Microbial Ecology Use of a Whole-Cell Biosensor and Flow Cytometry to Detect AHL Production by an Indigenous Soil Community During Decomposition of Litter Mette Burmølle, Lars Hestbjerg Hansen and Søren Johannes Sørensen Department of Microbiology, University of Copenhagen, Sølvgade 83H, 1307 Copenhagen K, Denmark Received: 28 May 2004 / Accepted: 29 October 2004 / Online publication: 6 October 2005 Abstract Quorum sensing, mediated by acylated homoserine lactones (AHLs), is well described for pure culture bacteria, but few studies report detection of AHL com- pounds in natural bacterial habitats. In this study, we detect AHL production during a degradation process in soil by use of whole-cell biosensor technology and flow cytometry analysis. An indigenous soil bacterium, be- longing to the family of Enterobacteriaceae, was isolated and transformed with a low-copy plasmid harboring a gene encoding an unstable variant of the green fluores- cent protein (gfpASV) fused to the AHL-regulated P luxI promoter originating from Vibrio fischeri. This resulted in a whole-cell biosensor, responding to the presence of AHL compounds. The biosensor was introduced to compost soil microcosms amended with nettle leaves. After 3 days of incubation, cells were extracted and analyzed by flow cytometry. All microcosms contained induced biosensors. From these microcosms, AHL producers were isolated and further identified as species previously shown to produce AHLs. The results demon- strate that AHL compounds are produced during degradation of litter in soil, indicating the presence of AHL-mediated quorum sensing in this environment. Introduction It is well known that some bacteria are able to sense their population density and regulate gene expression accord- ingly. This is described as quorum sensing. In more than 40 gram-negative species, quorum sensing is mediated by production and detection of acylated homoserine lac- tones (AHLs). Several phenotypic functions are regulated by AHL-mediated quorum sensing, for instance, produc- tion of enzymes, antibiotics, biotoxins, and virulence factors, cell/colony morphology and behavior, nodula- tion, and cell cycle status (for reviews, see [34, 44]). A common trait is that the functions activated by quorum sensing are favorable for bacterial cells present in dense populations. Quorum sensing is well described for pure culture bacteria, but only few studies report detection of AHL compounds in natural bacterial habitats. To evaluate the prevalence and significance of quorum sensing, it is important to demonstrate the presence of AHL compounds and thereby indicate the occurrence of quorum sensing in the natural habitats of bacteria. AHL compounds have previously been detected in, for instance, Pseudomonas aeruginosa-infected mouse lungs [46], spoiled food [6, 19], naturally occurring biofilms [33], and plant rhizosphere [35, 37]. In these studies, various AHL-inducible whole- cell bacterial biosensors were used. Those biosensors contain a construct in which an AHL-inducible promoter is fused to a reporter gene, resulting in production of a measurable product in the presence of AHLs. One advantage of using whole-cell biosensors is that only AHL concentrations, actually inducing quorum sensing- regulated gene expression in the bacteria, are detected. Therefore, AHL biosensor assays are useful in clarifying whether adequate AHL concentrations are present to induce a response. Very little is known about quorum sensing in bulk soil and litter. Soil is a heterogeneous environment containing many bacterial species in close proximity, which influence each other and constantly compete for nutrients. Several of these species are known either to produce or degrade AHL compounds [14, 15, 29, 44]. The nutrient status of bulk soil is generally considered to be low, but raises significantly when amended with, for instance, litter to be Correspondence to: Søren Johannes Sørensen; E-mail: sjs@bi.ku.dk DOI: 10.1007/s00248-004-0113-8  Volume 50, 221–229 (2005)  * Springer Science+Business Media, Inc. 2005 221