exchange for carbon. They are obligate fungi, relying on their host as their only carbon source. Much has been discovered in the last decade concerning the signaling events during initiation of the AM symbiosis, including the identification of signaling molecules generated by both partners. This signaling occurs through symbiosis-specific gene products in the host plant, which are indispensable for normal AM development. At the same time, plants have adapted complex mechanisms for avoiding infection by pathogenic fungi, including an innate immune response to general microbial molecules, such as chitin present in fungal cell walls. How it is that AM fungal colonization is maintained without eliciting a defensive response from the host is still uncertain. In this review, we present a summary of the molecular signals and their elicited responses during initiation of the AM symbiosis, including plant immune responses and their suppression.
Assuntos
Micorrizas/fisiologia , Transdução de Sinais , Simbiose/fisiologia , Quitina/metabolismo , Lipopolissacarídeos/metabolismo , Raízes de Plantas/metabolismoRESUMO
Medicago truncatula is used widely as a model system for studies of root symbioses, interactions with parasitic nematodes and fungal pathogens, as well as studies of development and secondary metabolism. In Medicago truncatula as well as other legumes, RNA interference (RNAi) coupled with Agrobacterium rhizogenes-mediated root transformation, has been used very successfully for analyses of gene function in roots. One of the major advantages of this approach is the ease and relative speed with which transgenic roots can be generated. There are several methods, both for the generation of the RNAi constructs and the root transformation. Here we provide details of an RNAi and root transformation protocol that has been used successfully in M. truncatula and which can be scaled up to enable the analysis of several hundred constructs.
Assuntos
Inativação Gênica , Medicago truncatula/genética , Raízes de Plantas/genética , Interferência de RNA , Regulação da Expressão Gênica de Plantas , Vetores Genéticos/genética , Medicago truncatula/crescimento & desenvolvimento , Medicago truncatula/microbiologia , Raízes de Plantas/microbiologia , Plantas Geneticamente Modificadas , Rhizobium/fisiologia , SimbioseRESUMO
UNLABELLED: Many Gram-negative bacteria utilize specialized secretion systems to inject proteins (effectors) directly into host cells. Little is known regarding how bacteria ensure that only small subsets of the thousands of proteins they encode are recognized as substrates of the secretion systems, limiting their identification through bioinformatic analyses. Many of these proteins require chaperones to direct their secretion. Here, using the newly described protein interaction platform assay, we demonstrate that type 3 secretion system class IB chaperones from one bacterium directly bind their own effectors as well as those from other species. In addition, we observe that expression of class IB homologs from seven species, including pathogens and endosymbionts, mediate the translocation of effectors from Shigella directly into host cells, demonstrating that class IB chaperones are often functionally interchangeable. Notably, class IB chaperones bind numerous effectors. However, as previously proposed, they are not promiscuous; rather they recognize a defined sequence that we designate the conserved chaperone-binding domain (CCBD) sequence [(LMIF)(1)XXX(IV)(5)XX(IV)(8)X(N)(10)]. This sequence is the first defined amino acid sequence to be identified for any interspecies bacterial secretion system, i.e., a system that delivers proteins directly into eukaryotic cells. This sequence provides a new means to identify substrates of type III secretion systems. Indeed, using a pattern search algorithm for the CCBD sequence, we have identified the first two probable effectors from an endosymbiont, Sodalis glossinidius. IMPORTANCE: Many Gram-negative pathogens utilize type 3 secretion systems to deliver tens of effectors into host cells. In order to understand the diverse ways that these organisms cause disease, it is necessary to identify their effectors, many of which require chaperones to be secreted. Here we establish that class IB chaperones are not promiscuous, as previously proposed, but rather recognize a conserved effector sequence. We demonstrate that pattern search algorithms based on this defined sequence can be used to identify previously unknown effectors. Furthermore, we observe that class IB chaperones from at least seven bacterial species are functionally interchangeable. Not only do they bind and mediate the delivery of their own set of effectors into host cells but they also bind to type 3 substrates from other bacteria, suggesting that inhibitors that block chaperone-effector interactions could provide a novel means to effectively treat infections due to Gram-negative pathogens, including organisms resistant to currently available antibiotics.
Assuntos
Sistemas de Secreção Bacterianos , Enterobacteriaceae/metabolismo , Chaperonas Moleculares/metabolismo , Mapeamento de Interação de Proteínas/métodos , Shigella/fisiologia , Algoritmos , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Biologia Computacional , Sequência Conservada , Enterobacteriaceae/fisiologia , Células HeLa , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Salmonella typhimurium/metabolismo , Salmonella typhimurium/fisiologia , Shigella/metabolismo , Especificidade da Espécie , Especificidade por Substrato , SimbioseRESUMO
Here we describe the protein interaction platform assay, a method for identifying interacting proteins in Saccharomyces cerevisiae. This assay relies on the reovirus scaffolding protein microNS, which forms large focal inclusions in living cells. When a query protein is fused to microNS and potential interaction partners are fused to a fluorescent reporter, interactors can be identified by screening for yeast that display fluorescent foci.