Search Results (304)

Copper ions serve as essential cofactors for many enzymes but exhibit toxicity at elevated concentrations. In Gram-negative bacteria, the Cop system, typically encoded by copABCD, plays a crucial role in maintaining copper homeostasis and detoxification. The chromosome of Pseudomonas putida harbors two copAB clusters but lacks copCD, along with two copR-copS clusters that encode the cognate two-component system. Here, the roles of these Cop components in countering copper toxicity were studied. We found that copAB2 was essential for full resistance to Cu²⁺ in P. putida, while copAB1 made only a minor contribution, partially due to its low expression. The two-component systems CopRS1 and CopRS2 both played significant regulatory roles in copper resistance. Although they could compensate for the absence of each other to mediate copper resistance, they exhibited distinct regulatory effects. CopR1 bound to all four cop promoters and activated their transcription under copper stress. In contrast, though CopR2 bound to the same sites as CopR1 in each cop promoter, it significantly activated only copAB2 and copRS2 expression. Its competitive binding at the copAB1 and copRS1 promoters likely impeded CopR1-mediated activation of these genes. Overall, this study reveals the distinct contributions of the two Cop systems to copper resistance and their regulatory interplay in P. putida. Full article

TtgR, a transcriptional repressor from Pseudomonas putida, plays a key role in regulating multidrug resistance by controlling the expression of genes in response to various ligands. Despite its broad specificity, TtgR represents a promising candidate for the development of transcription factor (TF)-based biosensors. In this study, we utilized TtgR and its native promoter region (P_ttgABC) as genetic components to construct TF-based biosensors in Escherichia coli. By coupling TtgR and P_ttgABC with egfp, we developed a biosensor responsive to diverse flavonoids. To enhance the selectivity and specificity of the biosensor, we genetically engineered a TtgR-binding pocket. Engineered TtgR variants exhibited altered sensing profiles, enabling the development of biosensors with tailored ligand responses. Computational structural analysis and ligand docking provided insights into the interaction mechanisms between TtgR variants and flavonoids. Notably, biosensors based on wild-type TtgR and its N110F mutant were capable of quantifying resveratrol and quercetin at 0.01 mM with >90% accuracy. Although the precise molecular mechanisms involved remain unclear and further optimization is needed, the biosensors developed herein demonstrate strong potential for applications in numerous fields. This study lays the foundation for future research that could extend the utility of TtgR-based biosensors to synthetic biology, metabolic engineering, and beyond. Full article

Due to its high bioconversion efficiency and nutritional value, the black soldier fly (Hermetia illucens L. 1758) is a promising insect species for sustainable animal feed production. However, concerns remain regarding microbial safety when larvae are reared on substrates contaminated by pathogenic or spoilage bacteria. This study investigated the effects of substrate inoculation with Bacillus subtilis 168 or Pseudomonas putida KT2440 on larval performance and gut microbiota composition. Larvae reared on contaminated diets showed no significant differences in survival or development time compared to controls. However, a short-term reduction in growth was observed in the Bacillus-exposed group. qPCR analyses confirmed the temporary presence of Bacillus taxa in larval guts, while Pseudomonas taxa were effectively excluded. Amplicon sequencing of the 16S rRNA gene revealed that the contamination did not affect gut bacterial microbiota richness and composition. Instead, the bacterial communities evolved naturally with Lactobacillales-related bacteria dominating early stages and Morganellaceae taxa becoming more abundant in prepupae. Our findings demonstrate the stability and resilience of H. illucens gut bacterial microbiota, reinforcing the safety and suitability of H. illucens as a feed ingredient, even when reared under challenging microbial conditions. Full article

The textile industry’s reliance on synthetic dyes contributes significantly to pollution, highlighting the need for sustainable alternatives like biopigments. This study investigates the production and application of the biopigment prodigiosin, which was produced by Pseudomonas putida with a yield of 1.85 g/L. Prodigiosin was prepared under acidic, neutral, and alkaline conditions, resulting in varying protonation states that influenced its affinity for cotton and polyester fibers. Three surfactants (anionic, cationic, non-ionic) were tested, with non-ionic Tween 80 yielding a promising color strength (above 4) and fastness results with neutral prodigiosin at 1.3 g/L. Cotton and polyester demonstrated good washing (color difference up to 14 for cotton, 5 for polyester) and light fastness (up to 15 for cotton, 16 for polyester). Cellulose acetate, used in the conventional printing process as a thickener, produced superior color properties compared to commercial thickeners. Neutral prodigiosin achieved higher color strength, and cotton fabrics displayed halochromic properties, distinguishing them from polyester, which showed excellent fastness. Prodigiosin-printed samples also exhibited strong antimicrobial activity against Pseudomonas aeruginosa and retained halochromic properties over 10 pH cycles. These findings suggest prodigiosin as a sustainable dye alternative and pH sensor, with potential applications in biomedical materials, such as antimicrobial and pH-responsive wound dressings. Full article

Photodynamic inactivation (aPDI) involves the interaction of three components: non-toxic photosensitizer molecules (PS), low-intensity visible light, and molecular oxygen. This interaction leads to the generation of toxic reactive oxygen species. The present work demonstrated the efficacy of light-induced antimicrobial photodynamic inactivation against Pseudomonas aeruginosa and Pseudomonas putida using 5-aminolevulinic acid (5-ALA) as a prodrug to produce the photosensitizer protoporphyrin IX. The photoeradication efficiency of these pathogens under blue (405 nm; 45 mW cm⁻²) and red (635 nm; 53 mW cm⁻²) light was investigated. Results showed that at least 30 min of blue light irradiation was necessary to achieve a 99.999% reduction of P. aeruginosa, whereas red light was less effective. P. putida exhibited limited susceptibility under similar conditions. To enhance aPDI efficiency, exogenous glucose was added alongside 5-ALA, which significantly increased the photodynamic efficacy—particularly against P. aeruginosa—leading to complete eradication after just 5 min of exposure. Spectroscopic analyses confirmed that glucose increased the levels of protoporphyrin IX, which correlated with enhanced photodynamic efficacy. Furthermore, multiple aPDI exposure reduced key virulence factors, including alkaline protease activity, biofilm formation, and swarming motility (in P. aeruginosa). These findings suggest that 5-ALA-mediated photodynamic inactivation offers a promising strategy to improve efficacy against resistant Gram-negative pathogens. Full article

The increasing release of nanoparticles into aquatic environments, particularly via wastewater, raises concerns about their biological effects on microbial communities. This study investigated the molecular response of Pseudomonas putida to aluminum oxide nanoparticles (Al₂O₃NPs) under controlled conditions and in synthetic wastewater, both before and after biological treatment. Acute toxicity was evaluated using growth inhibition assays, while the expression of katE, ahpC, and ctaD—genes associated with oxidative stress and energy metabolism—was quantified via RT-qPCR. Exposure to pristine Al₂O₃NPs induced a strong, time-dependent upregulation of all tested genes (e.g., katE and ahpC up to 4.5-fold). In untreated wastewater, this effect persisted but at a lower intensity; bulk Al₂O₃ caused only moderate changes. Treated wastewater samples showed markedly reduced gene expression, indicating partial detoxification. Nanoparticles elicited stronger biological responses than their bulk counterparts, confirming the material form-specific effects. Comparative analysis with Daphnia magna revealed similar patterns of oxidative stress gene activation. These findings highlight the influence of nanoparticle form and environmental matrix on microbial responses and support the use of gene expression analysis as a sensitive biomarker for nanoparticle-induced stress in environmental risk assessment. Full article

Regarding the trout microbiota, most information is focused on lactic acid bacteria, which can show beneficial properties. However, in trout farming, mostly pathogenic Gram-positive species were reported, such as Staphylococcus aureus, Listeria monocytogenes, and/or Clostridium spp. In this study, free-living trout were analyzed for Gram-negative microbiota that can cause loss as disease-stimulating agents. Bacteriocin postbiotics should be one of the approaches used to eliminate these agents. In total, 21 strains of different species isolated from the intestinal tract of 50 trout in Slovakia (Salmo trutta and Salmo gairdnerii) were taxonomically allotted into 13 species and 9 genera. This method showed variability in microbiota identified using MALDI-TOF mass spectrometry with the following species: Acinetobacter calcoaceticus, Citrobacter gillenii, Citrobacter freundii, Escherichia coli, Hafnia alvei, Kluyvera cryocrescens, K. intermedia, Leclercia adecarboxylata, Raoultella ornithinolytica, Pseudomonas fragi, Ps. putida, Ps. lundensis, Ps. teatrolens, and Serratia fonticola. Most strains were susceptible to the antibiotics used, reaching inhibitory zones up to 29 mm. On the other hand, 3 out of 21 strains (14%) were susceptible to nine enterocins- postbiotics (Hafnia alvei Hal281, Pseudomonas putida Pp391, and Ps. fragi Pf 284), with inhibitory activity in the range of 100–6400 AU/mL. Full article

Beneficial interactions between plants and bacteria are crucial in agricultural practices, as bacteria can improve soil fertility, promote plant growth, and protect plants from pathogens. This study aimed to molecularly identify and characterize soil bacterial isolates and evaluate their effect on the growth of maize (Zea mays L.), tomato (Solanum lycopersici L.), cucumber (Cucumis sativus L.), and pepper (Capsicum annuum L.) under greenhouse conditions. Plant growth parameters, including plant height, root length, and fresh (FW) and dry (DW) weights, were measured. Additionally, pigment extraction and element content analysis using leaves were performed. Among the isolates, the most effective strain in the greenhouse experiment was Bacillus safensis SCF6, which significantly enhanced plant height and fresh weight across all tested plants, with the greatest influence observed in maize plant height (439.42 ± 6.42 mm), fresh weight (14.07 ± 0.87 g plant⁻¹ FW), and dry weight (1.43 ± 0.17 g plant⁻¹ DW) compared to the control (364.67 ± 10.33 mm, 9.20 ± 1.16 g plant⁻¹ FW, and 0.92 ± 0.15 g plant⁻¹ DW, respectively). Other strains also demonstrated notable results, with Microbacterium testaceum SCF4, Bacillus mojavensis SCF8, and Pseudomonas putida SCF9 showing the highest plant growth-promoting effects on pepper, tomato, and cucumber, respectively. Pseudomonas putida SCF9 demonstrated strong antifungal activity against Monilinia laxa, with a percentage of mycelial growth inhibition (PGI) of 72.62 ± 2.06%, while Bacillus subtilis SCF1 exhibited effects against Botrytis cinerea (PGI = 69.57 ± 4.35%) and Cercospora sp. (PGI = 63.11 ± 1.12%). The development and application of beneficial bacterial inoculants or their formulated products can contribute to environmentally friendly farming practices and global food security. Full article

Importance of this study: Melanin synthesized through the oxidative polymerization of phenolic compounds exhibits a high molecular weight and has many physiological functions and activities. Main results: In this study, the key PKS1-1, PKS1-2, CMR1-1, and CMR1-2 genes for melanin biosynthesis and regulation from the highly genome-duplicated black yeast Hortaea werneckii Mo34, isolated from a deep-sea hydrothermal vent, were heterologously complemented in the ∆pks1 albino mutant K5 and the ∆cmr1 albino mutant CM7-2 of Aureobasidium melanogenum XJ5-1. Melanin formation in all the resulting transformants was restored, confirming that both the PKS1-1 and PKS1-2 genes from H. werneckii Mo34 were likely involved in the DHN melanin biosynthesis of A. melanogenum XJ5-1. Furthermore, the CMR1-1 and CMR1-2 genes from H. werneckii Mo34 could play significant roles in regulating melanin biosynthesis in A. melanogenum XJ5-1. Simultaneously, the expression of the PKS1 and THR1 genes involved in melanin biosynthesis was also enhanced in the transformants complemented with the CMR1-1 and CMR1-2 genes. The purified high-molecular-weight melanin from H. werneckii Mo34 exhibited excellent Fe²⁺-chelating, DPPH radical-scavenging, and superoxide radical-scavenging activities. Additionally, it actively inhibited the growth of Staphylococcus aureus and Pseudomonas putida. Conclusions: The black yeast H. werneckii Mo34 indeed had the DHN melanin biosynthesis pathway and the melanin produced by it had many potential applications. Full article

Endophytes, as an integral part of plants, form unique relationships with their hosts that go beyond classical definitions of symbiosis and influence plant development, immunity, and stress responses. The pepper endophyte strain Pseudomonas putida A32 has several plant growth-promoting properties and increases the tolerance of pepper to drought, but its biocontrol potential is unknown. In this study, we investigated the protective role of P. putida A32 against infection with the pathogenic bacterium P. syringae pv. aptata P21 in two pepper genotypes in laboratory experiments. The percentage of lesion reduction in genotype 26 treated with P. putida A32 was 46.62%. The results showed a significant reduction in hydrogen peroxide and malondialdehyde levels by 29.45 and 20.22%, respectively, in infected genotype 26. The treated but uninfected controls showed a significant increase in superoxide dismutase activity in genotype 26 by 41.26% and ascorbate peroxidase activity in genotype 19 by 40.28% in the treated infected plants. The tolerant genotype 19 was much less dependent on the bacterial treatment under stress conditions than the susceptible genotype 26. Future research will investigate the role of P. putida A32 in the induced systemic resistance of different pepper genotypes to protect against pathogens. Full article

Bacterial communities in diverse environmental niches respond to various external stimuli for survival. A primary means of communication between bacterial cells involves one-component (OC) and two-component signal transduction systems (TCSs). These systems are key for sensing environmental changes and regulating bacterial physiology. TCSs, which are the more complex of the two, consist of a sensor histidine kinase for receiving an external input and a response regulator to convey changes in bacterial cell physiology. For numerous reasons, TCSs have emerged as significant targets for antibacterial drug design due to their role in regulating expression level, bacterial viability, growth, and virulence. Diverse studies have shown the molecular mechanisms by which TCSs regulate virulence and antibiotic resistance in pathogenic bacteria. In this study, we performed a thorough analysis of the data from multiple public databases to assemble a comprehensive catalog of the principal detection systems present in both the non-pathogenic Pseudomonas putida KT2440 and the pathogenic Pseudomonas aeruginosa PAO1 strains. Additionally, we conducted a sequence analysis of regulatory elements associated with transcriptional proteins. These were classified into regulatory families based on Helix-turn-Helix (HTH) protein domain information, a common structural motif for DNA-binding proteins. Moreover, we highlight the function of bacterial TCSs and their involvement in functions essential for bacterial survival and virulence. This comparison aims to identify novel targets that can be exploited for the development of advanced biotherapeutic strategies, potentially leading to new treatments for bacterial infections. Full article

A pilot-scale treatment system was developed to manage winery wastewater (WWW) generated by small and medium wineries. The system incorporated three stages: pre-treatment for suspended solids removal and a two-step aerobic biotreatment. The biotreatment phase utilized a bioaugmented bioreactor with encapsulated Pseudomonas putida F1, employing the Small Bioreactor Platform (SBP) technology. This innovative encapsulation method enhanced the breakdown of recalcitrant compounds and accelerated the biodegradation process. The second reactor was operated as a Sequence Batch Bioreactor (SBR) to remove the remaining organics and solids. Over the 100 days of operation, the mean WWW flow rate was 0.5 m³/d with average organic loads of 3950 mg/L COD (chemical oxygen demand) and 2220 mg/L BOD (biological oxygen demand), operating with a hydraulic retention time (HRT) of 4 days. Reductions of up to 96% in BOD and 90% in COD values were observed with stable removal rates over time. The novelty of this study is that it offers a new, effective aerobic biological treatment process, embracing bioaugmentation of encapsulated biomass followed by SBR for WWW with a relatively short HRT, high organics removal, and a stable treatment process. The effluent quality from this treatment system met the regulatory requirements for release to a municipal wastewater treatment plant and potentially also for irrigation. Full article

Although the coking industry is a major polluter, it is still an important and irreplaceable industry in many countries. Wastewater from the coking industry typically contains large amounts of various hazardous substances, including phenols, cyanides, and thiocyanates; we conducted a comprehensive study on their ecotoxicity. This included five different toxicity tests with common species from different trophic levels: the bacteria Aliivibrio fischeri and Pseudomonas putida, the microalgae Chlorella sp., the duckweed Lemna minor, and the onion plant Allium cepa. These tests have rarely or never been used for these three toxicants. The results show that cyanide generally has the highest toxicity, while phenol has a relatively equal or higher toxicity than thiocyanate, depending on the test. Since no data on the joint toxic action of these three toxicants can be found in the literature, and although their joint occurrence in the aquatic environment is very likely, we performed joint toxic action analysis. The analysis was performed for binary and ternary mixtures of the toxicants using the Aliivibrio fischeri test. The concentration addition model was used as a reference model for the toxic behavior of these mixtures. The results obtained showed a synergistic deviation from the concentration addition model for combinations of phenol with cyanide and with thiocyanate, while the combination of cyanide and thiocyanate led to additive toxic behavior. Full article

Efficient utilization of lignin, a complex polymer in plant cell walls, is one of the key strategies for developing a green and sustainable bioeconomy. However, bioconversion of lignin poses a significant challenge due to its recalcitrant nature. Microorganisms, particularly fungi and bacteria, play a crucial role in lignin biodegradation, using various enzymatic pathways. Among bacteria, Pseudomonas putida is considered a promising host for lignin degradation and valorization, due to its robust and flexible metabolism and its tolerance to many noxious and toxic compounds. This review explores the various mechanisms of lignin breakdown by microorganisms, with a focus on P. putida’s metabolic versatility and genetic engineering potential. By leveraging advanced genetic tools and metabolic pathway optimization, P. putida can be engineered to efficiently convert lignin into valuable bioproducts, offering sustainable solutions for lignin valorization in industrial applications. Full article

Carbon quantum dots (CQD) are an emergent nanomaterial with unique optical and biological properties. However, the purification of CQD is one of the bottlenecks that makes it difficult to scale for application in different areas. In this work, we explore for the first time the potential of centrifugal partition chromatography (CPC) as an alternative preparative technology to achieve the purification of CQD at the gram scale. The hydrothermal method was used to synthesize CQD from avocado peels. After 6 h at 250 °C, a complex mix of strong blue-fluorescent CQDs were obtained and submitted to CPC fractionation without pretreatment. The best results were obtained with the solvent system n-hexane–ethyl acetate–methanol–water (1:2:1:2, v/v/v/v), in an elution-extrusion protocol. Nine fractions were obtained and were characterized by UV-VIS spectrophotometry, Fourier transform infrared (F-TIR), and field emission scanning electron microscopy (FESEM), confirming the presence of CQD of different sizes. CPC fractionations indicate that a polarity-based separation mechanism can be used to purify CQD. Interestingly, four fractions showed antibacterial and anti-biofilm effects on Pseudomonas putida and Listeria monocytogenes. Therefore, CPC allows for better refining of this type of nanomaterial, and in combination with other techniques, it would serve to obtain CQD of higher purity, facilitating the physicochemical and bioactivity characterization of these particles. CPC would also allow the use of waste, such as avocado peels, to obtain new materials. Full article