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ACS Synthetic Biology Mar 2024The advancement of metabolic engineering and synthetic biology has promoted in-depth research on the nonmodel microbial metabolism, and the potential of nonmodel...
The advancement of metabolic engineering and synthetic biology has promoted in-depth research on the nonmodel microbial metabolism, and the potential of nonmodel organisms in industrial biotechnology is becoming increasingly evident. The nonmodel organism is a safe plant growth promoting bacterium for the production of phenazine compounds; however, its application is seriously hindered due to the lack of an effective gene expression precise regulation toolkit. In this study, we constructed a library of 108 promoter-5'-UTR (PUTR) and characterized them through fluorescent protein detection. Then, 6 PUTRs with stable low, intermediate, and high intensities were further characterized by report genes encoding β-galactosidase from K12 and encoding PCA monooxygenase from GP72 and thus developed as a static gene expression regulation system. Furthermore, the stable and high-intensity expressed PUTR was fused with the LacO operator to construct an IPTG-induced plasmid, and a self-induced plasmid was constructed employing the high-intensity PUTR regulated by cell density, resulting in a dynamic gene expression regulation system. In summary, this study established two sets of static and dynamic regulatory systems for , providing an effective toolkit for fine-tuning gene expression and reprograming the metabolism flux.
Topics: Pseudomonas chlororaphis; Metabolic Engineering; Gene Expression Regulation, Bacterial; Promoter Regions, Genetic; Bacterial Proteins
PubMed: 38377538
DOI: 10.1021/acssynbio.3c00714 -
BMC Plant Biology Feb 2024Crown gall disease caused by Agrobacterium tumefaciens is a very destructive affliction that affects grapevines. Endophytic bacteria have been discovered to control...
Antivirulence effects of cell-free culture supernatant of endophytic bacteria against grapevine crown gall agent, Agrobacterium tumefaciens, and induction of defense responses in plantlets via intact bacterial cells.
BACKGROUND
Crown gall disease caused by Agrobacterium tumefaciens is a very destructive affliction that affects grapevines. Endophytic bacteria have been discovered to control plant diseases via the use of several mechanisms. This research examined the potential for controlling crown gall by three endophytic bacteria that were previously isolated from healthy cultivated and wild grapevines including Pseudomonas kilonensis Ba35, Pseudomonas chlororaphis Ba47, and Serratia liquefaciens Ou55.
RESULT
At various degrees, three endophytic bacteria suppressed the populations of A. tumefaciens Gh1 and greatly decreased the symptoms of crown gall. Furthermore, biofilm production and motility behaviors of A. tumefaciens Gh1were greatly inhibited by the Cell-free Culture Supernatant (CFCS) of endophytic bacteria. According to our findings, CFCS may reduce the adhesion of A. tumefaciens Gh1 cells to grapevine cv. Rashe root tissues as well as their chemotaxis motility toward the extract of the roots. When compared to the untreated control, statistical analysis showed that CFCS significantly reduced the swimming, twitching, and swarming motility of A. tumefaciens Gh1. The findings demonstrated that the endophytic bacteria effectively stimulated the production of plant defensive enzymes including superoxide dismutase (SOD), polyphenol oxidase (PPO), peroxidase (POD), phenylalanine ammonia lyase (PAL), and total soluble phenols at different time intervals in grapevine inoculated with A. tumefaciens Gh1. The Ba47 strain markedly increased the expression levels of defense genes associated with plant resistance. The up-regulation of PR1, PR2, VvACO1, and GAD1 genes in grapevine leaves indicates the activation of SA and JA pathways, which play a role in enhancing resistance to pathogen invasion. The results showed that treating grapevine with Ba47 increased antioxidant defense activities and defense-related gene expression, which reduced oxidative damage caused by A. tumefaciens and decreased the incidence of crown gall disease.
CONCLUSION
This is the first study on how A. tumefaciens, the grapevine crown gall agent, is affected by CFCS generated by endophytic bacteria in terms of growth and virulence features. To create safer plant disease management techniques, knowledge of the biocontrol processes mediated by CFCS during microbial interactions is crucial.
Topics: Plant Tumors; Agrobacterium tumefaciens; Plant Diseases; Bacteria
PubMed: 38336608
DOI: 10.1186/s12870-024-04779-1 -
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 -
Heliyon Feb 2024This study aimed to isolate and characterize plant growth-promoting rhizobacteria from the faba bean rhizosphere for future inoculum production. For this purpose,127...
This study aimed to isolate and characterize plant growth-promoting rhizobacteria from the faba bean rhizosphere for future inoculum production. For this purpose,127 dissimilar rhizobacterial colonies were isolated. All isolated colonies were tested for plant growth-promoting traits. Based on their multiple plant growth-promoting traits, eight potential isolates were selected and identified GY01, GY03, and GY08 are affiliated with Acinetobacter sp. GY04 and GY05 are affiliated with Chryseobacterium sp. GY06 and GY07 are affiliated with Pseudomonas costantinii and Pseudomonas chlororaphis, respectively; and GY02 is affiliated with the Bacterium strain. All eight isolates were evaluated for their effects on seed germination and vigor index and potential antagonism against Botrytis fabae. Selected isolates showed positive effects on seed germination and vigor index with different potentials. Isolate GY04 resulted in the highest vigor index (501), while isolate GY08 achieved the lowest (218). B. fabae radial growth inhibition was found in all eight isolates. The isolates inhibited the radial growth of the test pathogen with an inhibition efficacy of 72.38 % in GY04 to 25.57 % in GY-03. Generally, the results of this study indicated the potential of these isolates as a microbial inoculant with multiple functions for faba beans.
PubMed: 38318010
DOI: 10.1016/j.heliyon.2024.e25334 -
Journal of Xenobiotics Jan 2024Metsulfuron-methyl, a widely used herbicide, could cause damage to the sensitive plants in crop-rotation systems at extremely low levels in the soil. The potential of...
Metsulfuron-methyl, a widely used herbicide, could cause damage to the sensitive plants in crop-rotation systems at extremely low levels in the soil. The potential of plant growth-promoting bacteria (PGPB) for enhancing the resistance of plants against herbicide stress has been discovered recently. Therefore, it is poorly understood how physiological processes occur in plants, while PGPB reduce the phytotoxicity of herbicides for agricultural crops. In greenhouse studies, the effect of strains DA1.2 and 4CH on oxidative damage, acetolactate synthase (ALS), enzymatic and non-enzymatic antioxidants in canola ( L.), and wheat ( L.) were investigated under two levels (0.05 and 0.25 mg∙kg) of metsulfuron-methyl using spectrophotometric assays. The inoculation of herbicide-exposed wheat with bacteria significantly increased the shoots fresh weight (24-28%), amount of glutathione GSH (60-73%), and flavonoids (5-14%), as well as activity of ascorbate peroxidase (129-140%), superoxide dismutase SOD (35-49%), and ALS (50-57%). Bacterial treatment stimulated the activity of SOD (37-94%), ALS (65-73%), glutathione reductase (19-20%), and the accumulation of GSH (61-261%), flavonoids (17-22%), and shoots weight (27-33%) in herbicide-exposed canola. Simultaneous inoculation prevented lipid peroxidation induced by metsulfuron-methyl in sensitive plants. Based on the findings, it is possible that the protective role of bacterial strains against metsulfuron-metil is linked to antioxidant system activation.
PubMed: 38249102
DOI: 10.3390/jox14010005 -
Journal of Contaminant Hydrology Feb 2024Despite the serious health threats due to wide use of organophosphorus pesticides (OPPs) have been experimentally claimed to be remediated by probiotic microorganisms in...
Despite the serious health threats due to wide use of organophosphorus pesticides (OPPs) have been experimentally claimed to be remediated by probiotic microorganisms in various food and organism models, the interactions between OPPs and probiotics in the natural wetland ecosystem was rarely investigated. This study delves into the spatial and temporal distribution, contamination levels of OPPs in the Baiyangdian region, the diversity of probiotic communities in varying environmental contexts, and the potential connection with OPPs on these probiotics. In typical shallow lake wetland ecosystem-Baiyangdian lake in north China, eight OPPs were identified in the lake sediments, even though their detection rates were generally low. Malathion exhibited the highest average content among these pesticides (9.51 ng/g), followed by fenitrothion (6.70 ng/g). Conversely, chlorpyrifos had the lowest detection rate at only 2.14%. The region near Nanliu Zhuang (F10), significantly influenced by human activities, displayed the highest concentration of total OPPs (136.82 ng/g). A total of 145 probiotic species spanning 78 genera were identified in Baiyangdian sediments. Our analysis underscores the relations of environmental factors such as phosphatase activity, pH, and electrical conductivity (EC) with probiotic community. Notably, several high-abundance probiotics including Pseudomonas chlororaphis, Clostridium sp., Lactobacillus fermentum, and Pseudomonas putida, etc., which were reported to exhibit significant potential for the degradation of OPPs, showed strongly correlations with OPPs in the Baiyangdian lake sediments. The outcomes of this research offer valuable insights into the spatiotemporal dynamics of OPPs in natural large lake wetland and the probability of their in-situ residue bioremediation through the phosphatase pathway mediated by probiotic such as Lactic acid bacteria in soils/sediments contaminated with OPPs.
Topics: Humans; Pesticides; Organophosphorus Compounds; Ecosystem; Lakes; Phosphoric Monoester Hydrolases; China; Geologic Sediments; Environmental Monitoring
PubMed: 38244424
DOI: 10.1016/j.jconhyd.2024.104306 -
Polymers Nov 2023The use of biocidal agents is a common practice for protection against biofouling in biomass-rich environments. In this paper, oligohexamethyleneguanidine (OHMG)...
The use of biocidal agents is a common practice for protection against biofouling in biomass-rich environments. In this paper, oligohexamethyleneguanidine (OHMG) polymer, known for its biocidal properties, was further modified with para-aminosalicylic acid (PAS) to enhance its properties against microorganisms coated with a lipid membrane. The structure of the product was confirmed by H NMR, C NMR, and FTIR spectroscopy. The values of the minimum inhibitory concentration (MIC) against ATCC 607 and 449 were found to be 1.40 and 1.05 μg/mL, respectively. The synthesized substance was used as an additive to the polymer matrix of the composite optical oxygen sensor material. A series of samples with different contents of OHMG-PAS was prepared using a co-dissolution method implying the fabrication of a coating from a solution containing both polymers. It turned out that the mutual influence of the components significantly affects the distribution of the indicator in the matrix, surface morphology, and contact angle. The optimal polymer content turned out to be wt.3%, at which point the water contact angle reaches almost 122°, and the fouling rate decreases by almost five times, which is confirmed by both the respiratory MTT assay and confocal microscopy with staining. This opens up prospects for creating stable and biofouling-resistant sensor elements for use in air tanks or seawater.
PubMed: 38231936
DOI: 10.3390/polym15234508 -
Journal of Economic Entomology Feb 2024Pseudomonas fluorescens group, such as Pseudomonas protegens and Pseudomonas chlororaphis, can be utilized as insect-killing agents. Most insecticidal Pseudomonas...
Pseudomonas fluorescens group, such as Pseudomonas protegens and Pseudomonas chlororaphis, can be utilized as insect-killing agents. Most insecticidal Pseudomonas described so far have high toxicity for insects of the order Lepidoptera. In this study, Pseudomonas strain PcR3-3 was isolated from the willow root. It showed a high mortality for the coleopteran species Plagiodera versicolora (Coleoptera: Chrysomelidae), but not for the lepidopteran Helicoverpa armigera. Strain PcR3-3 displayed high colonization ability in the P. versicolora compared with P. chlororaphis PCL1391, indicating that the insecticidal activities correlated with the colonization ability of Pseudomonas strain in the host. Phylogenetic analysis of the genome revealed that PcR3-3 belonged to P. chlororaphis subsp. aureofaciens. Numerous insecticidal protein-encoding genes, typical biosynthetic gene clusters for some insecticidal metabolite and type VI secretion system, known to be involved in insect pathogenicity, were present in the P. chlororaphis PcR3-3 genome. However, the insecticidal toxin Fit-encoding gene which commonly presents in P. chlororaphis, was not found in the P. chlororaphis PcR3-3 genome. Furthermore, there are some divergent insecticidal genes between P. chlororaphis PcR3-3 and P. chlororaphis PCL1391. This finding implies that P. chlororaphis PcR3-3 is a promising biocontrol agent for pest management applications. The P. chlororaphis-P. versicolora association can be used as a model system to study the interaction between Pseudomonas and coleopteran insects.
Topics: Animals; Pseudomonas chlororaphis; Insecticides; Phylogeny; Genomics; Insecta; Coleoptera
PubMed: 38146627
DOI: 10.1093/jee/toad232 -
Microbial Biotechnology Jan 2024Rhamnolipids (RL) are biosurfactants naturally produced by the opportunistic pathogen Pseudomonas aeruginosa. Currently, RL are commercialized for various applications...
Rhamnolipids (RL) are biosurfactants naturally produced by the opportunistic pathogen Pseudomonas aeruginosa. Currently, RL are commercialized for various applications and produced by Pseudomonas putida due to the health risks associated with their large-scale production by P. aeruginosa. In this work, we show that RL containing one or two rhamnose moieties (mono-RL or di-RL, respectively) can be produced by the innocuous soil-bacterium Pseudomonas chlororaphis subsp chlororaphis ATCC 9446 at titres up to 66 mg/L (about 86% of the production of P. aeruginosa PAO1 in the same culture conditions). The production of RL depends on the expression of P. aeruginosa PAO1 genes encoding the enzymes RhlA, RhlB and RhlC. These genes were introduced in a plasmid, together with a transcriptional regulator (rhlR) forming part of the same operon, with and without RhlC. We show that the activation of rhlAB by RhlR depends on its interaction with P. chlororaphis endogenous acyl-homoserine lactones, which are synthetized by either PhzI or CsaI autoinducer synthases (producing 3-hydroxy-hexanoyl homoserine lactone, 3OH-C6-HSL, or 3-oxo-hexanoyl homoserine lactone, 3O-C6-HSL, respectively). P. chlororaphis transcriptional regulator couple with 3OH-C6-HSL is the primary activator of gene expression for phenazine-1-carboxylic acid (PCA) and phenazine-1-carboxamide (PCN) production in this soil bacterium. We show that RhlR coupled with 3OH-C6-HSL or 3O-C6-HSL promotes RL production and increases the production of PCA in P. chlororaphis. However, PhzR/3OH-C6-HSL or CsaR/3O-C6-HSL cannot activate the expression of the rhlAB operon to produce mono-RL. These results reveal a complex regulatory interaction between RhlR and P. chlororaphis quorum-sensing signals and highlight the biotechnology potential of P. chlororaphis ATCC 9446 expressing P. aeruginosa rhlAB-R or rhlAB-R-C for the industrial production of RL.
Topics: Pseudomonas chlororaphis; Acyl-Butyrolactones; Pseudomonas aeruginosa; Soil; Bacterial Proteins; Glycolipids; Pseudomonas; 4-Butyrolactone
PubMed: 38041625
DOI: 10.1111/1751-7915.14377 -
JACS Au Nov 2023Chemical fertilizers have been crucial for sustaining the current global population by supplementing overused farmland to support consistent food production, but their...
Chemical fertilizers have been crucial for sustaining the current global population by supplementing overused farmland to support consistent food production, but their use is unsustainable. is a nitrogen-fixing bacterium that could be used as a fertilizer replacement, but this microbe is delicate. It is sensitive to stressors, such as freeze-drying and high temperatures. Here, we demonstrate protection of from freeze-drying, high temperatures (50 C), and high humidity using self-assembling metal-phenolic network (MPN) coatings. The composition of the MPN is found to significantly impact its protective efficacy, and with optimized compositions, no viability loss is observed for MPN-coated microbes under conditions where uncoated cells do not survive. Further, we demonstrate that MPN-coated microbes improve germination of seeds by 150% as compared to those treated with fresh . Taken together, these results demonstrate the protective capabilities of MPNs against environmental stressors and represent a critical step towards enabling the production and storage of delicate microbes under nonideal conditions.
PubMed: 38034965
DOI: 10.1021/jacsau.3c00426