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ACS Synthetic Biology Feb 2024transcription-translation (TX-TL) can enable faster engineering of biological systems. This speed-up can be significant, especially in difficult-to-transform chassis....
transcription-translation (TX-TL) can enable faster engineering of biological systems. This speed-up can be significant, especially in difficult-to-transform chassis. This work shows the successful development of TX-TL systems using three soil-derived wild-type Pseudomonads known to promote plant growth: , , and . All three species demonstrated multiple sonication, runoff, and salt conditions producing detectable protein synthesis. One of these new TX-TL systems, , demonstrated a maximum protein yield of 2.5 μM at 125 proteins per DNA template, a maximum protein synthesis rate of 20 nM/min, and a range of DNA concentrations with a linear correspondence with the resulting protein synthesis. A set of different constitutive promoters driving mNeonGreen expression were tested in TX-TL and integrated into the genome, showing similar normalized strengths for and fluorescence. This correspondence between the TX-TL-derived promoter strength and the promoter strength indicates that these lysate-based cell-free systems can be used to characterize and engineer biological parts without genomic integration, enabling a faster design-build-test cycle.
Topics: Transcription, Genetic; Protein Biosynthesis; Cell-Free System; Escherichia coli; DNA
PubMed: 38319019
DOI: 10.1021/acssynbio.3c00468 -
Faraday Discussions Jun 2024The effective management of plastic waste has become a global imperative, given our reliance on a linear model in which plastics are manufactured, used once, and then...
The effective management of plastic waste has become a global imperative, given our reliance on a linear model in which plastics are manufactured, used once, and then discarded. This has led to the pervasive accumulation of plastic debris in landfills and environmental contamination. Recognizing this issue, numerous initiatives are underway to address the environmental repercussions associated with plastic disposal. In this study, we investigate the possible molecular mechanism of polyurethane esterase A (PueA), which has been previously identified as responsible for the degradation of a polyester polyurethane (PU) sample in , as an effort to develop enzymatic biodegradation solutions. After generating the unsolved 3D structure of the protein by AlphaFold2 from its known genome, the enzymatic hydrolysis of the same model PU compound previously used in experiments has been explored employing QM/MM molecular dynamics simulations. This required a preliminary analysis of the 3D structure of the apo-enzyme, identifying the putative active site, and the search for the optimal protein-substrate binding site. Finally, the resulting free energy landscape indicates that wild-type PueA can degrade PU chains, although with low-level activity. The reaction takes place by a characteristic four-step path of the serine hydrolases, involving an acylation followed by a diacylation step. Energetics and structural analysis of the evolution of the active site along the reaction suggests that PueA can be considered a promising protein scaffold for further development to achieve efficient biodegradation of PU.
PubMed: 38836643
DOI: 10.1039/d4fd00022f -
Journal of Plant Physiology Jun 2024Management of the plant microbiome may help support food needs for the human population. Bacteria influence plants through enhancing nutrient uptake, metabolism,...
Sucrose supplements modulate the Pseudomonas chlororaphis-Arabidopsis thaliana interaction via decreasing the production of phenazines and enhancing the root auxin response.
Management of the plant microbiome may help support food needs for the human population. Bacteria influence plants through enhancing nutrient uptake, metabolism, photosynthesis, biomass production and/or reinforcing immunity. However, information into how these microbes behave under different growth conditions is missing. In this work, we tested how carbon supplements modulate the interaction of Pseudomonas chlororaphis with Arabidopsis thaliana. P. chlororaphis streaks strongly repressed primary root growth, lateral root formation and ultimately, biomass production. Noteworthy, increasing sucrose availability into the media from 0 to 2.4% restored plant growth and promoted lateral root formation in bacterized seedlings. This effect could not be observed by supplementing sucrose to leaves only, indicating that the interaction was strongly modulated by bacterial access to sugar. Total phenazine content decreased in the bacteria grown in high (2.4%) sucrose medium, and conversely, the expression of phzH and pslA genes were diminished by sugar supply. Pyocyanin antagonized the promoting effects of sucrose in lateral root formation and biomass production in inoculated seedlings, indicating that this virulence factor accounts for growth repression during the plant-bacterial interaction. Defence reporter transgenes PR-1::GUS and LOX2::GUS were induced in leaves, while the expression of the auxin-inducible, synthetic reporter gene DR5::GUS was enhanced in the roots of bacterized seedlings at low and high sucrose treatments, which suggests that growth/defence trade-offs in plants are critically modulated by P. chlororaphis. Collectively, our data suggest that bacterial carbon nutrition controls the outcome of the relation with plants.
Topics: Sucrose; Arabidopsis; Plant Roots; Pseudomonas chlororaphis; Phenazines; Indoleacetic Acids
PubMed: 38705079
DOI: 10.1016/j.jplph.2024.154259 -
Microbiological Research Aug 2024The root microbiota plays a crucial role in plant performance. The use of microbial consortia is considered a very useful tool for studying microbial interactions in the...
The root microbiota plays a crucial role in plant performance. The use of microbial consortia is considered a very useful tool for studying microbial interactions in the rhizosphere of different agricultural crop plants. Thus, a consortium of 3 compatible beneficial rhizospheric Pseudomonas strains previously isolated from the avocado rhizosphere, was constructed. The consortium is composed of two compatible biocontrol P. chlororaphis strains (PCL1601 and PCL1606), and the biocontrol rhizobacterium Pseudomonas alcaligenes AVO110, which are all efficient root colonizers of avocado and tomato plants. These three strains were compatible with each other and reached stable levels both in liquid media and on plant roots. Bacterial strains were fluorescent tagged, and colonization-related traits were analyzed in vitro, revealing formation of mixed biofilm networks without exclusion of any of the strains. Additionally, bacterial colonization patterns compatible with the different strains were observed, with high survival traits on avocado and tomato roots. The bacteria composing the consortium shared the same root habitat and exhibited biocontrol activity against soil-borne fungal pathogens at similar levels to those displayed by the individual strains. As expected, because these strains were isolated from avocado roots, this Pseudomonas-based consortium had more stable bacterial counts on avocado roots than on tomato roots; however, inoculation of tomato roots with this consortium was shown to protect tomato plants under high-temperature stress. The results revealed that this consortium has side beneficial effect for tomato plants under high-temperature stress, thus improving the potential performance of the individual strains. We concluded that this rhizobacterial consortium do not improve the plant protection against soil-borne phytopathogenic fungi displayed by the single strains; however, its inoculation can show an specific improvement of plant performance on a horticultural non-host plant (such as tomato) when the plant was challenged by high temperature stress, thus extending the beneficial role of this bacterial consortium.
Topics: Plant Roots; Solanum lycopersicum; Rhizosphere; Pseudomonas; Soil Microbiology; Microbial Consortia; Persea; Plant Diseases; Biofilms; Hot Temperature; Biological Control Agents; Stress, Physiological
PubMed: 38761488
DOI: 10.1016/j.micres.2024.127761 -
Microbiological Research Jan 2024Pseudomonas chlororaphis PCL1606 (PcPCL1606) is a model rhizobacterium used to study beneficial bacterial interactions with the plant rhizosphere. Many of its beneficial...
Pseudomonas chlororaphis PCL1606 (PcPCL1606) is a model rhizobacterium used to study beneficial bacterial interactions with the plant rhizosphere. Many of its beneficial phenotypes depend on the production of the antifungal compound 2-hexyl, 5-propyl resorcinol (HPR). Transcriptomic analysis of PcPCL1606 and the deletional mutant in HPR production ΔdarB strain, assigned an additional regulatory role to HPR, and allowed the detection of differentially expressed genes during the bacterial interaction with the avocado rhizosphere. Interestingly, the putative genes phaG (PCL1606_46820) and phaI (PCL1606_56560), with a predicted role in polyhydroxyalkanoate biosynthesis, were detected to be under HPR control. Both putative genes were expressed in the HPR-producing wild-type strain, but strongly repressed in the derivative mutant ΔdarB, impaired in HPR production. Thus, a derivative mutant impaired in the phaG gene was constructed, characterized and compared with the wild-type strain PcPCL1606 and with the derivative mutant ΔdarB. The phaG mutant had strongly reduced PHA production by PcPCL1606, and displayed altered phenotypes involved in bacterial survival on the plant roots, such as tolerance to high temperature and hydrogen peroxide, and decreased root survival, in a similar way that the ΔdarB mutant. On the other hand, the phaG mutant does not have altered resistance to desiccation, motility, biofilm formation or adhesion phenotypes, as displayed by the HPR-defective ΔdarB mutant have. Interestingly, the mutant defective in PHA production also lacked a biocontrol phenotype against the soilborne pathogenic fungus Rosellinia necatrix, even when the derivative mutant still produced the antifungal HPR compound, demonstrating that the final biocontrol phenotype of PcPCL1606 first requires bacterial survival and adaptation traits to the soil and rhizosphere environment.
Topics: Pseudomonas chlororaphis; Antifungal Agents; Plant Roots; Bacterial Proteins
PubMed: 37863020
DOI: 10.1016/j.micres.2023.127527 -
Angewandte Chemie (International Ed. in... Jul 2023A non-canonical biosynthetic pathway furnishing the first natural brexane-type bishomosesquiterpene (chlororaphen, C H ) was elucidated in the γ-proteobacterium...
Non-canonical Biosynthesis of the Brexane-Type Bishomosesquiterpene Chlororaphen through Two Consecutive Methylation Steps in Pseudomonas chlororaphis O6 and Variovorax boronicumulans PHE5-4.
A non-canonical biosynthetic pathway furnishing the first natural brexane-type bishomosesquiterpene (chlororaphen, C H ) was elucidated in the γ-proteobacterium Pseudomonas chlororaphis O6. A combination of genome mining, pathway cloning, in vitro enzyme assays, and NMR spectroscopy revealed a three-step pathway initiated by C10 methylation of farnesyl pyrophosphate (FPP, C ) along with cyclization and ring contraction to furnish monocyclic γ-presodorifen pyrophosphate (γ-PSPP, C ). Subsequent C-methylation of γ-PSPP by a second C-methyltransferase furnishes the monocyclic α-prechlororaphen pyrophosphate (α-PCPP, C ), serving as the substrate for the terpene synthase. The same biosynthetic pathway was characterized in the β-proteobacterium Variovorax boronicumulans PHE5-4, demonstrating that non-canonical homosesquiterpene biosynthesis is more widespread in the bacterial domain than previously anticipated.
Topics: Methylation; Pseudomonas chlororaphis; Diphosphates; Comamonadaceae
PubMed: 37132448
DOI: 10.1002/anie.202303692 -
Journal of Natural Products Jun 2024Phenazines are aromatic compounds with antifungal and cytotoxic activities. Phenazines incorporating phenazine 1-carboxylic acid have widespread applications in...
Phenazines are aromatic compounds with antifungal and cytotoxic activities. Phenazines incorporating phenazine 1-carboxylic acid have widespread applications in agriculture, medicine, and industry. Griseoluteic acid is a cytotoxic compound secreted by P510, displaying potential medical applications. However, the biosynthetic pathway of griseoluteic acid has not been elucidated, limiting its development and application. In this study, a conserved phenazine biosynthetic gene cluster of P510 was identified through genomic analysis. Subsequently, its was confirmed that the four essential modification enzymes SgpH, SgpI, SgpK, and SgpL convert phenazine-1,6-dicarboxylic acid into griseoluteic acid by heterologous expression in . Moreover, the biosynthetic pathway of griseoluteic acid was established in characterized by a high growth rate and synthesis efficiency of phenazines, laying the foundation for the efficient production of griseoluteic acid.
Topics: Phenazines; Molecular Structure; Multigene Family; Biosynthetic Pathways; Streptomyces; Streptomyces griseus; Pseudomonas chlororaphis; Escherichia coli
PubMed: 38832657
DOI: 10.1021/acs.jnatprod.4c00017 -
Environmental Science & Technology Aug 2023Plant growth promoting rhizobacteria (PGPR) produce extracellular reactive oxygen species (ROS) to protect plants from external stresses. Fe-based nanomaterials can...
Plant growth promoting rhizobacteria (PGPR) produce extracellular reactive oxygen species (ROS) to protect plants from external stresses. Fe-based nanomaterials can potentially interact with PGPR and synergistically degrade organic pollutants, yet they have received no study. Here, we studied how the interaction between a typical PGPR (, JD37) and Fe-based nanomaterials facilitated the degradation of 2,4,4'-trichlorobiphenyl (PCB28), by comparing the zerovalent iron of 20 nm (nZVI), 100 nm (nZVI), and 5 μm; iron oxide nanomaterials (α-FeO, γ-FeO, and FeO) of ca. 20 nm; and ferrous and ferric salts. Although all Fe materials (0.1 g L) alone could not degrade aqueous PCB28 (0.1 mg L) under dark or aerobic conditions, nZVI, nZVI, α-FeO, and Fe promoted PCB28 degradation by JD37, with the half-life of PCB28 shortened from 16.5 h by JD37 alone to 8.1 h with nZVI cotreatment. Mechanistically, the nanomaterials stimulated JD37 to secrete phenazine-1-carboxylic acid and accelerated the NADH/NAD conversion, promoting O generation; JD37 increased Fe(II) dissolution from the nanomaterials, facilitating *OH generation; and the ROS gradually degraded PCB28 into benzoic acid through dihydroxy substitution, oxidation to quinone, and Michael addition. These findings provide a new strategy of nanoenabled biodegradation of organic pollutants by applying Fe-based nanomaterials and PGPR.
Topics: Polychlorinated Biphenyls; Reactive Oxygen Species; Iron; Nanostructures; Environmental Pollutants
PubMed: 37583057
DOI: 10.1021/acs.est.3c02495 -
Molecules (Basel, Switzerland) Nov 2023Polycyclic aromatic hydrocarbons (PAHs) are common xenobiotics that are detrimental to the environment and human health. Bacterial endophytes, having the capacity to...
Polycyclic aromatic hydrocarbons (PAHs) are common xenobiotics that are detrimental to the environment and human health. Bacterial endophytes, having the capacity to degrade PAHs, and plant growth promotion (PGP) may facilitate their biodegradation. In this study, phenanthrene (PHE) utilization of a newly isolated PGP endophytic strain of 23aP and factors affecting the process were evaluated. The data obtained showed that strain 23aP utilized PHE in a wide range of concentrations (6-100 ppm). Ethyl-acetate-extractable metabolites obtained from the PHE-enriched cultures were analyzed by gas chromatography-mass spectrometry (GC-MS) and thin-layer chromatography (HPTLC). The analysis identified phthalic acid, 3-(1-naphthyl)allyl alcohol, 2-hydroxybenzalpyruvic acid, -naphthol, and 2-phenylbenzaldehyde, and allowed us to propose that the PHE degradation pathway of strain 23aP is initiated at the 1,2-, 3,4-carbon positions, while the 9,10-C pathway starts with non-enzymatic oxidation and is continued by the downstream phthalic pathway. Moreover, the production of the biosurfactants, mono- (Rha-C-C, Rha-C-C, Rha-C-C, and Rha-C-C) and dirhamnolipids (Rha-Rha-C-C), was confirmed using direct injection-electrospray ionization-mass spectrometry (DI-ESI-MS) technique. Changes in the bacterial surface cell properties in the presence of PHE of increased hydrophobicity were assessed with the microbial adhesion to hydrocarbons (MATH) assay. Altogether, this suggests the strain 23aP might be used in bioaugmentation-a biological method supporting the removal of pollutants from contaminated environments.
Topics: Humans; Pseudomonas chlororaphis; Phenanthrenes; Polycyclic Aromatic Hydrocarbons; Spectrometry, Mass, Electrospray Ionization; Bacteria; Biodegradation, Environmental
PubMed: 38005303
DOI: 10.3390/molecules28227581 -
Microorganisms Jul 2023Causing major health and ecological disturbances, polychlorinated biphenyls (PCBs) are persistent organic pollutants still recovered all over the world. Microbial PCB...
Causing major health and ecological disturbances, polychlorinated biphenyls (PCBs) are persistent organic pollutants still recovered all over the world. Microbial PCB biotransformation is a promising technique for depollution, but the involved molecular mechanisms remain misunderstood. Ligninolytic enzymes are suspected to be involved in many PCB transformations, but their assessments remain scarce. To further inventory the capabilities of microbes to transform PCBs through their ligninolytic enzymes, we investigated the role of oxidase and peroxidase among a set of microorganisms isolated from a historically PCB-contaminated site. Among 29 isolated fungi and 17 bacteria, this work reports for the first time the PCB-transforming capabilities from fungi affiliated to , , , , , , and genera and bacteria affiliated to , , , , , sp., , and . In the same way, this is the first report of fungal isolates affiliated to the specie and genus that displayed oxidase (putatively laccase) and peroxidase activity, respectively, enhanced in the presence of PCBs (more than 4-fold and 20-fold, respectively, compared to controls). Based on these results, the observed activities are suspected to be involved in PCB transformation.
PubMed: 37630447
DOI: 10.3390/microorganisms11081887