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Microbial adhesion and spreading on surfaces are crucial aspects in environmental and industrial settings being also the early stage of complex surface-attached microbial communities known as biofilms. In this work, Pseudomonas fluorescens -laden droplets on hydrophilic substrates (glass coupons) are allowed to partially evaporate before running wetting measurements, to study the effect of evaporation on their interfacial behavior during spillover or splashing. Forced wetting is investigated by imposing controlled centrifugal forces, using a novel rotatory device ( Kerberos ). At a defined evaporation time, results for the critical tangential force required for the inception of sliding are presented. Microbe-laden droplets exhibit different wetting/spreading properties as a function of the imposed evaporation times. It is found that evaporation is slowed down in bacterial droplets with respect to nutrient medium ones. After sufficient drying times, bacteria accumulate at droplet edges, affecting the droplet shape and thus depinning during forced wetting tests. Droplet rear part does not pin during the rotation test, while only the front part advances and spreads along the force direction. Quantitative results obtained from the well-known Furmidge's equation reveal that force for sliding inception increases as evaporation time increases. This study can be of support for control of biofilm contamination and removal and possible design of antimicrobial/antibiofouling surfaces.

To explore the antifungal mechanisms of volatile organic compounds (VOCs) produced by Pseudomonas fluorescens ZX against Botrytis cinerea, biochemical analyses and transcriptomic techniques were employed in this work. The results showed that P. fluorescens ZX-producing VOCs can increase the cell membrane permeability of B. cinerea and disrupt cell membrane integrity, resulting in leakage of the pathogen's cellular contents, inhibition of ergosterol biosynthesis (about 76%), and an increase in malondialdehyde (MDA) content. Additionally, for B. cinerea respiration, P. fluorescens ZX-producing VOCs (1 × 10 9 CFU /mL) significantly inhibited the activities of ATPase (55.7%), malate dehydrogenase (MDH) (33.1%), and succinate dehydrogenase (SDH) (57.9%), seriously interfering with energy metabolism and causing accumulation of reactive oxygen species (ROS). Furthermore, transcriptome analysis of B. cinerea following exposure to VOCs revealed 4590 differentially expressed genes (DEGs) (1388 upregulated, 3202 downregulated). Through GO analysis, these DEGs were determined to be enriched in intrinsic components of membrane, integral components of membrane, and membrane parts, while KEGG analysis indicated that they were enriched in many amino acid metabolism pathways. Significantly, the DEGs related to ergosterol biosynthesis, ATPase, mitochondrial respiratory chain, malate dehydrogenase, and cell membrane showed down-regulation, corroborating the biochemical analyses. Taken together, these results suggest that the antifungal activity of P. fluorescens ZX-producing VOCs against B. cinerea occurs primary mechanisms: causing significant damage to the cell membrane, negatively affecting respiration, and interfering with amino acid metabolism., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of interest., (Copyright © 2022. Published by Elsevier GmbH.)

A biofungicide is a natural product that can be derived from various sources such as, among others, microorganisms, higher plants, animal products, phytochemicals, semiochemicals, and antagonist microorganisms. One of the most important approaches for the production of biofungicides is the combination of biocontrol agents. This study showed the inhibition growth of Alternaria alternata and Fusarium solani treated with cell-free extracts of P. fluorescens . Using thin-layer chromatography and plate assays it was also demonstrated that the cell-free extracts of P. fluorescens contained siderophores and derivates of 4-diacetylphloroglucinol and phenazine. Moreover, the combination of cell-free extracts of P. fluorescens and chitosan [50-1.5% ( v / v )] had a synergistic effect since they notably inhibited the mycelial growth of A. altenata and F. solani . Various morphological alterations to the mycelia and conidia of the treated fungi as a result of this combination were also observed. The present study could be a starting point to control other fungal phytopathogens using different cell-free extracts and chitosan as biocontrol agents.

The histidine brace (His-brace) is a copper-binding motif that is associated with both oxidative enzymes and proteinaceous copper chaperones. Here, we used biochemical and structural methods to characterize mutants of a His-brace-containing copper chaperone from Pseudomonas fluorescens (PfCopC). A total of 15 amino acid variants in primary and second-sphere residues were produced and characterized in terms of their copper binding and redox properties. PfCopC has a very high affinity for Cu(II) and also binds Cu(I). A high reorganization barrier likely prevents redox cycling and, thus, catalysis. In contrast, mutations in the conserved second-sphere Glu27 enable slow oxidation of ascorbate. The crystal structure of the variant E27A confirmed copper binding at the His-brace. Unexpectedly, Asp83 at the equatorial position was shown to be indispensable for Cu(II) binding in the His-brace of PfCopC. A PfCopC mutant that was designed to mimic the His-brace from lytic polysaccharide monooxygenase-like family X325 did not bind Cu(II), but was still able to bind Cu(I). These results highlight the importance of the proteinaceous environment around the copper His-brace for reactivity and, thus, the difference between enzyme and chaperone., (© 2021 Federation of European Biochemical Societies.)

Many volatile compounds secreted by bacteria play an important role in the interactions of microorganisms, can inhibit the growth of phytopathogenic bacteria and fungi, can suppress or stimulate plant growth and serve as infochemicals presenting a new type of interspecies communication. In this work, we investigated the effect of total pools of volatile substances and individual volatile organic compounds (VOCs) synthesized by the rhizosphere bacteria Pseudomonas chlororaphis 449 and Serratia plymuthica IC1270, the soil-borne strain P. fluorescens B-4117 and the spoiled meat isolate S. proteamaculans 94 on Arabidopsis thaliana plants. We showed that total gas mixtures secreted by these strains during their growth on Luria-Bertani agar inhibited A. thaliana growth. Hydrogen cyanide synthesis was unnecessary for the growth suppression. A decrease in the inhibition level was observed for the strain P. chlororaphis 449 with a mutation in the gacS gene, while inactivation of the rpoS gene had no effect. Individual VOCs synthesized by these bacteria (1-indecene, ketones 2-nonanone, 2-heptanone, 2-undecanone, and dimethyl disulfide) inhibited the growth of plants or killed them. Older A. thaliana seedlings were more resistant to VOCs than younger seedlings. The results indicated that the ability of some volatiles emitted by the rhizosphere and soil bacteria to inhibit plant growth should be considered when assessing the potential of such bacteria for the biocontrol of plant diseases.

<b>Background and Objective:</b> Nanoparticles with a little size to an enormous surface (1-100 nm) have expected clinical, mechanical and agricultural applications. This study aimed to produce nano Zinc Oxide (ZnO) and nano Copper Oxide (CuO) particles by green synthesis. <b>Materials and Methods:</b> Two strains of <i>Pseudomonas fluorescens</i> i.e., PSI and PSII, both cell culture supernatants and cell pellets from the two strains were examined separately in CuSO<sub>4</sub> or ZnSO<sub>4</sub> solutions. The supernatants from both strains produced color changes in both solutions referring to the formation of nano CuO or ZnO particles. The solutions were examined for nano-particle characteristics using UV-spectroscopy, particle size and morphology were tested using a scanning electron microscope and transmission electron microscopy. <b>Results:</b> UV-Vis absorption spectrum of solutions at a wavelength range 200-800 nm exhibits a distinct absorption peak in the region of 238-331 and at 303-366 nm for CuO or ZnO NPs, respectively. Absorption bands and the characteristic Surface Plasmon Resonance (SPR) spectra confirm the existence of CuO and ZnO NPs. SEM analysis micrographs indicated that CuO NPs were formed as spherical particles, while the exact shape of ZnO NPs could be identified as oval aggregates. <b>Conclusion:</b> Changes of color occurred in both solutions of two strains referring to the formation of nano CuO or ZnO particles.

Kynurenine 3-monoxygenase (KMO) catalyzes the conversion of l-kynurenine (L-Kyn) to 3-hydroxykynurenine (3-OHKyn) in the pathway for tryptophan catabolism. We have investigated the effects of pH and deuterium substitution on the oxidative half-reaction of KMO from P. fluorescens (PfKMO). The three phases observed during the oxidative half reaction are formation of the hydroperoxyflavin, hydroxylation and product release. The measured rate constants for these phases proved largely unchanging with pH, suggesting that the KMO active site is insulated from exchange with solvent during catalysis. A solvent inventory study indicated that a solvent isotope effect of 2-3 is observed for the hydroxylation phase and that two or more protons are in flight during this step. An inverse isotope effect of 0.84 ± 0.01 on the rate constant for the hydroxylation step with ring perdeutero-L-Kyn as a substrate indicates a shift from sp 2 to sp 3 hybridization in the transition state leading to the formation of a non-aromatic intermediate. The pH dependence of transient state data collected for the substrate analog meta-nitrobenzoylalanine indicate that groups proximal to the hydroperoxyflavin are titrated in the range pH 5-8.5 and can be described by a pKa of 8.8. That higher pH values do not slow the rate of hydroxylation precludes that the pKa measured pertains to the proton of the hydroperoxflavin. Together, these observations indicate that the C4a-hydroperoxyflavin has a pKa ≫ 8.5, that a non-aromatic species is the immediate product of hydroxylation and that at least two solvent derived protons are in-flight during oxygen insertion to the substrate aromatic ring. A unifying mechanistic proposal for these observations is proposed., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Motile bacteria follow gradients of nutrients or other environmental cues. Many bacterial chemoreceptors that sense exogenous amino acids contain a double Cache (dCache; calcium channels and chemotaxis receptors) ligand-binding domain (LBD). A growing number of studies suggest that broad-specificity dCache-type receptors that sense more than one amino acid are common. Here, we present an investigation into the mechanism by which the dCache LBD of the chemoreceptor CtaA from a plant growth-promoting rhizobacterium, Pseudomonas fluorescens , recognizes several chemically distinct amino acids. We established that amino acids that signal by directly binding to the CtaA LBD include ones with aliphatic (l-alanine, l-proline, l-leucine, l-isoleucine, l-valine), small polar (l-serine), and large charged (l - arginine) side chains. We determined the structure of CtaA LBD in complex with different amino acids, revealing that its ability to recognize a range of structurally and chemically distinct amino acids is afforded by its easily accessible plastic pocket, which can expand or contract according to the size of the ligand side chain. The amphipathic character of the pocket enables promiscuous interactions with both polar and nonpolar amino acids. The results not only clarify the means by which various amino acids are recognized by CtaA but also reveal that a conserved mobile lid over the ligand-binding pocket adopts the same conformation in all complexes, consistent with this being an important and invariant part of the signaling mechanism.

Extracellular polymeric substances (EPS) contain a vast number of functional groups which can provide sorption sites for heavy metal cations in solution, however, the mechanisms for the interaction of EPS with various metal cations were not well understood. In this study, the sorption potential of EPS from Pseudomonas fluorescens for different cations was investigated. The changes of electrokinetic properties that occurred on the surface of EPS once they adsorbed these cations were also studied using zeta potential measurements as a function of pH and cation concentration. The adsorption data fitted Freundlich isotherm better than Langmuir and D-R isotherms. The interactions of the cations with EPS were favourable with the separation factor K r < 1. Under different pH conditions, the zeta potential of EPS in the different cation solution followed the order: Fe(III) (at pH ≤ 5.0) > Al(III) > Cu(II) > Mn(II) > Ni(II)≈Cd(II) > Ca(II) > EPS, while with respect to the initial cation concentration, the zeta potential of EPS was in the order: Fe(III) > Al(III) > Cu(II) > Cd(II) > Ni(II)≈Mn(II)≈Ca(II). The effect of cation sorption on the surface charge of EPS increased with pH as well as cation concentration. The thermodynamic analysis demonstrated that besides the sorption of Fe which was exothermic, all the other cations were adsorbed through an endothermic process. The ΔS ads revealed that most of the cations interacted with EPS through the formation of inner-sphere complexes. The ATR-FTIR analyses confirmed that complexation occurred between the cations and functional groups on the surface of EPS. The zeta potential of EPS shifted to positive value direction due to sorption of cations on EPS, indicating that the specific interactions were involved in the sorption process. This study enhances our understanding of EPS aggregation and heavy metal bio-sorption through the electrokinetic mechanism. The results will provide useful references for immobilization of heavy metals and alleviation of Al toxicity in acidic soils., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The kinetic description of enzyme-catalyzed reactions is a core task in biotechnology and biochemical engineering. In particular, mechanistic kinetic models help from the discovery of the biocatalyst throughout its application. Chemo- or enantioselective enzyme reactions often undergo two alternative pathways for the release of two different products from a central intermediate. For these types of reaction, no explicit reaction equations have been derived so far. To this end, we extend the commonly used Cleland's notation for branched reaction pathways and explicitly derive the rate expressions for two-coupled ordered bi-uni reactions. This mechanism also leads to a ping-pong bi-bi mechanism for a transfer reaction between the two products via the same central intermediate of the reaction system. Using the cross-ligation of benzaldehyde and propanal catalyzed by the thiamine diphosphate-dependent enzyme benzaldehyde lyase from Pseudomonas fluorescens yielding (R)-2-hydroxy-1-phenylbutan-1-one as a case study, we performed model-based experimental analysis to show that such a reaction mechanism can be modeled mechanistically and leads to reasonable kinetic parameters., (© 2019 American Institute of Chemical Engineers.)

UCB-1 is the commercial rootstock of pistachio. Reproduction of this rootstock by tissue culture is limited by low levels of proliferation rate. Therefore, any compound that improves the proliferation rate and the quality of the shoots can be used in the process of commercial reproduction of this rootstock. Use of plant growth-promoting bacteria is one of the best ideas. Given the beneficial effects of nanoparticles in enhancement of the growth in plant tissue cultures, the aim of the present study was to investigate the effects of nanoencapsulation of plant growth-promoting rhizobacteria (using silica nanoparticles and carbon nanotubes) and their metabolites in improving UCB1 pistachio micropropagation. The experiment was conducted in a completely randomized design with three replications. Before planting, treatments on the DKW medium were added. The results showed that the use of Pseudomonas fluorescens VUPF5 and Bacillus subtilis VRU1 nanocapsules significantly enhanced the root length and proliferation. The nanoformulation of the VUPF5 metabolite led to the highest root length (6.26 cm) and the largest shoot (3.34 cm). Inoculation of explants with the formulation of the metabolites (both bacterial strains) significantly elevated the average shoot length and the fresh weight of plant compared to the control. The explants were dried completely using both bacterial strains directly and with capsule coating after the three days.

Nanotechnology is one of the most fascinating sciences with a great potential to improve many agricultural products. Use of nanoparticles in plant disease management is a novel area which may prove very effective in future. Use of nanomaterials and biocompatible compounds in nano-encapsulation of antagonist bacteria is an important step in enhancing the efficiency of these agents in adverse environmental conditions. Two strains of Pseudomonas fluorescens (VUPF5 and T17-4) were used for alginate-gelatin nanocomposite beads with different concentrations of gelatin. The moisture content, swelling, and releasing of encapsulated viable bacteria was investigated in vitro and in vivo conditions. The results of FT-IR and X-ray diffraction analysis revealed that when gelatin was added into sodium alginate, electrostatic interaction occurred. The swelling and moisture content of nanocomposite beads grew with gelatin enhancement. The maximum encapsulation efficiency at the gelatin concentration of 1.5% in VUPF5 and T17-4 was 91.23% and 87.23%, respectively. Further, the greenhouse experiment showed that inoculation of potato with bacterial strains and nanocomposite beads of these strains reduced disease incidence. The encapsulation method described in this study can be effectively used to protect the plant probiotic bacteria inoculum from harmful conditions of the soil for its successful establishment in the rhizosphere., (Copyright © 2019 Elsevier B.V. All rights reserved.)

In natural habitats, bacterial species often coexist in biofilms. They interact in synergetic or antagonistic ways and their interactions can influence the biofilm development and properties. Still, very little is known about how the coexistence of multiple organisms impact the multispecies biofilm properties. In this study, we examined the behaviour of a dual-species biofilm at the air-liquid interface composed by two environmental bacteria: Bacillus licheniformis and a phenazine mutant of Pseudomonas fluorescens. Study of the planktonic and biofilm growths for each species revealed that P. fluorescens grew faster than B. licheniformis and no bactericidal effect from P. fluorescens was detected, suggesting that the growth kinetics could be the main factor in the dual-species biofilm composition. To validate this hypothesis, the single- and dual-species biofilm were characterized by biomass quantification, microscopy and rheology. Bacterial counts and microscale architecture analysis showed that both bacterial populations coexist in the mature pellicle, with a dominance of P. fluorescens. Real-time measurement of the dual-species biofilms' viscoelastic (i.e. mechanical) properties using interfacial rheology confirmed that P. fluorescens was the main contributor of the biofilm properties. Evaluation of the dual-species pellicle viscoelasticity at longer time revealed that the biofilm, after reaching a first equilibrium, created a stronger and more cohesive network. Interfacial rheology proves to be a unique quantitative technique, which combined with microscale imaging, contributes to the understanding of the time-dependent properties within a polymicrobial community at various stages of biofilm development. This work demonstrates the importance of growth kinetics in the bacteria competition for the interface in a model dual-species biofilm.

The bacterial CopC family of proteins are periplasmic copper binding proteins that act in copper detoxification. These proteins contain Cu(I) and/or Cu(II) binding sites, with the family that binds Cu(II) only the most prevalent, based on sequence analyses. Here we present three crystal structures of the CopC protein from Pseudomonas fluorescens (Pf-CopC) that include the wild type protein bound to Cu(II) and two variant proteins, where Cu(II) coordinating ligands were mutated, in Cu-free states. We show that the Cu(II) atom in Pf-CopC is coordinated by two His residues, an Asp residue and the N-terminus of the protein (therefore a 3N + O site). This coordination structure is consistent with all structurally characterized proteins from the CopC family to date. Structural and sequence analyses of the CopC family allow a relationship between protein sequence and the Cu(II) binding affinity of these proteins to be proposed., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Aim: To observe the variation in accumulation of Fusarium and Alternaria mycotoxins across a topographically heterogeneous field and tested biotic (fungal and bacterial abundance) and abiotic (microclimate) parameters as explanatory variables., Methods and Results: We selected a wheat field characterized by a diversified topography, to be responsible for variations in productivity and in canopy-driven microclimate. Fusarium and Alternaria mycotoxins where quantified in wheat ears at three sampling dates between flowering and harvest at 40 points. Tenuazonic acid (TeA), alternariol (AOH), alternariol monomethyl ether (AME), tentoxin (TEN), deoxynivalenol (DON), zearalenone (ZEN) and deoxynivalenol-3-Glucoside (DON.3G) were quantified. In canopy temperature, air and soil humidity were recorded for each point with data-loggers. Fusarium spp. as trichothecene producers, Alternaria spp. and fungal abundances were assessed using qPCR. Pseudomonas fluorescens bacteria were quantified with a culture based method. We only found DON, DON.3G, TeA and TEN to be ubiquitous across the whole field, while AME, AOH and ZEN were only occasionally detected. Fusarium was more abundant in spots with high soil humidity, while Alternaria in warmer and drier spots. Mycotoxins correlated differently to the observed explanatory variables: positive correlations between DON accumulation, tri 5 gene and Fusarium abundance were clearly detected. The correlations among the others observed variables, such as microclimatic conditions, varied among the sampling dates. The results of statistical model identification do not exclude that species coexistence could influence mycotoxin production., Conclusions: Fusarium and Alternaria mycotoxins accumulation varies heavily across the field and the sampling dates, providing the realism of landscape-scale studies. Mycotoxin concentrations appear to be partially explained by biotic and abiotic variables., Significance and Impact of the Study: We provide a useful experimental design and useful data for understanding the dynamics of mycotoxin biosynthesis in wheat., (© 2018 The Society for Applied Microbiology.)

Quartz crystal microbalance with dissipation monitoring (QCM-D) is a powerful tool for studying adhesion, yet its use for analyzing the deposition of microparticles and living cells on surfaces has been hampered by difficulties in interpretation. Here we report a new quantitative model of QCM-D response, presented as an equivalent acoustic impedance circuit. As an essential feature, the particle interaction with surrounding fluid is modeled by relations for a freely oscillating rotating and translating sphere in an unbounded fluid, which is a valid approximation for microparticles. This helps deduce from the measured reponse the parameters pertinent to the contact mechanics. We use the model to analyze deposition of different microparticles as well as Pseudomonas fluorescens bacteria on several substrates using QCM-D combined with real-time microscopy. The parameter space is increased by varying particle type and size, substrate surface chemistry and rigidity, and ionic strength of the solution, which allows observation of diverse responses and transition from inertial to elastic loading, including rarely observed resonant regimes. Ultimately, we find that the model describes reasonably well the observed response for different microparticles and substrates, as well as for bacteria, and enables extraction of the contact characteristics in elastic and mixed loading regimes. It also reveals discrepancies between measured and anticipated parameters for large particles. The new model can be a useful tool for interpreting and quantifying QCM-D data on the adhesion of particles and living cells to surfaces, including time-dependent adhesion phenomena.

Take-all disease, caused by Gaeumannomyces graminis var. tritici (Ggt), is one of the most serious root diseases in wheat production. In this study, a proteomic platform based on 2-dimensional gel electrophoresis (2-DE) and Matrix-Assisted Laser Desorption/Ionization Time of Flight Tandem Mass Spectrometry (MALDI-TOF/TOF MS) was used to construct the first proteome database reference map of G. graminis var. tritici and to identify the response of the pathogen to 2,4-diacetylphloroglucinol (DAPG), which is a natural antibiotic produced by antagonistic Pseudomonas spp. in take-all suppressive soils. For mapping, a total of 240 spots was identified that represented 209 different proteins. The most abundant biological function categories in the Ggt proteome were related to carbohydrate metabolism (21%), amino acid metabolism (15%), protein folding and degradation (12%), translation (11%), and stress response (10%). In total, 51 Ggt proteins were affected by DAPG treatment. Based on gene ontology, carbohydrate metabolism, amino acid metabolism, stress response, and protein folding and degradation proteins were the ones most modulated by DAPG treatment. This study provides the first extensive proteomic reference map constructed for Ggt and represents the first time that the response of Ggt to DAPG has been characterized at the proteomic level., (Copyright © 2018 British Mycological Society. All rights reserved.)

The first total synthesis of the trisaccharide repeating unit of the O-specific polysaccharide of Pseudomonas fluorescens BIM B-582 is reported. This efficient synthesis involves consecutive 1,2- cis glycosylations including β-l-rhamnosylation and α selective coupling of rare 4-deoxy-d- xylo-hexose as the key steps. The synthetic trisaccharide is equipped with an aminopropyl linker at the reducing end to allow for conjugation to proteins and microarrays for further immunological studies.

The oxyvinylglycine 4-formylaminooxyvinylglycine (FVG) arrests the germination of weedy grasses and inhibits the growth of the bacterial plant pathogen Erwinia amylovora. Both biological and analytical methods have previously been used to detect the presence of FVG in crude and extracted culture filtrates of several Pseudomonas fluorescens strains. Although a combination of these techniques is adequate to detect FVG, none is amenable to high-throughput analysis. Likewise, filtrates often contain complex metabolite mixtures that prevent the detection of FVG using established chromatographic techniques. Here, we report the development of a new method that directly detects FVG in crude filtrates using laser ablation electrospray ionization-mass spectrometry (LAESI-MS). This approach overcomes limitations with our existing methodology and allows for the rapid analysis of complex crude culture filtrates. To validate the utility of the LAESI-MS method, we examined crude filtrates from Pantoea ananatis BRT175 and found that this strain also produces FVG. These findings are consistent with the antimicrobial activity of P. ananatis BRT175 and indicate that the spectrum of bacteria that produce FVG stretches beyond rhizosphere-associated Pseudomonas fluorescens., Competing Interests: The authors have read the journal’s policy and the authors of this manuscript have the following competing interests: CW was a paid scientist for Protea Biosciences, Inc. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Kynurenine 3-monooxygenase (KMO) inhibitors have been developed for the treatment of neurodegenerative disorders. The mechanisms of flavin reduction and hydrogen peroxide production by KMO inhibitors are unknown. Herein, we report the structure of human KMO and crystal structures of Saccharomyces cerevisiae (sc) and Pseudomonas fluorescens (pf) KMO with Ro 61-8048. Proton transfer in the hydrogen bond network triggers flavin reduction in p-hydroxybenzoate hydroxylase, but the mechanism triggering flavin reduction in KMO is different. Conformational changes via π-π interactions between the loop above the flavin and substrate or non-substrate effectors lead to disorder of the C-terminal α helix in scKMO and shifts of domain III in pfKMO, stimulating flavin reduction. Interestingly, Ro 61-8048 has two different binding modes. It acts as a competitive inhibitor in scKMO and as a non-substrate effector in pfKMO. These findings provide understanding of the catalytic cycle of KMO and insight for structure-based drug design of KMO inhibitors., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The aim of this study was to investigate the optimal conditions for the immobilization and stabilization of DyP1B dye decolorizing peroxidases type B (DyP1B) from Pseudomonas fluorescens Pf-5 immobilized in Ca-alginate ferromagnetic beads. The immobilized DyP1B was used in the degradation of the Reactive Blue 5 (RB5) synthetic dye. The enzyme was successfully entrapped in a Ca-alginate matrix and showed an encapsulation efficiency of 94%. The concentration of DyP1B (0.8 mg mL -1 ), 2% of alginate and magnetite (10.0 mg mL -1 ) was optimal for immobilization. The immobilized DyP1B showed optimum activity at pH 7.0 and 40 °C compared with pH 5.5 and 30 °C for free peroxidase. Reusability studies showed that after five cycles, the immobilized DyP1B system retained more than 58% of its initial activity. The immobilized DyP1B was able to decolorize RB5 at concentrations of 0.1, 0.05, and 0.01% (w v -1 ) with efficiency rates of about 20, 29, and 45%, respectively. The immobilization of DyP1B in alginate beads with the addition of Fe 3 O 4 increased its catalytic and applicative potential., (© 2017 International Union of Biochemistry and Molecular Biology, Inc.)

Pyoverdines are important siderophores that guarantee iron supply to important pathogenic and non-pathogenic pseudomonads in host habitats. A key characteristic of all pyoverdines is the fluorescent dihydroxyquinoline group that contributes two ligands to the iron complexes. Pyoverdines are derived from the non-ribosomally synthesized peptide ferribactin, and their fluorophore is generated by periplasmic oxidation and cyclization reactions of d-tyrosine and l-diaminobutyric acid. The formation of the fluorophore is known to be driven by the periplasmic tyrosinase PvdP. Here we report that the putative periplasmic oxidoreductase PvdO of Pseudomonas fluorescens A506 is required for the final oxidation of dihydropyoverdine to pyoverdine, which completes the fluorophore. The pvdO deletion mutant accumulates dihydropyoverdine, and this phenotype is fully complemented by recombinant PvdO. The autoxidation of dihydropyoverdine at alkaline pH and the presence of high copper concentrations can mask this phenotype. Mutagenesis of conserved residues with potential catalytic function identified Glu-260 as an essential residue whose mutation abolished function without affecting stability or transport. Glu-260 of PvdO is at the exact position of the active-site cysteine in the structurally related formylglycine-generating enzyme. Evolution thus used the same protein fold for two distinct functionalities. As purified PvdO was inactive, additional factors are required for catalysis., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

We describe a simple, scalable, modular, and frugal approach to create model ecosystems as millifluidic networks of interconnected habitats (hosting microbes or plants), which offers (i) quantitative and dynamic control over the exchange of chemicals between habitats, and (ii) independent control over their environment. Oscillatory laminar flows produce regions of vortex mixing around obstacles. When these overlap, rapid mass transport by dispersion occurs, which is quantitatively describable as diffusion, but is directional and tunable in rate over 3 orders of magnitude. This acceleration in the rate of diffusion is equivalent to reducing the distance between the habitats, and therefore, the organisms, down to the length scales characteristic of signaling in soil (<2 mm).

A profoundly time-efficient chemoenzymatic method for the synthesis of (S)-3-(4-chlorophenoxy)propan-1,2-diol and (S)-1-chloro-3-(2,5-dichlorophenoxy)propan-2-ol, two important pharmaceutical intermediates, was successfully developed using Pseudomonas fluorescens lipase (PFL). Kinetic resolution was successfully achieved using vinyl acetate as acylating agent, toluene/hexane as solvent, and reaction temperature of 30°C giving high enantioselectivity and conversion. Under optimized condition, PFL demonstrated 50.2% conversion, enantiomeric excess of 95.0%, enantioselectivity (E = 153) in an optimum time of 1 hour and 50.3% conversion, enantiomeric excess of 95.2%, enantioselectivity (E = 161) in an optimum time of 3 hours, for the two racemic alcohols, respectively. Docking of the R- and S-enantiomers of the intermediates demonstrated stronger H-bond interaction between the hydroxyl group of the R-enantiomer and the key binding residues of the catalytic site of the lipase, while the S-enantiomer demonstrated lesser interaction. Thus, docking study complemented the experimental outcome that PFL preferentially acylated the R form of the intermediates. The present study demonstrates a cost-effective and expeditious biocatalytic process that can be applied in the enantiopure synthesis of pharmaceutical intermediates and drugs., (© 2017 Wiley Periodicals, Inc.)

Cupins form one of the most functionally diverse superfamilies of proteins, with members performing a wide range of catalytic, non-catalytic, and regulatory functions. HutD is a predicted bicupin protein that is involved in histidine utilization (Hut) in Pseudomonas species. Previous genetic analyses have suggested that it limits the upper level of Hut pathway expression, but its mechanism of action is unknown. Here, we have determined the structure of PfluHutD at 1.74 Å resolution in several crystallization conditions, and identified N-formyl-l-glutamate (FG, a Hut pathway intermediate) as a potential ligand in vivo. Proteins 2017; 85:1580-1588. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

β-Lactone natural products occur infrequently in nature but possess a variety of potent and valuable biological activities. They are commonly derived from β-hydroxy-α-amino acids, which are themselves valuable chiral building blocks for chemical synthesis and precursors to numerous important medicines. However, despite a number of excellent synthetic methods for their asymmetric synthesis, few effective enzymatic tools exist for their preparation. Here we report cloning of the biosynthetic gene cluster for the β-lactone antibiotic obafluorin and delineate its biosynthetic pathway. We identify a nonribosomal peptide synthetase with an unusual domain architecture and an L-threonine:4-nitrophenylacetaldehyde transaldolase responsible for (2S,3R)-2-amino-3-hydroxy-4-(4-nitrophenyl)butanoate biosynthesis. Phylogenetic analysis sheds light on the evolutionary origin of this rare enzyme family and identifies further gene clusters encoding L-threonine transaldolases. We also present preliminary data suggesting that L-threonine transaldolases might be useful for the preparation of L-threo-β-hydroxy-α-amino acids.

Phytophthora infestans causes late blight of potato, one of the most devastating diseases affecting potato production. Alternative approaches for controlling late blight are being increasingly sought due to increasing environmental concerns over the use of chemical pesticides and the increasing resistance of P. infestans to fungicides. Our research group has isolated a new strain of Pseudomonas fluorescens (LBUM636) of biocontrol interest producing the antibiotic phenazine-1-carboxylic acid (PCA). Wild-type LBUM636 was shown to significantly inhibit the growth of Phytophthora infestans in in vitro confrontational assays whereas its isogenic mutant (phzC-; not producing PCA) only slightly altered the pathogen's growth. Wild-type LBUM636 but not the phzC- mutant also completely repressed disease symptom development on tubers. A pot experiment revealed that wild-type LBUM636 can significantly reduce P. infestans populations in the rhizosphere and in the roots of potato plants, as well as reduce in planta disease symptoms due to PCA production. The expression of eight common plant defense-related genes (ChtA, PR-1b, PR-2, PR-5, LOX, PIN2, PAL-2, and ERF3) was quantified in tubers, roots, and leaves by reverse-transcription quantitative polymerase chain reaction and revealed that the biocontrol observed was not associated with the induction of a plant defense response by LBUM636. Instead, a direct interaction between P. infestans and LBUM636 is required and PCA production appears to be a key factor for LBUM636's biocontrol ability.

When competing for space and resources, bacteria produce toxins known as bacteriocins to gain an advantage over competitors. Recent studies in the laboratory have confirmed theoretical predictions that bacteriocin production can determine coexistence, by eradicating sensitive competitors or driving the emergence of resistant genotypes. However, there is currently limited evidence that bacteriocin-mediated competition influences the coexistence and distribution of genotypes in natural environments, and what factors drive interactions towards inhibition remain unclear. Using natural soil populations of Pseudomonas fluorescens, we assessed the ability of the isolates to inhibit one another with respect to spatial proximity in the field, genetic similarity and niche overlap. The majority of isolates were found to produce bacteriocins; however, widespread resistance between coexisting isolates meant relatively few interactions resulted in inhibition. When inhibition did occur, it occurred more frequently between ecologically similar isolates. However, in contrast with results from other natural populations, we found no relationship between the frequency of inhibition and the genetic similarity of competitors. Our results suggest that bacteriocin production plays an important role in mediating competition over resources in natural settings and, by selecting for isolates resistant to local bacteriocin production, can influence the assembly of natural populations of P. fluorescens., (© 2016 European Society For Evolutionary Biology. Journal of Evolutionary Biology © 2016 European Society For Evolutionary Biology.)

Aldehyde dehydrogenase typically performs oxidation of aldehydes to their corresponding carboxylic acid while reducing NAD(P) + to NAD(P)H via covalent catalysis mediated by an active-site cysteine residue. One member of this superfamily, the enzyme 2-aminomuconate-6-semialdehyde dehydrogenase (AMSDH), is a component of the kynurenine pathway, which catabolizes tryptophan in mammals and certain bacteria. AMSDH catalyzes the NAD + -dependent oxidation of 2-aminomuconate semialdehyde to 2-aminomuconate. We recently determined the first crystal structure of AMSDH and several catalytic cycle intermediates. A conserved asparagine in the oxyanion hole, Asn-169, is found to be H-bonded to substrate-derived intermediates in the active site of AMSDH during catalysis, including both the covalently bound thiohemiacetal and thioacyl intermediates. To better interrogate the significance of the hydrogen bond provided by Asn-169 to the reaction mechanism of AMSDH, we created Ala, Ser, Asp, and Gln mutants and studied them using biochemical, kinetic, crystallographic, and computational studies. The in crystallo chemical reaction of the primary substrate with the co-crystalized complex of the N169D mutant and NAD + led to the successful trapping of a new catalytic intermediate that was not previously seen. The structural and computational data are consistent with a substrate imine/enol tautomer intermediate being formed prior to the formation of the covalent bond between the substrate and the active-site cysteine. Thus, AMSDH surprisingly includes an isomerization process within its known catalytic mechanism. These data establish a hidden intrinsic isomerization activity of the dehydrogenase and allow us to propose a pitcher-catcher type of catalytic mechanism for the isomerization., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The present attempt was made to determine the effects of untreated municipal wastewater (MW) on growth and physiology of maize and to evaluate the role of Ag nanoparticle and plant-growth-promoting rhizobacteria (PGPR) when interacting with MW used for irrigation. It was used for the isolation of PGPR. The isolates were identified and characterized based on the colony morphology, C/N source utilization pattern using miniaturized identification system (QTS 24), catalase (CAT) and oxidase tests, and 16S rRNA sequence analyses. The three PGPR isolates were Planomicrobium chinense (accession no. NR042259), Bacillus cereus (accession no. CP003187) and Pseudomonas fluorescens (accession no. GU198110). The isolates solubilized phosphate and exhibited antibacterial activities against pathogenic bacteria i.e., Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Klebsiella pneumoniae and Escherichia coli and antifungal activities against Helminthosporium sativum and Fusarium solani. The untreated MW irrigation as well as Ag nanoparticle treatment resulted in significant accumulation of Ni in the rhizosphere soil. PGPR induced accumulation of Ni and Pb in the rhizosphere soil and maize shoot. Ag nanoparticle also caused Ni and Pb accumulation in maize shoot. Combined treatment with PGPR, Ag nanoparticle and MW resulted in decreased accumulation of Pb and Ni both in the rhizosphere soil and maize shoot. Combined treatment of Ag nanoparticle, MW and PGPR decreased Na accumulation and increased K accumulation. Ag nanoparticle increased Fe and Co accumulation but decreased Zn and Cu accumulation in MW treatment; in combined treatment, it reduced PGPR-induced accumulation of Co and Fe in the rhizosphere and Co accumulation in shoot. PGPR significantly increased root weight, shoot weight, root length, shoot length, leaf area, and proline, chlorophyll and carotenoid content of the maize plant. Ag nanoparticle also enhanced the leaf area, fresh weight, root length and antioxidant activities of maize. Treatment with Ag nanoparticle increased the gibberellic acid (GA) and abscisic acid (ABA) content of maize leaves but decreased the accumulation of GA in the presence of PGPR and MW.

Background: Mineral iron(III) recognition by bacteria is considered a matter of debate. The peculiar surface chemistry of novel naked magnetic nanoparticles, called SAMNs (surface active maghemite nanoparticles) characterized by solvent exposed Fe(3+) sites on their surface, was exploited for studying mineral iron sensing in Pseudomonas fluorescens., Methods: SAMNs were applied for mimicking Fe(3+) ions in solution, acting as magnetically drivable probes to evaluate putative Fe(3+) recognition sites on the microorganism surface. Culture broths and nano-bio-conjugates were characterized by UV-Vis spectroscopy and mass spectrometry., Results: The whole heritage of a membrane porin (OprF) of P. fluorescens Ps&#95;22 cells was recognized and firmly bound by SAMNs. The binding of nanoparticles to OprF porin was correlated to a drastic inhibition of a siderophore (pyoverdine) biosynthesis and to the stimulation of the production and rate of formation of a secondary siderophore. The analysis of metabolic pathways, based on P. fluorescens Ps&#95;22 genomic information, evidenced that this putative secondary siderophore does not belong to a selection of the most common siderophores., Conclusions: In the scenario of an adhesion mechanism, it is plausible to consider OprF as the biological component deputed to the mineral iron sensing in P. fluorescens Ps&#95;22, as well as one key of siderophore regulation., General Significance: The present work sheds light on mineral iron sensing in microorganisms. Peculiar colloidal naked iron oxide nanoparticles offer a useful approach for probing the adhesion of bacterial surface on mineral iron for the identification of the specific recognition site for this iron uptake regulation in microorganisms., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Chemotaxis towards nutrients plays a crucial role in root colonization by Pseudomonas fluorescens. The P. fluorescens chemotactic transducer of amino acids type A (CtaA) mediates movement towards amino acids present in root exudates. In this study, the periplasmic sensory domain of CtaA has been crystallized by the hanging-drop vapor diffusion method using ammonium sulfate as a precipitating agent. A complete data set was collected to 1.9 Å resolution using cryocooling conditions and synchrotron radiation. The crystals belong to space group I222 or I212121, with unit-cell parameters a = 67.2, b = 76.0, c = 113.3 Å. This is an important step towards elucidation of the structural basis of how CtaA recognizes its signal molecules and transduces the signal across the membrane.

Quantitative characterization of metalloproteins at molecular and atomic levels generally requires tens of milligrams of highly purified samples, a situation frequently challenged by problems in generating unmodified native forms. A variety of affinity tags, such as the popular poly-histidine tag, have been developed to facilitate purification but they generally rely on expensive affinity resins and their presence may interfere with protein characterization. This paper documents that addition of a poly-lysine tag to the C-terminus enables, for the copper-binding proteins examined, ready purification in large scale via cost-effective cation-exchange chromatography. The tag may be removed readily by the enzyme carboxypeptidase B to generate the native protein with no extra residues. However, this cleavage step is normally not necessary since the poly-lysine tag is shown to have no detectable affinity for either Cu(I) or Cu(II) and imposes no interference to the copper binding properties of the target proteins. In contrast, the poly-histidine tag possesses a sub-picomolar affinity for Cu(I) and -nanomolar affinity for Cu(II) and may need to be removed for reliable characterization of the target proteins. These conclusions may be extended to the study of other metallo-proteins and metallo-enzymes., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Bacterial defense mechanisms have evolved to protect bacteria against predation by nematodes, predatory bacteria, or amoebae. We identified novel bacterial alkaloids (pyreudiones A-D) that protect the producer, Pseudomonas fluorescens HKI0770, against amoebal predation. Isolation, structure elucidation, total synthesis, and a proposed biosynthetic pathway for these structures are presented. The generation of P. fluorescens gene-deletion mutants unable to produce pyreudiones rendered the bacterium edible to a variety of soil-dwelling amoebae., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Knowledge of the relative contributions of different water sources to intracellular fluids and body water is important for many fields of study, ranging from animal physiology to paleoclimate. The intracellular fluid environment of cells is challenging to study due to the difficulties of accessing and sampling the contents of intact cells. Previous studies of multicelled organisms, mostly mammals, have estimated body water composition-including metabolic water produced as a byproduct of metabolism-based on indirect measurements of fluids averaged over the whole organism (e.g., blood) combined with modeling calculations. In microbial cells and aquatic organisms, metabolic water is not generally considered to be a significant component of intracellular water, due to the assumed unimpeded diffusion of water across cell membranes. Here we show that the (18)O/(16)O ratio of PO4 in intracellular biomolecules (e.g., DNA) directly reflects the O isotopic composition of intracellular water and thus may serve as a probe allowing direct sampling of the intracellular environment. We present two independent lines of evidence showing a significant contribution of metabolic water to the intracellular water of three environmentally diverse strains of bacteria. Our results indicate that ∼30-40% of O in PO4 comprising DNA/biomass in early stationary phase cells is derived from metabolic water, which bolsters previous results and also further suggests a constant metabolic water value for cells grown under similar conditions. These results suggest that previous studies assuming identical isotopic compositions for intracellular/extracellular water may need to be reconsidered.

Pyoverdines are siderophores synthesized by fluorescent Pseudomonas spp. Under iron-limiting conditions, these high-affinity ferric iron chelators are excreted by bacteria in the soil to acquire iron. Pyoverdines produced by beneficial Pseudomonas spp. ameliorate plant growth. Here, we investigate the physiological incidence and mode of action of pyoverdine from Pseudomonas fluorescens C7R12 on Arabidopsis (Arabidopsis thaliana) plants grown under iron-sufficient or iron-deficient conditions. Pyoverdine was provided to the medium in its iron-free structure (apo-pyoverdine), thus mimicking a situation in which it is produced by bacteria. Remarkably, apo-pyoverdine abolished the iron-deficiency phenotype and restored the growth of plants maintained in the iron-deprived medium. In contrast to a P. fluorescens C7R12 strain impaired in apo-pyoverdine production, the wild-type C7R12 reduced the accumulation of anthocyanins in plants grown in iron-deficient conditions. Under this condition, apo-pyoverdine modulated the expression of around 2,000 genes. Notably, apo-pyoverdine positively regulated the expression of genes related to development and iron acquisition/redistribution while it repressed the expression of defense-related genes. Accordingly, the growth-promoting effect of apo-pyoverdine in plants grown under iron-deficient conditions was impaired in iron-regulated transporter1 and ferric chelate reductase2 knockout mutants and was prioritized over immunity, as highlighted by an increased susceptibility to Botrytis cinerea This process was accompanied by an overexpression of the transcription factor HBI1, a key node for the cross talk between growth and immunity. This study reveals an unprecedented mode of action of pyoverdine in Arabidopsis and demonstrates that its incidence on physiological traits depends on the plant iron status., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

Background: Pseudomonas fluorescens lectin (PFL) belongs to a recently discovered anti-HIV lectin family and induces anoikis-like cell death of MKN28 gastric cancer cells by causing α2 integrin internalization through recognition of high mannose glycans; however, the detailed anti-cancer mechanism is not fully elucidated., Methods: Cell adherence potency of MKN28 upon PFL treatment was assessed using a colorimetric assay. Cell surface molecules to which PFL bound were identified by peptide mass finger printing with Matrix Assisted Laser Desorption/Ionization-time of flight mass spectrometry and their cellular localization determined by immunofluorescence microscopy. Gene and protein expression in PFL-treated MKN28 cells were evaluated by microarray analysis and western blot, and the function of these genes was evaluated by siRNA knock-down. A proliferation assay measured the sensitivity of PFL-treated cancer cells to anti-cancer drugs. The effect of PFL on subcutaneous MKN28 tumor growth and hepatic tumor formation in BALB/c nude mice was evaluated., Results: The strength of MKN28 cell adherence in vitro to the extracellular matrix was impaired by PFL treatment, consistent with the observation that PFL induces rapid downregulation of surface integrins. PFL also was found to bind to cell surface epidermal growth factor receptor (EGFR). Surface EGFR molecules were endocytosed following PFL binding, and were degraded in a time-dependent fashion. This degradation process was largely the result of autophagy, as revealed by the increased expression of autophagic proteins. PFL-induced EGFR degradation was partly inhibited by RAB7 siRNA as well as LC3 siRNA, and internalized EGFR colocalized with ATG9 at 48 h post-PFL treatment, suggesting that these proteins contribute to dynamic degradation induced by PFL. PFL-induced decrease in surface EGFR rendered MKN28 cells susceptible to gefitinib, a selective inhibitor of EGFR tyrosine kinase. In vivo experiments showed that PFL-treated MKN28-EGFP cells injected in the portal vein of BALB/c nude mice failed to form tumor colonies on the liver, and intratumoral injection of PFL significantly inhibited tumor growth., Conclusion: PFL-mediated downregulation of integrin and EGFR contributes to the inhibition of tumor growth in vitro and in vivo. This novel anti-cancer mechanism of PFL suggests that this lectin would be useful as an anti-cancer drug or an adjuvant for other drugs.

Against the increase of bacterial resistance to traditional antibiotics, antimicrobial peptides (AMP) are considered as promising alternatives. Bacterial biofilms are more resistant to antibiotics that their planktonic counterpart. The purpose of this study was to investigate the action of an AMP against a nascent bacterial biofilm. The activity of dermaseptin S4 derivative S4(1-16)M4Ka against 6 h-old Pseudomonas fluorescens biofilms was assessed by using a combination of Attenuated Total Reflectance-Fourier Transform InfraRed (ATR-FTIR) spectroscopy in situ and in real time, fluorescence microscopy using the Baclight™ kit, and Atomic Force Microscopy (AFM, imaging and force spectroscopy). After exposure to the peptide at three concentrations, different dramatic and fast changes over time were observed in the ATR-FTIR fingerprints reflecting a concentration-dependent action of the AMP. The ATR-FTIR spectra revealed major biochemical and physiological changes, adsorption/accumulation of the AMP on the bacteria, loss of membrane lipids, bacterial detachment, bacterial regrowth, or inhibition of biofilm growth. AFM allowed estimating at the nanoscale the effect of the AMP on the nanomechanical properties of the sessile bacteria. The bacterial membrane elasticity data measured by force spectroscopy were consistent with ATR-FTIR spectra, and they allowed suggesting a mechanism of action of this AMP on sessile P. fluorescens. The combination of these three techniques is a powerful tool for in situ and in real time monitoring the activity of AMPs against bacteria in a biofilm., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The effect of temperature on the growth kinetics and proteolytic activity of Chryseobacterium joostei and Chryseobacterium bovis was determined during this study. The results were compared with the activities of Pseudomonas fluorescens, which is regarded to be a major food spoilage psychrotolerant microorganism. For the growth studies, cultures were incubated in nutrient broth in a temperature gradient incubator (from 9 to 50 °C) and separately at 4 °C, and the optical density was measured at different time intervals. Growth temperature profiles for each organism were constructed. For determination of proteolytic activity, the cultures were incubated in fat-free ultra-high temperature processed milk in the temperature gradient incubator for 72 h (temperature range as above). Cell-free extracts were used to determine the proteolytic activity using the azocasein method. Results of the growth studies showed that C. joostei had the ability to grow over a wider temperature range than C. bovis and P. fluorescens without being affected by changes in the temperature. For the proteolytic activity, C. joostei had significantly (p < 0.001) higher activity per milligram of protein at 15.5 °C, followed by C. bovis and P. fluorescens. The results showed that C. joostei potentially has an even greater spoilage capacity in milk on the basis of growth rate and proteolytic activity than did P. fluorescens.

Peroxiredoxins (Prxs) are enzymatic antioxidants widely distributed in biological kingdoms, which constitute a family of heme-free peroxidases that reduce alkyl hydroperoxides and hydrogen peroxide. In this paper, an open reading frame (ORF) of 639 bp, which encoded a protein of 213 amino acid residues, was cloned from Pseudomonas fluorescens GcM5-1A carried by pine wood nematode. Amino acid sequence alignment showed that the encoded protein shared 99, 97, and 97 % identity with the thiol-specific antioxidant protein LsfA of P. fluorescens Q2-87, the peroxiredoxin of Pseudomonas sp. GM17 and 1-Cys peroxiredoxin of P. fluorescens Pf 0-1, respectively. The ORF was cloned into expressing vector pET-15b and introduced into Escherichia coli BL21 (DE3). Overexpression of a 27-kDa protein was achieved by IPTG induction. The recombinant protein was purified by affinity chromatography on a Ni(2+) matrix column. Non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicated that part of the recombinant appeared in dimer form. Bioassay results showed that purified recombinant protein had both peroxidase and thioredoxin activity. Furthermore, E. coli expressing the ORF showed tolerance to hydrogen peroxide stress, which indicated that the gene might help P. fluorescens GcM5-1A resist hydrogen peroxide generated by host pines after pine wood nematode associated with this bacterium infected pine trees.

A PP (pea)-HASApf-redoxin complex eluted from encapsulated PP gel with aeration displays asymmetric oxidation activity over 200 times greater than that of a similar protein expressed by E. coli cells. The intermediate spin, identified in the ESR spectrum, appears at g = 4.3 and g = 2.0, suggesting that an iron electron-transfer system for the asymmetric oxidation of secondary alcohols may be successfully created by the PP-HASApf-redoxin complex (39 kDa). FTIR experiments provided values νs(SO2) ≈ 950-1050 cm(-1) and νas(SO2) ≈ 1100-1200 cm(-1) for metal-bound sulfinate S-O and Fe-O vibrations. The sulfur and iron detected by physicochemical inspection (IC/ICP-AES) may facilitate the electron transport of a sulfate-iron complex (e.g., rubredoxin (6 kDa) or ferredoxin (9 kDa)) to the HASApf (21 kDa). The observations are consistently acceptable; i.e., the oxygen-driven PP-HASApf-redoxin complex functions regenerate via the successive asymmetric catalytic event - Fe(ii) + O2 → Fe(iii)-O-O(-) → Fe(iv) = O (oxidizing rac- or rac-) → Fe(ii) + H2O. Therefore, the use of a raw biomaterial as a PP-HASApf-redoxin complex-catalytic system for asymmetric oxidation is an important novelty, despite the apparent difficulties in working with pure dehydrogenase enzymatic/redox-cofactor systems for biotransformation.

Volatile organic compounds (VOCs) from plant growth-promoting rhizobacteria (PGPR) play key roles in modulating plant growth and induced systemic resistance (ISR) to pathogens. Despite their significance, the physiological functions of the specific VOCs produced by Pseudomonas fluorescens SS101 (Pf.SS101) have not been precisely elucidated. The effects of Pf.SS101 and its VOCs on augmentation of plant growth promotion were investigated in vitro and in planta. A significant growth promotion was observed in plants exposed Pf.SS101 under both conditions, suggesting that its VOCs play a key role in promoting plant growth. Solid-phase micro-extraction (SPME) and a gas chromatography-mass spectrophotometer (GC-MS) system were used to characterize the VOCs emitted by Pf.SS101 and 11 different compounds were detected in samples inoculated this bacterium, including 13-Tetradecadien-1-ol, 2-butanone and 2-Methyl-n-1-tridecene. Application of these compounds resulted in enhanced plant growth. This study suggests that Pf.SS101 promotes the growth of plants via the release of VOCs including 13-Tetradecadien-1-ol, 2-butanone and 2-Methyl-n-1-tridecene, thus increasing understanding of the role of VOCs in plant-bacterial inter-communication., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The interaction between the Pseudomonas fluorescens biofilm and U(VI) were studied using extended X-ray absorption fine structure spectroscopy (EXAFS), and time-resolved laser fluorescence spectroscopy (TRLFS). In EXAFS studies, the formation of a stable uranyl phosphate mineral, similar to autunite (Ca[UO2]2[PO4]2•2-6H2O) or meta-autunite (Ca[UO2]2[PO4]2•10-12H2O) was observed. This is the first time such a biomineralization process has been observed in P. fluorescens. Biomineralization occurs due to phosphate release from the cellular polyphosphate, likely as a cell's response to the added uranium. It differs significantly from the biosorption process occurring in the planktonic cells of the same strain. TRLFS studies of the uranium-contaminated nutrient medium identified aqueous Ca2UO2(CO3)3 and UO2(CO3)3 (4-) species, which in contrast to the biomineralization in the P. fluorescens biofilm, may contribute to the transport and migration of U(VI). The obtained results reveal that biofilms of P. fluorescens may play an important role in predicting the transport behavior of uranium in the environment. They will also contribute to the improvement of remediation methods in uranium-contaminated sites.

Biofilm formation in natural and engineered porous systems can significantly impact hydrodynamics by reducing porosity and permeability. To better understand and characterize how biofilms influence hydrodynamic properties in porous systems, the genetically engineered bioluminescent bacterial strain Pseudomonas fluorescens HK44 was used to quantify microbial population characteristics and biofilm properties in a translucent porous medium. Power law relationships were found to exist between bacterial bioluminescence and cell density, fraction of void space occupied by biofilm (i.e. biofilm saturation), and hydraulic conductivity. The simultaneous evaluation of biofilm saturation and porous medium hydraulic conductivity in real time using a non-destructive approach enabled the construction of relative hydraulic conductivity curves. Such information can facilitate simulation studies related to biological activity in porous structures, and support the development of new models to describe the dynamic behavior of biofilm and fluid flow in porous media. The bioluminescence based approach described here will allow for improved understanding and control of industrially relevant processes such as biofiltration and bioremediation., (Copyright © 2014. Published by Elsevier B.V.)

In Pseudomonas species, production of secondary metabolites and exoenzymes is regulated by the GacS/GacA two-component regulatory system. In Pseudomonas fluorescens SBW25, mutations in the Gac-system cause major transcriptional changes and abolished production of the lipopeptide viscosin and of an exoprotease. In contrast to many other Pseudomonas species and strains, inactivation of the Gac-system in strain SBW25 significantly enhanced its antimicrobial activities against oomycete, fungal and bacterial pathogens. Here, random plasposon mutagenesis of the gacS mutant led to the identification of seven mutants with reduced or loss of antimicrobial activity. In four mutants, the plasposon insertion was located in genes of the pyrroloquinoline quinone (PQQ) biosynthesis pathway. Genetic complementation, ectopic expression, activity bioassays and Reversed-phase high-performance liquid chromatography (RP-HPLC) analyses revealed that a gacS mutation in SBW25 leads to enhanced expression of pqq genes, resulting in an increase in gluconic and 2-ketogluconic acid production, which in turn acidified the extracellular medium to levels that inhibit growth of other microorganisms. We also showed that PQQ-mediated acidification comes with a growth penalty for the gacS mutant in the stationary phase. In conclusion, PQQ-mediated acidification compensates for the loss of several antimicrobial traits in P. fluorescens SBW25 and may help gac mutants to withstand competitors., (© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Lipids are major functional components of bacterial cells that play fundamental roles in bacterial metabolism and the barrier function between cells and the environment. In an effort to investigate the bacterial lipidome, we adopted a protocol using MALDI-TOF MS imaging coupled to HPTLC to screen a large number of phospholipid classes in a short span of time. With this method, phospholipids of airborne Pseudomonas fluorescens MFAF76a were visualized and identified in sample extracts (measurement accuracy below 0.1 Da, phospholipid identification by means of four characteristic fragment peaks). Via this technique, the P. fluorescens lipidome was shown to comprise three major lipid classes: phosphatidylethanolamine, phosphatidylglycerol and phosphatidylcholine. The protocol described herein is simple, rapid and effective for screening of bacterial phospholipid classes. The remarkable presence of a eukaryotic phospholipid, phosphatidylcholine, was observed in P. fluorescens MFAF76a. This lipid is known to play a role in bacteria-host interactions and had not been known to be found in P. fluorescens cells., (Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Two of the potential bacterial isolates, viz., Pseudomonas fluorescens (VCRC B-426) and Bacillus subtilis (VCRC B-471) whose toxins kill the mosquito pupae/larvae have been identified at our center. As the mode of action of these bacteria are not known, an attempt was made to find out the binding sites of the toxic proteins through immunological methods. Antibodies were raised in BALB/c mice and egg yolk system of chicken layers against the mosquitocidal proteins. The antibodies showed specific binding on to the cephalic and thoracic cuticle of the pupae as well as the paddles of the larvae, indicating the binding of the mosquitocidal proteins.

The viscosin group covers a series of cyclic lipodepsipeptides (CLPs) produced by Pseudomonas bacteria, with a range of biological functions and antimicrobial activities. Their oligopeptide moieties are composed of both L- and D-amino acids. Remarkably, the Leu5 amino acid-centrally located in the nonapeptide sequence-is the sole residue found to possess either an L or D configuration, depending on the producing strain. The impact of this D/L switch on the solution conformation was investigated by NMR-restrained molecular modelling of the epimers pseudodesmin A and viscosinamide A. Although the backbone fold remained unaffected, the D/L switch adjusted the segregation between hydrophobic and hydrophilic residues, and thus the amphipathicity. It also influenced the self-assembly capacity in organic solvents. Additionally, several new minor variants of viscosinamide A from Pseudomonas fluorescens DR54 were identified, and an NMR assay is proposed to assess the presence of either an L- or D-Leu5., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

It has been recently reported that in presence of low Reynolds number (Re ≪ 1) transport, preformed bacterial biofilms, several hours after their formation, may degenerate in form of filamentous structures, known as streamers. In this work, we explain that such streamers form as the highly viscous liquid states of the intrinsically viscoelastic biofilms. Such "viscous liquid" state can be hypothesized by noting that the time of appearance of the streamers is substantially larger than the viscoelastic relaxation time scale of the biofilms, and this appearance is explained by the inability of a viscous liquid to withstand external shear. Further, by identifying the post formation dynamics of the streamers as that of a viscous liquid jet in a surrounding flow field, we can interpret several unexplained issues associated with the post-formation dynamics of streamers, such as the clogging of the flow passage or the exponential time growth of streamer dimensions. Overall our manuscript provides a biophysical basis for understanding the evolution of biofilm streamers in creeping flows.

The Fusarium head blight of grain cereals is a significant disease worldwide. In Argentina, high levels of contamination with Fusarium proliferatum have been found in crops. Many strains of the Pseudomonas genus antagonize the growth of fungi by different mechanisms, such as the production of antibiotics, siderophores, volatiles, and extracellular enzymes. In this work, we have designed a new system for studying the growth inhibition of F. proliferatum-namely by volatile compounds produced by Pseudomonas fluorescens MGR12. In both rich and minimal media, the bacterium released volatiles that negatively affected the mycelial growth of that phytopathogenic fungus. These bacterial compounds were analyzed by gas chromatography-mass spectrometry, but only a few could be identified by comparing their mass spectra with the libraries of the National Institutes of Standards and Technology MS search.