Pseudomonas aeruginosa and Burkholderia cepacia are capable of forming mixed biofilms in the lungs of cystic fibrosis patients. Both bacteria employ quorum-sensing systems, which rely on N-acylhomoserine lactone (AHL) signal molecules, to co-ordinate expression of virulence factors with the formation of biofilms. As both bacteria utilize the same class of signal molecules the authors investigated whether communication between the species occurs. To address this issue, novel Gfp-based biosensors for non-destructive, in situ detection of AHLs were constructed and characterized. These sensors were used to visualize AHL-mediated communication in mixed biofilms, which were cultivated either in artificial flow chambers or in alginate beads in mouse lung tissue. In both model systems B. cepacia was capable of perceiving the AHL signals produced by P. aeruginosa, while the latter strain did not respond to the molecules produced by B. cepacia. Measurements of extracellular proteolytic activities of defined quorum-sensing mutants grown in media complemented with AHL extracts prepared from culture supernatants of various wild-type and mutant strains supported the view of unidirectional signalling between the two strains.

In order to perform single-cell analysis and online studies of N-acyl homoserine lactone (AHL)-mediated communication among bacteria, components of the Vibrio fischeri quorum sensor encoded by luxR-P luxI have been fused to modified versions of gfpmut3‫ء‬ genes encoding unstable green fluorescent proteins. Bacterial strains harboring this green fluorescent sensor detected a broad spectrum of AHL molecules and were capable of sensing the presence of 5 nM N-3-oxohexanoyl-L-homoserine lactone in the surroundings. In combination with epifluorescent microscopy, the sensitivity of the sensor enabled AHL detection at the single-cell level and allowed for real-time measurements of fluctuations in AHL concentrations. This green fluorescent AHL sensor provides a state-of-the-art tool for studies of communication between the individuals present in mixed bacterial communities.In recent years it has become apparent that bacteria coordinate their interaction and association with higher organisms by intercellular communication systems. In gram-negative bacteria, one type of communication system functions via small, diffusible N-acyl homoserine lactone (AHL) signal molecules. The signals are synthesized from precursors by a synthase protein, "I," and once they have reached a certain threshold concentration, they interact with a transcriptional activating "R" protein to induce expression of different target genes (for reviews see references 11, 13, and 43). Such regulatory systems operate as a quorum-sensing mechanism that allows bacteria to sense and express target genes in relation to their cell density.Several methods to detect the presence of AHL have been described. AHLs can be extracted from liquid cultures, purified to homogeneity by semipreparative high-performance liquid chromatography (HPLC), and identified by mass spectrometry and 1 H nuclear magnetic resonance (NMR) spectroscopy (10). A number of bacterial sensor systems such as the pigment-developing Chromobacterium violaceum (30) and luxABand lacZ-based systems have been described (36, 50). Bioluminescent sensor systems have been conveniently used in Escherichia coli and have enabled the isolation and cloning of a number of I genes (31, 41, 42). A simple and convincing method for separation and tentative identification of AHL molecules in extracts of whole cultures has been developed; it consists of thin-layer chromatography (TLC) followed by detection of AHL molecules by means of agar overlay with sensor bacteria (36).Although these methods are very useful and highly sensitive, they do not allow for detection at the single-cell level or at the local environment. Furthermore, investigation of AHL expression based on population level analysis does not give information about local concentrations. A live bacterial AHL sensor that signals the presence of AHL molecules by expressing a reporter such as green fluorescent protein (GFP) can fulfil these requirements. GFP, obtained from the jellyfish Aequorea victoria, requires only trace amounts of oxygen to mature, i.e., n...

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A GFPmut3b-tagged derivative of broad host-range plasmid RP4 was used to monitor the conjugative transfer of the plasmid from a Pseudomonas putida donor strain to indigenous bacteria in activated sludge. Transfer frequencies were determined to be in the range of 4U103T to 1U10 3S transconjugants per recipient. In situ hybridisation with fluorescently labeled, rRNA-targeted oligonucleotides was used to phylogenetically affiliate the bacteria that had received the plasmid. z

A GFPmut3b-tagged derivative of broad host-range plasmid RP4 was used to monitor the conjugative transfer of the plasmid from a Pseudomonas putida donor strain to indigenous bacteria in activated sludge. Transfer frequencies were determined to be in the range of 4 x 10(-6) to 1 x 10(-5) transconjugants per recipient. In situ hybridisation with fluorescently labeled, rRNA-targeted oligonucleotides was used to phylogenetically affiliate the bacteria that had received the plasmid.

The potential of the green fluorescent protein (GFP) as a marker gene for ecological investigations of an activated sludge community was assessed. By inserting the hybrid transposon mini‐Tn5 gfp into the chromosome of Pseudomonas putida KT2442 a strongly fluorescent mutant was obtained. This strain was used for in vivo tracking of individual cells after introduction into a simple sludge microcosm. It is demonstrated that the observed reduction of introduced bacteria from sewage is mainly the result of predation by protozoa. The feasibility of combining detection of GFP fluorescence with whole cell hybridization employing fluorescently labeled, rRNA‐targeted oligonucleotides in paraformaldehyde fixed samples is demonstrated.

The N-acyl-L-homoserine lactones (AHLs) produced by sequential Pseudomonas aeruginosa isolates from chronically infected patients with cystic fibrosis were analyzed by thin-layer chromatography. It is demonstrated that both the amounts and the types of molecules synthesized by isolates from patients who were monitored over periods of up to 11 years do not change significantly during chronic colonization. However, in the case of a patient who became co-infected with an AHL-producing Burkholderia cepacia strain a dramatic reduction in the amounts of AHLs produced by the co-residing P. aeruginosa isolates was observed.

Tetrahymena sp. was found to graze extensively on Serratia liquefaciens MG1 swim cells (1.5-3 microns long rods) resulting in the rapid elimination of the bacterial strain. However, when S. liquefaciens cells are exposed to certain surfaces they differentiate into elongated, highly motile swarm cells and these cells were found to be grazing-resistant provided their length exceeded 15 microns.