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The adsorption of flagellin monomers from Pseudomonas fluorescens on Au(111) has been studied by Atomic Force Microscopy (AFM), Scanning Tunneling Microscopy (STM), X-ray Photoelectron Spectroscopy (XPS), Surface Plasmon Resonance (SPR), and electrochemical techniques. Results show that flagellin monomers spontaneously self-assemble forming a monolayer thick protein film bounded to the Au surface by the more hydrophobic subunit and exposed to the environment the hydrophilic subunit. The films are conductive and allow allocation of electrochemically active cytochrome C. The self-assembled films could be used as biological platforms to build 3D complex molecular structures on planar metal surfaces and to functionalize metal nanoparticles., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: The use of biocatalysts has become an increasingly attractive alternative to traditional chemical methods, due to the high selectivity, mild reaction conditions and environmentally-friendly processes in nonaqueous catalysis of nucleosids. However, the extensive use of organic solvents may generally suffer from sever drawbacks such as volatileness and toxicity to the environment and lower activity of the biocatalyst. Recently, ionic liquids are considered promising solvents for nonaqueous biocatalysis of polyhydroxyl compounds as ILs are environmental-friendly., Results: In this research, we developed new IL-containing reaction systems for synthesis of long chain nucleoside ester catalyzed by Pseudomonas fluorescens whole-cells. Various ILs exerted significant but different effects on the bio-reaction. And their effects were closely related with both the anions and cations of the ILs. Use of 10% [BMI][PF6]/THF gave high reaction efficiency of arabinocytosine laurate synthesis, in which the initial rate, product yield and 5'-regioselectivity reached 2.34 mmol/L·h, 81.1% and >99%, respectively. Furthermore, SEM analysis revealed that ILs can alter the cell surface morphology, improve the permeability of cell envelopes and thus facilitate the mass transfer of substrates to the active sites of cell-bound enzymes., Conclusion: Our research demonstrated the potential of ILs as promising reaction medium for achieving highly efficient and regioselective whole-cell catalysis.

Aims: To explore specific mechanisms of endophytic Pseudomonas fluorescens antagonizing Athelia rolfsii, causing southern blight of Atractylodes lancea and to evaluate the potential of this Ps. fluorescens strain to control southern blight., Methods and Results: Endophytic Ps. fluorescens strain ALEB 7B isolated from A. lancea can significantly inhibit the growth of A. rolfsii strain SY4. Pre-inoculating A. lancea seedlings with Ps. fluorescens ALEB 7B reduces the southern blight morbidity rate significantly. In situ observation using scanning electron microscopy shows Ps. fluorescens ALEB 7B colonizing the plant cells. Volatile organic compounds (VOCs) from Ps. fluorescens ALEB 7B can kill A. rolfsii SY4 and dimethyl disulphide (DMDS) plays a major role. 2-Piperidinone is a unique substance having antifungal activity in dichloromethane extracts of bacterial cell-free culture filtrates. Other antagonistic mechanisms include ecological niche occupation, antibiotic production and lytic exoenzymes secretion., Conclusions: Specific antagonistic mechanisms of Ps. fluorescens ALEB 7B on A. rolfsii SY4 were detailed, including release of DMDS, production of 2-piperidone, secretion of antibiotics and lytic exoenzymes and competition for spaces and nutrients., Significance and Impact of the Study: This work firstly reports the significant inhibition of VOCs released by Ps. fluorescens on the growth of A. rolfsii. 2-Piperidinone is firstly found synthesized by Ps. fluorescens, having antifungal activity. This work provides an antagonistic bacterium with practical convenience and ecologically amity, which has potential for control to A. rolfsii in vitro., (© 2014 The Society for Applied Microbiology.)

Cytidine 2',3'-cyclic monophosphate (2',3'-cCMP) and uridine 2',3'-cyclic monophosphate (2',3'-cUMP) were isolated from Pseudomonas fluorescens pfo-1 cell extracts by semi-preparative reverse phase HPLC. The structures of the two compounds were confirmed by NMR and mass spectroscopy against commercially available authentic samples. Concentrations of both intracellular and extracellular 2',3'-cCMP and 2',3'-cUMP were determined. Addition of 2',3'-cCMP and 2',3'-cUMP to P. fluorescens pfo-1 culture did not significantly affect the level of biofilm formation in static liquid cultures., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

A bacterial strain (JH 70-4) exhibiting plant growth promoting characteristics (indoleacetic acid production and 1-aminocyclopropane-1-carboxylate deaminase activity), as well as heavy metal(loid) (HM) tolerance and Pb precipitation, was isolated from HM-contaminated soil at an abandoned mine site. The bacterium was identified as Pseudomonas fluorescens based on 16S rDNA sequencing. The JH 70-4 strain induced precipitation of Pb as PbS nanoparticles, confirmed by X-ray diffraction. Solution pH, incubation time, and Pb concentration influenced removal and PbS formation. Inoculating contaminated soil with JH 70-4 decreased Pb availability; exchangeable Pb decreased while organic- and sulphide-bound Pb increased. The toxicity characteristic leaching procedure showed a 65% decrease in Pb in leachate 60 d after inoculating soil with JH 70-4. Shoot and root lengths of Sudan grass grown in the inoculated soil were greater than in the uninoculated soil. Findings suggest that microbial Pb fixation is a viable strategy for remediating soil and promoting plant growth for phytostabilization of contaminated sites.

The identification of Pseudomonas aeruginosa from samples of bottled natural mineral water by the analysis of subcultures is time consuming and other species of the authentic Pseudomonas group can be a problem. Therefore, this study aimed to investigate the influence of different aquatic environmental conditions (pH, mineral content) and growth phases on the cultivation-free differentiation between water-conditioned Pseudomonas spp. by applying Raman microspectroscopy. The final dataset was comprised of over 7500 single-cell Raman spectra, including the species Pseudomonas aeruginosa, P. fluorescens and P. putida, in order to prove the feasibility of the introduced approach. The collection of spectra was standardized by automated measurements of viable stained bacterial cells. The discrimination was influenced by the growth phase at the beginning of the water adaptation period and by the type of mineral water. Different combinations of the parameters were tested and they resulted in accuracies of up to 85% for the identification of P. aeruginosa from independent samples by applying chemometric analysis., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

The kynurenine pathway is the major pathway of l-tryptophan catabolism in eukaryotes and some bacteria. In this pathway, kynureninase catalyzes the hydrolysis of l-kynurenine (in bacteria) or 3-hydroxy-l-kynurenine (in eukaryotes) to give anthranilic acid or 3-hydroxyanthranilic acid, respectively, and l-alanine. Kynureninase is a member of the aminotransferase superfamily and contains pyridoxal-5'-phosphate (PLP) as cofactor. The enzyme is a dimer of two identical subunits, with the active site containing residues contributed from both subunits. The reaction of kynureninase is formally a retro-Claisen reaction, and thus requires extensive acid-base catalysis. The pH dependence of the reaction of Pseudomonas fluorescens kynureninase shows two pKa's, a base with 6.5 and an acid with 8.8, on kcat/Km, and one pKa of 6.8 on kcat. The effects of mutagenesis of Tyr-226 and (31)P NMR results suggest that the basic group with pKa of 6.5 is the phosphate group of the PLP, which accepts a proton from the amino acid substrate zwitterion to initiate transaldimination. The external aldimine of kynurenine and PLP is then deprotonated by the ε-amino group of Lys-227 to give a quinonoid intermediate, which is reprotonated at C-4' to give a ketimine. Addition of water to the γ-carbonyl, assisted by Lys-227, then gives a gem-diol, which undergoes Cβ-Cγ cleavage to give the first product, anthranilic acid, and an enamine intermediate. The enamine is protonated at the β-carbon, resulting in a pyruvate ketimine. Deprotonation at C-4' and reprotonation of the α-carbon gives the external aldimine of l-alanine, which releases the second product, l-alanine. The reaction specificity of kynureninases is determined in part by active site residues, Trp64, Gly281, and Thr282 in P. fluorescens, and the homologous His102, Ser332, and Asn333 in human kynureninase. Asn333 can form a hydrogen bond to the 3-OH of 3-hydroxykynurenine in the human enzyme. Halogenation of kynurenine at C-5 increases activity with both enzymes, but halogenation at C-3 only increases activity for human kynureninase. The effect of halogenation at C-5 may be due to hydrophobic or van der Waals effects, and the effect of halogenation at C-3 for the human enzyme may be due to halogen bonding., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Antagonistic bacteria are common soil inhabitants with potential to be developed into biofungicides for the management of seedling damping-off, root rot, and other soil-borne diseases of various crops. In this study, antagonistic bacteria were isolated from a commercial potato field and screened for their growth inhibition of fungal and oomycete pathogens in laboratory tests. The biocontrol potential of the 3 most effective antagonistic bacteria from the in vitro tests was evaluated against seedling damping-off and root rot of cucumber caused by Pythium ultimum. Based on phenotypic characteristics, biochemical tests, and sequence analysis of 16S-23S rDNA gene, the 3 antagonistic bacteria were identified as Pseudomonas fluorescens (isolate 9A-14), Pseudomonas sp. (isolate 8D-45), and Bacillus subtilis (isolate 8B-1). All 3 bacteria promoted plant growth and suppressed Pythium damping-off and root rot of cucumber seedlings in growth-room assays. Both pre- and post-planting application of these bacteria to an infested peat mix significantly increased plant fresh masses by 113%-184% and percentage of healthy seedlings by 100%-290%, and decreased damping-off and root rot severity by 27%-50%. The peat and talc formulations of these antagonistic bacteria applied as seed or amendment treatments to the infested peat mix effectively controlled Pythium damping-off and root rot of cucumber seedlings and enhanced plant growth. The survival of all 3 antagonistic bacteria in peat and talc formulations decreased over time at room temperature, but the populations remained above 10(8) CFU/g during the 180-day storage period. The peat formulation of a mixture of 3 bacteria was the best seed treatment, significantly increasing the plant fresh masses by 245% as compared with the Pythium control, and by 61.4% as compared with the noninfested control. This study suggests that the indigenous bacteria from agricultural soils can be developed and formulated as biofungicides for minimizing the early crop losses caused by seedling damping-off and root rot diseases.

This study was aimed at isolation and characterization of natural antifungal compounds for grain mold, a key parasitic fungal disease of sorghum. Pseudomonas fluorescens strain isolated from rhizosphere of groundnut crop was selected as a source. Its biocontrolling ability was assessed by testing some biochemical attributes such as phosphate-solubilization, and HCN, NH3 , indole-3-acetic acid, and siderophore production. The strain showed positive result for all except indole-3-acetic acid, revealing its suitability for a further study. The antibiotic-sensitivity pattern of the strain against 43 antibiotics was also established, which showed resistance to 15 antibiotics. The efficacy of P. fluorescens strain against grain mold was identified by dual culture technique. Hundred percent inhibition was found against Fusarium moniliforme, an important causative agent of this disease. The strain was fermented for secondary metabolites and extracted with AcOEt. Chromatographic separation of the extract yielded four known compounds, cyclo(L-Pro-L-Phe) (1), cyclo(trans-4-hydroxy-L-Pro-L-Leu) (2), cyclo(trans-4-hydroxy-L-Pro-L-Phe) (3), and cyclo(Gly-L-Pro) (4), which were characterized by spectral analysis and optical rotation. The crude extract, a mixture of 2 and 3, and isolated 1 were proved to be significantly effective against grain mold fungi. This is the first report on production of these cyclic dipeptides by P. fluorescens and their antagonistic properties., (Copyright © 2014 Verlag Helvetica Chimica Acta AG, Zürich.)

A series of substituted kynurenines (3-bromo-DL, 3-chloro-DL, 3-fluoro-DL, 3-methyl-DL, 5-bromo-L, 5-chloro-L, 3,5-dibromo-L and 5-bromo-3-chloro-DL) have been synthesized and tested for their substrate activity with human and Pseudomonas fluorescens kynureninase. All of the substituted kynurenines examined have substrate activity with both human as well as P. fluorescens kynureninase. For the human enzyme, 3- and 5-substituted kynurenines have kcat and kcat/Km values higher than L-kynurenine, but less than that of the physiological substrate, 3-hydroxykynurenine. However, 3,5-dibromo- and 5-bromo-3-chlorokynurenine have kcat and kcat/Km values close to that of 3-hydroxykynurenine with human kynureninase. The effects of the 3-halo substituents on the reactivity with human kynureninase may be due to electronic effects and/or halogen bonding. In contrast, for the bacterial enzyme, 3-methyl, 3-halo and 3,5-dihalokynurenines are much poorer substrates, while 3-fluoro, 5-bromo, and 5-chlorokynurenine have kcat and kcat/Km values comparable to that of its physiological substrate, L-kynurenine. Thus, 5-bromo and 5-chloro-L-kynurenine are good substrates for both human as well as bacterial enzyme, indicating that both enzymes have space for substituents in the active site near C-5. The increased activity of the 5-halokynurenines may be due to van der Waals contacts or hydrophobic effects. These results may be useful in the design of potent and/or selective inhibitors of human and bacterial kynureninase., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Aryloxyalkanoate dioxygenase-12 (AAD-12) was discovered from the soil bacterium Delftia acidovorans MC1 and is a nonheme Fe(II)/α-ketoglutarate-dependent dioxygenase, which can impart herbicide tolerance to transgenic plants by catalyzing the degradation of certain phenoxyacetate, pyridyloxyacetate, and aryloxyphenoxypropionate herbicides. (1) The development of commercial herbicide-tolerant crops, in particular AAD-12-containing soybean, has prompted the need for large quantities of the enzyme for safety testing. To accomplish this, the enzyme was produced in Pseudomonas fluorescens (Pf) and purified to near homogeneity. A small amount of AAD-12 was partially purified from transgenic soybean and through various analytical, biochemical, and in vitro activity analyses demonstrated to be equivalent to the Pf-generated enzyme. Furthermore, results from in vitro kinetic analyses using a variety of plant endogenous compounds revealed activity with trans-cinnamate and indole-3-acetic acid (IAA). The catalytic efficiencies (kcat/Km) of AAD-12 using trans-cinnamate (51.5 M(-1) s(-1)) and IAA (8.2 M(-1) s(-1)) as substrates were very poor when compared to the efficiencies of plant endogenous enzymes. The results suggest that the presence of AAD-12 in transgenic soybean would not likely have an impact on major plant metabolic pathways.

The fatty acid composition of cell lipids of 15 strains isolated from the affected plants of rape and five collection strains has been studied. According to the results of chemotaxonomic analysis it has been found that 9 isolated strains are similar to representatives of species Pseudomonas marginalis and Pseudomonas fluorescens, and 6 - to those of Xanthomonas campestris pv. campestris. The authors have established the efficiency of certain methods for the extraction of fatty acids used for the identification of bacteria pathogenic for rape which belong to the genera Pseudomonas and Xanthomonas.

The method of protein-based stable isotope probing (protein-SIP) has previously been shown to allow the modeling of carbon fluxes in microbial communities, thus tackling one of the key questions in microbial ecology. The method allows the analysis of stable isotope distribution in peptides, revealing metabolic activities of the species present in an ecosystem. Besides carbon, an application of protein-SIP with nitrogen is of interest for resolving the nitrogen fluxes in microbial communities. Thus, the sensitivity and reliability of a protein-SIP approach employing (15)N was analyzed. For this, cultivations of Pseudomonas fluorescens ATCC 17483 with different ratios of (14)N/(15)N were performed, from 10 % down to 0.1 % (15)N. After incubation leading to complete labeling of biomass, proteins were extracted and separated by one-dimensional gel electrophoresis, followed by tryptic digest and UPLC Orbitrap MS/MS analysis. (15)N relative isotope abundance (RIA) was calculated based on isotopic patterns from identified peptides in mass spectra. Proteomics data have been deposited to ProteomeXchange with identifier PXD000127. The distribution of (15)N RIA values among peptides was analyzed in samples with different (15)N amount, and potential causes for variations within individual samples of either technical or biological origin were investigated. Using a number of 50 peptides, significant differences (p ≤ 0.05) in (15)N incorporation were found between samples of different (15)N RIA down to 0.1 %. The study demonstrates that protein-SIP using (15)N is sufficiently sensitive for quantitative investigation of microbial activity in nitrogen cycling processes.

EXPANDING OUR KNOWLEDGE: Natural lipocyclocarbamate natural products have provided the inspiration for the first-in-class synthetic phospholipase inhibitor darapladib, currently in phase III clinical trials for the treatment of atherosclerosis. Here, we discuss their biosynthesis by a nonribosomal peptide synthetase., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Arsenate and phosphate are abundant on Earth and have striking similarities: nearly identical pK(a) values, similarly charged oxygen atoms, and thermochemical radii that differ by only 4% (ref. 3). Phosphate is indispensable and arsenate is toxic, but this extensive similarity raises the question whether arsenate may substitute for phosphate in certain niches. However, whether it is used or excluded, discriminating phosphate from arsenate is a paramount challenge. Enzymes that utilize phosphate, for example, have the same binding mode and kinetic parameters as arsenate, and the latter's presence therefore decouples metabolism. Can proteins discriminate between these two anions, and how would they do so? In particular, cellular phosphate uptake systems face a challenge in arsenate-rich environments. Here we describe a molecular mechanism for this process. We examined the periplasmic phosphate-binding proteins (PBPs) of the ABC-type transport system that mediates phosphate uptake into bacterial cells, including two PBPs from the arsenate-rich Mono Lake Halomonas strain GFAJ-1. All PBPs tested are capable of discriminating phosphate over arsenate at least 500-fold. The exception is one of the PBPs of GFAJ-1 that shows roughly 4,500-fold discrimination and its gene is highly expressed under phosphate-limiting conditions. Sub-ångström-resolution structures of Pseudomonas fluorescens PBP with both arsenate and phosphate show a unique mode of binding that mediates discrimination. An extensive network of dipole-anion interactions, and of repulsive interactions, results in the 4% larger arsenate distorting a unique low-barrier hydrogen bond. These features enable the phosphate transport system to bind phosphate selectively over arsenate (at least 10(3) excess) even in highly arsenate-rich environments.

The extracellular polysaccharide produced by a newly isolated strain Pseudomonas fluorescens WR-1 was purified and characterized and its production was optimized using response surface methodology. The results showed that the strain WR-1 produced one kind of EPS that was composed of arabinose, glucose and uronic acid. The molecular weight of the EPS was determined to be 6.78 × 10(6)Da. The preferable culture conditions for EPS production were pH 7.0, temperature 28°C for 72 h with peptone and maltose as best N and C sources, respectively. The model predicted that the maximum EPS production (39.6 gL(-1)) was appeared with maltose 48.65 gL(-1), Mn(2+) 1118 μM and Zn(2+) 901 μM. The EPS also showed good H(2)O(2) scavenging activity while moderate free radical scavenging activity and reductive ability were determined. The EPS from WR-1 may be a new source of natural antioxidants with potential value for health, food and industry., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Cold shock proteins perform vital functions, such as mRNA masking, coupling of transcription to translation and developmental timing and regulation, which aids in survival of microbes in cold stress. Pseudomonas fluorescens is an ecologically important bacterium which helps in plant growth promotion. Since the cold tolerant mutant of the bacterium is able to grow at the temperature ranges from 30 to 4°C, it is of interest to study the process of its survival in the extreme temperatures. Therefore, this study is focused on the three dimensional structure and molecular modeling of cold resistant protein (CRP) from P. fluorescens to predict its molecular mechanism. Investigating the structure of CRP confirmed the presence of a conserved domain characteristic of the cold shock domain (CSD) family and a single nucleotide binding domain. When 3D structure of CRP was compared with the existing cold shock proteins, major deviations were found in the loop regions connecting the β2-β3, β3-β4 and β4-β5 sheets. Docking studies showed that CRP forms a significant clamp like structure at the substrate binding cleft which stabilizes the ligand. Therefore, it can be concluded that CRP has a strong affinity for the poly thymidine (poly T) stretch and can be considered a candidate for transcription regulation., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The previously reported crystal structures of α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD) show a five-coordinate Zn(II)(His)(3)(Asp)(OH(2)) active site. The water ligand is H-bonded to a conserved His228 residue adjacent to the metal center in ACMSD from Pseudomonas fluorescens (PfACMSD). Site-directed mutagenesis of His228 to tyrosine and glycine in this study results in a complete or significant loss of activity. Metal analysis shows that H228Y and H228G contain iron rather than zinc, indicating that this residue plays a role in the metal selectivity of the protein. As-isolated H228Y displays a blue color, which is not seen in wild-type ACMSD. Quinone staining and resonance Raman analyses indicate that the blue color originates from Fe(III)-tyrosinate ligand-to-metal charge transfer. Co(II)-substituted H228Y ACMSD is brown in color and exhibits an electron paramagnetic resonance spectrum showing a high-spin Co(II) center with a well-resolved (59)Co (I = 7/2) eight-line hyperfine splitting pattern. The X-ray crystal structures of as-isolated Fe-H228Y (2.8 Å) and Co-substituted (2.4 Å) and Zn-substituted H228Y (2.0 Å resolution) support the spectroscopic assignment of metal ligation of the Tyr228 residue. The crystal structure of Zn-H228G (2.6 Å) was also determined. These four structures show that the water ligand present in WT Zn-ACMSD is either missing (Fe-H228Y, Co-H228Y, and Zn-H228G) or disrupted (Zn-H228Y) in response to the His228 mutation. Together, these results highlight the importance of His228 for PfACMSD's metal specificity as well as maintaining a water molecule as a ligand of the metal center. His228 is thus proposed to play a role in activating the metal-bound water ligand for subsequent nucleophilic attack on the substrate.

Rapid phenotype characterization and identification of cultured cells, which is needed for progress in tissue engineering and drug testing, requires an experimental technique that measures physical properties of cells with sub-micron resolution. Recently, band excitation piezoresponse force microscopy (BEPFM) has been proven useful for recognition and imaging of bacteria of different types in pure water. Here, the BEPFM method is performed for the first time on physiologically relevant electrolyte media, such as Dulbecco's phosphate-buffered saline (DPBS) and Dulbecco's modified Eagle's medium (DMEM). Distinct electromechanical responses for Micrococcus lysodeikticus (Gram-positive) and Pseudomonas fluorescens (Gram-negative) bacteria in DPBS are demonstrated. The results suggest that mechanical properties of the outer surface coating each bacterium, as well as the electrical double layer around them, are responsible for the BEPFM image formation mechanism in electrolyte media.

The lyophilized Pseudomonas fluorescens cell was an efficient alternative catalyst to enzymes for highly regioselective acylation of a polar nucleoside, 1-β-D-arabinofuranosylcytosine (ara-C). The cells showed an evident solvent dependence in the reaction. Among the tested solvents except for acetonitrile-pyridine, catalytic activity of the cells clearly increased with increasing the polarity of the organic solvents used. Among all the tested solvents both pure and binary, the best results were observed in isopropyl ether-pyridine system, in which the catalyst also showed good thermal and operational stabilities. For the biocataylsis in isopropyl ether-pyridine, the optimal isopropyl ether concentration, water content, acyl donor/ara-C ratio, biocatalyst dosage and reaction temperature were 30% (v/v), 4%, 45, 50mg/mL and 30 °C, respectively, under which the initial rate, yield and 5'-regioselectivity were 2.93 mM/h, 77.1% and 97.3%, respectively. The bacterial cells exhibited comparable 5'-regioselectivity to the expensive immobilized enzyme, which could also have environmental and cost advantages., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

R-type and F-type pyocins are high-molecular-mass bacteriocins produced by Pseudomonas aeruginosa that resemble bacteriophage tails. They contain no head structures and no DNA, and are used as defence systems. In this report, we show that Pseudomonas fluorescens SF4c, a strain isolated from the wheat rhizosphere, produces a high-molecular-mass bacteriocin which inhibits the growth of closely related bacteria. A mutant deficient in production of this antimicrobial compound was obtained by transposon mutagenesis. Sequence analysis revealed that the transposon had disrupted a gene that we have named ptm, since it is homologous to that encoding phage tape-measure protein in P. fluorescens Pf0-1, a gene belonging to a prophage similar to phage-like pyocin from P. aeruginosa PAO1. In addition, we have identified genes from the SF4c pyocin cluster that encode a lytic system and regulatory genes. We constructed a non-polar ptm mutant of P. fluorescens SF4c. Heterologous complementation of this mutation restored the production of bacteriocin. Real-time PCR was used to analyse the expression of pyocin under different stress conditions. Bacteriocin was upregulated by mitomycin C, UV light and hydrogen peroxide, and was downregulated by saline stress. This report constitutes, to our knowledge, the first genetic characterization of a phage tail-like bacteriocin in a rhizosphere Pseudomonas strain.

Background: TliA is a thermostable lipase secreted by the type 1 secretion system (T1SS) of Pseudomonas fluorescens. The secretion is promoted by its secretion/chaperone domain located near the C-terminus, which is composed mainly of four Repeat-in-Toxin (RTX) repeats. In order to identify the minimal region of TliA responsible for its secretion, five different copies of the secretion/chaperone domain, each involving truncated N-terminal residues and a common C-terminus, were acquired and named as lipase ABC transporter recognition domains (LARDs). Each LARD was fused to epidermal growth factor (EGF) or green fluorescent protein (GFP), and the secretion of EGF-LARD or GFP-LARD fusion proteins was assessed in Escherichia coli with ABC transporter., Results: Among the fusion proteins, GFP or EGF with 105-residue LARD3 was most efficiently secreted. In addition, GFP-LARD3 emitted wild type GFP fluorescence. Structurally, LARD3 had the 4 RTX repeats exposed at the N-terminus, while other LARDs had additional residues prior to them or missed some of the RTX repeats. LARD3 was both necessary and sufficient for efficient secretion and maintenance of GFP fluorescence in E. coli, which was also confirmed in P. fluorescens and P. fluorescens ▵tliA, a knock-out mutant of tliA., Conclusion: LARD3 was a potent secretion signal in T1SS for its fusion flanking RTX motif, which enhanced secretion and preserved the fluorescence of GFP. LARD3-mediated secretion in E. coli or P. fluorescens will enable the development of enhanced protein manufacturing factory and recombinant microbe secreting protein of interest in situ.

Lipopeptides (LPs) are a structurally diverse class of amphipathic natural products that were in the past mainly known for their surfactant properties. However, the recent discovery of their antimicrobial and cytotoxic bioactivities have fueled and renewed the interest in this compound class. Propelled by the antimicrobial potential of this compound class, in this study a range of six underinvestigated LPs from Pseudomonads were examined with respect to their antibiotic activities towards bacteria. The assays revealed that only the glycosylated lipodipeptide SB-253514, produced by Pseudomonas strain SH-C52, showed significant antibacterial activity. Since the bioactivity of LPs is commonly attributed to membrane interactions, we analyzed the molecular interactions between the LPs and bacteria-like lipid model membranes in more detail via complementary biophysical approaches. Application of the quartz crystal microbalance (QCM) showed that all LPs possess a high binding affinity towards the model membranes. Despite their similar membrane affinity, monolayer studies displayed different tendencies of LPs to incorporate into the membrane. The degree of membrane incorporation could be correlated with specific structural features of the investigated LPs, such as distance between the peptidic macrocycle and the fatty acid, but did not fully reflect their respective antibacterial activity. Cyclic voltammetry (CV) experiments further demonstrated that SB-253514 showed no membrane permeabilization effects at inhibitory concentrations. Collectively, these results suggests that the antibacterial activity of SB-253514 cannot be explained by an unspecific detergent-like mechanism generally proposed for amphiphilic molecules but instead appears to occur via a defined structural target., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Novel anti-HIV lectin family which shows a strict binding specificity for high mannose glycans has been found in lower organisms. The bacterial orthologue has been identified in the genome of Pseudomonas fluorescens Pf0-1 and the gene coding a putative lectin was cloned, expressed in Escherichia coli and purified by one step gel filtration. Glycan array screening of the recombinant lectin, termed PFL, has revealed that PFL preferentially recognizes high mannose glycans with α1-3 Man that was highly exposed at the D2 position. In contrast, masking of this α1-3 Man with α1-2 Man dramatically impaired lectin-carbohydrate interactions. Reducing terminal disaccharide, GlcNAc-GlcNAc of high mannose glycans was also essential for PFL-binding. PFL showed a potent anti-influenza virus activity by inhibiting the virus entry into cells at doses of low nanomolar concentration. At micromolar concentration or higher, PFL showed a cytotoxicity accompanying loss of the cell adhesion against human gastric cancer MKN28 cells. The cell surface molecule to which PFL bound was co-precipitated with biotin-labeled PFL and identified as integrin α2 by peptide mass fingerprinting using MALDI-TOF mass spectrometry. Intriguingly, upon treatment with exogenous PFL, integrin α2 on the cell surface underwent rapid internalization to the cytoplasm and accumulated to perinuclear region, together with the bound PFL. The resulting loss of cell adherence would trigger a signaling pathway that induced anoikis-like cell death. These events were effectively inhibited by pretreatment of PFL with mannnan, indicating the involvement of high mannose glycans on PFL-induced cell death that was triggered by PFL-integrin α2 interactions.

A novel constituent of bacterial polysaccharides, 4-deoxy-D-xylo-hexose (D-4dxylHex), was found in the major O-specific polysaccharide from the lipopolysaccharide of Pseudomonas fluorescens BIM B-582. D-4dxylHex was isolated in the free state by paper chromatography after full acid hydrolysis of the polysaccharide and identified by GLC-mass spectrometry, 1H and 13C NMR spectroscopy, and specific rotation. It occurs as a lateral substituent in ∼40% of the oligosaccharide repeating units, making the polysaccharide devoid of strict regularity. The structure of the polysaccharide was established by sugar analysis, Smith degradation, and two-dimensional 1H and 13C NMR spectroscopy. In addition, a minor polysaccharide was isolated from the same lipopolysaccharide and found to contain 4-O-methylrhamnose.

Pyochelin (Pch) and enantiopyochelin (EPch) are enantiomeric siderophores, with three chiral centers, produced under iron limitation conditions by Pseudomonas aeruginosa and Pseudomonas fluorescens , respectively. After iron chelation in the extracellular medium, Pch-Fe and EPch-Fe are recognized and transported by their specific outer-membrane transporters: FptA in P. aeruginosa and FetA in P. fluorescens . Structural analysis of FetA-EPch-Fe and FptA-Pch-Fe, combined with mutagenesis and docking studies revealed the structural basis of the stereospecific recognition of these enantiomers by their respective transporters. Whereas FetA and FptA have a low sequence identity but high structural homology, the Pch and EPch binding pockets do not share any structural homology, but display similar physicochemical properties. The stereospecific recognition of both enantiomers by their corresponding transporters is imposed by the configuration of the siderophore's C4'' and C2'' chiral centers. This recognition involves specific hydrogen bonds between the Arg91 guanidinium group and EPch-Fe for FetA and between the Leu117-Leu116 main chain and Pch-Fe for FptA. FetA and FptA are the first membrane receptors to be structurally described with opposite binding enantioselectivities for their ligands, giving insights into the structural basis of their enantiospecificity.

Volatile organic compounds (VOCs), characterized by low molecular weight and high vapor pressure, are produced by all organisms as part of normal metabolism, and play important roles in communication within and between organisms. We examined the effects of VOCs released by three species of plant growth-promoting rhizobacteria (Pseudomonas fluorescens, Bacillus subtilis, Azospirillum brasilense) on growth parameters and composition of essential oils (EO) in the aromatic plant Mentha piperita (peppermint). The bacteria and plants were grown on the same Petri dish, but were separated by a physical barrier such that the plants were exposed only to VOCs but not to solutes from the bacteria. Growth parameters of plants exposed to VOCs of P. fluorescens or B. subtilis were significantly higher than those of controls or A. brasilense-treated plants. Production of EOs (monoterpenes) was increased 2-fold in P. fluorescens-treated plants. Two major EOs, (+)pulegone and (-)menthone, showed increased biosynthesis in P. fluorescens-treated plants. Menthol in A. brasilense-treated plants was the only major EO that showed a significant decrease. These findings suggest that VOCs of rhizobacteria, besides inducing biosynthesis of secondary metabolites, affect pathway flux or specific steps of monoterpene metabolism. Bacterial VOCs are a rich source for new natural compounds that may increase crop productivity and EO yield of this economically important plant species., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

The influence of total surface energy on bacterial adhesion has been investigated intensively with the frequent conclusion that bacterial adhesion is less on low-energy surfaces. However, there are also a number of contrary findings that high-energy surfaces have a smaller biofouling tendency. Recently, it was found that the CQ ratio, which is defined as the ratio of Lifshitz-van der Waals (LW) apolar to electron donor surface-energy components of substrates, has a strong correlation to bacterial adhesion. However, the electron donor surface-energy components of substrates varied over only a very limited range. In this article, a series of Ni-P-TiO(2)-PTFE nanocomposite coatings with wide range of surface-energy components were prepared using an electroless plating technique. The bacterial adhesion and removal on the coatings were evaluated with different bacteria under both static and flow conditions. The experimental results demonstrated that there was a strong correlation between bacterial attachment (or removal) and the CQ ratio. The coatings with the lowest CQ ratio had the lowest bacterial adhesion or the highest bacterial removal, which was explained using the extented DLVO theory.

Because most "low fouling" polymers resisting bacterial attachment are hydrophilic, they are usually also significantly swollen. Swelling leads to purely physical dilution of interaction and weakens attachment; however, these nonspecific contributions are usually not separated from the specific effect of polymer chemistry. Taking advantage of the fact that chemistry and swelling of hydrogels may be independently varied through the fraction of a cross-linker, the roles of chemistry and physical dilution (swelling) in bacterial attachment are analyzed for selected hydrogels. Using as a quantitative indicator the rate of bacterial deposition in a parallel plate setup under defined flow conditions, the observed correlation of deposition rate with swelling provides a straightforward comparison of gels with different chemistries that can factor out the effect of swelling. In particular, it is found that chemistry appears to contribute similarly to bacterial deposition on hydrogels prepared from acrylamide and a zwitterioninic monomer 2-(methacryloyloxy)ethyl) dimethyl-(3-sulfopropyl) ammonium hydroxide so that the observed differences may be related to swelling only. In contrast, these gels were inferior to PEG-based hydrogels, even when swelling of the latter was lower, indicating a greater contribution of PEG chemistry to reduced bacterial deposition. This demonstrates that swelling must be accounted for when comparing different biofouling-resistant materials. Chemical and physical principles may be combined in hydrogel coatings to develop efficient antibiofouling surfaces.

Non-trophic interactions are increasingly recognised as a key parameter of predator-prey interactions. In soil, predation by bacterivorous nematodes is a major selective pressure shaping soil bacterial communities, and many bacteria have evolved defence mechanisms such as toxicity. In this study, we show that extracellular secondary metabolites produced by the model soil bacterium Pseudomonas fluorescens CHA0 function as a complex defence strategy against bacterivorous nematodes. Using a collection of functional mutants lacking genes for the biosynthesis of one or several extracellular metabolites, we evaluated the impact of bacterial secondary metabolites on the survival and chemotactic behaviour of the nematode Caenorhabditis elegans. Additionally, we followed up the stress status of the nematodes by measuring the activation of the abnormal DAuer Formation (DAF) stress cascade. All studied secondary metabolites contributed to the toxicity of the bacteria, with hydrogen cyanide efficiently repelling the nematodes, and both hydrogen cyanide and 2,4-DAPG functioning as nematicides. Moreover, these metabolites elicited the DAF stress response cascade of C. elegans, showing that they affect nematode physiology already at sublethal concentrations. The results suggest that bacterial secondary metabolites responsible for the suppression of plant pathogens strongly inhibit bacterivorous nematodes and thus likely contribute to the resistance of bacteria against predators in soil.

The biosorption characteristics of Cs(I) ions from aqueous solution using exopolymers (PFC02) produced from Pseudomonas fluorescens C-2 were investigated as a function of pH, biosorbent dosage, contact time and initial concentration. pH played a major role in the adsorption process, and the optimum pH for the removal of Cs(I) was 8.0. Langmuir, Freundlich and Dubinin-Radushkevich (D-R) models were applied to describe the biosorption isotherm of the Cs(I) ions by PFC02. The Lagergren first-order, pseudo second-order kinetic and intraparticle diffusion models were used to test the kinetic data. Langmuir model and D-R model fitted the equilibrium data better than the Freundlich isotherm. The monolayer adsorption capacities of PFC02 as obtained from Langmuir isotherm at 25 degrees C was found to be 32.63 mg/g. From the D-R isotherm model, the mean free energy was calculated as 26.73 kJ/mol, indicating that the biosorption of cesium was chemisorption. The biosorption process was rapid, and the kinetic rates were best fitted to the pseudo second-order model, which indicated the biosorption process operated through chemisorption mechanism. FT-IR analysis of PFC02 showed the possible functional groups responsible for cesium adsorption were hydroxyl, carboxyl, carbonyl and sulphonate groups. SEM analysis showed the porous structure of the material while EDX analysis confirmed the adsorption of Cs(I) on PFC02. Cesium adsorbed onto the PFC02 could be desorbed efficiently using 1 mol/L HNO3, and the enrichment factor was 50.0. Furthermore, PFC02 could be reused five times with only about 8.25% regeneration loss. The developed method was successfully utilized for the removal of Cs(I) ions from aqueous solution.

DING proteins constitute an intriguing family of phosphate-binding proteins that was identified in a wide range of organisms, from prokaryotes and archae to eukaryotes. Despite their seemingly ubiquitous occurrence in eukaryotes, their encoding genes are missing from sequenced genomes. Such a lack has considerably hampered functional studies. In humans, these proteins have been related to several diseases, like atherosclerosis, kidney stones, inflammation processes and HIV inhibition. The human phosphate binding protein is a human representative of the DING family that was serendipitously discovered from human plasma. An original approach was developed to determine ab initio the complete and exact sequence of this 38 kDa protein by utilizing mass spectrometry and X-ray data in tandem. Taking advantage of this first complete eukaryotic DING sequence, a immunohistochemistry study was undertaken to check the presence of DING proteins in various mice tissues, revealing that these proteins are widely expressed. Finally, the structure of a bacterial representative from Pseudomonas fluorescens was solved at sub-angstrom resolution, allowing the molecular mechanism of the phosphate binding in these high-affinity proteins to be elucidated.

A novel enzyme, induced by choline, ethanolamine, glycine betaine or dimethylglycine, was released at low temperature and phosphate from Pseudomonas fluorescens (CECT 7229) suspensions at low cell densities. It is a CDP-ethanolamine pyrophosphatase/(dihexanoyl)glycerophosphoethanolamine phosphodiesterase (CGDEase) less active on choline derivatives, and inactive on long-chain phospholipids, CDP-glycerol and other NDP-X compounds. The reaction pattern was typical of phospholipase C (PLC), as either phosphoethanolamine or phosphocholine was produced. Peptide-mass analyses, gene cloning and expression provided a molecular identity for CGDEase. Bioinformatic studies assigned it to the PLC branch of the phospholipase C/acid phosphatase (PLC/APase) superfamily, revealed an irregular phylogenetic distribution of close CGDEase relatives, and suggested their genes are not in operons or conserved contexts. A theoretical CGDEase structure was supported by mutagenesis of two predicted active-site residues, which yielded essentially inactive mutants. Biological relevance is supported by comparisons with CGDEase relatives, induction by osmoprotectants (not by osmotic stress itself) and repression by micromolar phosphate. The low bacterial density requirement was related to phosphate liberation from lysed bacteria in denser populations, rather than to a classical quorum-sensing effect. The results fit better a CGDEase role in phosphate scavenging than in osmoprotection., (© 2010 Blackwell Publishing Ltd.)

Soluble microbial products (SMP) in the wastewater treatment process not only cause fouling to the membrane, but also generate disinfection by-products (DBP) in the effluent, thus get increasing attention. In this study, SMP produced by activated sludge and isolates under different stressful conditions, i.e. starvation, salinity, heavy metals, low pH and high temperature, were characterized to investigate the effects of these conditions on the amount of SMP and their compositions. The analysis results using size exclusion chromatography (SEC), high pressure liquid chromatography (HPLC) and fluorescence excitation emission matrix (FEEM) showed that activated sludge and isolates suffered with the same stressful condition contained almost the same concentration and composition of SMP, indicating that the stressful condition instead of the microbial species played the crucial role in the production of SMP. Among of stressful conditions tested, high temperature had stimulated the production of polysaccharides and polycarboxylate-type humic acid with high hydrophilicity, which is in positive proportion to the foulants formation potential, thus should be avoided in membrane bioreactors. Low pH had promoted the generation of hydrophobic humic acid-like or protein-like organics, which had been proved as the main disinfection byproduct (DBP) precursor, thus should be avoided in the biological treatment. Starvation had less effect on SMP production as the seeding microbes had no substrates., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

A biosurfactant with a low critical micelle concentration, CMC (290 mg L(-1)), was produced by a Pseudomonas fluorescens strain using olive oil. Measurements of surface tension and emulsification index (E24) showed a positive effect on alkaline pH and a high level of tolerance to ionic strength of the product. Above the CMC, naphthalene solubility was affected by biosurfactant concentration (3-7 times its aqueous solubility), pH and salinity; for 0.5-1.5 g L(-1) of biosurfactant, pH 7 and no salinity, naphthalene solubility was about 7-fold its aqueous solubility. The solubility reached a saturation value (205 mg L(-1)) when biosurfactant concentration exceeded 1.5 g L(-1). For alkaline pH or high salinity (above 10%) the solubility decreased by more than 50%. The weight solubilization ratio decreased from 0.63 to 0.015 for increasing biosurfactant concentration up to 1.5 g L(-1), alkaline pH or high salinity; and reached a constant value for 4.0 g L(-1) biosurfactant irrespective of pH and salinity (in the range of 0.02-0.05 g L(-1)). In all cases, the solubility of naphthalene in water was enhanced by the biosurfactant addition, showing its potential for application in bioremediation of polycyclic aromatic hydrocarbons contamination in extreme environments., (Copyright 2010 Elsevier B.V. All rights reserved.)

The bacteria fatty acid profile has been extensively studied for taxonomic classification purposes, since bacteria, in general, contain particular and rare fatty acids, compared with animal and plant tissues. As for any real-world sample type, the development of rapid and reliable methods for (i) sample identification (in this case, bacterium type), and (ii) constituent identification (in this instance, the fatty acid profile) is desirable. In this research, a half-an-hour procedure, to analyze bacteria, was developed: a 2-min one-step sample preparation step was followed by a relatively fast comprehensive 2D GC-MS separation (25 min). Furthermore, dedicated MS libraries were constructed for the identification of bacteria and fatty acids. Finally, data processing, only qualitative at this stage, was carried out with the support of a novel comprehensive 2D GC software.

The balance of essential elements (e.g. carbon [C], nitrogen [N], and phosphorus [P]) between consumers and their resources influences not only the growth and reproduction of the consumers but also the nutrients they regenerate. Flagellate protists are significant predators of aquatic bacteria and directly influence nutrient flow to higher trophic levels and, through excretion, influence the mineral element composition of dissolved nutrients. Because the element stoichiometry of protists is poorly characterized, we varied the resource composition of the bacterium Pseudomonas fluorescens and used it to grow the mixotrophic bacterivorous flagellate Ochromonas danica. Using a mass balance approach, the element composition of O. danica was found to vary depending upon the nutrient composition of the prey and ranged between 482:36:1 and 80:12:1 (C:N:P molar). Homeostasis plots suggested that flagellate protists weakly regulate their element composition and are likely to regenerate different elements depending upon the nature of the element limiting growth of their prey.

A study was conducted to understand the role of cell concentration and metabolic state in the transport and deposition behaviour of Pseudomonas fluorescens with and without substrate addition. Column experiments using the short-pulse technique (pulse was equivalent to 0.028 pore volume) were performed in quartz sand operating under saturated conditions. For comparison, experiments with microspheres and inactive (killed) bacteria were also conducted. The effluent concentrations, the retained particle concentrations and the cell shape were determined by fluorescent microscopy. For the transport of metabolically-active P. fluorescens without substrate addition a bimodal breakthrough curve was observed, which could be explained by the different breakthrough behaviour of the rod-shaped and coccoidal cells of P. fluorescens. The 70:30 rod/coccoid ratio in the influent drastically changed during the transport and it was about 20:80 in the effluent and in the quartz sand packing. It was assumed that the active rod-shaped cells were subjected to shrinkage into coccoidal cells. The change from active rod-shaped cells to coccoidal cells could be explained by oxygen deficiency which occurs in column experiments under saturated conditions. Also the substrate addition led to two consecutive breakthrough peaks and to more bacteria being retained in the column. In general, the presence of substrate made the assumed stress effects more pronounced. In comparison to microspheres and inactive (killed) bacteria, the transport of metabolically-active bacteria with and without substrate addition is affected by differences in physiological state between rod-shaped and the formed stress-resistant coccoidal cells of P. fluorescens., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Aims: This study examined the effect of microbial cell-free meat extract (CFME) derived from spoiled meat, in which quorum sensing (QS) compounds were present, on the growth kinetics (lag phase, and growth rate) of two spoilage bacteria, Pseudomonas fluorescens and Serratia marcescens., Methods and Results: Aliquots of CFME from spoiled meat were transferred to Brain Heart Infusion broth inoculated with 10(3) CFU ml(-1) of 18 h cultures of Ps. fluorescens or Ser. marcescens, both fresh meat isolates; CFME derived from unspoiled fresh meat ('clean' meat) served as a control. Changes in impedance measurements were monitored for 48 h, and the detection time (Tdet) was recorded. It was found that in the absence of CFME containing QS compounds the Tdet was shorter (P < 0.05) than that in broth samples with added CFME from spoiled meat. The rate of growth of Ps. fluorescens, recorded as the maximum slope rate of conductance changes (MSrCC), after Tdet, was higher (P < 0.05) in samples with CFME containing QS compounds compared to samples without CFME or CFME derived from 'clean' meat. Similar results in MSrCC of impedance changes were obtained for Ser. marcescens., Conclusions: The study indicated that the growth rate (expressed in MSrCC units) of meat spoilage bacteria in vitro was enhanced in samples supplemented with CFME containing QS compounds compared to control samples (i.e., without CFME or with CFME from 'clean' meat). This behaviour may explain the dominant role of these two bacteria in the spoilage of meat., Significance and Impact of the Study: These results illustrate the potential effect of signalling compounds released during storage of meat on the behaviour of meat spoilage bacteria. Understanding such interactions may assist in the control of fresh meat quality and the extension of its shelf life.

Two types of bacterial biosensor were constructed by immobilization of Gluconobacter oxydans and Pseudomonas fluorescens cells on graphite electrodes modified with the conducting polymer; poly(1-(4-nitrophenyl)-2,5-di(2-thienyl)-1 H-pyrrole) [SNS(NO(2))]. The measurement was based on the respiratory activity of cells estimated by the oxygen consumption at -0.7 V due to the metabolic activity in the presence of substrate. As well as analytical characterization, the linear detection ranges, effects of electropolymerization time, pH and cell amount were examined by using glucose as the substrate. The linear relationships were observed in the range of 0.25-4.0 mM and 0.2-1.0 mM for G. oxydans and P. fluorescens based sensors, respectively.

Ingestion and growth rates of the nanoflagellate predator Ochromonas danica feeding on the bacterium Pseudomonas fluorescens were quantified in laboratory cultures. Bacterial prey were grown under four nutritional conditions with respect to macronutrient elements: C-limited, N-limited, P-limited, and balanced. Ingestion and growth rates were saturating functions of prey abundance when preying upon nutritionally balanced, C-limited, and P-limited bacteria but were unimodal functions of abundance when preying on N-limited bacteria. At saturating prey concentrations, the ingestion rate of C-limited prey was about twice that of prey in other nutritional states, while at subsaturating prey concentrations, the ingestion rates of both C- and N-limited prey were higher than those of prey in other nutritional states. Over all prey concentrations, growth was most rapid on balanced and C-limited prey and generally lowest for P-limited prey. Due to the unimodal response of growth rate to abundance of N-limited prey, growth rate on N-limited prey approached that obtained on balanced and C-limited prey when prey were available at intermediate abundances. The accumulation of recycled N increased with the growth rate of O. danica. Recycling of N was highest when O. danica was feeding upon P-limited prey. The accumulation of recycled P increased with growth rate for balanced and N-limited prey, but not for P-limited prey, which consistently had low accumulation of recycled P. The low growth rate and negligible recycling of P for O. danica preying on P-limited prey is consistent with the theory of ecological stoichiometry and resembles results found for crustacean zooplankton, especially in the genus Daphnia. Potentially, the major predators of bacterioplankton and a major predator of phytoplankton play analogous roles in the trophic dynamics and biogeochemistry of aquatic ecosystems.

PfluDING is a bacterial protein isolated from Pseudomonas fluorescens that belongs to the DING protein family, which is ubiquitous in eukaryotes and extends to prokaryotes. DING proteins and PfluDING have very similar topologies to phosphate Solute Binding Proteins (SBPs). The three-dimensional structure of PfluDING was obtained at subangstrom resolution (0.88 and 0.98 A) at two different pH's (4.5 and 8.5), allowing us to discuss the hydrogen bond network that sequesters the phosphate ion in the binding site. From this high resolution data, we experimentally elucidated the molecular basis of phosphate binding in phosphate SBPs. The phosphate ion is tightly bound to the protein via 12 hydrogen bonds between phosphate oxygen atoms and OH and NH groups of the protein. The proton on one oxygen atom of the phosphate dianion forms a 2.5 A low barrier hydrogen bond with an aspartate, with the energy released by forming this strong bond ensuring the specificity for the dianion even at pH 4.5. In particular, contrary to previous theories on phosphate SBPs, accurate electrostatic potential calculations show that the binding cleft is positively charged. PfluDING structures reveal that only dibasic phosphate binds to the protein at both acidic and basic phosphate, suggesting that the protein binding site environment stabilizes the HPO(4)(2-) form of phosphate.

Pseudomonads are able to form a variety of biofilms that colonize the air-liquid (A-L) interface of static liquid microcosms, and differ in matrix composition, strength, resilience and degrees of attachment to the microcosm walls. From Pseudomonas fluorescens SBW25, mutants have evolved during prolonged adaptation-evolution experiments which produce robust biofilms of the physically cohesive class at the A-L interface, and which have been well characterized. In this study we describe a novel A-L interface biofilm produced by SBW25 that is categorized as a viscous mass (VM)-class biofilm. Several metals were found to induce this biofilm in static King's B microcosms, including copper, iron, lead and manganese, and we have used iron to allow further examination of this structure. Iron was demonstrated to induce SBW25 to express cellulose, which provided the matrix of the biofilm, a weak structure that was readily destroyed by physical disturbance. This was confirmed in situ by a low (0.023-0.047 g) maximum deformation mass and relatively poor attachment as measured by crystal violet staining. Biofilm strength increased with increasing iron concentration, in contrast to attachment levels, which decreased with increasing iron. Furthermore, iron added to mature biofilms significantly increased strength, suggesting that iron also promotes interactions between cellulose fibres that increase matrix interconnectivity. Whilst weak attachment is important in maintaining the biofilm at the A-L interface, surface-interaction effects involving cellulose, which reduced surface tension by approximately 3.8 mN m(-1), may also contribute towards this localization. The fragility and viscoelastic nature of the biofilm were confirmed by controlled-stress amplitude sweep tests to characterize critical rheological parameters, which included a shear modulus of 0.75 Pa, a zero shear viscosity of 0.24 Pa s(-1) and a flow point of 0.028 Pa. Growth and morphological data thus far support a non-specific metal-associated physiological, rather than mutational, origin for production of the SBW25 VM biofilm, which is an example of the versatility of bacteria to inhabit optimal niches within their environment.

Nitrile groups are catabolized to the corresponding acid and ammonia through one-step reaction involving a nitrilase. Here, we report the use of bioinformatic and biochemical tools to identify and characterize the nitrilase (NitPf5) from Pseudomonas fluorescens Pf-5. The nitPf5 gene was identified via sequence analysis of the whole genome of P. fluorescens Pf-5 and subsequently cloned and overexpressed in Escherichia coli. DNA sequence analysis revealed an open-reading frame of 921 bp, capable of encoding a polypeptide of 307 amino acids residues with a calculated isoelectric point of pH 5.4. The enzyme had an optimal pH and temperature of 7.0 degrees C and 45 degrees C, respectively, with a specific activity of 1.7 and 1.9 micromol min(-1) mg protein(-1) for succinonitrile and fumaronitrile, respectively. The molecular weight of the nitrilase as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography was 33,000 and 138,000 Da, respectively, suggesting that the enzyme is homotetrameric. Among various nitriles, dinitriles were the preferred substrate of NitPf5 with a K (m) = 17.9 mM and k (cat)/K (m) = 0.5 mM(-1) s(-1) for succinonitrile. Homology modeling and docking studies of dinitrile and mononitrile substrate into the active site of NitPf5 shed light on the substrate specificity of NitPf5. Although nitrilases have been characterized from several other sources, P. fluorescens Pf-5 nitrilase NitPf5 is distinguished from other nitrilases by its high specific activity toward dinitriles, which make P. fluorescens NitPf5 useful for industrial applications, including enzymatic synthesis of various cyanocarboxylic acids.

The rhizobacterium Pseudomonas fluorescens CHA0 is able to antagonize fungal phytopathogens on a variety of crop plants, mainly due to the production of secondary metabolites that are coordinately upregulated by the global regulatory Gac/Rsm cascade. The two-component system GacS/GacA activates transcription of the three small regulatory RNAs RsmX, RsmY, and RsmZ, which counteract translational repression of target mRNAs by RsmA and RsmE proteins. In a search for novel Gac/Rsm targets based on the minimal sequence on mRNA leaders required for RsmA/RsmE control, the leader region of the major porin OprF emerged as a candidate. Although an isogenic CHA0 oprF mutant showed a reduced ability to attach to cucumber and tomato roots, suggesting a role for OprF in root colonization as a requisite for pathogen antagonism, a translational oprF'-'lacZ fusion was weakly regulated by Gac/Rsm despite its high sequence similarity to the hcnA leader. A single base substitution put the modified oprF 5'-UTR under strict control by Gac/Rsm. The results highlight the subtle sequence requirements of Gac/Rsm targets.

The Baeyer-Villiger monooxygenase (BVMO) BmoF1 from Pseudomonas fluorescens DSM 50106 was shown before to enantioselectively oxidize different 4-hydroxy-2-ketones to the corresponding hydroxyalkyl acetates, being the first example of a BVMO-catalyzed kinetic resolution of aliphatic acyclic ketones. However, the wild-type enzyme exhibited only moderate E values (E approximately 55). Thus, the enantioselectivity was enhanced by means of directed evolution and optimization of reaction conditions since it was found that higher E values (E approximately 70 for wild-type BmoF1) could already be obtained when performing biotransformations in shake flasks rather than small tubes. In a first step, random mutations were introduced by error-prone polymerase chain reaction, and BmoF1 mutants (>3,500 clones) were screened for improved activity and enantioselectivity using a microtiter-plate-based screening method. Mutations S136L and L252Q were found to increase conversion compared to wild type, while several mutations (H51L, F225Y, S305C, and E308V) were identified enhancing the enantioselectivity to a varying extent (E approximately 75-90). In a second step, beneficial mutations were recombined by consecutive cycles of QuikChange site-directed mutagenesis resulting in a double mutant (H51L/S136L) showing both improved conversion and enantioselectivity (E approximately 86).

Production of biosurfactant by free and alginate-entrapped cells of Pseudomonas fluorescens Migula 1895-DSMZ was investigated using olive oil as the sole carbon and energy source. Biosurfactant synthesis was followed by measuring surface tension and emulsifying index E24 over 5 days at ambient temperature and at neutral pH. Diffusional limitations in alginate beads affected the kinetics of biosurfactant production when compared to that obtained with free cells culture. Nevertheless, the emulsion stability was improved and fewer by-products interfered with the biosurfactant activity. A decrease in pH down to 5 in the case of immobilized cells was observed during the first 3 days, after which it returned to its initial value. The minimum values of surface tension were 30 and 35 dynes cm(-1) achieved after 40 and 72 h with free and immobilized cells, respectively, while the corresponding maximum E24 values were 67 and 62%, respectively. After separation by acetone precipitation, the biosurfactant showed a rhamnolipid-type in nature, and had a good foaming and emulsifying activities. The critical micellar concentration was found to be 290 mg l(-1). The biosurfactant also showed good stability during exposure to high temperatures (up to 120 degrees C for 15 min), to high salinity (10% NaCl) and to a wide range of pH (4-9).

The cells of psychrotrophic Pseudomonas fluorescens BM07 were found to secrete large amounts of exobiopolymer (EBP) composed of mainly hydrophobic (water insoluble) polypeptide(s) (as contain approximately 50 mol% hydrophobic amino acids, lacking cysteine residue) when grown on fructose containing limited M1 medium at the temperatures as low as 0-10 degrees C but trace amount at high (30 degrees C, optimum growth) temperature. Two types of nonliving BM07 cells (i.e., cells grown at 30 degrees C and 10 degrees C) as well as the freeze-dried EBP were compared for biosorption of mercury (Hg(II)) and cadmium (Cd(II)). The optimum adsorption pH was found 7 for Hg(II) but 6 for Cd(II), irrespective of the type of biomass. Equilibrium adsorption data well fitted the Langmuir adsorption model. The maximum adsorption (Q(max)) was 72.3, 97.4, and 286.2 mg Hg(II)/g dry biomass and 18.9, 27.0, and 61.5 mg Cd(II)/g dry biomass for cells grown at 30 degrees C and 10 degrees C and EBP, respectively, indicating major contribution of heavy metal adsorption by cold-induced EBP. Mercury(II) binding induced a significant shift of infrared (IR) amide I and II absorption of EBP whereas cadmium(II) binding showed only a very little shift. These IR shifts demonstrate that mercury(II) and cadmium(II) might have different binding sites in EBP, which was supported by X-ray diffraction and differential scanning calorimetric analysis and sorption results of chemically modified biomasses. This study implies that the psychrotrophs like BM07 strain may play an important role in the bioremediation of heavy metals in the temperate regions especially in the inactive cold season.

The use of biosurfactants is a promising alternative in biological control of zoospore-producing plant pathogens. In the present study, biosurfactant production by the indigenous population of fluorescent pseudomonads in a soilless plant cultivation system was studied during the growing season. A total of 600 strains was screened and of these 18.5% were observed to produce biosurfactants. Production of both antibiotics and biosurfactant was uncommon among the isolated strains. A selective effect of the cultivation system filter was observed on the biosurfactant-producing strains and these strains were only occasionally observed after the filter, despite having a significantly higher motility than the nonbiosurfactant-producing strains. The majority of biosurfactant-producing strains were isolated from the filter skin, which suggests that this is a suitable surface for inoculation with biocontrol strains.

The products synthesized from a hybrid polyketide synthase/nonribosomal peptide synthetase gene cluster in the genome of Pseudomonas fluorescens Pf-5 were identified using a genomics-guided strategy involving insertional mutagenesis and subsequent metabolite profiling. Five analogs of rhizoxin, a 16-member macrolide with antifungal, phytotoxic, and antitumor activities, were produced by Pf-5, but not by a mutant with an insertion in the gene cluster. The five rhizoxin analogs, one of which had not been described previously, were differentially toxic to two agriculturally important plant pathogens, Botrytis cinerea and Phytophthora ramorum. The rhizoxin analogs also caused swelling of rice roots, a symptom characteristic of rhizoxin itself, but were less toxic to pea and cucumber roots. Of the rhizoxin analogs produced by Pf-5, the predominant compound, WF-1360 F, and the newly described compound 22Z-WF-1360 F were most toxic against the two plant pathogens and three plant species. These rhizoxin analogs were tested against a panel of human cancer lines, and they exhibited potent but nonselective cytotoxicity. This study highlights the value of the genomic sequence of the soil bacterium P. fluorescens Pf-5 in providing leads for the discovery of novel metabolites with significant biological properties.