Final Activity Report Summary - QUORUM SENSING (Inter-species cell-cell signalling in bacteria) The project's laboratory focussed on the molecular mechanisms bacteria use for cell-cell communication involving a process called quorum sensing. This process enables a bacterial population to regulate behaviours which are only productive when many bacteria act in concert as a group. Our ultimate goal is to understand how bacteria use inter-species cell-cell communication to coordinate population-wide behaviours in consortia and in microbial-host interactions. Bacteria communication is mediated by the production, release, and detection of quorum sensing signal molecules called auto-inducers. Most auto-inducers are species-specific, however one signal named autoinducer-2 (AI-2), is produced and detected by a wide variety of species of bacteria and allows inter-species communication. We have shown that certain bacteria have a mechanism that enables them to interfere with other species' ability to sense and respond to auto-inducer signals and thereby correctly regulate their group behaviours according to changes in cell population densities. Specifically, by studying inter-species signalling in consortia, I have previously demonstrated that using the mechanism of interference with AI-2-mediated cell-cell communication Escherichia coli can manipulate AI-2 signalling of the marine bacterium V. harveyi and the human pathogen V. cholerae and thereby interfere with these species' ability to regulate bioluminescence production and virulence. We predict that this type of inter-species interactions have important consequence in the maintenance of normal gut microflora in eukaryotes. The project has previously shown that in E. coli the interference mechanism is mediated by the AI-2-inducible lsr operon encoding for the proteins involved in AI-2 uptake and degradation. These studies revealed that the Lsr operon is regulated by a transcriptional repressor, LsrR, and suggested that de-repression of lsr occurs in the presence of phosphorylated AI-2 (AI-2-P). Here, we characterize the series of biochemical events involved in regulating this operon. We showed that AI-2-P binds specifically to LsrR consistent with the genetic studies indicating that AI-2-P is the inducer of the Lsr operon. With a combined effort of different biochemical techniques we have characterised the reaction catalysed by LsrG a cytoplasmic enzyme involved in cleaving AI-2-P. The products of the reaction catalysed by LsrG no longer bind to LsrR. Thus, cleavage of AI-2-P by LsrG terminates induction of the Lsr operon closing the AI-2 signalling cycle. Additionally, we show that the AI-2 interference mechanism is also present in organisms that colonise the rizosphere of several plants. We demonstrated that Sinorhizobium meliloti, a plant symbiont well known for its ability to fixate nitrogen, has an AI-2-inducible operon encoding a functional AI-2 interference mechanism. We showed that in mixed-species cultures S. meliloti can use this system to clear the AI-2 signal produced by Erwinia carotovora, a plant pathogen that can co-exist with S. meliloti in the rhizosphere and that has been reported to regulate virulence via AI-2. Importantly, the mechanism of interference functions somewhat differently for S. meliloti than for the enteric species. S. meliloti is incapable of producing AI-2, thus the S. meliloti can only induce the AI-2 interference mechanism in response to the presence of AI-2 produced by other bacterial species in the vicinity. Thus, unlike E. coli presumably S. meliloti can induce its mechanism of interference without allowing the other species to detect its presence via AI-2 mediated signalling, effectively eavesdropping on its neighbours. We hypothesise that the ability of S. meliloti to interfere with the quorum sensing of plant pathogens that use AI-2 to regulate virulence of pathogens like Erwinia carotovora could be beneficial to the plant. Thus identification and characterization of the molecular mechanism of the S. meliloti interference system has provided us an excellent tool to begin studying the influence of inter-species bacterial signalling on bacteria-plant interactions, both symbiotic and pathogenic.
