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Water Research Nov 2023Bacterial communication interruption based on quorum quenching (QQ) has been proven its potential in biofilm formation inhibition and biofouling control. However, it...
Bacterial communication interruption based on quorum quenching (QQ) has been proven its potential in biofilm formation inhibition and biofouling control. However, it would be more satisfying if QQ could be combined with the efficient degradation of contaminants in environmental engineering. In this study, we engineered a biofilm of Pseudomonas putida through introducing a QQ synthetic gene, which achieved both biofilm formation inhibition and efficient degradation of benzene series in wastewater. The aiiO gene introduced into the P. putida by heat shock method was highly expressed to produce QQ enzyme to degrade AHL-based signal molecules. The addition of this engineered P. putida reduced the AHLs concentration, quorum sensing gene expression, and connections of the microbial community network in activated sludge and therefore inhibited the biofilm formation. Meanwhile, the sodium benzoate degradation assay indicated an enhanced benzene series removal ability of the engineering bacteria on activated sludge. Besides, we also demonstrated a controllable environmental risk of this engineered bacteria through monitoring its abundance and horizontal gene transfer test. Overall, the results of this study suggest an alternative strategy to solve multiple environmental problems through genetic engineering means and provide support for the application of engineered bacteria in environmental biotechnology.
Topics: Sewage; Pseudomonas putida; Benzene; Biofilms; Quorum Sensing; Bioreactors
PubMed: 37804807
DOI: 10.1016/j.watres.2023.120690 -
BMC Veterinary Research Apr 2024Pseudomonas putida is a pathogenic bacterium that induces great losses in fishes, including Nile tilapia (Oreochromis niloticus). Currently, the application of...
Pseudomonas putida infection induces immune-antioxidant, hepato-renal, ethological, and histopathological/immunohistochemical disruptions in Oreochromis niloticus: the palliative role of titanium dioxide nanogel.
BACKGROUND
Pseudomonas putida is a pathogenic bacterium that induces great losses in fishes, including Nile tilapia (Oreochromis niloticus). Currently, the application of nanomaterials in aquaculture practices has gained more success as it endows promising results in therapies compared to traditional protocols.
OBJECTIVE
Therefore, the current perspective is considered the first report to assess the anti-bacterial efficacy of titanium dioxide nanogel (TDNG) against Pseudomonas putida (P. putida) in Nile tilapia.
METHODS
The fish (n = 200; average body weight: 47.50±1.32 g) were allocated into four random groups (control, TDNG, P. putida, and TDNG + P. putida), where 0.9 mg/L of TDNG was applied as bath treatment for ten days.
RESULTS
Outcomes revealed that P. putida infection caused ethological alterations (surfacing, abnormal movement, and aggression) and depression of immune-antioxidant variables (complement 3, lysozyme activity, total antioxidant capacity, superoxide dismutase, and reduced glutathione content). Additionally, a substantial elevation in hepatorenal biomarkers (aspartate and alanine aminotransferases and creatinine) with clear histopathological changes and immuno-histochemical alterations (very weak BCL-2 and potent caspase-3 immuno-expressions) were seen. Surprisingly, treating P. putida-infected fish with TDNG improved these variables and obvious restoration of the tissue architectures.
CONCLUSION
Overall, this report encompasses the key role of TDNG as an anti-bacterial agent for controlling P. putida infection and improving the health status of Nile tilapia.
Topics: Animals; Antioxidants; Pseudomonas putida; Cichlids; Nanogels; Diet; Dietary Supplements; Animal Feed; Fish Diseases; Polyethylene Glycols; Polyethyleneimine; Titanium
PubMed: 38561720
DOI: 10.1186/s12917-024-03972-6 -
International Microbiology : the... Jan 2024Pseudomonas spp., such as P. fluorescens group, P. fragi, and P. putida, are the major psychrophilic spoilage bacteria in the food industry. Bacteriophages (phages) are...
Pseudomonas spp., such as P. fluorescens group, P. fragi, and P. putida, are the major psychrophilic spoilage bacteria in the food industry. Bacteriophages (phages) are a promising tool for controlling food-spoilage and food-poisoning bacteria; however, there are few reports on phages effective on food-spoilage bacteria such as Pseudomonas spp. In this study, 12 Pseudomonas phages were isolated from chicken and soil samples. Based on the host range and lytic activity at 30 °C and 4 °C and various combinations of phages, phages vB_PflP-PCS4 and vB_PflP-PCW2 were selected to prepare phage cocktails to control Pseudomonas spp. The phage cocktail consisting of vB_PflP-PCS4 and vB_PflP-PCW2 showed the strongest lytic activity and retarded regrowth of P. fluorescens and P. putida at 30 °C, 8 °C, and 4 °C at a multiplicity of infection of 100. Nucleotide sequence analysis of the genomic DNA indicated that vB_PflP-PCS4 and vB_PflP-PCW2 phages were lytic phages of the Podoviridae family and lacked tRNA, toxin, or virulence genes. A novel endolysin gene was found in the genomic DNA of phage vB_PflP-PCS4. The results of this study suggest that the phage cocktail consisting of vB_PflP-PCS4 and vB_PflP-PCW2 is a promising tool for the biocontrol of psychrophilic food-spoilage pseudomonads during cold storage and distribution.
PubMed: 38206524
DOI: 10.1007/s10123-023-00479-2 -
Astrobiology Oct 2023Since the Viking Labeled Release experiments were carried out on Mars in the 1970s, it has been evident that the martian surface regolith has a strong oxidizing capacity...
Since the Viking Labeled Release experiments were carried out on Mars in the 1970s, it has been evident that the martian surface regolith has a strong oxidizing capacity that can convert organic compounds into CO and probably water. While HO was suggested originally for being the oxidizing agent responsible for the outcome of the Viking experiments, recent analyses of the martian regolith by the Phoenix lander and by consecutive missions point toward radiation-mediated decomposition products of perchlorate salts as the primary oxidant. In a series of experiments, we have shown that abrasion and triboelectric charging of basalt by simulated saltation could be an additional way of activating regolith. We have also shown that abraded basalt with a chemical composition close to that of martian regolith is toxic to several bacterial species and thus may affect the habitability of the martian surface. In the present study, we investigated the effect of the quantitatively most important minerals (olivine, augite, and plagioclase) and iron oxides (hematite, magnetite, and maghemite) on the survival of bacterial cells to elucidate whether a specific mineral that constitutes basalt is responsible for our observations. We observed that suspensions of iron-containing minerals olivine and augite in phosphate-buffered saline (1 × PBS) significantly reduce the number of surviving cells of our model organism after 24 h of incubation. In contrast, the iron-free mineral plagioclase showed no effect. We also observed that suspending abraded olivine and augite in 1 × PBS led to a dramatic increase in pH compared to the pH of 1 × PBS alone. The sudden increase in pH caused by the presence of these minerals may partly explain the observed cytotoxicity. The cytotoxic effect of augite could be relieved when a strong buffer (20 × PBS) was used. In contrast, olivine, despite the stronger buffer, maintained its cytotoxicity. Iron oxides have no negative effect on the survival of our test organism. Overall, our experiments confirm the cytotoxicity of basalt and show that no single constituent mineral of the basalt can account for its toxicity. We could show that abraded iron-containing minerals (olivine and augite) change the pH of water when brought into suspension and thereby could affect the habitability of martian regolith.
PubMed: 37672600
DOI: 10.1089/ast.2023.0015 -
Nature Communications Sep 2023Plastic pollution is rapidly increasing worldwide, causing adverse impacts on the environment, wildlife and human health. One tempting solution to this crisis is...
Plastic pollution is rapidly increasing worldwide, causing adverse impacts on the environment, wildlife and human health. One tempting solution to this crisis is upcycling plastics into products with engineered microorganisms; however, this remains challenging due to complexity in conversion. Here we present a synthetic microbial consortium that efficiently degrades polyethylene terephthalate hydrolysate and subsequently produces desired chemicals through division of labor. The consortium involves two Pseudomonas putida strains, specializing in terephthalic acid and ethylene glycol utilization respectively, to achieve complete substrate assimilation. Compared with its monoculture counterpart, the consortium exhibits reduced catabolic crosstalk and faster deconstruction, particularly when substrate concentrations are high or crude hydrolysate is used. It also outperforms monoculture when polyhydroxyalkanoates serves as a target product and confers flexible tuning through population modulation for cis-cis muconate synthesis. This work demonstrates engineered consortia as a promising, effective platform that may facilitate polymer upcycling and environmental sustainability.
Topics: Humans; Pregnancy; Female; Animals; Engineering; Labor, Obstetric; Animals, Wild; Cross Reactions; Polyhydroxyalkanoates
PubMed: 37752119
DOI: 10.1038/s41467-023-40777-x -
Botanical Studies Oct 2023Ralstonia solanacearum causes potato brown rot disease, resulting in lower crop's production and quality. A sustainable and eco-friendly method for controlling the...
BACKGROUND
Ralstonia solanacearum causes potato brown rot disease, resulting in lower crop's production and quality. A sustainable and eco-friendly method for controlling the disease is required. Algae's bioactive chemicals have shown the potential to enhance plant defenses. For the first time, the efficacy of foliar application of Acanthophora spicifera and Spirulina platensis seaweed extracts, along with the utilization of dried algal biomasses (DABs) of Turbinaria ornata and a mixture of Caulerpa racemosa and Cystoseira myrica (1:1)on potato yield and brown rot suppression were investigated under field conditions. Field experiments were conducted in three locations: Location 1 (Kafr Shukr district, Kaliobeya governorate), Location 2 (Moneira district, Kaliobeya governorate), and Location 3 (Talia district, Minufyia governorate). Locations 1 and 2 were naturally infested with the pathogen, while location 3 was not. The study evaluated potato yield, plant nutritive status and antioxidants, soil available nitrogen-phosphorus-potassium (N-P-K), and organic matter percentage. Additionally, the shift in soil microbial diversity related to R. solanacearum suppression was examined for the most effective treatment.
RESULTS
The results revealed that seaweed extracts significantly increased potato yield at all locations, which correlated with higher phosphorus absorption, while T. ornate DAB increased potato yield only at location 2, accompanied by noticeable increases in soil nitrogen and plant phosphorus. The mixed DABs of C. racemosa and C. myrica demonstrated greater disease suppression than foliar applications. The disease-suppressive effect of the mixed DABs was accompanied by significant increases in flavonoids and total antioxidant capacity (TAC). Moreover, the application of mixed DABs increased soil bacterial biodiversity, with a higher abundance of oligotrophic marine bacterial species such as Sphingopyxis alaskensis and growth-promoting species like Glutamicibacter arilaitensis, Promicromonospora sp., and Paenarthrobacter nitroguajacolicus in all three locations compared to the untreated control. Klebsiella sp., Pseudomonas putida, and P. brassicacearum abundances were increased by the mixed DABs in Location 1. These species were less abundant in locations 2 and 3, where Streptomyces sp., Bacillus sp., and Sphingobium vermicomposti were prevalent.
CONCLUSIONS
The results demonstrated that the used seaweed extracts improved potato yield and phosphorous absorption, while the mixed DABs potentially contributed in disease suppression and improved soil microbial diversity.
PubMed: 37843648
DOI: 10.1186/s40529-023-00402-y -
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 -
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 -
ACS Synthetic Biology Jul 2023Genome editing tools, through the disruption of an organism's native genetic material or the introduction of non-native DNA, facilitate functional investigations to link...
Genome editing tools, through the disruption of an organism's native genetic material or the introduction of non-native DNA, facilitate functional investigations to link genotypes to phenotypes. Transposons have been instrumental genetic tools in microbiology, enabling genome-wide, randomized disruption of genes and insertions of new genetic elements. Due to this randomness, identifying and isolating particular transposon mutants (i.e., those with modifications at a genetic locus of interest) can be laborious, often requiring one to sift through hundreds or thousands of mutants. Programmable, site-specific targeting of transposons became possible with recently described CRISPR-associated transposase (CASTs) systems, allowing the streamlined recovery of desired mutants in a single step. Like other CRISPR-derived systems, CASTs can be programmed by guide-RNA that is transcribed from short DNA sequence(s). Here, we describe a CAST system and demonstrate its function in bacteria from three classes of Proteobacteria. A dual plasmid strategy is demonstrated: (i) CAST genes are expressed from a broad-host-range replicative plasmid and (ii) guide-RNA and transposon are encoded on a high-copy, suicidal pUC plasmid. Using our CAST system, single-gene disruptions were performed with on-target efficiencies approaching 100% in Beta- and Gammaproteobacteria ( and , respectively). We also report a peak efficiency of 45% in the Alphaproteobacterium . In , we performed simultaneous co-integration of transposons at two different target sites, demonstrating CAST's utility in multilocus strategies. The CAST system is also capable of high-efficiency large transposon insertion totaling over 11 kbp in all three bacteria tested. Lastly, the dual plasmid system allowed for iterative transposon mutagenesis in all three bacteria without loss of efficiency. Given these iterative capabilities and large payload capacity, this system will be helpful for genome engineering experiments across several fields of research.
Topics: Clustered Regularly Interspaced Short Palindromic Repeats; Transposases; DNA Transposable Elements; Proteobacteria; Mutagenesis; Gene Editing; Bacteria; RNA; CRISPR-Cas Systems
PubMed: 37368499
DOI: 10.1021/acssynbio.3c00065 -
Metabolic Engineering Jun 2024Biological conversion of lignin from biomass offers a promising strategy for sustainable production of fuels and chemicals. However, aromatic compounds derived from...
Biological conversion of lignin from biomass offers a promising strategy for sustainable production of fuels and chemicals. However, aromatic compounds derived from lignin commonly contain methoxy groups, and O-demethylation of these substrates is often a rate-limiting reaction that influences catabolic efficiency. Several enzyme families catalyze aromatic O-demethylation, but they are rarely compared in vivo to determine an optimal biocatalytic strategy. Here, two pathways for aromatic O-demethylation were compared in Pseudomonas putida KT2440. The native Rieske non-heme iron monooxygenase (VanAB) and, separately, a heterologous tetrahydrofolate-dependent demethylase (LigM) were constitutively expressed in P. putida, and the strains were optimized via adaptive laboratory evolution (ALE) with vanillate as a model substrate. All evolved strains displayed improved growth phenotypes, with the evolved strains harboring the native VanAB pathway exhibiting growth rates ∼1.8x faster than those harboring the heterologous LigM pathway. Enzyme kinetics and transcriptomics studies investigated the contribution of selected mutations toward enhanced utilization of vanillate. The VanAB-overexpressing strains contained the most impactful mutations, including those in VanB, the reductase for vanillate O-demethylase, PP_3494, a global regulator of vanillate catabolism, and fghA, involved in formaldehyde detoxification. These three mutations were combined into a single strain, which exhibited approximately 5x faster vanillate consumption than the wild-type strain in the first 8 h of cultivation. Overall, this study illuminates the details of vanillate catabolism in the context of two distinct enzymatic mechanisms, yielding a platform strain for efficient O-demethylation of lignin-related aromatic compounds to value-added products.
PubMed: 38936762
DOI: 10.1016/j.ymben.2024.06.009