Both Gram-negative and Gram-positive bacteria use a

BothGram-negative and Gram-positive bacteria use a form of chemical communication,known as quorum sensing, to sense and respond to variations in cell density intheir environment, enabling them to synchronize gene expression and coordinate behaviours.This phenomenon is critical for bacterial survival as it allows individualcells to undertake tasks that would only be effective in a social context, suchas biofilm formation or bioluminescence. Not only are cells able to detect presenceand quantity of neighbouring cells but they are also able to differentiatebetween kin and non kin and thus discern species variety in their environment. Thisallows them to discriminate against competing non kin by, for example, triggeringcollective inhibition of production of public goods so that the competitorswill not take advantage of them. Themechanism of quorum sensing is almost analogous across all species of bacteria,with slight variations in the nature of the signalling molecules and receptorsused to elicit a response. In general, small molecules, known as autoinducers,are produced within individual cells and exported.

As cell density increases,the production and release of autoinducers, by individual cells, grows, causingtheir extracellular concentration to rise. Once this concentration has passed acertain threshold, the autoinducer will bind to its receptor which, by avariety of pathways, activates heterogeneous changes in gene expressionpatterns, triggering a specific response. This enables cells to collectively reactto changes in cell density.

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 Cellsmust be able to respond to a variety of autoinducers in order to adapt tochanging environments and quorum sensing network architectures provide themeans by which this can be achieved. The existence of these communicationnetworks allows for more flexible signal-response dynamics and improved signalfidelity, as well as providing adaptability to deal with fluctuations intemperature, pH and the presence of non kin species. The details of thesefundamental steps in Gram-positive and Gram-negative bacteria will be discussedin further detail, with emphasis on the network architectures of the quorumsensing circuits.  Communicationand collective behaviours mediated by quorum sensing were first observed andunderstood in Gram-negative bacteria. The most distinctive feature in theirquorum sensing mechanism is the use of derivatives of S-adenosylmethionine(SAM) as autoinducers, most commonly, acyl homoserine lactones (AHL).

These aresmall molecules that are synthesized intracellularly (e.g. by a LuxI synthase)and released passively outside the cell. They have an N-acylatedhomoserine-lactone ring and a variable 4/18 carbon acyl chain, derived fromfatty acid synthesis, the length of which contributes to stability andtherefore longevity of elicited response. AHLs will accumulate as cell densityincreases and diffuse back into cells, through the cell membrane, where theywill bind to their cognate receptor. It is common for multiple AHLs to bepresent in the environment at once and thus a cellular response may be based onthe blend of autoinducers which it detects.

To accommodate this, complex quorumsensing networks exist within cells, containing a variety of receptors, each ofwhich must be highly specific for their cognate AHL. Amongst other feedbacksystems that are present in these networks, autoinduction provides a feedforward loop within which quorum sensing, which has been activated by anautoinducer, itself induces the production of more secreted autoinducer inorder to amplify the signal.  Mostcommonly, the receptor of a Gram-negative bacterium is a cytosolictranscription factor (TF), though further on it will be discussed how this isnot always the case. In general, receptors tend to have an N-terminal AHLbinding domain, which contains 3 highly conserved tryptophan residues, and aC-terminal helix-turn-helix DNA binding domain.

Upon autoinducer binding, thereceptors form homodimers and bind to regulatory regions of DNA. This structureallows the signal to be directly coupled to a response. Moreover, each receptoris highly specific for its cognate autoinducer, achieved by specific amino acidresidues within a flexible binding pocket, however some receptors, such as LuxRsolo receptors (so named as they do not have an accompanying LuxI synthase)have lower specificities in order to detect autoinducers of non kin species. Thereare three main classes of such receptors, which differ in AHL binding affinityand requirement of ligand for folding. Class 1 receptors, such as LasR of Pseudomonas aeruginosa, fail to foldwithout AHL and degrade, however in the presence of AHL will bind tightly. Thishigh binding affinity allows the receptor to continue being active for sometime even after AHL concentrations inside cell decrease. Class 2 receptors,such as LuxR of Vibrio fischeri (FIG11), alsorequire AHL to fold stably, but bind reversible, allowing signal to be moretransient. Class 3 receptors, such as EsaR of Pantoea stewartii, bind reversibly however do not require AHL forfolding and work in reverse to the other 2 whereby they act as transcriptionfactors prior to AHL binding, and degrade when it does.

Havingsensed that cell density is above the threshold, different species of bacteriawill respond differently. For example, activation of LasR in P. aeruginosa (FIG 22) willcause bacteria to begin virulence factor secretion, whilst activation of LuxRin V. fischeri initiates productionof luciferase.

Other responses based on cell density might be cell adhesion, sporulation,biofilm formation, antibiotic production or public goods production, to name afew.        Otherstrains of Gram-negative bacteria, such as Vibrioharveyi, have a 2 component quorum sensing system, the details of which willbe further explored in relation to Gram-positive strains. V. harveyi displays features of both Gram-negative and Gram-positivequorum sensing: although it uses AHLs characteristic of a G-ive strain, its membranebound TM spanning helix receptor is more closely analogous to that of a G+ivebacterium.

Quorum sensing in this organism activates bioluminescence, just likein V. fischeri, but by a verydifferent method of signal transduction. Morespecifically, V. harveyi uses 3 pathwaysin parallel, each with its own receptor, AHL and an AHL synthase. The autoinducerHAI-1 is synthesized by LuxM synthase and detected by LuxN histidine kinase.AI-2 is synthesized by LuxS synthase and detected by LuxP-LuxQ histidine kinasecomplex. And CAI-1 is synthesized by CqsA synthase and detected by CqsS. Allthree membrane bound receptors, LuxN, LuxPQ and CqsS, have kinase and phosphataseactivity and this intrinsic property allows the extracellular AHL to signal tocytoplasmic transcription factors by regulating phosphorylation.

At low celldensity (FIG 33),autoinducer concentration is low and the receptor is free. In this unboundstate, it has a high kinase activity which allows it to carry out an ATPdependent autophosphorylation of its conserved Histidine residue. Thisphosphate group is then transferred to a phosphotransfer protein LuxU fromwhich it moves to LuxO. When Lux O is phosphorylated, it acts as atranscription factor and activates 5 sRNA genes, collectively known as QrrsRNAs. Once transcribed, these sRNAs target and degrade the mRNA of a differenttranscription factor, LuxR, by an RNA inhibition mechanism. At high celldensity conditions, high concentrations of autoinducer are available, and AHLsbind to their cognate receptor outside the cell. This induces a conformationalchange in the receptor which reduces its kinase activity whilst promoting itsphosphatase activity. This inhibits autophosphorylation and as a consequence,LuxO is dephosphorylated which inhibits its function as a TF.

Now, because noQrr sRNAs are transcribed to degrade LuxR mRNA, it is translated and LuxR canact as a TF, regulating the expression of a variety of quorum sensing targetgenes.         Gram-positivebacteria were long thought to be unable to undergo quorum sensing, however now,these fundamental pathways are better understood (FIG 44). Themain difference between quorum sensing in G+ive and G-ive strains is the natureof autoinducers. Gram-positive bacteria synthesize oligopeptides, known as autoinducing peptides (AIP), which are impermeable to the cell membrane and requirespecialized transporters (often an ATP binding cassette) for secretion. TheseAIPs have more variability than AHLs, as they are genetically encoded, ratherthan synthesized from a common derivative, and undergo various modifications,such as cyclization, prior to secretion.  Thereceptors, commonly found in Gram-positive strains, are membrane bound, with anextracellular AIP binding site.

Thus, like in V. harveyi, signal transduction relies on a 2 component system (FIG55)mediated by a series of phosphorylation events. The2 component system is made up of the membrane bound histidine kinase dimer receptor(HK) and a cytoplasmic response regulator (RR) that connects extracellularsignal detection with a cellular response. Both of these domains are highlyvariable due to the diversity of incoming signals and required responses,however, the signally pathway can be broken down into 4 fundamental steps:signal detection, kinase activation, phosphotransfer and response generation.The extracellular signal is detected by the extracytoplasmic N terminus sensor domainof HK which undergoes a conformational change it its helix-periplasmic-helixdomain (connecting ligand binding site to TM helices) in response to ligandbinding. The catalytic core of the HK is made up of a dimerization andhistidine phosphotransfer domain (DHp), and a catalytic and ATP binding domain(CA).

The conformational change caused by ligand binding is thought to arrangethe two domains asymmetrically such that the ATP and conserved histidine areclose enough for autophosphorylation to occur. This is known as kinaseactivation. Depending on the nature of the HK monomer, the ? phosphate of thesame monomer’s ATP will be used to phosphorylate the N of the histidine (cisphosphorylation), or that of the other (trans phosphorylation).

        Next RR binds to the DHp domain of HK andundergoes phosphotransfer, using its conserved REC N-terminal receiver domainto catalyse the phosphorylation of its Aspartyl residue. The response ismediated by the variable C-terminal effector domain of the RR which isactivated upon phosphorylation. This can have: a DNA binding domain and act asa transcription factor, an RNA binding domain and trigger mRNA decay or aprotein binding domain. Alternatively it can have its own enzymatic activitywhich allows it to generate a response. The RR also has an impact on thetransience of the response as dephosphorylation is required to terminate it andthe rate of dephosphorylation depends on active/inactive conformations andnature of RR. Often the inactive HK receptor will have phosphatase activity andenhance this rate as soon as the AIP concentration goes below threshold. Staphylococcus aureus (FIG 66)is a Gram-positive bacterium that grows on human epithelial cells and is ofgreat medical relevance due its pathogenic potential under specific conditions(MRSA). This strain’s quorum sensing circuitis mediated by accessory gene regulators (Agr), consisting of an AgrC membranebound histidine kinase receptor, an AgrA cytoplasmic response regulator, an AgrDtranslated AIP precursor and an AgrB transporter for it.

It senses and respondsto changes in local cell density as described in the fundamental steps above,using phosphorylated AgrA as transcription factor to regulate genes associatedwith the quorum sensing circuit, as part of a feedback loop, as well asinducing the transcription of RNAIII which acts as the effector for the quorumsensing response, instead of AgrA, and upregulates expression of delta-haemolysinand other virulence factor and toxin associated genes. At high cell density,this strain will respond by secreting virulence factors and decreasing expressionof certain surface proteins as well as in some cases producing antibodies andforming biofilms.                              Thus,bacteria are able to sense changes in cell density by highly specific autoinducerand receptor systems, allowing then to transduce the signals such that theygenerate a global response, mediated by changes in gene expression. Despitecomplications by factors such as fluid flow, non-uniform growth conditions andthe cohabitation of multiple species in one environment, bacteria still manageto thrive and quorum sensing mediated communication remains effective due tothe highly specialized and organized network architectures present in thesecells.


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