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QS is often described as density-dependent cell-to-cell signalling. The definition of QS is a mechanism for the co-ordination of gene expression in a bacterial population which depends on the production and response to a self-generated signal [73, 92-94]. This allows individual bacteria to initiate social interactions and exhibit multicellular behaviour [93-95]. Diggle et al define a signal as “any act or structure that alters the behaviours of other organisms, which evolved owing to that effect, and which is effective because the receivers response has also evolved” [96].

Many bacteria synthesise QS signal molecules or autoinducers [93]. Within a defined area, such as an organ, as the bacterial cell density increases the concentration of autoinducer will rise

(Figure 6) [61, 93]. By detecting the concentration of signal molecule an individual bacterium ‘senses’ when a threshold signal concentration and thereby population level has been reached, leading to a response. These changes occur within a bacterium on an individual level, but occurs within multiple cells at the same time producing a concerted population response. QS is a multicellular behaviour modulating a range of physiological processes [95, 97]. Binding of a specific QS molecule with a receptor either directly or indirectly through a signal transduction cascade initiates repression or induction of target genes or regulons [73, 91, 93, 98, 99]. Examples of population-wide responses to QS include bioluminescence, sporulation, biofilm production, and expression of virulence factors including siderophores and adhesion molecules [91, 97, 100-103].

There is no defined quorum size as the QS effects are dependent on the rates of synthesis and consumption of the QS molecules, which are affected by external conditions [93, 94, 98, 103]. Some bacterial species require a relatively low amount of signal to activate the transduction cascade, other species need a much higher concentration and some require a variable concentration with responses activated in a dose-dependent manner [94].

QS has been shown to play a role in food spoilage and biofouling [90, 91, 104]. Biofouling describes growth of algae, bacteria and animals, such as crustaceans, on surfaces with prolonged exposure to water [104]. Such surfaces include pipes, tanks and the hulls and keels of ships [104]. Many opportunistic pathogens are able to utilise QS to aid biofilm formation on surfaces and increase specific virulence characteristics [104].

In addition, QS has been shown to be an important factor in the medical and dental areas. Opportunistic pathogens, such as Pseudomonas aeruginosa produce highly complex biofilms on medical catheters and other instruments. Research is currently underway to determine the effect of QS inhibitors on biofilm formation and degradation of different materials to reduce the risk of infection in susceptible patients [100, 104].

Figure 6 – Schematic diagram illustrating the basis of bacterial quorum sensing Blue ovals (●) represent the bacterial cells and the red triangles (▲) represent the QS signalling molecule.

In a true QS system there are four key components which must be present:

1) A signal synthase responsible for producing the specific signals, such as LuxI in Vibrio fischeri or LasI in Pseudomonas aeruginosa [61, 91, 93, 94, 98]

2) The signal, such as AHL or AI-2 [61, 91, 93, 94, 98]

3) A signal regulator, e.g. LuxR in Vibrio fischeri, LasR in Ps. aeruginosa and SdiA in E. coli [61, 91, 93, 94, 98]

4) A QS regulon, i.e. the genes to be regulated, for example the lux operon in Vibrio fischeri [61, 91, 93, 94, 98]

During the first half of the 20th _{Century it was determined that bacteria communicated via}

chemical signals [105]. By 1965 information on genes and gene regulation was widespread, but it was believed that changes were in response to chemical fluctuations within the individual bacterium, with no influence from other sources [105]. Kempner and Hanson suggested the bioluminescence produced by Vibrio fischeri was due to the removal of inhibitor found within the growth medium due to the late on-set of light production [106]. Nealson and Hastings suggested that bioluminescence was as a result of autoinduction and V. fischeri was communicating population-wide, introducing the idea of quorum sensing [107]. In 1981 Eberhard determined the structure of 3-oxo-C6-HSL, an N-acylhomoserine lactone responsible for communication in Vibrio fischeri [108]. Dunny et al identified bacterial ‘sex pheromones’, which in 1984 were redefined as autoinducing polypeptides affecting aggregation in Enterococcus faecalis [80, 105]. This type of chemical signalling was found to be density dependent, acting to coordinate the bacterial population as a whole. In 1994 Fuqua et al termed the bacterial unit affected by this type of chemical signalling as a quorum in order to recognise that the size of the population was critical to whether the signals had an effect. Later, this became known as quorum sensing (QS) [103, 105, 109-111].

Three main bacterial QS systems have been discovered so far, each relying on the production and release of different types of signal; N-acylhomoserine lactones (AHL), autoinducer-2 (AI-2) and auto-inducing peptides (AIP) (Table 1) [94, 110]. These molecules are chemically diverse, and different cognate regulators are often found in combination within a bacterial species working together in a hierarchical system [75, 93].

Briefly, AHLs are produced by some gram negative bacteria (Section 1.3) and AI-2 is often termed a universal signal as it is produced by some Gram negative and positive bacteria (Section 1.4). AIPs are only produced by Gram positive bacteria, such as Staphylococcus aureus [93]. These peptide signals, recognised by cell surface transmembrane histidine kinases, activate transcriptional regulators through a phospho-relay system [98, 110].

Other QS systems do exist, but appear to be found in a smaller range of bacterial species such as the Pseudomonas quinolone signal (PQS) and 2-heptyl-4-quinolone (HHQ), both of which are produced by Pseudomonas aeruginosa (Table 1) [93]. AI-3 is a controversial signalling molecule with a structure similar to mammalian catecholamines, the structure of which is still largely unknown. Recent research has suggested a role in metabolism rather than QS for AI-3 [24, 73, 112, 113]. Research showed AI-2 interacts with QseABC to regulate expression of the LEE locus [24, 113-118]. Other molecules such as indole and diamino acids have been suggested as quorum sensing signals, but the evidence suggests that these are more likely to be associated with cell status and age, although this hypothesis has yet to be confirmed [119-123].

Social networking is an expression utilised to describe the relationships between types of molecules produced by bacteria using economic terms such as “public goods” and “private goods”. Public goods are factors released by the bacteria into the external milieu constituting social products which can be beneficial to multiple species present in the QS niche [124]. By comparison, “private goods” remain within the cell, thereby constituting non-social products

Table 1 - Examples of chemical structures of different classes of quorum sensing signals Structures taken from [93, 94, 125] and [125]

Signal family Structure Example of

producers N-acylhomoserine lactone (AHL) Gram negative bacteria, e.g. Pseudomonas, Vibrio and Yersinia Autoinducer-2 (AI-2)

Many species both gram positive and negative, e.g. E. coli,

Salmonella and Enterococcus faecalis Autoinducing Polypeptide (AIP) Gram positive bacteria, e.g. Staphylococcus aureus Pseudomonas Quinolone Signal (PQS) Pseudomonas aeruginosa only

which are often required for metabolic processes [124, 126]. As these factors remain intracellular, concentration is not as important and therefore quorum sensing regulation is unlikely to be involved [124].

Although quorum sensing signals may be construed as public goods by voyeuristic bacteria such as E. coli, it is actually used to communicate and coordinate expression of external factors, and thereby control pathogenic phenotypes [96, 111, 124, 127]. These extracellular factors have been found to be utilised in the control of various biological processes, such as motility, biofilm production, nutrient acquisition and host immune evasion and/or suppression [124].

QS signals are rarely species specific, for example AHLs produced by one gram negative species can be detected and utilised by another species, thereby making the environment much more competitive and complex [93, 94, 110]. The interactions between bacterial species and QS signals can be accidental, antagonistic, agonistic or symbiotic in nature, thereby increasing the complexity of these systems further [110].