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Nucleic Acids Research Jan 2024The ubiquitous bacterial second messenger cyclic diguanylate (c-di-GMP) coordinates diverse cellular processes through its downstream receptors. However, whether...
The ubiquitous bacterial second messenger cyclic diguanylate (c-di-GMP) coordinates diverse cellular processes through its downstream receptors. However, whether c-di-GMP participates in regulating nitrate assimilation is unclear. Here, we found that NasT, an antiterminator involved in nitrate assimilation in Pseudomonas putida, specifically bound c-di-GMP. NasT was essential for expressing the nirBD operon encoding nitrite reductase during nitrate assimilation. High-level c-di-GMP inhibited the binding of NasT to the leading RNA of nirBD operon (NalA), thus attenuating the antitermination function of NasT, resulting in decreased nirBD expression and nitrite reductase activity, which in turn led to increased nitrite accumulation in cells and its export. Molecular docking and point mutation assays revealed five residues in NasT (R70, Q72, D123, K127 and R140) involved in c-di-GMP-binding, of which R140 was essential for both c-di-GMP-binding and NalA-binding. Three diguanylate cyclases (c-di-GMP synthetases) were found to interact with NasT and inhibited nirBD expression, including WspR, PP_2557, and PP_4405. Besides, the c-di-GMP-binding ability of NasT was conserved in the other three representative Pseudomonas species, including P. aeruginosa, P. fluorescens and P. syringae. Our findings provide new insights into nitrate assimilation regulation by revealing the mechanism by which c-di-GMP inhibits nitrate assimilation via NasT.
Topics: Bacterial Proteins; Cyclic GMP; Gene Expression Regulation, Bacterial; Molecular Docking Simulation; Nitrates; Nitrite Reductases; Pseudomonas aeruginosa; Pseudomonas putida
PubMed: 38000372
DOI: 10.1093/nar/gkad1117 -
Science Advances Nov 2023Cross-linked elastomers are stretchable materials that typically are not recyclable or biodegradable. Medium-chain-length polyhydroxyalkanoates (mcl-PHAs) are soft and...
Cross-linked elastomers are stretchable materials that typically are not recyclable or biodegradable. Medium-chain-length polyhydroxyalkanoates (mcl-PHAs) are soft and ductile, making these bio-based polymers good candidates for biodegradable elastomers. Elasticity is commonly imparted by a cross-linked network structure, and covalent adaptable networks have emerged as a solution to prepare recyclable thermosets via triggered rearrangement of dynamic covalent bonds. Here, we develop biodegradable and recyclable elastomers by chemically installing the covalent adaptable network within biologically produced mcl-PHAs. Specifically, an engineered strain of was used to produce mcl-PHAs containing pendent terminal alkenes as chemical handles for postfunctionalization. Thiol-ene chemistry was used to incorporate boronic ester (BE) cross-links, resulting in PHA-based vitrimers. mcl-PHAs cross-linked with BE at low density (<6 mole %) affords a soft, elastomeric material that demonstrates thermal reprocessability, biodegradability, and denetworking at end of life. The mechanical properties show potential for applications including adhesives and soft, biodegradable robotics and electronics.
Topics: Polyhydroxyalkanoates; Pseudomonas putida; Elasticity; Elastomers
PubMed: 37992173
DOI: 10.1126/sciadv.adi1735 -
Microbial Biotechnology Jan 2024Providing an anodic potential in a bio-electrochemical system to the obligate aerobe Pseudomonas putida enables anaerobic survival and allows the cells to overcome redox...
Providing an anodic potential in a bio-electrochemical system to the obligate aerobe Pseudomonas putida enables anaerobic survival and allows the cells to overcome redox imbalances. In this setup, the bacteria could be exploited to produce chemicals via oxidative pathways at high yield. However, the absence of anaerobic growth and low carbon turnover rates remain as obstacles for the application of such an electro-fermentation technology. Growth and carbon turnover start with carbon uptake into the periplasm and cytosol. P. putida KT2440 has three native transporting systems for glucose, each differing in energy and redox demand. This architecture previously led to the hypothesis that internal redox and energy constraints ultimately limit cytoplasmic carbon utilization in a bio-electrochemical system. However, it remains largely unclear which uptake route is predominantly used by P. putida under electro-fermentative conditions. To elucidate this, we created three gene deletion mutants of P. putida KT2440, forcing the cells to exclusively utilize one of the routes. When grown in a bio-electrochemical system, the pathway mutants were heavily affected in terms of sugar consumption, current output and product formation. Surprisingly, however, we found that about half of the acetate formed in the cytoplasm originated from carbon that was put into the system via the inoculation biomass, while the other half came from the consumption of substrate. The deletion of individual sugar uptake routes did not alter significantly the secreted acetate concentrations among different strains even with different carbon sources. This means that the stoichiometry of the sugar uptake routes is not a limiting factor during electro-fermentation and that the low rates might be caused by other reasons, for example energy limitations or a yet-to-be-identified oxygen-dependent regulatory mechanism.
Topics: Pseudomonas putida; Anaerobiosis; Glucose; Carbon; Acetates
PubMed: 37990843
DOI: 10.1111/1751-7915.14375 -
Water Research Jan 2024One of the main problems in contaminated soils is that many toxic substances, such as PAHs, which are found in areas close to aquifers and groundwater, are difficult to...
One of the main problems in contaminated soils is that many toxic substances, such as PAHs, which are found in areas close to aquifers and groundwater, are difficult to access and degrade via traditional methods of remediation. The use of controlled bacterial mobility through chemotaxis has been shown to be efficient in increasing the dispersion of pollutant-degrading organisms, increasing the biodegradation rates of pollutants. In this study, using percolation columns as model aquifers, the mobilization of the Pseudomonas putida G7 strain to a distant pyrene source was demonstrated using γ-aminobutyric acid and artificial root exudates as strong chemoeffectors. An increase in the biodegradation rates of the pollutant was observed relative to columns in which the tactic effector was not added. The presence of different metabolites was detected via a fraction collector associated with an HPLC system, providing evidence for the cometabolic capacity of strain G7. The use of chemotactic organisms can be an effective approach for the remediation of polluted sediments associated with aquifers and groundwaters, offering new possibilities for the treatment of contaminated aqueous areas.
Topics: Pyrenes; Groundwater; Environmental Pollutants; Biodegradation, Environmental; Pseudomonas putida
PubMed: 37976951
DOI: 10.1016/j.watres.2023.120850 -
Microbiology Spectrum Dec 2023The CRISPR-Cas3 editing system as presented here facilitates the creation of genomic alterations in and in a straightforward manner. By providing the Cas3 system as a...
The CRISPR-Cas3 editing system as presented here facilitates the creation of genomic alterations in and in a straightforward manner. By providing the Cas3 system as a vector set with Golden Gate compatibility and different antibiotic markers, as well as by employing the established Standard European Vector Architecture (SEVA) vector set to provide the homology repair template, this system is flexible and can readily be ported to a multitude of Gram-negative hosts. Besides genome editing, the Cas3 system can also be used as an effective and universal tool for vector curing. This is achieved by introducing a spacer that targets the origin-of-transfer, present on the majority of established (SEVA) vectors. Based on this, the Cas3 system efficiently removes up to three vectors in only a few days. As such, this curing approach may also benefit other genomic engineering methods or remove naturally occurring plasmids from bacteria.
Topics: CRISPR-Cas Systems; Pseudomonas; Plasmids; Pseudomonas putida; CRISPR-Associated Proteins
PubMed: 37975669
DOI: 10.1128/spectrum.02707-23 -
MicroLife 2023This study presents an inexpensive approach for the macro- and microscopic observation of fungal mycelial growth. The 'fungal drops' method allows to investigate the...
This study presents an inexpensive approach for the macro- and microscopic observation of fungal mycelial growth. The 'fungal drops' method allows to investigate the development of a mycelial network in filamentous microorganisms at the colony and hyphal scales. A heterogeneous environment is created by depositing 15-20 µl drops on a hydrophobic surface at a fixed distance. This system is akin to a two-dimensional (2D) soil-like structure in which aqueous-pockets are intermixed with air-filled pores. The fungus (spores or mycelia) is inoculated into one of the drops, from which hyphal growth and exploration take place. Hyphal structures are assessed at different scales using stereoscopic and microscopic imaging. The former allows to evaluate the local response of regions within the colony (modular behaviour), while the latter can be used for fractal dimension analyses to describe the hyphal network architecture. The method was tested with several species to underpin the transferability to multiple species. In addition, two sets of experiments were carried out to demonstrate its use in fungal biology. First, mycelial reorganization of was assessed as a response to patches containing different nutrient concentrations. Second, the effect of interactions with the soil bacterium on habitat colonization by the same fungus was assessed. This method appeared as fast and accessible, allowed for a high level of replication, and complements more complex experimental platforms. Coupled with image analysis, the fungal drops method provides new insights into the study of fungal modularity both macroscopically and at a single-hypha level.
PubMed: 37965130
DOI: 10.1093/femsml/uqad042 -
Plants (Basel, Switzerland) Nov 2023Currently, one of the most serious global problems is the increasing incidence of infectious diseases. This is closely related to the increase in antibiotic use, which...
Currently, one of the most serious global problems is the increasing incidence of infectious diseases. This is closely related to the increase in antibiotic use, which has resulted in the development of multidrug resistance in microorganisms. Another problem is the numerous microbiological contaminations of cosmetic products, which can lead to dangerous bacterial infections in humans. Natural fragrance raw materials exhibit a wide spectrum of biological properties, including antimicrobial properties. Despite their prevalence and availability on the commercial market, there is little research into their effects on multidrug-resistant microorganisms. This study examines the inhibitory effect of natural substances on Gram-positive and Gram-negative bacteria. For this purpose, screening and appropriate assays were carried out to determine the minimum inhibitory concentration (MIC) value of individual substances, using the alamarBlue reagent. The lowest MIC values were observed for (black seed () expressed oil, MIC = 25 µg/mL), (fir balsam absolute, MIC = 12.5 µg/mL), and (cubeb oil and fir balsam absolute, MIC = 12.5 µg/mL). The most resistant Gram-negative species was , while was the most resistant Gram-positive species.
PubMed: 37960133
DOI: 10.3390/plants12213777 -
Scientific Reports Nov 2023Wastewater malodour is the proverbial 'elephant in the room' notwithstanding its severe implications on sanitation, health, and hygiene. The predominant malodorous...
Wastewater malodour is the proverbial 'elephant in the room' notwithstanding its severe implications on sanitation, health, and hygiene. The predominant malodorous compounds associated with wastewater treatment plants and toilets are volatile organic compounds, such as hydrogen sulphide, ammonia, methanethiol, and organic acids. Among them, methanethiol warrants more attention owing to its relatively low olfactory threshold and associated cytotoxicity. This requires an efficient odour-abatement method since conventional techniques are either cost-prohibitive or leave recalcitrant byproducts. Bacteriophage-based methodology holds promise, and the described work explores the potential. In this study, a non-lysogenous Pseudomonas putida strain is used as a model organism that produces methanethiol in the presence of methionine. Two double-stranded DNA phages of genome sizes > 10 Kb were isolated from sewage. ɸPh_PP01 and ɸPh_PP02 were stable at suboptimal pH, temperature, and at 10% chloroform. Moreover, they showed adsorption efficiencies of 53% and 89% in 12 min and burst sizes of 507 ± 187 and 105 ± 7 virions per cell, respectively. In augmented synthetic wastewater, ɸPh_PP01 and ɸPh_PP02 reduced methanethiol production by 52% and 47%, respectively, with the concomitant reduction in P. putida by 3 logs in 6 h. On extension of the study in P. putida spiked-sewage sample, maximum reduction in methanethiol production was achieved in 3 h, with 49% and 48% for ɸPh_PP01 and ɸPh_PP02, respectively. But at 6 h, efficiency reduced to 36% with both the phages. The study clearly demonstrates the potential of phages as biocontrol agents in the reduction of malodour in wastewater.
Topics: Bacteriophages; Pseudomonas putida; Wastewater; Sewage; Sulfhydryl Compounds
PubMed: 37945592
DOI: 10.1038/s41598-023-46938-8 -
Nucleic Acids Research Dec 2023The widespread Pseudomonas genus comprises a collection of related species with remarkable abilities to degrade plastics and polluted wastes and to produce a broad...
The widespread Pseudomonas genus comprises a collection of related species with remarkable abilities to degrade plastics and polluted wastes and to produce a broad set of valuable compounds, ranging from bulk chemicals to pharmaceuticals. Pseudomonas possess characteristics of tolerance and stress resistance making them valuable hosts for industrial and environmental biotechnology. However, efficient and high-throughput genetic engineering tools have limited metabolic engineering efforts and applications. To improve their genome editing capabilities, we first employed a computational biology workflow to generate a genus-specific library of potential single-stranded DNA-annealing proteins (SSAPs). Assessment of the library was performed in different Pseudomonas using a high-throughput pooled recombinase screen followed by Oxford Nanopore NGS analysis. Among different active variants with variable levels of allelic replacement frequency (ARF), efficient SSAPs were found and characterized for mediating recombineering in the four tested species. New variants yielded higher ARFs than existing ones in Pseudomonas putida and Pseudomonas aeruginosa, and expanded the field of recombineering in Pseudomonas taiwanensisand Pseudomonas fluorescens. These findings will enhance the mutagenesis capabilities of these members of the Pseudomonas genus, increasing the possibilities for biotransformation and enhancing their potential for synthetic biology applications. .
Topics: DNA, Single-Stranded; Gene Editing; Metabolic Engineering; Pseudomonas; Pseudomonas putida
PubMed: 37941137
DOI: 10.1093/nar/gkad1024 -
Journal of Hazardous Materials Feb 2024Microorganisms from L. terrestris gut previously exposed to different types of plastic (PET, LDPE, LLDPE, and PS) were studied to be used as probiotics of earthworms in...
Microorganisms from L. terrestris gut previously exposed to different types of plastic (PET, LDPE, LLDPE, and PS) were studied to be used as probiotics of earthworms in plastic-contaminated soils (LDPE, LLDPE and recycled mulching film) at mesocosm-scale trials. The most abundant morphotypes with enzymatic capacities of interest were identified. Pseudomonas alkylphenolica (PL4) and Pseudomonas putida (PL5) strains were selected to be used as inoculants using Morus alba leaves as carriers to strengthen the intestinal microbiota of earthworms. Culture (selective cetrimide agar medium) and molecular (qPCR) techniques were used to trace the presence of the inoculum in the intestine of the earthworms. Additionally, a metataxonomic analysis was carried out to study the biodiversity and functionality of the earthworm microbiome, and their measure of survival and weight. Probiotics improved the survival rates of earthworms exposed to plastics, which also increased the abundance of microbial groups of interest in plastic bioremediation tasks.
Topics: Animals; Oligochaeta; Gastrointestinal Microbiome; Polyethylene; Biodiversity; Soil
PubMed: 37931339
DOI: 10.1016/j.jhazmat.2023.132836