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Acta Biologica Hungarica Sep 2016An alginate lyase with high specific enzyme activity was purified from Pseudomonas stutzeri MSEA04, isolated from marine brown algae. The alginate lyase was purified by...
An alginate lyase with high specific enzyme activity was purified from Pseudomonas stutzeri MSEA04, isolated from marine brown algae. The alginate lyase was purified by precipitation with ammonium sulphate, acetone and ethanol individually. 70% ethanol fraction showed maximum specific activity (133.3 U/mg). This fraction was re-purified by anion exchange chromatography DEAE- Cellulose A-52. The loaded protein was separated into 3 peaks. The second protein peak was the major one which contained 48.2% of the total protein recovered and 79.4% of the total recovered activity. The collected fractions of this peak were subjected to further purification by re-chromatography on Sephadex G-100. Alginate lyase activity was fractionated in the Sephadex column into one major peak, and the specific activity of this fraction reached 116 U/mg. The optimal substrate concentration, pH and temperature for alginate lyase activity were 8 mg/ml, pH 7.5 and 37 °C, respectively. While, Km and Vmax values were 1.07 mg alginate/ ml and 128.2 U/mg protein, respectively. The enzyme was partially stable below 50 °C, and the activity of the enzyme was strongly enhanced by K(+), and strongly inhibited by Ba(+2), Cd(+2), Fe(+2) and Zn(+2). The purified enzyme yielded a single band on SDS-PAGE with molecular weight (40.0 kDa).
Topics: Alginates; Bacterial Proteins; Chromatography, DEAE-Cellulose; Chromatography, Gel; DEAE-Cellulose; Dextrans; Electrophoresis, Polyacrylamide Gel; Enzyme Stability; Glucuronic Acid; Hexuronic Acids; Hydrogen-Ion Concentration; Kinetics; Molecular Weight; Polysaccharide-Lyases; Protein Denaturation; Pseudomonas stutzeri; Temperature; Water Microbiology
PubMed: 27630053
DOI: 10.1556/018.67.2016.3.8 -
BMC Microbiology Nov 2016Microbial antimonite [Sb(III)] oxidation converts toxic Sb(III) into less toxic antimonate [Sb(V)] and plays an important role in the biogeochemical Sb cycle. Currently,...
BACKGROUND
Microbial antimonite [Sb(III)] oxidation converts toxic Sb(III) into less toxic antimonate [Sb(V)] and plays an important role in the biogeochemical Sb cycle. Currently, little is known about the mechanisms underlying bacterial Sb(III) resistance and oxidation.
RESULTS
In this study, Tn5 transposon mutagenesis was conducted in the Sb(III)-oxidizing strain Pseudomonas stutzeri TS44 to isolate the genes responsible for Sb(III) resistance and oxidation. An insertion mutation into gshA, encoding a glutamate cysteine ligase involved in glutathione biosynthesis, generated a strain called P. stutzeri TS44-gshA. This mutant strain was complemented with a plasmid carrying gshA to generate strain P. stutzeri TS44-gshA-C. The transcription of gshA, the two superoxide dismutase (SOD)-encoding genes sodB and sodC as well as the catalase-encoding gene katE was monitored because gshA-encoded glutamate cysteine ligase is responsible for the biosynthesis of glutathione (GSH) and involved in the cellular stress defense system as are superoxide dismutase and catalase responsible for the conversion of ROS. In addition, the cellular content of total ROS and in particular HO was analyzed. Compared to the wild type P. stutzeri TS44 and TS44-gshA-C, the mutant P. stutzeri TS44-gshA had a lower GSH content and exhibited an increased content of total ROS and HO and increased the Sb(III) oxidation rate. Furthermore, the transcription of sodB, sodC and katE was induced by Sb(III). A positive linear correlation was found between the Sb(III) oxidation rate and the HO content (R = 0.97), indicating that the accumulated HO is correlated to the increased Sb(III) oxidation rate.
CONCLUSIONS
Based on the results, we propose that a disruption of the pathway involved in ROS-protection allowed HO to accumulate. In addition to the previously reported enzyme mediated Sb(III) oxidation, the mechanism of bacterial oxidation of Sb(III) to Sb(V) includes a non-enzymatic mediated step using HO as the oxidant.
Topics: Antimony; Bacterial Proteins; Catalase; DNA Transposable Elements; Gene Expression Regulation, Bacterial; Genes, Bacterial; Glutamate-Cysteine Ligase; Glutathione; Hydrogen Peroxide; Mutagenesis; Mutation; Oxidants; Oxidation-Reduction; Pseudomonas stutzeri; Reactive Oxygen Species; Stress, Psychological; Superoxide Dismutase
PubMed: 27884113
DOI: 10.1186/s12866-016-0902-5 -
Applied and Environmental Microbiology Apr 2022Aerobic methanotrophic activity is highly dependent on copper availability, and methanotrophs have developed multiple strategies to collect copper. Specifically, when...
Aerobic methanotrophic activity is highly dependent on copper availability, and methanotrophs have developed multiple strategies to collect copper. Specifically, when copper is limiting (ambient concentrations less than 1 μM), some methanotrophs produce and secret a small modified peptide (less than 1,300 Da) termed methanobactin (MB) that binds copper with high affinity. As MB is secreted into the environment, other microbes that require copper for their metabolism may be inhibited as MB may make copper unavailable; e.g., inhibition of denitrifiers as complete conversion nitrate to dinitrogen involves multiple enzymes, some of which are copper-dependent. Of key concern is inhibition of the copper-dependent nitrous oxide reductase (NosZ), the only known enzyme capable of converting nitrous oxide (NO) to dinitrogen. Herein, we show that different forms of MB differentially affect copper uptake and NO reduction by Pseudomonas stutzeri strain DCP-Ps1 (that expresses clade I NosZ) and Dechloromonas aromatica strain RCB (that expresses clade II NosZ). Specifically, in the presence of MB from Methylocystis sp. strain SB2 (SB2-MB), copper uptake and expression were more significantly reduced than in the presence of MB from Methylosinus trichosporium OB3b (OB3b-MB). Further, NO accumulation increased more significantly for both P. stutzeri strain DCP-Ps1 and D. aromatica strain RCB in the presence of SB2-MB versus OB3b-MB. These data illustrate that copper competition between methanotrophs and denitrifying bacteria can be significant and that the extent of such competition is dependent on the form of MB that methanotrophs produce. Herein, it was demonstrated that the different forms of methanobactin differentially enhance NO emissions from Pseudomonas stutzeri strain DCP-Ps1 (harboring clade I nitrous oxide reductase) and Dechloromonas aromatica strain RCB (harboring clade II nitrous oxide reductase). This work contributes to our understanding of how aerobic methanotrophs compete with denitrifiers for the copper uptake and also suggests how MBs prevent copper collection by denitrifiers, thus downregulating expression of nitrous oxide reductase. This study provides critical information for enhanced understanding of microbe-microbe interactions that are important for the development of better predictive models of net greenhouse gas emissions (i.e., methane and nitrous oxide) that are significantly controlled by microbial activity.
Topics: Betaproteobacteria; Copper; Imidazoles; Methylocystaceae; Methylosinus trichosporium; Nitrous Oxide; Oligopeptides; Pseudomonas stutzeri
PubMed: 35285718
DOI: 10.1128/aem.02346-21 -
Microbes and Environments 2022Excess nitrate (NO) and nitrite (NO) in surface waters adversely affect human and environmental health. Bacteria with the ability to remove nitrogen (N) have been...
Excess nitrate (NO) and nitrite (NO) in surface waters adversely affect human and environmental health. Bacteria with the ability to remove nitrogen (N) have been isolated to reduce water pollution caused by the excessive use of N fertilizer. To obtain plant growth-promoting rhizobacteria (PGPR) with salt tolerance and NO-N removal abilities, bacterial strains were isolated from plant rhizosphere soils, their plant growth-promoting effects were evaluated using tomato in plate assays, and their NO-N removal abilities were tested under different salinity, initial pH, carbon source, and agriculture wastewater conditions. The results obtained showed that among the seven strains examined, five significantly increased the dry weight of tomato plants. Two strains, Pseudomonas stutzeri NRCB010 and Bacillus velezensis NRCB026, showed good plant growth-promoting effects, salinity resistance, and NO-N removal abilities. The maximum NO-N removal rates from denitrifying medium were recorded by NRCB010 (90.6%) and NRCB026 (92.0%) at pH 7.0. Higher NO-N removal rates were achieved using glucose or glycerin as the sole carbon source. The total N (TN) removal rates of NRCB010 and NRCB026 were 90.6 and 66.7% in farmland effluents, respectively, and 79.9 and 81.6% in aquaculture water, respectively. These results demonstrate the potential of NRCB010 and NRCB026 in the development of novel biofertilizers and their use in reducing N pollution in water.
Topics: Agriculture; Bacteria; Carbon; Denitrification; Fertilizers; Glucose; Glycerol; Humans; Nitrates; Nitrites; Nitrogen; Nitrogen Dioxide; Soil; Wastewater; Water
PubMed: 36123022
DOI: 10.1264/jsme2.ME22025 -
Nature Microbiology Feb 2020Legumes obtain nitrogen from air through rhizobia residing in root nodules. Some species of rhizobia can colonize cereals but do not fix nitrogen on them. Disabling...
Legumes obtain nitrogen from air through rhizobia residing in root nodules. Some species of rhizobia can colonize cereals but do not fix nitrogen on them. Disabling native regulation can turn on nitrogenase expression, even in the presence of nitrogenous fertilizer and low oxygen, but continuous nitrogenase production confers an energy burden. Here, we engineer inducible nitrogenase activity in two cereal endophytes (Azorhizobium caulinodans ORS571 and Rhizobium sp. IRBG74) and the well-characterized plant epiphyte Pseudomonas protegens Pf-5, a maize seed inoculant. For each organism, different strategies were taken to eliminate ammonium repression and place nitrogenase expression under the control of agriculturally relevant signals, including root exudates, biocontrol agents and phytohormones. We demonstrate that R. sp. IRBG74 can be engineered to result in nitrogenase activity under free-living conditions by transferring a nif cluster from either Rhodobacter sphaeroides or Klebsiella oxytoca. For P. protegens Pf-5, the transfer of an inducible cluster from Pseudomonas stutzeri and Azotobacter vinelandii yields ammonium tolerance and higher oxygen tolerance of nitrogenase activity than that from K. oxytoca. Collectively, the data from the transfer of 12 nif gene clusters between 15 diverse species (including Escherichia coli and 12 rhizobia) help identify the barriers that must be overcome to engineer a bacterium to deliver a high nitrogen flux to a cereal crop.
Topics: Azorhizobium caulinodans; Bacterial Proteins; Edible Grain; Escherichia coli; Genes, Bacterial; Metabolic Engineering; Multigene Family; Nitrogen Fixation; Nitrogenase; Plant Root Nodulation; Pseudomonas; Rhizobium; Symbiosis
PubMed: 31844298
DOI: 10.1038/s41564-019-0631-2 -
Biofilm Dec 2023Biofilms are complex microbial communities embedded in extracellular matrix. Pathogens within the biofilm become more resistant to the antibiotics than planktonic...
Biofilms are complex microbial communities embedded in extracellular matrix. Pathogens within the biofilm become more resistant to the antibiotics than planktonic counterparts. Novel strategies are required to encounter biofilms. Exopolysaccharides are one of the major components of biofilm matrix and play a vital role in biofilm architecture. In previous studies, a glycosyl hydrolase, PslG, from was found to be able to inhibit biofilm formation by disintegrating exopolysaccharide in biofilms. Here, we investigate the potential spectrum of PslG homologous protein with anti-biofilm activity. One glycosyl hydrolase from , PslG, exhibits anti-biofilm activities and the key catalytic residues of PslG are conserved with those of PslG. PslG at concentrations as low as 50 nM efficiently inhibits the biofilm formation of and disassemble its preformed biofilm. Furthermore, PslG exhibits anti-biofilm activity on a series of , including and pv. . PslG stays active under various temperatures. Our findings suggest that glycosyl hydrolase PslG has potential to be a broad spectrum inhibitor on biofilm formation of a wide range of .
PubMed: 37928620
DOI: 10.1016/j.bioflm.2023.100155 -
Journal of Bacteriology Oct 2019A1501 is a versatile nitrogen-fixing bacterium capable of living in diverse environments and coping with various oxidative stresses. NfiS, a regulatory noncoding RNA...
A1501 is a versatile nitrogen-fixing bacterium capable of living in diverse environments and coping with various oxidative stresses. NfiS, a regulatory noncoding RNA (ncRNA) involved in the control of nitrogen fixation in A1501, was previously shown to be required for optimal resistance to HO; however, the precise role of NfiS and the target genes involved in the oxidative stress response is entirely unknown. In this work, we systematically investigated the NfiS-based mechanisms underlying the response of this bacterium to HO at the cellular and molecular levels. A mutant strain carrying a deletion of showed significant downregulation of oxidative stress response genes, especially , a catalase gene, and , an essential regulator for transcription of catalase genes. Secondary structure prediction revealed two binding sites in NfiS for mRNA. Complementation experiments using truncated genes showed that each of two sites is functional, but not sufficient, for NfiS-mediated regulation of oxidative stress resistance and nitrogenase activities. Microscale thermophoresis assays further indicated direct base pairing between mRNA and NfiS at both sites 1 and 2, thus enhancing the half-life of the transcript. We also demonstrated that expression is dependent on OxyR and that both OxyR and KatB are essential for optimal oxidative stress resistance and nitrogenase activities. HO at low concentrations was detoxified by KatB, leaving O as a by-product to support nitrogen fixation under O-insufficient conditions. Moreover, our data suggest that the direct interaction between NfiS and mRNA is a conserved and widespread mechanism among strains. Protection against oxygen damage is crucial for survival of nitrogen-fixing bacteria due to the extreme oxygen sensitivity of nitrogenase. This work exemplifies how the small ncRNA NfiS coordinates oxidative stress response and nitrogen fixation via base pairing with mRNA and mRNA. Hence, NfiS acts as a molecular link to coordinate the expression of genes involved in oxidative stress response and nitrogen fixation. Our study provides the first insight into the biological functions of NfiS in oxidative stress regulation and adds a new regulation level to the mechanisms that contribute to the oxygen protection of the MoFe nitrogenase.
Topics: Bacterial Proteins; Base Pairing; Catalase; Gene Expression Regulation, Bacterial; Hydrogen Peroxide; Mutation; Nitrogen Fixation; Oxidative Stress; Pseudomonas stutzeri; RNA, Bacterial; RNA, Untranslated; Repressor Proteins
PubMed: 31262840
DOI: 10.1128/JB.00334-19 -
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 -
Nanomaterials (Basel, Switzerland) Apr 2019Production water is the largest byproduct of the oil industry and must be treated before disposal, either by reinjection or shedding processes, with the purpose of...
Production water is the largest byproduct of the oil industry and must be treated before disposal, either by reinjection or shedding processes, with the purpose of eliminating emulsified crude oil and avoiding the operational and toxic problems associated with it. The objective of this work was to immobilize a hydrocarbon-degrading strain on activated carbons, to evaluate the biocomplex's capacity for catalyzing hydrocarbons from Oil in Brine emulsions (O/W) simulating produced waters. Activated carbons were prepared and their chemical and porous properties were estimated by XPS, pH and SEM, N₂ adsorption, and mercury porosimetry. Biomaterials were synthesized and hydrocarbon removal tests were performed. The basic and neutral carbons immobilized by physisorption in the macroporous space and electrostatic interactions (10⁸⁻10⁸ UFC∙g), while acid materials inhibited bacterial growth. Removal of aromatic hydrocarbons was more efficient using materials (60%⁻93%) and biomaterials (16%⁻84%) than using free (1%⁻47%), and the removal efficiencies of crude oil were 22%, 48% and 37% for and two biomaterials, respectively. The presence of minor hydrocarbons only when was present confirmed the biotransformation process.
PubMed: 30939741
DOI: 10.3390/nano9040500 -
Scientific Reports May 2023Pomegranate crops are prone to wilt complex disease, which is known to severely hamper the crop yield. There have been limited studies that have explored...
Pomegranate crops are prone to wilt complex disease, which is known to severely hamper the crop yield. There have been limited studies that have explored bacteria-plant-host associations in wilt complex disease affecting pomegranate crops. In the present study, wilt infected rhizosphere soil samples (ISI, ASI) in pomegranate were studied in comparison to a healthy control (HSC). The 16S metagenomics sequencing approach using the MinION platform was employed for screening of bacterial communities and predictive functional pathways. Altered physicochemical properties in the soil samples were recorded showing a comparatively acidic pH in the ISI (6.35) and ASI (6.63) soil samples to the HSC soil (7.66), along with higher electrical conductivity in the ISI (139.5 µS/cm), ASI soil (180 µS/cm), HSC soil sample (123.33 µS/cm). While concentration of micronutrients such as Cl and B were significantly higher in the ISI and ASI soil as compared to the HSC, Cu and Zn were significantly higher in the ASI soil. The effectiveness and accuracy of 16S metagenomics studies in identifying beneficial and pathogenic bacterial communities in multi-pathogen-host systems depend on the completeness and consistency of the available 16S rRNA sequence repositories. Enhancing these repositories could significantly improve the exploratory potential of such studies. Thus, multiple 16S rRNA data repositories (RDP, GTDB, EzBioCloud, SILVA, and GreenGenes) were benchmarked, and the findings indicated that SILVA yields the most reliable matches. Consequently, SILVA was chosen for further analysis at the species level. Relative abundance estimates of bacterial species showed variations of growth promoting bacteria, namely, Staphylococcus epidermidis, Bacillus subtilis, Bacillus megatarium, Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas stutzeri and Micrococcus luteus. Functional profiling predictions employing PICRUSt2 revealed a number of enriched pathways such as transporter protein families involved in signalling and cellular processes, iron complex transport system substrate binding protein, peptidoglycan biosynthesis II (staphylococci) and TCA cycle VII (acetate-producers). In line with past reports, results suggest that an acidic pH along with the bioavailability of micronutrients such as Fe and Mn could be facilitating the prevalence and virulence of Fusarium oxysporum, a known causative pathogen, against the host and beneficial bacterial communities. This study identifies bacterial communities taking into account the physicochemical and other abiotic soil parameters in wilt-affected pomegranate crops. The insights obtained could be instrumental in developing effective management strategies to enhance crop yield and mitigate the impact of wilt complex disease on pomegranate crops.
Topics: Soil; Pomegranate; RNA, Ribosomal, 16S; Rhizosphere; Bacteria; Soil Microbiology; Plant Diseases
PubMed: 37244920
DOI: 10.1038/s41598-023-35219-z