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Plants (Basel, Switzerland) Dec 2023Salinity inhibits plant growth by affecting physiological processes, but soil microorganisms like plant growth-promoting rhizobacteria (PGPR) can alleviate abiotic...
Salinity inhibits plant growth by affecting physiological processes, but soil microorganisms like plant growth-promoting rhizobacteria (PGPR) can alleviate abiotic stress and enhance crop productivity. However, it should be noted that rhizobacteria employ different approaches to deal with salt stress conditions and successfully colonize roots. The objective of this study was to investigate the effect of salt stress on bacterial survival mechanisms such as mobility, biofilm formation, and the autoaggregation capacity of three plant growth-promoting strains: SJ04, WCS417r, and GB03. These strains were grown in diluted LB medium supplemented with 0, 100, 200, or 300 mM NaCl. Swimming and swarming mobility were evaluated in media supplemented with 0.3 and 0.5% agar, respectively. Biofilm formation capacity was quantified using the crystal violet method, and the autoaggregation capacity was measured spectrophotometrically. In addition, we evaluated in vitro the capacity of the strains to ameliorate the effects of saline stress in . The study found that the GB03 strain exhibited enhanced swarming mobility when the salt concentration in the medium increased, resulting in a two-fold increase in the halo diameter at 300 mM. However, high concentrations of NaCl did not affect the swimming mobility. In contrast, swimming motility was reduced in WCS417r and SJ04 under salt stress. On the other hand, exposure to 300 mM NaCl resulted in a 180% increase in biofilm formation and a 30% rise in the percentage of autoaggregation in WCS417r. Conversely, the autoaggregation percentage of the strains SJ04 and GB03 remained unaffected by saline stress. However, for GB03, biofilm formation decreased by 80% at 300 mM. Simultaneously, inoculation with the three evaluated strains alleviated the detrimental effects of salinity on plant growth. Under 150 mM salt stress, all strains showed increased fresh weight, with GB03 and WCS417r improving by 40% and SJ04 exhibiting the most remarkable effect with a 70% rise compared to non-inoculated plants. Despite their different strategies for mitigating salt stress, the application of these strains presents a promising strategy for effectively mitigating the negative consequences of salt stress on plant cultivation.
PubMed: 38068694
DOI: 10.3390/plants12234059 -
Current Opinion in Biotechnology Feb 2024The soil bacterium Pseudomonas putida, especially the KT2440 strain, is increasingly being utilized as a host for biotransformations of both industrial and environmental... (Review)
Review
The soil bacterium Pseudomonas putida, especially the KT2440 strain, is increasingly being utilized as a host for biotransformations of both industrial and environmental interest. The foundations of such performance include its robust redox metabolism, ability to tolerate a wide range of physicochemical stresses, rapid growth, versatile metabolism, nonpathogenic nature, and the availability of molecular tools for advanced genetic programming. These attributes have been leveraged for hosting engineered pathways for production of valuable chemicals or degradation/valorization of environmental pollutants. This has in turn pushed the boundaries of conventional enzymology toward previously unexplored reactions in nature. Furthermore, modifications to the physical properties of the cells have been made to enhance their catalytic performance. These advancements establish P. putida as bona fide chassis for synthetic biology, on par with more traditional metabolic engineering platforms.
Topics: Metabolic Engineering; Pseudomonas putida; Synthetic Biology; Biotransformation; Oxidation-Reduction
PubMed: 38061264
DOI: 10.1016/j.copbio.2023.103025 -
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 -
Heliyon Nov 2023Chitinases are hydrolytic enzymes that dissolve the glycosidic linkages in chitin. Chitin is a cell wall component of fungi and fund in exoskeleten of worms and...
Chitinases are hydrolytic enzymes that dissolve the glycosidic linkages in chitin. Chitin is a cell wall component of fungi and fund in exoskeleten of worms and arthropods. Chitinase has been applied in agriculture, as a biopesticide for the control of plant fungal infections, in medicine, and in waste management. This research aimed to isolate, screen, and identification of chitinase-producing bacteria from riverbank soils. Twenty nine chitinolytic bacteria were isolated from the river bank soil samples, from which 9 of them had strong chitinolytic properties. Chitinase production was determined by zones of hydrolysis produced after 96 h of incubation at 37 °C. The different bacterial isolates were characterized morphologically, microscopically, and biochemically and finally eight strain were identified at species level by Matrix Assisted Laser Desorption Ionization - Time of Flight Mass Spectrometry (MALDI-TOF MS). From the eight, bacterial isolates investigated in this study showed the highest chitinase enzyme activity (625 μg/mL) followed by with the enzyme activity of (553 μg/mL) and the least enzyme activity was recorded for (80 μg/mL). An incubation temperature of 45 °C, neutral pH and an incubation period of 96 h are found to be the optimum condition for the chitinase enzyme production from . The results of this study indicated the possibility of the production of chitinase from the chitinolytic bacterial isolates, which was highly useful for a variety of applications, including biocontrol of harmful insects and pathogenic fungi as well as in the biochemical, pharmaceutical, and medical sectors.
PubMed: 38027800
DOI: 10.1016/j.heliyon.2023.e21643 -
Heliyon Nov 2023In this work, it is presented a first approach of a mathematical and kinetic analysis for improving the decoloration and further degradation process of an azo dye named...
In this work, it is presented a first approach of a mathematical and kinetic analysis for improving the decoloration and further degradation process of an azo dye named acid red 27 (AR27), by means of a novel microbial consortium formed by the fungus and the bacterium . A multivariate analysis was carried out by simulating scenarios with different operating conditions and developing a specific mathematical model based on kinetic equations describing all stages of the biological process, from microbial growth and substrate consuming to decoloration and degradation of intermediate compounds. Additionally, a sensitivity analysis was performed by using a factorial design and the Response Surface Method (RSM), for determining individual and interactive effects of variables like, initial glucose concentration, initial dye concentration and the moment in time for bacterial inoculation, on response variables assessed in terms of the minimum time for: full decoloration of AR27 (R = 2.375 days); maximum production of aromatic metabolites (R = 1.575 days); and full depletion of aromatic metabolites (R = 12.9 days). Using RSM the following conditions improved the biological process, being: an initial glucose concentration of 20 g l, an initial AR27 concentration of 0.2 g l and an inoculation moment in time of at day 1. The mathematical model is a feasible tool for describing AR27 decoloration and its further degradation by the microbial consortium of and , this model will also work as a mathematical basis for designing novel bio-reaction systems than can operate with the same principle of the described consortium.
PubMed: 38027625
DOI: 10.1016/j.heliyon.2023.e21793 -
Frontiers in Bioengineering and... 2023Designing cell factories for the production of novel polyhydroxyalkanoates (PHAs) via smart metabolic engineering is key to obtain materials with tailored...
Designing cell factories for the production of novel polyhydroxyalkanoates (PHAs) via smart metabolic engineering is key to obtain materials with tailored physicochemical properties. To this end, we used the model medium-chain-length-PHA producing bacterium, KT2440 as a chassis, which is characterized by its metabolic versatility and stress tolerance. Different PHA biosynthetic modules were assembled in expression plasmids using the Golden gate/MoClo modular assembly technique to implement an orthogonal short-chain-lengh-PHA (scl-PHA) switch in a "deaf" PHA mutant. This was specifically constructed to override endogenous multilevel regulation of PHA synthesis in the native strain. We generated a panel of engineered approaches carrying the genes from and demonstrating that diverse scl-PHAs can be constitutively produced in the chassis strain to varying yields from 23% to 84% PHA/CDW. Co-feeding assays of the most promising engineered strain harboring the PHA machinery from resulted to a panel of PHBV from 0.6% to 19% C5 monomeric incorporation. Chromosomally integrated PHA machineries with high PhaC synthase dosage successfully resulted in 68% PHA/CDW production. Interestingly, an inverse relationship between PhaC synthase dosage and granule size distribution was demonstrated in the heterologous host. In this vein, it is proposed the key involvement of inclusion body protein IbpA to the heterologous production of tailored PHA in KT2440.
PubMed: 38026847
DOI: 10.3389/fbioe.2023.1275036 -
Metabolic Engineering Jan 2024Pseudomonas putida KT2440 is a robust, aromatic catabolic bacterium that has been widely engineered to convert bio-based and waste-based feedstocks to target products....
Pseudomonas putida KT2440 is a robust, aromatic catabolic bacterium that has been widely engineered to convert bio-based and waste-based feedstocks to target products. Towards industrial domestication of P. putida KT2440, rational genome reduction has been previously conducted, resulting in P. putida strain EM42, which exhibited characteristics that could be advantageous for production strains. Here, we compared P. putida KT2440- and EM42-derived strains for cis,cis-muconic acid production from an aromatic compound, p-coumarate, and in separate strains, from glucose. To our surprise, the EM42-derived strains did not outperform the KT2440-derived strains in muconate production from either substrate. In bioreactor cultivations, KT2440- and EM42-derived strains produced muconate from p-coumarate at titers of 45 g/L and 37 g/L, respectively, and from glucose at 20 g/L and 13 g/L, respectively. To provide additional insights about the differences in the parent strains, we analyzed growth profiles of KT2440 and EM42 on aromatic compounds as the sole carbon and energy sources. In general, the EM42 strain exhibited reduced growth rates but shorter growth lags than KT2440. We also observed that EM42-derived strains resulted in higher growth rates on glucose compared to KT2440-derived strains, but only at the lowest glucose concentrations tested. Transcriptomics revealed that genome reduction in EM42 had global effects on transcript levels and showed that the EM42-derived strains that produce muconate from glucose exhibit reduced modulation of gene expression in response to changes in glucose concentrations. Overall, our results highlight that additional studies are warranted to understand the effects of genome reduction on microbial metabolism and physiology, especially when intended for use in production strains.
Topics: Pseudomonas putida; Glucose; Bioreactors
PubMed: 38000549
DOI: 10.1016/j.ymben.2023.11.004 -
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