RESULTS:
1 - 5 of 5 for "Arne Heydorn"
Inhibition of quorum sensing in Pseudomonas aeruginosa biofilm bacteria by a halogenated furanone compound
Novel molecular tools have been constructed which allow for in situ detection of N-acyl homoserine lactone (AHL)-mediated quorum sensing in Pseudomonas aeruginosa biofilms. The reporter responds to AHL activation of LasR by expression of an unstable version of the green-fluorescent protein (Gfp). Gfp-based reporter technology has been applied for non-destructive single-cell-level detection of quorum sensing in laboratory-based P. aeruginosa biofilms. It is reported that a synthetic halogenated furanone compound which is a derivative of the secondary metabolites produced by the Australian macroalga Delisea pulchra is capable of interfering with AHL-mediated quorum sensing in P. aeruginosa. It is demonstrated that the furanone compound specifically represses expression of a PlasB-gfp reporter fusion without affecting growth or protein synthesis. In addition it reduces the production of important virulence factors indicating a general effect on target genes of the las quorum sensing circuit. The furanone was applied to P. aeruginosa biofilms established in biofilm flow chambers. The Gfp-based analysis reveals that the compound penetrates microcolonies and blocks cell signalling and quorum sensing in most biofilm cells. The compound did not affect initial attachment to the abiotic substratum. It does however affect the architecture of the biofilm and enhances the process of bacterial detachment leading to a loss of bacterial biomass from the substratum.
The cep quorum-sensing system of Burkholderia cepacia H111 controls biofilm formation and swarming motility
Burkholderia cepacia and Pseudomonas aeruginosa often co-exist as mixed biofilms in the lungs of patients suffering from cystic fibrosis (CF). Here the isolation of random mini-Tn5 insertion mutants of B. cepacia H111 defective in biofilm formation on an abiotic surface is reported. It is demonstrated that one of these mutants no longer produces N-acylhomoserine lactones (AHLs) due to an inactivation of the cepR gene. cepR and the cepI AHL synthase gene together constitute the cep quorum-sensing system of B. cepacia. By using a gene replacement method two defined mutants H111-I and H111-R were constructed in which cepI and cepR respectively had been inactivated. These mutants were used to demonstrate that biofilm formation by B. cepacia H111 requires a functional cep quorum-sensing system. A detailed quantitative analysis of the biofilm structures formed by wild-type and mutant strains suggested that the quorum-sensing system is not involved in the regulation of initial cell attachment but rather controls the maturation of the biofilm. Furthermore it is shown that B. cepacia is capable of swarming motility a form of surface translocation utilized by various bacteria to rapidly colonize appropriate substrata. Evidence is provided that swarming motility of B. cepacia is quorum-sensing-regulated possibly through the control of biosurfactant production. Complementation of the cepR mutant H111-R with different biosurfactants restored swarming motility while biofilm formation was not significantly increased. This result suggests that swarming motility per se is not essential for biofilm formation on abiotic surfaces.
Quantification of biofilm structures by the novel computer program comstat
The structural organization of four microbial communities was analysed by a novel computer program COMSTAT which comprises ten features for quantifying three-dimensional biofilm image stacks. Monospecies biofilms of each of the four bacteria Pseudomonas putida P. aureofaciens P. fluorescens and P. aeruginosa tagged with the green fluorescent protein (GFP) were grown in flow chambers with a defined minimal medium as substrate. Analysis by the COMSTAT program of four variables describing biofilm structure – mean thickness roughness substratum coverage and surface to volume ratio – showed that the four Pseudomonas strains represent different modes of biofilm growth. P. putida had a unique developmental pattern starting with single cells on the substratum growing into micro-colonies which were eventually succeeded by long filaments and elongated cell clusters. P. aeruginosa colonized the entire substratum and formed flat uniform biofilms. P. aureofaciens resembled P. aeruginosa but had a stronger tendency to form micro-colonies. Finally the biofilm structures of P. fluorescens had a phenotype intermediate between those of P. putida and P. aureofaciens. Analysis of biofilms of P. aureofaciens growing on 0·03 mM 0·1 mM or 0·5 mM citrate minimal media showed that mean biofilm thickness increased with increasing citrate concentration. Moreover biofilm roughness increased with lower citrate concentrations whereas surface to volume ratio increased with higher citrate concentrations.
Experimental reproducibility in flow-chamber biofilms
The structural organization of microbial communities is influenced by many factors e.g. nutrient composition shear stress and temperature. This paper presents a general method for quantitative comparison of biofilm structures and assessment of experimental reproducibility between independent biofilm experiments. By using a novel computer program COMSTAT biofilm structures of Pseudomonas aeruginosa and an isogenic rpoS mutant were quantified. The strains were tagged with the green fluorescent protein (GFP) and grown in flow chambers with a defined minimal medium as substrate. Three independent rounds of biofilm experiments were performed and in each round each of the two variants was grown in two separate channels. Nine image stacks were acquired in each channel 146 h after inoculation. An analysis of variance model incorporating the factors experiment round bacterial strain channel number and image stack number was used to analyse the data calculated by COMSTAT. Experimental reproducibility was verified by estimating the magnitude of the variance of the effects round () and the interaction between bacterial strain and round (). Mean thickness of the wild-type and rpoS mutant biofilms was estimated at 6·31 μm (SE 0·81 μm) and 16·85 μm (SE 0·87 μm) respectively.
Detection of N-acylhomoserine lactones in lung tissues of mice infected with Pseudomonas aeruginosa
The pathogenesis of Pseudomonas aeruginosa is associated with expression of virulence factors many of which are controlled by two N-acylhomoserine lactone (AHL)-based quorum-sensing systems. Escherichia coli strains equipped with a luxR-based monitor system expressing green fluorescent protein (GFP) in the presence of exogenous AHL molecules were used to detect the production of AHLs from P. aeruginosa in vivo. Mice were challenged intratracheally with alginate beads containing P. aeruginosa and E. coli and killed on different days after the challenge. By means of confocal scanning laser microscopy GFP-expressing E. coli bacteria could be detected in the lung tissues indicating production and excretion of AHL molecules in vivo by the infecting P. aeruginosa. AHL signals were detected mainly in lung tissues exhibiting severe pathological changes. These findings support the view that expression of AHL molecules by P. aeruginosa during infection coincides with its pathogenesis.