APPLIED AND ENVIRONMENTAL MICROBIOLOGY
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2021
·Vol. 87
·期 5
·
IF 4.792
Shanshan Chen,Xianyue Jing,Yongliang Yan,Shaofu Huang,Xing Liu,Piao Chen,Shungui Zhou
Diazotrophs can produce bioavailable nitrogen from inert N2 gas by bioelectrochemical nitrogen fixation (e-BNF), which is emerging as an energy-saving and highly selective strategy for agriculture and industry. However, current e-BNF technology is impeded by requirements for NH4+ assimilation inhibitors to facilitate intracellular ammonia secretion and precious metal catalysts to generate H2 as the energy-carrying intermediate. Here, we initially demonstrate inhibitor- and catalystless extracellular NH4+ production by the diazotroph Pseudomonas stutzeri A1501 using an electrode as the sole electron donor. Multiple lines of evidence revealed that P. stutzeri produced 2.32 ± 0.25 mg/liter extracellular NH4+ at a poised potential of −0.3 V (versus standard hydrogen electrode [SHE]) without the addition of inhibitors or expensive catalysts. The electron uptake mechanism was attributed to the endogenous electron shuttle phenazine-1-carboxylic acid, which was excreted by P. stutzeri and mediated electron transfer from electrodes into cells to directly drive N2 fixation. The faradaic efficiency was 20% ± 3%, which was 2 to 4 times that of previous e-BNF attempts using the H2-mediated pathway. This study reports a diazotroph capable of producing secretable NH4+ via extracellular electron uptake, which has important implications for optimizing the performance of e-BNF systems and exploring the novel nitrogen-fixing mode of syntrophic microbial communities in the natural environment.IMPORTANCE Ammonia greatly affects global ecology, agriculture, and the food industry. Diazotrophs with an enhanced capacity of extracellular NH4+ excretion have been proven to be more beneficial to the growth of microalgae and plants, whereas most previously reported diazotrophs produce intracellular organic nitrogen in the absence of chemical suppression and genetic manipulation. Here, we demonstrate that Pseudomonas stutzeri A1501 is capable of extracellular NH4+ production without chemical suppression or genetic manipulation when the extracellular electrode is used as the sole electron donor. We also reveal the electron uptake pathway from the extracellular electron-donating partner to P. stutzeri A1501 via redox electron shuttle phenazines. Since both P. stutzeri A1501 and potential electron-donating partners (such as electroactive microbes and natural semiconductor minerals) are abundant in diverse soils and sediments, P. stutzeri A1501 has broader implications on the improvement of nitrogen fertilization in the natural environment.
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Frontiers in Bioengineering and Biotechnology
·
2020
·
IF 3.644
Sun Jun,Lu Li-Bing,Liang Tian-Xin,Yang Li-Rong,Wu Jian-Ping
Pseudomonas putida (P. putida) KT2440 is a paradigmatic environmental-bacterium that possesses significant potential in synthetic biology, metabolic engineering and biodegradation applications. However, most genome editing methods of P. putida KT2440 depend on heterologous repair proteins and the provision of donor DNA templates, which is laborious and inefficient. In this report, an efficient cytosine base editing system was established by using cytidine deaminase (APOBEC1), enhanced specificity Cas9 nickase (eSpCas9ppD10A) and the uracil DNA glycosylase inhibitor (UGI). This constructed base editor converts C-G into T-A in the absence of DNA strands breaks and donor DNA templates. By introducing a premature stop codon in target spacers, we successfully applied this system for gene inactivation with an efficiency of 25–100% in various Pseudomonas species, including P. putida KT2440, P. aeruginosa PAO1, P. fluorescens Pf-5 and P. entomophila L48. We engineered an eSpCas9ppD10A-NG variant with a NG protospacer adjacent motif to expand base editing candidate sites. By modifying the APOBEC1 domain, we successfully narrowed the editable window to increase gene inactivation efficiency in cytidine-rich spacers. Additionally, multiplex base editing in double and triple loci was achieved with mutation efficiencies of 90–100% and 25–35%, respectively. Taken together, the establishment of a fast, convenient and universal base editing system will accelerate the pace of future research undertaken with P. putida KT2440 and other Pseudomonas species.
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Scientific Reports
·
2020
·Vol. 10
·期 1
·1-11
·
IF 3.998
Song Jinlong,Han Gang,Wang Yani,Jiang Xu,Zhao Dongxue,Li Miaomiao,Yang Zhen,Ma Qingyun,Parales Rebecca E.,Ruan Zhiyong,Mu Yingchun
Malachite green is a common environmental pollutant that poses a great threat to non-target organisms, including humans. This study reports the characterization of a bacterial strain, Pseudomonas veronii JW3-6, which was isolated from a malachite green enrichment culture. This strain degraded malachite green efficiently in a wide range of temperature and pH levels. Under optimal degradation conditions (32.4 °C, pH 7.1, and inoculum amount of 2.5 × 10 7 cfu/mL), P. veronii JW3-6 could degrade 93.5% of 50 mg/L malachite green within seven days. Five intermediate products from the degradation of malachite green were identified: leucomalachite green, 4-(dimethylamino) benzophenone, 4-dimethylaminophenol, benzaldehyde, and hydroquinone. We propose a possible degradation pathway based on these findings. The present study is the first to report the degradation of malachite green by P. veronii and the identification of hydroquinone as a metabolite in the degradation pathway.
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Food Science & Nutrition
·
2021
·Vol. 9
·期 7
·3873-3884
·
IF 2.863
Mingcan Qin,Xiaoyi Ma,Shihui Fan,Hangjie Wu,Wanli Yan,Xiaopeng Tian,Jing Lu,Mingsheng Lyu,Shujun Wang
In the present study, a DNAzyme was screened in vitro through the use of a DNA library and crude extracellular mixture (CEM) of Pseudomonas aeruginosa . Following eight rounds of selection, a DNAzyme termed PAE-1 was obtained, which displayed high rates of cleavage with strong specificity. A fluorescent biosensor was designed for the detection of P. aeruginosa in combination with the DNAzyme. A detection limit as low as 1.2 cfu/ml was observed. Using proteases and filtration, it was determined that the target was a protein with a molecular weight of 10 kDa–50 kDa. The DNAzyme was combined with a polystyrene board to construct a simple indicator plate sensor which produced a color that identified the target within 10 min. The results were reliable when tap water and food samples were tested. The present study provides a novel experimental strategy for the development of sensors based on a DNAzyme to rapidly detect P. aeruginosa in the field.
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ARCHIVES OF MICROBIOLOGY
·
2026
·Vol. 208
·期 5
·235
·
IF 3.4
Zhou Yong,Wu Lei,Rubo Lv,Szpyrka Ewa,Podbielska Magdalena,Amirian Veghar,Russel Mohammad
This study investigated the aerobic biodegradation potential of two novel indigenous bacterial strains, Pseudomonas aeruginosa DUT-Pa and Rhodococcus erythropolis DUT-Re, utilizing benzene, toluene, ethylbenzene, and o-xylene (BTEX) as the sole carbon source. The optimal metabolic activity for both strains was identified at neutral pH (7.0) and mesophilic conditions (30 °C), establishing a critical baseline for enhancing bioremediation protocols. Biochemical oxygen demand (BOD) analysis revealed a direct correlation between the substrate concentration (up to 400 mg/L) and microbial respiratory activity. Dissolved oxygen (DO) depletion from 7.77 mg/L (pre-experiment) to 2.97 mg/L (DUT-Pa) and 2.15 mg/L (DUT-Re) further confirmed the oxygen-dependent degradation. In the gas phase, the highest degradation rate was recorded for benzene, with 98.43, 97.34, and 98.91% by DUT-Pa, DUT-Re, and mixed bacteria, respectively. Meanwhile, the liquid phase demonstrated superior toluene degradation efficiency by DUT-Pa, DUT-Re, and mixed bacteria with 91.99, 83.54, and 93.27% respectively. Bacterial combinations enhanced BTEX degradation, achieving the shortest half-lives and the highest degradation rate constants. Meanwhile, the SOD (325 U/mL) level and MDA (1.5 to 2.59 nmol/g) increased in the mixed system. This study demonstrates that an additive effect of DUT-Pa and DUT-Re combination promotes efficient, complete degradation of BTEX and offers a scalable, eco-friendly solution based on strain-specific cooperative microbial dynamics. Graphical
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Scientific Reports
·
2022
·Vol. 12
·期 1
·1-13
·
IF 4.996
Li Miaomiao,Li Qingqing,Yao Jun,Sunahara Geoffrey,Duran Robert,Zhang Qinghua,Ruan Zhiyong
The overuse of the herbicide nicosulfuron has become a global environmental concern. As a potential bioremediation technology, the microbial degradation of nicosulfuron shows much promise; however, the mechanism by which microorganisms respond to nicosulfuron exposure requires further study. An isolated soil-borne bacteria Pseudomonas nicosulfuronedens LAM1902 displaying nicosulfuron, chlorimuron-ethyl, and cinosulfuron degradabilities in the presence of glucose, was used to determine the transcriptional responses to nicosulfuron exposure. RNA-Seq results indicated that 1102 differentially expressed genes (DEGs) were up-regulated and 702 down-regulated under nicosulfuron stress. DEGs were significantly enriched in “ABC transporters”, “sulfur metabolism”, and “ribosome” pathways ( p ≤ 0.05). Several pathways (glycolysis and pentose phosphate pathways, a two-component regulation system, as well as in bacterial chemotaxis metabolisms) were affected by nicosulfuron exposure. Surprisingly, nicosulfuron exposure showed positive effects on the production of oxalic acid that is synthesized by genes encoding glycolate oxidase through the glyoxylate cycle pathway. The results suggest that P. nicosulfuronedens LAM1902 adopt acid metabolites production strategies in response to nicosulfuron, with concomitant nicosulfuron degradation. Data indicates that glucose metabolism is required during the degradation and adaptation of strain LAM1902 to nicosulfuron stress. The present studies provide a glimpse at the molecular response of microorganisms to sulfonylurea pesticide toxicity and a potential framework for future mechanistic studies.
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Frontiers in Microbiology
·
2023
·
IF 5.2
Li Wen-Ru,Zeng Tao-Hua,Zhang Zhi-Qing,Shi Qing-Shan,Xie Xiao-Bao
Pseudomonas aeruginosa is a ubiquitous opportunistic pathogen that can cause severe respiratory tract infections. Geraniol, a chemical component of essential oils, has antimicrobial and anti-inflammatory activities, along with low toxicity. However, the effect and mechanism of geraniol against P. aeruginosa virulence factors are rarely studied. In this study, we investigated the quorum sensing (QS) inhibitory effects and mechanisms of geraniol against P. aeruginosa PAO1, using physiological and biochemical techniques, quantitative reverse transcription polymerase chain reaction, and transcriptomics. Geraniol slightly affected P. aeruginosa PAO1 growth, prolonged the lag phase, and delayed growth periods in a concentration-dependent manner. Geraniol inhibited three QS systems of P. aeruginosa, las, rhl, and pqs by suppressing the expression level of their key genes, including the three signal synthetase encoding genes of lasI, rhlI, and pqsABCDEH, and the corresponding signal receptor encoding genes of lasR, rhlR, and pqsR. Geraniol also suppressed certain virulence genes regulated by these three QS systems, including rhlABC, lasAB, lecAB, phzABMS, and pelABG, resulting in the attenuation of the related virulence factors, rhamnolipids, exoprotease LasA, elastase, lectin, pyocyanin, and biofilm. In conclusion, geraniol can suppress the virulence factors of P. aeruginosa PAO1 by inhibiting the three QS systems of las, rhl, and pqs. This study is significant for improving the treatment of bacterial infections caused by P. aeruginosa.
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SENSORS AND ACTUATORS B-CHEMICAL
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2020
·
IF 7.1
Yujie Gao,Yu Chen,Miaomiao Li,Lizhen Jia,Lianbin Zhang,Jintao Zhu
Identification or detection of pathogenic microbes is one of the most important aspects to prevent and/or treat bacterial infection. In this study, we report a facile yet versatile strategy for preparation of gelatin-based photonic hydrogel sensors for visual detection of Pseudomonas aeruginosa ( P. aeruginosa ). The photonic hydrogels are generated by in-situ photopolymerization of acrylamide (AM), N , N ’-methylenebis-(acrylamide) (BIS), and gelatin methacrylate (GelMA) with the self-assembled one-dimensional (1D) chain-like structures of carbon-encapsulated Fe 3 O 4 nanoparticles (Fe 3 O 4 @C NPs). In the presence of P. aeruginosa , the hydrolysis of gelatin triggered by gelatinase reduces the crosslinking density of photonic hydrogel, expands the hydrogel in volume, increases the lattice spacing of the colloidal particles, and red-shifts the reflection spectra of the photonic hydrogel. The optical appearance variation of the photonic hydrogel, resulting from the reflection peak shift, can be observed by naked eyes. Besides, we show that the photonic hydrogels exhibit negligible appearance changes with the presence of Escherichia coli ( E. coli ), indicating their potential for distinguishing various bacteria by a simple structural color variation. This finding provides a proof-of-concept for a colorimetric sensor for visual detection of P. aeruginosa and demonstrates the great potential of the photonic hydrogels in visual detection of pathogens.
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JOURNAL OF POLYMERS AND THE ENVIRONMENT
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2016
·Vol. 25
·期 2
·156-164
·
IF 1.969
Liu Huifang,Gao Zhaoying,Hu Xueyan,Wang Zhanyong,Su Tingting,Yang Lei,Yan Song
Poly(3-hydroxybutyrate-co-hydroxyvalerate) (PHBV) is a biodegradable polymer synthesized in microorganisms. The application of PHBV is limited by certain material disadvantages. Poly(ε-caprolactone) (PCL) possesses excellent thermodynamic and mechanical properties and was used to modify PHBV in the presence of triethyl citrate (TEC) and dicumyl peroxide (DCP), which was used as plasticizer and grafting agent, respectively. The effects of PCL and additive agents on the mechanical, thermal, amphipathic and degradability behaviors of the blends were investigated. The results showed that the mechanical properties of the PHBV blends improved by PCL incorporation and improved even further after TEC and DCP addition. The addition of DCP could not induce an increase in crystallization temperature but improved the crystallization degree of the blends. The presence of hydrophilic groups in TEC leads to an apparent increases in the hydrophilicity of the PHBV blends. A PHBV/PCL blend (40/60) with TEC (20 wt.%) and DCP (0.5 wt.%) was chosen for its good mechanical properties and hydrophilicity. The chosen ratio of the blends was also shown a preferable degradation activity by biodegradation assay using Pseudomonas mendocina. The addition of TEC and DCP has no conspicuous negative effect on the biodegradation.
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ELECTRONIC JOURNAL OF BIOTECHNOLOGY
·
2025
·
IF 2.5
Yi Li,Jiacheng Tang,Kaixiang Xin,Zongda Chen,Lele Zhao,Yifan Zhao,Yinbiao Xu,Pei Zhou,Yang Sun,Yupeng Liu,Hua Li
Background Nicotinic acid dehydrogenase possesses the capability to convert nicotinic acid into 6-hydroxynicotinic acid, a compound of significant research value as a pharmaceutical intermediate. The extraction of nicotinic acid dehydrogenase is primarily performed by strains. However, the enzyme activity of the strains reported currently is relatively low, and their potential to catalyze the production of 6-hydroxynicotinic acid is insufficient to meet industrial requirements. Results Due to the revealing properties of 6-hydroxynicotinic acid, this study proposes a technique for calculating the luminescence intensity of colonies, which is based on a fluorescence spectrometer. The developed method establishes a reliable linear relationship (88.2%) between the luminescence intensity and enzyme activity. Consequently, it has been employed to screen strains that produce nicotinate dehydrogenase. This screening approach allows for the evaluation of about 500 enzyme-producing strains daily, presenting an efficient strategy for screening. Conclusions Through this approach, a novel high enzyme activity strain producing nicotinic acid dehydrogenase, Pseudomonas poae have been obtained, which designated as HD530. After process optimization, it was utilized to produce 6-hydroxynicotinic acid, achieving a high yield of 155.45 g/L within 72 h, meeting the requirements for industrial production. The effectiveness and potential of this technique lie in its application for strain screening and improvement.
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PROTEIN EXPRESSION AND PURIFICATION
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2025
·
IF 1.2
Yipeng Chen,Bin Huai,Jin Chuan Wu,Ning Zhang,Yong Wang,Qingxin Li
The structural and functional investigation of bacterial membrane proteins is crucial to the development of antibiotics. Diacylglycerol kinase (DAGK) from Escherichia coli ( E. coli ) has been extensively studied as a model membrane protein. However, the DAGK from Pseudomonas aeruginosa (PAO1-DAGK) with a 44% sequence identity to E. coli -DAGK is not well characterized. To explore the properties of PAO1-DAGK, it was successfully expressed in E. coli and was purified in Decyl-β-D-maltoside (DM) micelles followed with characterizations. Chemical cross-linking studies revealed that PAO1-DAGK in DM micelles could form dimers and trimers. The kinase activity of PAO1-DAGK was determined to be 24.2 ± 2.2 U/mg protein in a mixed-micelle system. The effects of pH and temperature on the activity of PAO1-DAGK were also investigated, respectively. PAO1-DAGK in DM micelles exhibited good stability at pH 6.0–10.0 and below 45°C. Substrate specificity measurements indicated that PAO1-DAGK demonstrated a clear preference for medium-chain diacylglycerols (DAGs) in the mixed-micelle system, with sn-1,2-Dihexanoylglycerol (DiC6) being the most favored substrate. Molecular docking results demonstrated the interactions between DAGs and PAO1-DAGK.
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iScience
·
2025
·
IF 4.1
Jie Liu,Youzhi Li,Kairuo Yan,Jinghua Han,Ziqi Wu,Ying Li
Pseudomonas aeruginosa ( P. aeruginosa ) biofilms pose substantial challenges in clinical settings due to their resistance to conventional antibiotic treatments. This study investigated the influence of reductive stress on the formation and eradication of P. aeruginosa biofilms. A series of redox-active compounds were employed to assess their impact on both biofilm development and disruption. Among them, hydrazine (HZ) showed potent activity. Mechanistic studies revealed that these compounds suppressed pyocyanin production and impaired bacterial metabolic activity. A structure–activity relationship analysis highlighted hydroxylamine (HA) as a promising candidate, owing to its favorable balance between efficacy and cytotoxicity. Notably, quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of HA-treated P. aeruginosa suggested inhibition of matrix biosynthesis, quorum sensing, and oxidative stress defenses. This study provides insights into biofilm management by targeting the redox environment, offering potential strategies for the development of redox-based therapies to combat biofilm-associated infections.
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