CH391L/S14/QuorumSensing

Introduction
Quorum Sensing refers to the cell to cell communication within bacterial populations facilitated by chemical signaling that results in a change in gene expression. Certain bacterial strains continuously produce chemical signals in the form of small molecules known as autoinducers that are excreted and enter the extracellular environment. Under conditions of low population density, the level of autoinducers present in this environment is not high enough to produce a change in gene expression. However, in very dense populations, the level surpasses the threshold necessary to induce such a change. The resultant gene expression favors the production of proteins that benefit cells living in a colony and tends to increase factors such as virulence and antibiotic resistance. Quorum sensing has been shown to occur in both gram negative and gram positive bacterial strains, and can occur in monoclonal populations and between bacterial populations of different species Williams2007.

Benefits of Quorum Sensing
Quorum sensing provides a vast array of benefits to bacterial populations, including increasing virulence and antibiotic resistance, the ability to survive and reproduce, motility and attachment including biofilm formation, as well as species specific benefits like sporulation, antibiotic production and symbiosis. One of the most widely studied instances of quorum sensing was discovered to be integral in the symbiotic relationship between Vibrio fischeri and squid such as Euprymna scolopes. In the dense populations of bacterial cells within the light pockets of the squid, quorum sensing results in bioluminescence which helps disguise the presence of the squid in the dark by preventing visibility of its shadow on the sea floor Verma2013. It has also been discovered in recent years that bacterial/eukaryotic signaling is important in pathogenic bacterial strains, and that this may be in part facilitated by a quorum sensing-like process Javaraman2008.

Autoinducers and Gene Expression
Autoinducers in both gram negative and gram positive bacterial strains are constantly produced at a basal level, and upon reaching a threshold concentration in the extracellular environment, initiate production of both additional autoinducer molecules as well as certain proteins that benefit the propagation and sustainability of the bacterial colony. There are two main classes of autoinducers, Autoinducer 1 (AI-1) and Autoinducer 2 (AI-2). AI-1's are used for intraspecies communication and differ between gram negative and gram positive bacteria. AI-2's are used mainly in interspecies communication and are used by both gram negative and gram positive bacteria. All autoinducer systems utilize either a variant of Lux proteins or a phosphorylation cascade as the mechanism of production/recognition within the cell Jacobi2012. AI-1 AI-2
 * Gram Negative- Homoserine Lactone (HSL) and Acylated Homoserine Lactone (AHL) molecules are small neutral lipid based signaling molecules used by gram negative bacteria as autoinducers in quorum sensing. The production and recognition of these molecules within the cell is regulated by a LuxI/LuxR system. LuxI proteins are responsible for the activation of synthesis of these lipids which are then excreted into the extracellular environment. AHLs and HSLs are able to diffuse across cell membranes of adjacent cells and at above-threshold levels, sufficient accumulation within the cell initiates binding by the LuxR protein. This complex binds to the promoter region of target genes and results in the formation of various target proteins Schauder2001.
 * Gram Positive- Conversly, in gram positive bacteria, oligopeptides are used to facilitate quorum sensing. These oligopeptides also accumulate in high density populations, but do so on the outside of the cell. A receptor kinase at the cell surface is activated upon binding the oligopeptide, and an auto-phosphorylation event occurs at this receptor kinase on the inside of the cell. The phosphate group is subsequently transferred to a second protein that, when phosphorylated, binds to the promoter region of the target genes, similar to the LuxR/AHL complex in gram negatives. This in turn results in the production of the target proteins  Schauder2001.
 * Autoinducers formed from the precursor molecule 5-dihydroxy-2,3-pentane-dione (DPD) are used by both gram negative and gram positive bacteria to facilitate intraspecies communication. After the cell-specific modifications to this precursor molecule are performed by LuxS, the product, commonly furanosyl borate diester, is excreted and following extracellular accumulation also induces a phosphorylation cascade with the help of LuxP Jacobi2012.

In Synthetic Biology


A popular focus in modern synthetic biology with respect to quorum sensing is the engineering of exact biological circuitry which can function as a highly sensitive biosensor using bacterial populations. In 2011, a complex study was published detailing a highly reliable and sensitive array of bacterial populations that could be used to sense arsenic through the harnessing of the quorum sensing cell signaling pathway. This system was augmented through the use of hydrogen peroxide as a component of the system, which allowed cellular signaling to travel further than the diffusible AHL molecules Prindle2011. The benefit of these types of sensors is that due to the nature of the quorum sensing pathways the system can have a very high degree of spatiotemporal predictability and can be tuned to suit the particular application. Due to the nature of quorum sensing, the ability to manipulate genetic expression can be utilized to program whole populations of cells on a large scale. Now that the manipulation of bacterial cell genetic expression has been simplified, there are a number of current applications being considered. Two popular applications are engineering population control as well as harnessing and directing bacterial activity. Population control can be done in a number of ways. In 2004, a collaborative research effort resulted in the development of a method of coupling gene expression and cell death using quorum sensing by installing a “population control circuit” that caused regulation of Escherichia coli populations, limiting their ability to proliferate. As populations reached density levels sufficient to induce quorum sensing, the programmed genetics induced cell death You2004. In 2010, another effort to control population was made using an additional species of bacteria. Young mice were inoculated with a strain of E. coli engineered to express autoinducer molecule cholera autoinducer 1 (CAI-1) which prevents virulence of Vibrio cholerae under quorum sensing conditions. Subsequent inoculation with V. cholerae resulted in populations with decreased virulence and the survival rate of the mice increased compared to controls Duan2010. Harnessing bacterial activity is another popular area of investigation. A review published in 2012 focused on the engineering of synthetic microbial ecosystems which could serve a variety of functions including tissue engineering, drug production, and intestinal flora control Mee2012. One of the most important steps in this process is being able to replace existing colonies or existing colony functions on demand. This ability to tune and control bacterial consortia was recently invesitgated by a research group at Texas A&M. This team developed a novel method in which they were able to introduce E.coli into an existing population of Bacillus subtilis and subsequently through the use of synthetic quorum sensing molecules replace the monoclonal biofilm with a functioning dual biofilm. Furthermore, upon addition of a second synthetic signal, the B. subtilis was able to displace the E. coli, rendering this process completely reversible Hong2012 .. This has extremely important implications for industrial bacterial applications as it enables the replacement and tuning of bacterial populations in an efficient and reversible manner.
 * Biosensors
 * Population Programming

iGEM
Components of the quorum sensing pathways in both AI-1 and AI-2 systems have been the target of iGEM. There are multiple BioBricks for the various Lux genes, AHLs, kinases, phosphatases and other constituents, as well as their promoters, activators, and suppressors. As of 2012, Boulder iGEM team alone had created three BioBricks coding for various proteins that disrupted quorum sensing, including Part:BBa_C0060 which codes for Aiia, an enzyme that hydrolyzes AHL, along with a Biobrick for a protein that is highly sensitive to AHLs, and a third that codes for a protein that degrades preexisting biofilms. In 2013, the Rutgers team submitted an additional BioBrick coding for an AHL degrading enzyme, Bba_K1206000. Additionally the team at TU Delft has made it an ongoing goal to achieve the detection and destruction of restistant Staphylococcus aureus strains by controlling various steps in the quorum signaling pathway. They have an ongoing list of dozens of BioBricks they have created on their website. Finally, also in 2013, the teams BIT and BIT-China received two gold rewards, one of which was for a BioBrick coding for a heat shock protein under the control of quorum sensing pathway products that resulted in programmed cell death.