Quorum Sensing and Population Control
4.1 Introduction
4.1.2 Quorum Sensing in Bacteria
61 measurements of microbial population properties under steady-state conditions with single cell resolving power and advanced automation. Equally important, the microchemostat’s miniaturized working volume of ~10 nL is capable of culturing extremely small populations of bacteria (~102 to ~104 cells versus at least ~109 in macroscale cultures). The microsized population reduces the number of cell-division events per unit time and hence slows down microbial evolution (10). This aspect facilitates monitoring of programmed behavior of bacterial populations for hundreds of hours despite strong selection pressure to evade population control, something that may not be achieved in macroscopic reactors (6).
62 culture of V. fisheri at very high cell density. In these symbiotic associations, the eukaryotic host supplies V. fischeri with a nutrient rich environment in which to live, while V. fisheri provides the host with light. Each eukaryotic host uses the light provided by the bacteria for a specific purpose. For example in the squid Euprymna scolopes―V.
fischeri association, the squid has evolved an antipredation strategy in which it counter- illuminates itself using light from V. fischeri. Counter illumination enables the squid to avoid casting a shadow beneath it on bright clear nights when the light from the moon and stars penetrates sea water. In contrast, the fish Monocentris japonicus uses the light produced by V. fisheri to attract a mate (60). Quorum sensing relationships are not always as amicable as the ones characterized by symbiotic bacteria. On the contrary, they can take on an adversarial role, as seen with pathogenic bacteria. For example, virulent bacteria like Pseudomonas aeruginosa use quorum sensing to sustain their pathogenic lifestyle. Evading host defenses is a major goal of pathogens,and as such, quorum sensing is an important asset because it enablesbacteria to appropriately time expression of immune-response activating products. Using quorum sensing, bacteria can innocuously grow within a host without expressing virulence determinants. Once they amass a high celldensity, they become aggressive; their numbers sufficient to produce ample virulence factors to overwhelm the hostdefenses, launch a successful infection and form an antibiotic-resistant biofilm, leading to disease (61, 62).
Quorum sensing works by allowing bacteria to communicate and regulate their gene expression in response to fluctuations in cell-population density (60). In the past decade, quorum sensing circuits have been identified in over 25 bacterial species. In
63 most cases, the quorum sensing circuits identified resemble the canonical quorum sensing circuit of the symbiotic bacterium, Vibrio fischeri in the above example.
Specifically, quorum sensing circuits contain, at a minimum, homologues of two Vibrio fischeri proteins called LuxI and LuxR. The LuxI-like (or ‘I’) proteins are responsible for the biosynthesis of a chemical signaling molecule called acyl-homoserine lactones (AHL), which is small enough to freely diffuse across the cell membrane (60, 63) into the surrounding medium and back into the cell. Accordingly, the intracellular (and extracellular) AHL concentration changes as a function of the cell density. When the AHL concentration achieves a critical threshold concentration, it becomes bound to its cognate ‘R’ protein (64), to form an ‘R’-AHL complex, which can activate target gene expression (65-67). When only a few other bacteria of the same kind are in the vicinity, diffusion reduces the concentration of AHL in the surrounding medium to almost zero.
For this reason, there is a low likelihood for a bacterium to detect its own secreted AHL.
With many bacteria of the same kind, however, the concentration of AHL can reach (or exceed) the required threshold, whereupon ‘R’ receptor becomes activated to initiate transcription of specific genes, such as luciferase in V. fishcheri. Using such quorum sensing mechanisms, bacteria can efficiently couple gene expression to fluctuations in cell population density.
In the natural environment, there are many different bacterial species living together, communicating via a variety of LuxI/LuxR-type circuits with their respective signaling molecules or “languages”. For example the LuxI/LuxR bioluminescence system in Vibrio fischeri, the LasI/LasR-Rh1I/Rh1R virulence system in Pseudomonas
64 aeruginosa, the TraI/TraR virulence system in Agrobacterium tumefaciens, and the ExpI/ExpR-CarI/CarR virulence/antibiotic system in Erwinia carotovora (60). There is evidence that interspecies communication via quorum sensing or quorum sensing cross talk can occur (68, 69). Nevertheless, the languages themselves are generally mutually exclusive and, therefore, as one species employs a specific language, it does not necessarily talk to all other species.
Because many important animal andplant pathogens use quorum sensing to regulate virulence, strategiesdesigned to interfere with these signaling systems will likely have broad applicability for biological control of disease-causingorganisms (69). For example, the discovery that P. aeruginosa uses quorum sensing to regulate biofilm production suggests that agents capable of blocking quorumsensing may also be useful for preventing biofilm formation. Therecent production of AHLs in plants represents an exciting new approach to controlling crop diseases as well as to manipulatingplant- microbe interactions for improved crop production in thefuture (61).
The principle of quorum sensing can also be used in synthetic biology programs to control the dynamics of an entire population despite variability in the behavior of individual cells. In this chapter, we used the microchemostat to characterize a synthetic population control circuit (5), programmed to autonomously regulate the cell density of an Escherichia coli population using quorum sensing.
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