Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

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    Listeria monocytogenes repellence by enzymatically modified PES surfaces
    Veen, S. van der; Nady, N. ; Franssen, M.C.R. ; Zuilhof, H. ; Boom, R.M. ; Abee, T. ; Schroën, C.G.P.H. - \ 2015
    Journal of Applied Polymer Science 132 (2015)10. - ISSN 0021-8995 - 6 p.
    stainless-steel - catalyzed modification - biofilm formation - attachment - growth - membranes - water - acid - functionalization - hydrophobicity
    : The effect of enzyme-catalyzed modification of poly(ethersulfone) (PES) on the adhesion and biofilm formation of two Listeria monocytogenes strains is evaluated under static and dynamic flow conditions. PES has been modified with gallic acid, ferulic acid and 4-hydroxybenzoic acid. The surfaces modified with any of these compounds show up to 70% reduced adhesion of L. mono-cytogenes under static conditions and up to 95% under dynamic flow conditions compared with unmodified surfaces. Also, under static conditions the formation of biofilms is reduced by 70%. These results indicate that the brush structures that are formed by the polymers on the PES surface directly influence the ability of microorganisms to interact with the surface, thereby reducing attachment and biofilm formation of L. monocytogenes. Based on these results, it is expected that enzyme-catalyzed surface modification is a promising tool to reduce microbial adhesion and biofilm formation
    Chemical characterization and biological activity of Chaga (Inonotus obliquus) a medicinal "mushroom"
    Glamoclija, J. ; Ciric, A. ; Nikolic, M. ; Fernandes, A. ; Barros, L. ; Calhelha, R.C. ; Ferreira, I.C.F.R. ; Sokovic, M. ; Griensven, L.J.L.D. van - \ 2015
    Journal of Ethnopharmacology 162 (2015). - ISSN 0378-8741 - p. 323 - 332.
    pseudomonas-aeruginosa - swarming motility - in-vitro - biofilm formation - edible mushrooms - social evolution - aqueous extract - gene-expression - hot-water - antioxidant
    ETHNOPHARMACOLOGICAL RELEVANCE: In Russian traditional medicine, an extract from the mushroom Inonotus obliquus (Fr.) Pil´at is used as an anti-tumor medicine and diuretic. It has been reported that Inonotus obliquus has therapeutic effects, such as anti-inflammatory, immuno-modulatory and hepatoprotective effects. This study was designed to investigate the chemical composition and biological properties of aqueous and ethanolic extracts of Inonotus obliquus from Finland, Russia, and Thailand. Their antioxidative, antimicrobial, and antiquorum properties were tested as well as the cytotoxicity on various tumor cell lines. MATERIALS AND METHODS: The tested extract was subjected to conventional chemical study to identified organic acids and phenolic compounds. Antioxidative activity was measured by several different assays. Antimicrobial potential of extracts was tested by microdilution method, and antiquorum sensing activity and antibiofilm formation of Inonotus obliquus extracts was tested on Pseudomonas aeruginosa. Cytotoxicity of the extracts was tested on tumor cells (MCF-7, NCI-H460, HeLa and HepG2) and non-tumor liver cells primary cultures. RESULTS: Oxalic acid was found as the main organic acid, with the highest amount in the aqueous extract from Russia. Gallic, protocatechuic and p-hydroxybenzoic acids were detected in all samples. Inonotus obliquus extracts showed high antioxidant and antimicrobial activity. Extracts were tested at subMIC for anti-quorum sensing (AQS) activity in Pseudomonas aeruginosa and all extracts showed definite AQS activity. The assays were done using twitching and swarming of bacterial cultures, and the amount of produced pyocyanin as QS parameters. All the extracts demonstrated cytotoxic effect on four tumor cell lines and not on primary porcine liver cells PLP2. CONCLUSIONS: As the Inonotus obliquus presence in Chaga conks is limited, further purification is necessary to draw quantitative conclusions. The presence of AQS activity in medicinal mushrooms suggests a broader anti-infectious disease protection than only immunomodulatory effects.
    The Freshwater Sponge Ephydatia fluviatilis Harbours Diverse Pseudomonas Species (Gammaproteobacteria, Pseudomonadales) with Broad-Spectrum Antimicrobial Activity
    Keller-Costa, T. ; Jousset, A. ; Overbeek, L.S. van; Elsas, J.D. ; Costa, R. - \ 2014
    PLoS ONE 9 (2014)2. - ISSN 1932-6203
    fluorescent pseudomonads - secondary metabolites - phenotypic variation - bacterial symbiont - biological-control - biofilm formation - small rnas - sp nov. - rhizosphere - community
    Bacteria are believed to play an important role in the fitness and biochemistry of sponges (Porifera). Pseudomonas species (Gammaproteobacteria, Pseudomonadales) are capable of colonizing a broad range of eukaryotic hosts, but knowledge of their diversity and function in freshwater invertebrates is rudimentary. We assessed the diversity, structure and antimicrobial activities of Pseudomonas spp. in the freshwater sponge Ephydatia fluviatilis. Polymerase Chain Reaction - Denaturing Gradient Gel Electrophoresis (PCR-DGGE) fingerprints of the global regulator gene gacA revealed distinct structures between sponge-associated and free-living Pseudomonas communities, unveiling previously unsuspected diversity of these assemblages in freshwater. Community structures varied across E. fluviatilis specimens, yet specific gacA phylotypes could be detected by PCR-DGGE in almost all sponge individuals sampled over two consecutive years. By means of whole-genome fingerprinting, 39 distinct genotypes were found within 90 fluorescent Pseudomonas isolates retrieved from E. fluviatilis. High frequency of in vitro antibacterial (49%), antiprotozoan (35%) and anti-oomycetal (32%) activities was found among these isolates, contrasting less-pronounced basidiomycetal (17%) and ascomycetal (8%) antagonism. Culture extracts of highly predation-resistant isolates rapidly caused complete immobility or lysis of cells of the protozoan Colpoda steinii. Isolates tentatively identified as P. jessenii, P. protegens and P. oryzihabitans showed conspicuous inhibitory traits and correspondence with dominant sponge-associated phylotypes registered by cultivation-independent analysis. Our findings suggest that E. fluviatilis hosts both transient and persistent Pseudomonas symbionts displaying antimicrobial activities of potential ecological and biotechnological value.
    Agaricus Blazei Hot Water Extract Shows Anti Quorum Sensing Activity in the Nosocomial Human PathogenPseudomonas Aeruginosa
    Sokovic, M. ; Ciric, A. ; Glamoclija, J. ; Nicolic, M. ; Griensven, L.J.L.D. van - \ 2014
    Molecules 19 (2014). - ISSN 1420-3049 - p. 4189 - 4199.
    medicinal mushroom - biofilm formation - inhibition - resistance - infection - virulence - impact - mice
    The edible mushroom Agaricus blazei Murill is known to induce protective immunomodulatory action against a variety of infectious diseases. In the present study we report potential anti-quorum sensing properties of A. blazei hot water extract. Quorum sensing (QS) plays an important role in virulence, biofilm formation and survival of many pathogenic bacteria, including the Gram negative Pseudomonas aeruginosa, and is considered as a novel and promising target for anti-infectious agents. In this study, the effect of the sub-MICs of Agaricus blazei water extract on QS regulated virulence factors and biofilm formation was evaluated against P. aeruginosa PAO1. Sub-MIC concentrations of the extract which did not kill P. aeruginosa nor inhibited its growth, demonstrated a statistically significant reduction of virulence factors of P. aeruginosa, such as pyocyanin production, twitching and swimming motility. The biofilm forming capability of P. aeruginosa was also reduced in a concentration-dependent manner at sub-MIC values. Water extract of A. blazei is a promising source of antiquorum sensing and antibacterial compounds. Keywords: Agaricus blazei; mushroom; antiqourum sensing activity; antimicrobial activity; antibiofilm activity; Pseudomonas aeruginosa.
    Explaining Bacterial Dispersion on Leaf Surfaces with an Individual-Based Model (PHYLLOSIM)
    Wal, A. van der; Tecon, R. ; Kreft, J.U. ; Mooij, W.M. ; Leveau, J.H.J. - \ 2013
    PLoS ONE 8 (2013)10. - ISSN 1932-6203
    plant-microbe interactions - pseudomonas-syringae - biofilm formation - water availability - growth - detachment - diffusion - motility - colonization - wettability
    We developed the individual-based model PHYLLOSIM to explain observed variation in the size of bacterial clusters on plant leaf surfaces (the phyllosphere). Specifically, we tested how different 'waterscapes' impacted the diffusion of nutrients from the leaf interior to the surface and the growth of individual bacteria on these nutrients. In the 'null' model or more complex 'patchy' models, the surface was covered with a continuous water film or with water drops of equal or different volumes, respectively. While these models predicted the growth of individual bacterial immigrants into clusters of variable sizes, they were unable to reproduce experimentally derived, previously published patterns of dispersion which were characterized by a much larger variation in cluster sizes and a disproportionate occurrence of clusters consisting of only one or two bacteria. The fit of model predictions to experimental data was about equally poor (
    Functional analysis of the ComK protein of Bacillus coagulans
    Kovács, Á.T. ; Eckhardt, T.H. ; Hartskamp, M. van; Kranenburg, R. van; Kuipers, O.P. - \ 2013
    PLoS ONE 8 (2013)1. - ISSN 1932-6203
    competence transcription factor - gram-positive bacteria - subtilis k-state - gene-expression - natural competence - biofilm formation - genome sequence - lactic-acid - dna uptake - transformation
    The genes for DNA uptake and recombination in Bacilli are commonly regulated by the transcriptional factor ComK. We have identified a ComK homologue in Bacillus coagulans, an industrial relevant organism that is recalcitrant for transformation. Introduction of B. coagulans comK gene under its own promoter region into Bacillus subtilis comK strain results in low transcriptional induction of the late competence gene comGA, but lacking bistable expression. The promoter regions of B. coagulans comK and the comGA genes are recognized in B. subtilis and expression from these promoters is activated by B. subtilis ComK. Purified ComK protein of B. coagulans showed DNA-binding ability in gel retardation assays with B. subtilis- and B. coagulans-derived probes. These experiments suggest that the function of B. coagulans ComK is similar to that of ComK of B. subtilis. When its own comK is overexpressed in B. coagulans the comGA gene expression increases 40-fold, while the expression of another late competence gene, comC is not elevated and no reproducible DNA-uptake could be observed under these conditions. Our results demonstrate that B. coagulans ComK can recognize several B.subtilis comK-responsive elements, and vice versa, but indicate that the activation of the transcription of complete sets of genes coding for a putative DNA uptake apparatus in B. coagulans might differ from that of B. subtilis
    Ex vivo transcriptional profiling reveals a common set of genes important for the adaptation of Pseudomonas aeruginosa to chronically infected host sites
    Bielecki, P. ; Komor, U. ; Bielecka, A. ; Müsken, M. ; Puchalka, J. ; Pletz, M.W. ; Ballmann, M. ; Martins Dos Santos, V.A.P. ; Weiss, S. ; Häussler, S. - \ 2013
    Environmental Microbiology 15 (2013)2. - ISSN 1462-2912 - p. 570 - 587.
    burn wound infections - biofilm formation - cystic-fibrosis - therapeutic strategies - expression - motility - mutants - protein - system - identification
    The opportunistic bacterium Pseudomonas aeruginosa is a major nosocomial pathogen causing both devastating acute and chronic persistent infections. During the course of an infection, P.¿ aeruginosa rapidly adapts to the specific conditions within the host. In the present study, we aimed at the identification of genes that are highly expressed during biofilm infections such as in chronically infected lungs of patients with cystic fibrosis (CF), burn wounds and subcutaneous mouse tumours. We found a common subset of differentially regulated genes in all three in vivo habitats and evaluated whether their inactivation impacts on the bacterial capability to form biofilms in vitro and to establish biofilm-associated infections in a murine model. Additive effects on biofilm formation and host colonization were discovered by the combined inactivation of several highly expressed genes. However, even combined inactivation was not sufficient to abolish the establishment of an infection completely. These findings can be interpreted as evidence that either redundant traits encode functions that are essential for in vivo survival and chronic biofilm infections and/or bacterial adaptation is considerably achieved independently of transcription levels. Supplemental screens, will have to be applied in order to identify the minimal set of key genes essential for the establishment of chronic infectious diseases
    Bacterial canker on kiwifruit in Italy: Anatomical changes in the wood and in the primary infection sites
    Renzi, M. ; Copini, P. ; Taddei, A.R. ; Rossetti, A. ; Gallipoli, L. ; Mazzaglia, A. ; Balestra, G.M. - \ 2012
    Phytopathology 102 (2012)9. - ISSN 0031-949X - p. 827 - 840.
    syringae pv. actinidiae - pseudomonas-syringae - biofilm formation - innate immunity - diseases - trees - expression - dynamics - tyloses - stomata
    The bacterial canker of kiwifruit caused by Pseudomonas syringae pv. actinidiae is a severe threat to kiwifruit production worldwide. Many aspects of P. syringae pv. actinidiae biology and epidemiology still require in-depth investigation. The infection by and spread of P. syringae pv. actinidiae in xylem and phloem was investigated by carrying out artificial inoculation experiments with histological and dendrochronological analyses of naturally diseased plants in Italy. We found that the bacterium can infect host plants by entering natural openings and lesions. In naturally infected kiwifruit plants, P. syringae pv. actinidiae is present in the lenticels as well as in the dead phloem tissue beneath the lenticels, surrounded by a lesion in the periderm which appears to indicate the importance of lenticels to kiwifruit infection. Biofilm formation was observed outside and inside plants. In cases of advanced stages of P. syringae pv. actinidiae infection, neuroses of the phloem occur, which are followed by cambial dieback and most likely by infection of the xylem. Anatomical changes in wood such as reduced ring width, a drastic reduction in vessel size, and the presence of tyloses were observed within several infected sites. In the field, these changes occur only a year after the first leaf symptoms are observed suggesting a significant time lapse between primary and secondary symptoms. It was possible to study the temporal development of P. syringae pv. actinidiae-induced cambial dieback by applying dendrochronology methods which revealed that cambial dieback occurs only during the growing season.
    Functional Analysis of Lactobacillus rhamnosus GG Pili in Relation to Adhesion and Immunomodulatory Interactions with Intestinal Epithelial Cells
    Lebeer, S. ; Claes, I.J. ; Tytgat, H.L.P. ; Verhoeven, T.L.A. ; Marien, E. ; Ossowski, I. von; Reunanen, J. ; Palva, A. ; Vos, W.M. de; Keersmaecker, S.C. de; Vanderleyden, J. - \ 2012
    Applied and Environmental Microbiology 78 (2012)1. - ISSN 0099-2240 - p. 185 - 193.
    gram-positive bacteria - gene-expression - salmonella-typhimurium - lipoteichoic acid - biofilm formation - reveals - protein - inflammation - molecules - pathogens
    Lactobacillus rhamnosus GG, a probiotic with good survival capacity in the human gut, has well-documented adhesion properties and health effects. Recently, spaCBA-encoded pili that bind to human intestinal mucus were identified on its cell surface. Here, we report on the phenotypic analysis of a spaCBA pilus knockout mutant in comparison with the wild type and other adhesin mutants. The SpaCBA pilus of L. rhamnosus GG showed to be key for efficient adherence to the Caco-2 intestinal epithelial cell (IEC) line and biofilm formation. Moreover, the spaCBA mutant induces an elevated level of interleukin-8 (IL-8) mRNA in Caco-2 cells compared to the wild type, possibly involving an interaction of lipoteichoic acid with Toll-like receptor 2. In contrast, an L. rhamnosus GG mutant without exopolysaccharides but with an increased exposure of pili leads to the reduced expression of IL-8. Using Transwells to partition bacteria from Caco-2 cells, IL-8 induction is blocked completely regardless of whether wild-type or mutant L. rhamnosus GG cells are used. Taken together, our data suggest that L. rhamnosus GG SpaCBA pili, while promoting strong adhesive interactions with IECs, have a functional role in balancing IL-8 mRNA expression induced by surface molecules such as lipoteichoic acid
    Involvement of phenazines and lipopeptides in interactions between Pseudomonas species and Sclerotium rolfsii, causal agent of stem rot disease on groundnut
    Le, N.C. ; Kruijt, M. ; Raaijmakers, J. - \ 2012
    Journal of Applied Microbiology 112 (2012)2. - ISSN 1364-5072 - p. 390 - 403.
    rhizoctonia root-rot - arachis-hypogaea l. - biological-control - fluorescent pseudomonads - antibiotic production - biocontrol agents - biofilm formation - in-vitro - biosynthesis - peanut
    Aims: To determine the role of phenazines (PHZ) and lipopeptide surfactants (LPs) produced by Pseudomonas in suppression of stem rot disease of groundnut, caused by the fungal pathogen Sclerotium rolfsii. Methods and Results: In vitro assays showed that PHZ-producing Pseudomonas chlororaphis strain Phz24 significantly inhibited hyphal growth of S. rolfsii and suppressed stem rot disease of groundnut under field conditions. Biosynthesis and regulatory mutants of Phz24 deficient in PHZ production were less effective in pathogen suppression. Pseudomonas strains SS101, SBW25, and 267, producing viscosin or putisolvin-like LPs, only marginally inhibited hyphal growth of S. rolfsii and did not suppress stem rot disease. In contrast, Pseudomonas strain SH-C52, producing the chlorinated LP thanamycin, inhibited hyphal growth of S. rolfsii and significantly reduced stem rot disease of groundnut in nethouse and field experiments, whereas its thanamycin-deficient mutant was less effective. Conclusions: Phenazines and specific lipopeptides play an important role in suppression of stem rot disease of groundnut by root-colonizing Pseudomonas strains. Significance and Impact of the Study: Pseudomonas strains Phz24 and SH-C52 showed significant control of stem rot disease. Treatment of seeds or soil with these strains provides a promising supplementary strategy to control stem rot disease of groundnut
    Effect of conventional chemical treatment on the microbial population in a biofouling layer of reverse osmosis systems
    Bereschenko, L.A. ; Prummel, H. ; Euverink, G.J.W. ; Stams, A.J.M. ; Loosdrecht, M.C.M. van - \ 2011
    Water Research 45 (2011)2. - ISSN 0043-1354 - p. 405 - 416.
    biofilm formation - sphingomonas-paucimobilis - community structure - membrane systems - exopolysaccharide - bacterium - pressure
    The impact of conventional chemical treatment on initiation and spatiotemporal development of biofilms on reverse osmosis (RO) membranes was investigated in situ using flow cells placed in parallel with the RO system of a full-scale water treatment plant. The flow cells got the same feed (extensively pre-treated fresh surface water) and operational conditions (temperature, pressure and membrane flux) as the full-scale installation. With regular intervals both the full-scale RO membrane modules and the flow cells were cleaned using conventional chemical treatment. For comparison some flow cells were not cleaned. Sampling was done at different time periods of flow cell operation (i.e., 1, 5, 10 and 17 days and 1, 3, 6 and 12 months). The combination of molecular (FISH, DGGE, clone libraries and sequencing) and microscopic (field emission scanning electron, epifluorescence and confocal laser scanning microscopy) techniques made it possible to thoroughly analyze the abundance, composition and 3D architecture of the emerged microbial layers. The results suggest that chemical treatment facilitates initiation and subsequent maturation of biofilm structures on the RO membrane and feed-side spacer surfaces. Biofouling control might be possible only if the cleaning procedures are adapted to effectively remove the (dead) biomass from the RO modules after chemical treatment
    In-Vivo Expression Profiling of Pseudomonas aeruginosa Infections Reveals Niche-Specific and Strain-Independent Transcriptional Programs
    Bielecki, P. ; Puchalka, J. ; Wos-Oxley, M.L. ; Martins Dos Santos, V.A.P. - \ 2011
    PLoS ONE 6 (2011). - ISSN 1932-6203 - 11 p.
    metabolic network analysis - burn wound infections - gene-expression - opportunistic pathogen - virulence factors - biofilm formation - microarray data - plant hosts - identification - carbon
    Pseudomonas aeruginosa is a threatening, opportunistic pathogen causing disease in immunocompromised individuals. The hallmark of P. aeruginosa virulence is its multi-factorial and combinatorial nature. It renders such bacteria infectious for many organisms and it is often resistant to antibiotics. To gain insights into the physiology of P. aeruginosa during infection, we assessed the transcriptional programs of three different P. aeruginosa strains directly after isolation from burn wounds of humans. We compared the programs to those of the same strains using two infection models: a plant model, which consisted of the infection of the midrib of lettuce leaves, and a murine tumor model, which was obtained by infection of mice with an induced tumor in the abdomen. All control conditions of P. aeruginosa cells growing in suspension and as a biofilm were added to the analysis. We found that these different P. aeruginosa strains express a pool of distinct genetic traits that are activated under particular infection conditions regardless of their genetic variability. The knowledge herein generated will advance our understanding of P. aeruginosa virulence and provide valuable cues for the definition of prospective targets to develop novel intervention strategies
    Utilization of oligo- and polysaccharides at microgram-per-litre levels in freshwater by Flavobacterium johnsoniae
    Sack, E.L.W. ; Wielen, P.W.J.J. van der; Kooij, D. van der - \ 2010
    Journal of Applied Microbiology 108 (2010)4. - ISSN 1364-5072 - p. 1430 - 1440.
    assimilable organic-carbon - drinking-water - extracellular polysaccharides - biofilm formation - potable water - phytoplankton - matter - bacteria - enumeration - dynamics
    Aims: To obtain a bacterial strain that can be used to quantify biodegradable polysaccharides at concentrations of a few micrograms per litre in freshwater. Methods and Results: Flavobacterium johnsoniae strain A3 was isolated from tap water supplemented with laminarin, pectin or amylopectin at 100 µg C l-1 and river Rhine water. The organism utilized 14 of 23 oligo- and polysaccharides, and 1 of 9 monosaccharides, but none of the sugar acids, sugar alcohols, carboxylic acids or aromatic acids tested at 10 µg C l-1. Amino acids promoted growth of strain A3, but not in coculture with assimilable organic carbon (AOC) test strain Pseudomonas fluorescens P17, which utilized these compounds more rapidly than strain A3. Compounds released by strain P17 and AOC test strain Spirillum sp. NOX grown on acetate promoted the growth of strain A3 at Nmax values of = 2 × 105 CFU ml-1 of strain P17 and = 5 × 105 CFU ml-1 of strain NOX. Significant growth of strain A3 was observed in surface water and in tap water in the presence of strain P17 (Nmax P17 <2 × 105 CFU ml-1). Conclusions: Strain A3 utilizes oligo- and polysaccharides at microgram-per-litre levels. In surface water and in tap water, the organism was able to utilize compounds that were not utilized by strain P17. These compounds may include oligo- and/or polysaccharides. Significance and Impact of the Study: Phytoplanktonic and bacterial polysaccharides can constitute an important biodegradable fraction of natural organic matter in water and may promote growth of heterotrophic bacteria during water treatment and drinking water distribution. Strain A3 can be used to quantify a group of compounds that includes oligo- and polysaccharides at microgram-per-litre levels in freshwater
    Protozoan-induced regulation of cyclic lipopeptide biosynthesis Is an effective predation defense mechanism for Pseudomonas fluorescens
    Mazzola, M. ; Bruijn, I. de; Cohen, M.F. ; Raaijmakers, J.M. - \ 2009
    Applied and Environmental Microbiology 75 (2009)21. - ISSN 0099-2240 - p. 6804 - 6811.
    amebas acanthamoeba-castellanii - planktonic bacteria - biological-control - biofilm formation - plant-disease - putisolvin-ii - sugar-beet - soil - biocontrol - growth
    Environmental bacteria are exposed to a myriad of biotic interactions that influence their function and survival. The grazing activity of protozoan predators significantly impacts the dynamics, diversification, and evolution of bacterial communities in soil ecosystems. To evade protozoan predation, bacteria employ various defense strategies. Soil-dwelling Pseudomonas fluorescens strains SS101 and SBW25 produce the cyclic lipopeptide surfactants (CLPs) massetolide and viscosin, respectively, in a quorum-sensing-independent manner. In this study, CLP production was shown to protect these bacteria from protozoan predation as, compared to CLP-deficient mutants, strains SS101 and SBW25 exhibited resistance to grazing by Naegleria americana in vitro and superior persistence in soil in the presence of this bacterial predator. In the wheat rhizosphere, CLP-producing strains had a direct deleterious impact on the survival of N. americana. In vitro assays further showed that N. americana was three times more sensitive to viscosin than to massetolide and that exposure of strain SS101 or SBW25 to this protozoan resulted in upregulation of CLP biosynthesis genes. Enhanced expression of the massABC and viscABC genes did not require physical contact between the two organisms as gene expression levels were up to threefold higher in bacterial cells harvested 1 cm from feeding protozoans than in cells collected 4 cm from feeding protozoans. These findings document a new natural function of CLPs and highlight that bacterium-protozoan interactions can result in activation of an antipredator response in prey populations
    Regulation of cyclic lipopeptide biosynthesis in Pseudomonas fluorescens by the ClpP protease
    Bruijn, I. de; Raaijmakers, J.M. - \ 2009
    Journal of Bacteriology 191 (2009)6. - ISSN 0021-9193 - p. 1910 - 1923.
    syringae pv.-syringae - atp-dependent proteases - escherichia-coli - biofilm formation - bacillus-subtilis - stress tolerance - staphylococcus-aureus - putisolvin-ii - gene-cluster - swarming motility
    Cyclic lipopeptides produced by Pseudomonas species exhibit potent surfactant and broad-spectrum antibiotic properties. Their biosynthesis is governed by large multimodular nonribosomal peptide synthetases, but little is known about the genetic regulatory network. This study provides, for the first time, evidence that the serine protease ClpP regulates the biosynthesis of massetolides, cyclic lipopeptides involved in swarming motility, biofilm formation, and antimicrobial activity of Pseudomonas fluorescens SS101. The results show that ClpP affects the expression of luxR(mA), the transcriptional regulator of the massetolide biosynthesis genes massABC, thereby regulating biofilm formation and swarming motility of P. fluorescens SS101. Transcription of luxR(mA) was significantly repressed in the clpP mutant, and introduction of luxR(mA) restored, in part, massetolide biosynthesis and swarming motility of the clpP mutant. Site-directed mutagenesis and expression analyses indicated that the chaperone subunit ClpX and the Lon protease are not involved in regulation of massetolide biosynthesis and are transcribed independently of clpP. Addition of Casamino Acids enhanced the transcription of luxR(mA) and massABC in the clpP mutant, leading to a partial rescue of massetolide production and swarming motility. The results further suggested that, at the transcriptional level, ClpP-mediated regulation of massetolide biosynthesis operates independently of regulation by the GacA/GacS two-component system. The role of amino acid metabolism and the putative mechanisms underlying ClpP-mediated regulation of cyclic lipopeptide biosynthesis, swarming motility, and growth in P. fluorescens are discussed.
    Functional, genetic and chemical characterization of biosurfactants produced by plant growth-promoting Pseudomonas putida 267
    Kruijt, M. ; Tran, H. ; Raaijmakers, J.M. - \ 2009
    Journal of Applied Microbiology 107 (2009)2. - ISSN 1364-5072 - p. 546 - 556.
    cyclic lipopeptide production - phytophthora-infestans - biological-control - bacillus-subtilis - biofilm formation - fluorescens dr54 - pythium-ultimum - putisolvin-ii - biocontrol - viscosinamide
    Aims: Plant growth-promoting Pseudomonas putida strain 267, originally isolated from the rhizosphere of black pepper, produces biosurfactants that cause lysis of zoospores of the oomycete pathogen Phytophthora capsici. The biosurfactants were characterized, the biosynthesis gene(s) partially identified, and their role in control of Phytophthora damping-off of cucumber evaluated. Methods and Results: The biosurfactants were shown to lyse zoospores of Phy. capsici and inhibit growth of the fungal pathogens Botrytis cinerea and Rhizoctonia solani. In vitro assays further showed that the biosurfactants of strain 267 are essential in swarming motility and biofilm formation. In spite of the zoosporicidal activity, the biosurfactants did not play a significant role in control of Phytophthora damping-off of cucumber, since both wild type strain 267 and its biosurfactant-deficient mutant were equally effective, and addition of the biosurfactants did not provide control. Genetic characterization revealed that surfactant biosynthesis in strain 267 is governed by homologues of PsoA and PsoB, two nonribosomal peptide synthetases involved in production of the cyclic lipopeptides (CLPs) putisolvin I and II. The structural relatedness of the biosurfactants of strain 267 to putisolvins I and II was supported by LC-MS and MS-MS analyses. Conclusions: The biosurfactants produced by Ps. putida 267 were identified as putisolvin-like CLPs; they are essential in swarming motility and biofilm formation, and have zoosporicidal and antifungal activities. Strain 267 provides excellent biocontrol activity against Phytophthora damping-off of cucumber, but the lipopeptide surfactants are not involved in disease suppression. Significance and Impact of the Study: Pseudomonas putida 267 suppresses Phy. capsici damping-off of cucumber and provides a potential supplementary strategy to control this economically important oomycete pathogen. The putisolvin-like biosurfactants exhibit zoosporicidal and antifungal activities, yet they do not contribute to biocontrol of Phy. capsici and colonization of cucumber roots by Ps. putida 267. These results suggest that Ps. putida 267 employs other, yet uncharacterized, mechanisms to suppress Phy. capsici.
    Genetic and functional characterization of the gene cluster directing the biosynthesis of putisolvin I and II in Pseudomonas putida strain PCL1445
    Dubern, J.F. ; Coppoolse, E.R. ; Stiekema, W.J. ; Bloemberg, G.V. - \ 2008
    Microbiology 154 (2008). - ISSN 1350-0872 - p. 2070 - 2083.
    nonribosomal peptide-synthesis - escherichia-coli - lipopeptide production - rhizobium-meliloti - biofilm formation - system - synthetases - syringae - sequence - bacteria
    Pseudomonas putida PCL1445 secretes two cyclic lipopeptides, putisolvin I and putisolvin II, which possess a surface-tension-reducing ability, and are able to inhibit biofilm formation and to break down biofilms of Pseudomonas species including Pseudomonas aeruginosa. The putisolvin synthetase gene cluster (pso) and its surrounding region were isolated, sequenced and characterized. Three genes, termed psoA, psoB and psoC, were identified and shown to be involved in putisolvin biosynthesis. The gene products encode the 12 modules responsible for the binding of the 12 amino acids of the putisolvin peptide moiety. Sequence data indicate that the adenylation domain of the 11th module prioritizes the recognition of Val instead of Leu or Ile and consequently favours putisolvin I production over putisolvin II. Detailed analysis of the thiolation domains suggests that the first nine modules recognize the D form of the amino acid residues while the two following modules recognize the L form and the last module the L or D form, indifferently. The psoR gene, which is located upstream of psoA, shows high similarity to luxR-type regulatory genes and is required for the expression of the pso cluster. In addition, two genes, macA and macB, located downstream of psoC were identified and shown to be involved in putisolvin production or export.
    Two homologous Agr-like quorum-sensing systems cooperatively control adherence, cell morphology, and cell viability properties in Lactobacillus plantarum WCFS1
    Fujii, T. ; Ingham, C.J. ; Nakayama, J. ; Beerthuyzen, M.M. ; Kunuki, R. ; Molenaar, D. ; Sturme, M.H.J. ; Vaughan, E.E. ; Kleerebezem, M. ; Vos, W.M. de - \ 2008
    Journal of Bacteriology 190 (2008)23. - ISSN 0021-9193 - p. 7655 - 7665.
    gram-positive bacteria - staphylococcus-aureus - bacillus-subtilis - biofilm formation - escherichia-coli - transcriptional regulators - enterococcus-faecalis - streptococcus-mutans - signal-transduction - lactococcus-lactis
    A two-component regulatory system of Lactobacillus plantarum, encoded by genes designated lamK and lamR (hpk10 and rrp10), was studied. The lamK and lamR genes encode proteins which are highly homologous to the quorum-sensing histidine kinase LamC and the response regulator LamA, respectively. Transcription analysis of the lamKR operon and the lamBDCA operon and liquid chromatography-mass spectrometry analysis of production of the LamD558 autoinducing peptide were performed for DeltalamA, DeltalamR, DeltalamA DeltalamR deletion mutants and a wild-type strain. The results suggested that lamA and lamR are cooperating genes. In addition, typical phenotypes of the DeltalamA mutant, such as reduced adherence to glass surfaces and filamentous cell morphology, were enhanced in the DeltalamA DeltalamR mutant. Microarray analysis suggested that the same cell wall polysaccharide synthesis genes, stress response-related genes, and cell wall protein-encoding genes were affected in the DeltalamA and DeltalamA DeltalamR mutants. However, the regulation ratio was more significant for the DeltalamA DeltalamR mutant, indicating the cooperative effect of LamA and LamR
    Massetolide A biosynthesis in Pseudomonas fluorescens
    Bruijn, I. de; Kock, M.J.D. de; Waard, P. de; Beek, T.A. van; Raaijmakers, J.M. - \ 2008
    Journal of Bacteriology 190 (2008)8. - ISSN 0021-9193 - p. 2777 - 2789.
    syringae pv.-syringae - nonribosomal peptide-synthesis - n-acylhomoserine lactones - quorum-sensing system - gene-cluster - biofilm formation - putisolvin-ii - arthrofactin synthetase - adenylation domains - mutational analysis
    Massetolide A is a cyclic lipopeptide (CLP) antibiotic produced by various Pseudomonas strains from diverse environments. Cloning, sequencing, site-directed mutagenesis, and complementation showed that massetolide A biosynthesis in P. fluorescens SS101 is governed by three nonribosomal peptide synthetase (NRPS) genes, designated massA, massB, and massC, spanning approximately 30 kb. Prediction of the nature and configuration of the amino acids by in silico analysis of adenylation and condensation domains of the NRPSs was consistent with the chemically determined structure of the peptide moiety of massetolide A. Structural analysis of massetolide A derivatives produced by SS101 indicated that most of the variations in the peptide moiety occur at amino acid positions 4 and 9. Regions flanking the mass genes contained several genes found in other Pseudomonas CLP biosynthesis clusters, which encode LuxR-type transcriptional regulators, ABC transporters, and an RND-like outer membrane protein. In contrast to most Pseudomonas CLP gene clusters known to date, the mass genes are not physically linked but are organized in two separate clusters, with massA disconnected from massB and massC. Quantitative real-time PCR analysis indicated that transcription of massC is strongly reduced when massB is mutated, suggesting that these two genes function in an operon, whereas transcription of massA is independent of massBC and vice versa. Massetolide A is produced in the early exponential growth phase, and biosynthesis appears not to be regulated by N-acylhomoserine lactone-based quorum sensing. Massetolide A production is essential in swarming motility of P. fluorescens SS101 and plays an important role in biofilm formation.
    Genome-based discovery, structure prediction and functional analysis of cyclic lipopeptide antibiotics in Pseudomonas species
    Bruijn, I. de; Kock, M.J.D. de; Meng, Y. ; Waard, P. de; Beek, T.A. van; Raaijmakers, J.M. - \ 2007
    Molecular Microbiology 63 (2007)2. - ISSN 0950-382X - p. 417 - 428.
    nonribosomal peptide synthetases - streptomyces-coelicolor genome - biofilm formation - fluorescent pseudomonads - adenylation domains - biological-control - damping-off - biosynthesis - syringae - strain
    Analysis of microbial genome sequences have revealed numerous genes involved in antibiotic biosynthesis. In Pseudomonads, several gene clusters encoding non-ribosomal peptide synthetases (NRPSs) were predicted to be involved in the synthesis of cyclic lipopeptide (CLP) antibiotics. Most of these predictions, however, are untested and the association between genome sequence and biological function of the predicted metabolite is lacking. Here we report the genome-based identification of previously unknown CLP gene clusters in plant pathogenic Pseudomonas syringae strains B728a and DC3000 and in plant beneficial Pseudomonas fluorescens Pf0-1 and SBW25. For P. fluorescens SBW25, a model strain in studying bacterial evolution and adaptation, the structure of the CLP with a predicted 9-amino acid peptide moiety was confirmed by chemical analyses. Mutagenesis confirmed that the three identified NRPS genes are essential for CLP synthesis in strain SBW25. CLP production was shown to play a key role in motility, biofilm formation and in activity of SBW25 against zoospores of Phytophthora infestans. This is the first time that an antimicrobial metabolite is identified from strain SBW25. The results indicate that genome mining may enable the discovery of unknown gene clusters and traits that are highly relevant in the lifestyle of plant beneficial and plant pathogenic bacteria
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