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Journal of Advanced Research Dec 2023Transcription of biological nitrogen fixation (nif) genes is activated by the NifA protein which recognizes specific activating sequences upstream of σ-dependent nif...
INTRODUCTION
Transcription of biological nitrogen fixation (nif) genes is activated by the NifA protein which recognizes specific activating sequences upstream of σ-dependent nif promoters. The large quantities of nitrogenase which can make up 20% of the total proteins in the cell indicates high transcription activating efficiency of NifA and high transcription level of nifHDK nitrogenase genes.
OBJECTIVES
Development of an efficient gene transcription activating strategy in bacteria based on positive transcription regulatory proteins and their regulating DNA sequences.
METHODS
We designed a highly efficient gene transcription activating strategy in which the nifA gene was placed directly downstream of its regulating sequences. The NifA protein binds its regulating sequences and stimulates transcription of itself and downstream genes. Overexpressed NifA causes transcription activation by positive reinforcement.
RESULTS
When this gene transcription activating strategy was used to overexpress NifA in Pseudomonas stutzeri DSM4166 containing the nif gene cluster, the nitrogenase activity was increased by 368 folds which was 16 times higher than that obtained by nifA driven by the strongest endogenous constitutive promoter. When this strategy was used to activate transcription of exogenous biosynthetic genes for the plant auxin indole-3-acetic acid and the antitumor alkaloid pigment prodigiosin in DSM4166, both of them resulted in better performance than the strongest endogenous constitutive promoter and the highest reported productions in heterologous hosts to date. Finally, we demonstrated the universality of this strategy using the positive transcriptional regulator of the psp operon, PspF, in E. coli and the pathway-specific positive transcription regulator of the polyene antibiotic salinomycin biosynthesis, SlnR, in Streptomyces albus.
CONCLUSION
Many positive transcription regulatory proteins and their regulating DNA sequences have been identified in bacteria. The gene transcription activating strategy developed in this study will have broad applications in molecular biology and biotechnology.
PubMed: 38123018
DOI: 10.1016/j.jare.2023.12.015 -
Journal of Microbiology (Seoul, Korea) Dec 2023Pseudomonas stutzeri strain AJR13 was isolated for growth on the related compounds biphenyl (BPH) and diphenylmethane (DPM). The BPH and DPM degradative pathway genes...
Pseudomonas stutzeri strain AJR13 was isolated for growth on the related compounds biphenyl (BPH) and diphenylmethane (DPM). The BPH and DPM degradative pathway genes are present on an integrative and conjugative element (ICE) in the chromosome. Examination of the genome sequence of AJR13 revealed a gene encoding a salicylate 1-monooxygenase (salA) associated with the ICE even though AJR13 did not grow on salicylate. Transfer of the ICE to the well-studied Pseudomonas putida KT2440 resulted in a KT2440 strain that could grow on salicylate. Knockout mutagenesis of the salA gene on the ICE in KT2440 eliminated the ability to grow on salicylate. Complementation of the knockout with the cloned salA gene restored growth on salicylate. Transfer of the cloned salA gene under control of the lac promoter to KT2440 resulted in a strain that could grow on salicylate. Heterologous expression of the salA gene in E. coli BL21 DE3 resulted in the production of catechol from salicylate, confirming that it is indeed a salicylate 1-monooxygenase. Interestingly, transfer of the cloned salA gene under control of the lac promoter to AJR13 resulted in a strain that could now grow on salicylate, suggesting that gene expression for the downstream catechol pathway is intact.
Topics: Pseudomonas stutzeri; Escherichia coli; Salicylates; Catechols
PubMed: 38100000
DOI: 10.1007/s12275-023-00093-x -
Bioresource Technology Feb 2024Aerobic denitrification and its mechanism by P. stutzeri was investigated in the presence of nanoscale zero-valent iron (nZVI). The removal of nitrate and ammonia was...
Aerobic denitrification and its mechanism by P. stutzeri was investigated in the presence of nanoscale zero-valent iron (nZVI). The removal of nitrate and ammonia was accelerated and the nitrite nitrogen accumulation was reduced by nZVI. The particle size and dosage of nZVI were key factors for enhancing aerobic denitrification. nZVI reduced the negative effects of low carbon/nitrogen, heavy metals, surfactants and salts to aerobic denitrification. nZVI and its dissolved irons were adsorbed into the bacteria cells, enhancing the transfer of electrons from nicotinamide adenine dinucleotide (NADH) to nitrate reductase. Moreover, the activities of NADH-ubiquinone reductase involved in the respiratory system, and the denitrifying enzymes were increased. The expression of denitrifying enzyme genes napA and nirS, as well as the iron metabolism gene fur, were promoted in the presence of nZVI. This work provides a strategy for enhancing the biological denitrification of wastewater using the bio-stimulation of nanomaterials.
Topics: Iron; Pseudomonas stutzeri; Denitrification; Electrons; Nitrates; Nitrogen; Gene Expression
PubMed: 38092073
DOI: 10.1016/j.biortech.2023.130202 -
Bioresource Technology Feb 2024Research on microalgae has surged due to its diverse biotechnological applications and capacity for accumulating bioactive compounds. Despite considerable advancements,...
Application of response surface Methodology coupled with Artificial Neural network and genetic algorithm to model and optimize symbiotic interactions between Chlorella vulgaris and Stutzerimonas stutzeri strain J3BG for chlorophyll accumulation.
Research on microalgae has surged due to its diverse biotechnological applications and capacity for accumulating bioactive compounds. Despite considerable advancements, microalgal cultivation remains costly, prompting efforts to reduce expenses while enhancing productivity. This study proposes a cost-effective approach through the coculture of microalgae and bacteria, exploiting mutualistic interactions. An engineered consortium of Chlorella vulgaris and Stutzerimonas stutzeri strain J3BG demonstrated biofilm-like arrangements, indicative of direct cell-to-cell interactions and metabolite exchange. Strain J3BG's enzymatic characterization revealed amylase, lipase, and protease production, sustaining mutual growth. Employing Response Surface Methodology (RSM), Artificial Neural Network (ANN), and Genetic Algorithm (GA) in a hybrid modeling approach resulted in a 2.1-fold increase in chlorophyll production. Optimized conditions included a NaNO3 concentration of 128.52 mg/l, a 1:2 (Algae:Bacteria) ratio, a 6-day cultivation period, and a pH of 5.4, yielding 10.92 ± 0.88 mg/l chlorophyll concentration.
Topics: Chlorella vulgaris; Chlorophyll; Neural Networks, Computer; Bacteria; Biotechnology; Microalgae; Pseudomonas stutzeri; Biomass
PubMed: 38086458
DOI: 10.1016/j.biortech.2023.130148 -
Ecotoxicology and Environmental Safety Jan 2024Silver nanoparticles (AgNPs) are widely used in daily life and industry because of their excellent antibacterial properties. AgNPs can exist in wastewater in various...
Silver nanoparticles (AgNPs) are widely used in daily life and industry because of their excellent antibacterial properties. AgNPs can exist in wastewater in various forms, such as Ag, AgSO, AgCO, AgS, AgO, and AgCl. To assess the potential environmental risk of AgNPs and various forms of Ag, their toxic effects were investigated using the common denitrifier species Pseudomonas stutzeri (P. stutzeri). The inhibitory effect of AgNPs and various forms of Ag on P. stutzeri growth and its denitrification performance occurred in a concentration-dependent manner. The denitrification efficiency of P. stutzeri decreased from 95%∼97% to 89∼95%, 74∼95%, and 56∼85% under low, medium, and high exposure doses, respectively, of AgNPs and various forms of Ag. The changes in cell membrane morphology and increases in lactate dehydrogenase (LDH) release indicated that AgNPs and various forms of Ag damaged the cell membrane of P. stutzeri. Oxidative stress caused by excessive accumulation of reactive oxygen species (ROS) increased superoxide dismutase (SOD) and catalase (CAT) activities and decreased glutathione (GSH) levels. Overall, this study will help elucidate the impact of AgNPs and their transformation products on nitrogen removal efficiency in wastewater biological treatment systems.
Topics: Silver; Pseudomonas stutzeri; Metal Nanoparticles; Denitrification; Wastewater; Nitrogen; Antioxidants
PubMed: 38056119
DOI: 10.1016/j.ecoenv.2023.115785 -
Frontiers in Bioengineering and... 2023Acetate is a low-cost feedstock for the production of different bio-chemicals. Electrochemical reduction of CO into acetate and subsequent acetate fermentation is a...
Acetate is a low-cost feedstock for the production of different bio-chemicals. Electrochemical reduction of CO into acetate and subsequent acetate fermentation is a promising method for transforming CO into value-added chemicals. However, the significant inhibitory effect of acetate on microbial growth remains a barrier for acetate-based biorefinery. In this study, the deletion of genes involved in L-leucine degradation was found to be beneficial for the growth of A1501 in acetate. (Δ), in which the hydroxymethylglutaryl-CoA lyase catalyzing -hydroxy--methylglutaryl-CoA into acetyl-CoA and acetoacetate was deleted, grew faster than other mutants and exhibited increased tolerance to acetate. Then, the genes from H16 for poly-3-hydroxybutyrate (PHB) biosynthesis were overexpressed in (∆) and the recombinant strain (∆-) can accumulate 0.11 g L PHB from commercial acetate. Importantly, (∆-) can also use CO-derived acetate to produce PHB and the accumulated PHB accounted for 5.42% (w/w) of dried cell weight of (∆-).
PubMed: 38026858
DOI: 10.3389/fbioe.2023.1297431 -
Environmental Research Feb 2024Polycyclic aromatic hydrocarbons (PAHs) are potentially hazardous compounds that could cause a severe impact on many ecosystems. They are very challenging to remove...
Polycyclic aromatic hydrocarbons (PAHs) are potentially hazardous compounds that could cause a severe impact on many ecosystems. They are very challenging to remove using conventional methods due to their hydrophobic nature. However, this issue can be resolved by utilizing surface-active molecules to increase their bioavailability. In this study, pyrene was chosen as the PAH compound to explore its degradability by the effect of individual bacterial strains (Pseudomonas stutzeri NA3 and Acinetobacter baumannii MN3) and mixed consortia (MC) along with natural surfactant derived from Sapindus mukorossi and iron oxide nanoparticles (NPs). Additionally, fatty acids esters, dipeptides, and sugar derivative groups were identified as potent bioactive components of natural surfactants. Various techniques, such as XRD, VSM, TEM, and FE-SEM with EDX, were utilized to characterize the pristine and Fenton-treated iron oxide NPs. The analytical results confirmed that the FeO crystal phase and spherical-shaped NPs exhibited excellent magnetic properties. The impact of natural surfactants and iron oxide NPs has significantly contributed to the biodegradation process, resulting in a prominent decrease in chemical oxygen demand (COD) levels. Gas chromatography-mass spectrometry (GC-MS) analysis showed that biodegradation systems produced primary hydrocarbon intermediates, which underwent oxidative degradation through Fenton treatment. Interestingly, synthesized iron oxide NPs effectively produced hydroxyl radical (•OH) during the Fenton reaction, which was confirmed by electron paramagnetic resonance (EPR) spectra, and the pristine iron oxide NPs underwent a material transformation observed. The study demonstrated an integrated approach for biodegradation and the Fenton reaction process to enhance the pyrene degradation efficiency (90%) compared to other systems. Using natural surfactants and iron oxide NPs in aquatic environments serves as a crucial platform at the interface of microorganisms and contaminated oil products. This interaction offers a promising solution for PAHs bioremediation.
Topics: Surface-Active Agents; Biodegradation, Environmental; Ecosystem; Soil Pollutants; Pyrenes; Polycyclic Aromatic Hydrocarbons; Bacteria; Magnetic Iron Oxide Nanoparticles; Ferric Compounds
PubMed: 38008204
DOI: 10.1016/j.envres.2023.117753 -
ACS Synthetic Biology Dec 2023The soil environment adjacent to plant roots, termed the rhizosphere, is home to a wide variety of microorganisms that can significantly affect the physiology of nearby...
The soil environment adjacent to plant roots, termed the rhizosphere, is home to a wide variety of microorganisms that can significantly affect the physiology of nearby plants. Microbes in the rhizosphere can provide nutrients, secrete signaling compounds, and inhibit pathogens. These processes could be manipulated with synthetic biology to enhance the agricultural performance of crops grown for food, energy, or environmental remediation, if methods can be implemented in these nonmodel microbes. A common first step for domesticating nonmodel organisms is the development of a set of genetic engineering tools, termed a synthetic biology toolbox. A toolbox comprises transformation protocols, replicating vectors, genome engineering (e.g., CRISPR/Cas9), constitutive and inducible promoter systems, and other gene expression control elements. This work validated synthetic biology toolboxes in three nitrogen-fixing soil bacteria: , (), and a new isolate of . All three organisms were amenable to transformation and reporter protein expression, with several functional inducible systems available for each organism. and showed more reliable plasmid-based expression, resulting in successful Cas9 recombineering to create scarless deletions and insertions. Using these tools, we generated mutants with inducible nitrogenase activity and introduced heterologous genes to produce resorcinol products with relevant biological activity in the rhizosphere.
Topics: Nitrogen; Soil; Synthetic Biology; Plasmids; Genetic Engineering; CRISPR-Cas Systems
PubMed: 37988619
DOI: 10.1021/acssynbio.3c00414 -
Journal of Hazardous Materials Feb 2024Ibuprofen (IBP) is a widely used drug of environmental concern as emerging contaminant due to its low elimination rates by wastewater treatment plants (WWTPs), leading...
Ibuprofen (IBP) is a widely used drug of environmental concern as emerging contaminant due to its low elimination rates by wastewater treatment plants (WWTPs), leading to the contamination of the environment, where IBP is introduced mainly from wastewater discharge and sewage sludge used as fertilizer. This study describes the application of a consortium from sewage sludge and acclimated with ibuprofen (consortium C7) to accelerate its biodegradation both in solution and sewage sludge. 500 mg L IBP was degraded in solution in 28 h, and 66% mineralized in 3 days. IBP adsorbed in sewage sludge (10 mg kg) was removed after bioaugmentation with C7 up to 90% in 16 days, with a 5-fold increase in degradation rate. This is the first time that bioaugmentation with bacterial consortia or isolated bacterial strains have been used for IBP degradation in sewage sludge. The bacterial community of consortium C7 was significantly enriched in Sphingomonas wittichii, Bordetella petrii, Pseudomonas stutzeri and Bosea genosp. after IBP degradation, with a special increase in abundance of S. wittichii, probably the main potential bacterial specie responsible for IBP mineralization. Thirteen bacterial strains were isolated from C7 consortium. All of them degraded IBP in presence of glucose, especially Labrys neptuniae. Eight of these bacterial strains (B. tritici, L. neptuniae, S. zoogloeoides, B. petrii, A. denitrificans, S. acidaminiphila, P. nitroreducens, C. flaccumfaciens) had not been previously described as IBP-degraders. The bacterial community that makes up the indigenous consortium C7 appears to have a highly efficient biotic degradation potential to facilitate bioremediation of ibuprofen in contaminated effluents as well as in sewage sludge generated in WWTPs.
Topics: Sewage; Biodegradation, Environmental; Ibuprofen; Microbial Consortia; Wastewater; Bacteria
PubMed: 37976863
DOI: 10.1016/j.jhazmat.2023.132970 -
International Journal of Molecular... Oct 2023The healing properties of silver have been used since ancient times. The main aim of the study was to collect and review the literature on the clinical potential of... (Review)
Review
The healing properties of silver have been used since ancient times. The main aim of the study was to collect and review the literature on the clinical potential of silver, its salts and complex compounds. The second goal was to present an outline of the historical use of silver in medicine and pharmacy, taking into account the possibility of producing pharmaceutical drug forms on the premises of pharmacies. In the context of the growing resistance of microorganisms to available, widely used antibiotics, silver plays a key role. There is only one known case of bacterial resistance to silver-the strain, which naturally occurs in silver mines. The development of research in the field of coordination chemistry offers great opportunities in the design of new substances in which silver ions can be incorporated. These substances exhibit increased potency and often an extended antimicrobial spectrum. Silver-based compounds are, however, only limited to external applications, as opposed to their historic oral administration. Advanced studies of their physicochemical, microbiological, cytotoxic and genotoxic properties are ongoing and full of challenges. The improvement of the methods of synthesis gives the possibility of applying the newly synthesized compounds , as was the case with the complex of metronidazole with silver (I) nitrate. Some of these experimental efforts performed in vitro are followed with clinical trials. The third and final goal of this study was to present the possibility of obtaining an ointment under the conditions of an actual pharmacy using silver (I) salts and a ligand, both of which are active substances with antimicrobial properties.
Topics: Silver; Salts; Pharmacies; Anti-Infective Agents; Anti-Bacterial Agents; Silver Compounds; Pharmaceutical Preparations; Pharmacy
PubMed: 37958707
DOI: 10.3390/ijms242115723