Quorum sensing

Phenomenon of quorum sensing

The phenomenon of quorum sensing was first characterized in the bioluminescent bacterium, Vibrio fisheri. Vibrio fisheri is a marine bacterium that inhabits the light organ of certain fish and squid, thus forming a symbiotic relationship. V. fisheri can also be found free-living in seawater. Light production by V. fisheri does not occur until the cells reach a certain density (see graph). Since this density of cells of V. fisheri is only reached in the light organs of the host, planktonic (free-living) V. fisheri are not bioluminescent.

• Bioluminescence webpage
• Graph of luminscence 

Light production in bioluminescent bacteria is catalyzed by an enzyme called luciferase. Luciferase catalyzes the oxidation of a reduced flavin and a long chain aldehyde, thus emitting light, and the production of an oxidized flavin and a long chain fatty acid.

• luciferase and light production

When V. fisheri is bioluminescent, luciferase can make up 5% of the cellular protein and use up more than 10% of the energy of the cell in the light emission process. Light production only occurs when cells reach a certain density and is mediated by a small sensory molecule, called an autoinducer. Dilution of cells that are luminescent to a lower density results in loss of bioluminescence. The addition of the supernatant from a bioluminescent culture to a non-luminescent culture at lower cell density results in the premature induction of bioluminescence. A compound was isolated from the culture supernatant and identified as N-(3-oxohexanoyl)-homoserine lactone (or N-acyl homoserine lactone or AHL). The addition of AHL could induce luminescence in a bacterial culture that were below the normally required cell density for bioluminescence to occur.

• AHL

Homoserine lactones are derived from S-adenosyl methionine. The acyl group is contributed by acyl carrier proteins or acyl coenzyme A. A transporter for AHL to get out of the cell and a receptor to bring AHL into a neighboring cell are not required to carry AHL across the membrane because of AHL permeability to membranes.

These observations showed that: (1) bioluminescence is a highly regulated process in V. fisheri, (2) bioluminescence is regulated by cell density or quorum sensing and is essentially a "social behavior" in bacteria, (3) quorum sensing is regulated by a diffusible factor.

Since the identification of quorum sensing in V. fisheri, it has been described in over 30 species of gram negative bacteria. Quorum sensing also occurs in gram positive bacteria, such as Bacillus subtilis. Quorum sensing in B. subtilis regulates entry into sporulation and competency for natural transformation. Unlike in gram-negative species of bacteria, quorum sensing in gram positive bacteria, such as Bacillus, is mediated by peptide pheromones.

• Engebrecht and Silverman Fig. 2

The genes for bioluminescence from V. fisheri were introduced onto a plasmid and transformed into E. coli and transformants capable of producing light under high cell densities were identified. These experiments were performed because of the ease of genetic analysis in E. coli, as compared to V. fisheri. Mutational analysis of the introduced clones by Tn5 mutagenesis and hydroxylamine treatment was used to identify the function of genes necessary for bioluminescence. Why were both Tn5 and hydroxylamine mutagenesis used to characterize the bioluminescence genes?

• Genetic analysis identified 7 gene products necessary for bioluminescence, luxR, luxI, luxC, luxD, luxA, luxB and luxE that are organized as two divergently transcribed operons. Functions were assigned to these genes based on the phenotype of mutants.

  luxA and luxB encode the alpha and beta subunits of luciferase.

  luxA and luxB mutants cannot be complemented by addition of exogenous aldehyde

  lux C, D and E encode genes involved in either the synthesis or recycling of the aldehyde substrate

  luxC, luxD and lux E mutants can be complemented by addition of exogenous aldehyde

  luxI encodes a gene involved in synthesizing AHL.

  luxI mutants can be complemented by addition of autoinducer (AHL), but do not produce any autoinducer themselves (culture supernatants cannot induce bioluminescence in a different culture).

  luxR encodes a transcriptional regulator.

  luxR mutants could not be complemented by addition of autoinducer, nor did they produce detectable levels of autoinducer. What does this result tell you about the function of LuxR?

LuxR is a transcriptional activator in the helix-turn-helix family. Binding between LuxR and the autoinducer is required to activate DNA binding activity of LuxR. Binding of AHL to LuxR results in a conformational change that alters the DNA binding affinity of LuxR. LuxR activates transcription of the lux operon, which includes both the genes for luciferase production (luxABCDE) and luxI.

LuxR possesses a membrane bound N-terminal domain that binds AHL. Mutants have been identified in the N-terminal region that require larger amounts of AHL for induction. Mutants that are missing the N-terminal domain are constituitively active.

The carboxy terminal portion of LuxR is required for DNA binding. Interaction with AHL is required for LuxR dimerization. LuxR binds DNA as a dimer at promoter sites designated lux boxes. Binding of the AHL by LuxR is thought to release LuxR from the membrane to the cytoplasm, where activation then occurs. Mutants missing the C-terminal domain abolish DNA binding and transcriptional activation. Loss of only 40 aa from the carboxy terminus results in DNA binding (as shown by repression), but failure to activate transcription.

It is believed that LuxR interacts with the sigma 70 subunit of RNA polymerase to activate transcription at the lux box.

 lux operon

 Autoinduction: binding of the LuxR-AHL to the promoter of the lux operon results in the activation of transcription. The lux operon includes the genes for luciferase production, but also luxI, which is involved in formation of AHL. Thus, binding of LuxR to AHL results in activation of the lux operon, more LuxI, and thus more autoinducer, and thus more LuxR-AHL binding and activation. This results in a very rapid and dramatic increase in light production (1000-fold increase).

Model for quorum sensing in V. fisheri

Quorum sensing has been identified in a large number of bacteria, both gram negative and gram positive, and regulates a plethora of responses.

gram negative bacteria

Quorum sensing has been reported in over 30 species of gram negative bacteria which control cellular functions based on cell density. Although luxR/luxI orthologs are present in all of these species, the target genes that are controlled by quorum sensing differ. In V. fisheri, LuxR/LuxI regulates transcription of the lux operon. In the plant pathogen, Agrobacterium tumefaciens, LuxR/LuxI homologs regulate conjugative transfer of a virulence plasmid called the Ti plasmid. Bacteria may also contain more than one quorum sensing pathways that respond to different AHLs (see structures). For example, the human pathogen, Pseudomonas aeruginosa, has two quorum sensing pathways that coordinately regulate the production of virulence factors. Regulation by quorum sensing may either be by activation or by repression of target pathways.

Screens have been performed that show bacteria can cross-talk to each other with AHLs.

Quorum sensing can also occur via two-component systems, where the receptor kinase binds the autoinducer. Binding of autoinducer results in autophosphorylation of a histidine residue and subsequent transfer to the response regulator. Bioluminescence in a related Vibrio species, V. harveyi is mediated in a cell density dependent manner by a two component system.

• quorum sensing and two component systems

gram positive bacteria

• Gram positive bacteria also exhibit quorum sensing that can regulate the decision to sporulate, compentency for transformation, virulence, conjugation and antibiotic production. However, in species that have been investigated, quorum sensing is mediated by short peptides. These small peptides are transported from inside the cell to the outside via dedicated transporters (ABC transporters) present in the cell membrane. The peptides in the extracellular compartment are recognized by a receptor kinase present in the membrane of the cell. Auotphosphorylation of the receptor kinase upon ligand binding results in the transfer of the phospho group to a cytoplasmic response regulator, thus activating it and resulting in either transcriptional activation or repression of target genes.

• model for quorum sensing gram positive bacteria

• Eukaryotic organisms may produce compounds that interfere with quorum sensing as a means of defense against pathogen invasion. An example is Delisea pulchara, an alga that produces compounds that interfere with quorum sensing, and thus colonization by Serratia liquefaciens. One of these compounds from D. pulchara has been shown to bind to LuxR and displace AHL, thus inactivating LuxR.

The green alga, Entermorpha, senses AHL to attach to bacterial cells in marine biofilms (Science 298:1207), particularly biofilms associated with fish pathogenesis. Bacterial biofilms play an important role in the development of algal communities.

Integration of quorum sensing with other genetic regulatory pathways

Quorum sensing can be affected by additional regulatory components, such as entry into stationary phase. Genes involved in adaptation to stationary phase are regulated by the stationary phase sigma factor, RpoS.

• quorum pathways and integration

An example of the integration of quorum sensing with other regulatory pathways is shown by Pseudomonas aeruginosa, an opportunistic human pathogen. P. aeruginosa is capable of causing infections of the eye, lungs (especially in cystic fibrosis patients) and burn wound victims. P. aeruginosa has at least two quorum sensing systems, both regulated by diffusible substances structurally similar to AHLs. Many of the genes requlated by quorum sensing in P. aeruginosa are involved in virulence, such as the production of enzymes that cause damage to cells, (elastase), toxins (exotoxin), lipases and pathogen defense molecules, such as catalase and superoxide dismutase. The stationary phase sigma factor RpoS, also plays a role in quorum sensing regulation.

In addition to quorum sensing affecting requlation within a single species, the fact that different species make similar AHLs and will respond appropriately to an AHL formed by a different species suggests that quorum sensing may also play a critical role in interactions between different bacterial species in nature.

AHL mediated control of gene expression occurs through the activity of the LuxI and LuxR family of proteins. Structural variations in the acyl side chains of AHL have a profound effect upon activation of LuxR. For example, in P. aeruginosa, N-3-Oxododecanoly-l-homoserine lactone is the activator of LasR, while N-butanoyl-L-homoserine lactone is the activator for RhlR.

The use of biolumenescent sensors based on the LuxRI regulatory cassette has been useful for identifying quorum sensing in gram-negative bacteria. Genes encoding LuxR and LuxI homologs show limited nucleotide sequence similarity, so homolog searches have had limited successs in identifying LuxR/LuxI homologs. Promoter probe plasmids based on luxCDABE have been used extensively to identify quorum sensing mechanisms in diverse bacterial species.