Quorum Sensing: Communication on a microbial level

Quorum sensing is the mechanism in which bacteria cells are able to communicate and co-ordinate their behaviour within population densities. Bacteria are able to sense the presence of other members of the species and in some cases other species, by using signal molecules known as autoinducers (Als). These signalling molecules can accumulate in the environment and once they reach a certain concentration they trigger a specific response, for instance bioluminescence. The higher the densities of bacteria, the larger concentrations of auto inducers are present. Quorum sensing ensures at low densities bacteria are individuals and in high cell densities bacteria exhibit group behaviour. This allows bacteria to express a gene product only in high densities, reducing the risk of wasting energy releasing the gene product in low densities, for instance luciferase in Virbro fisheri. Non-coding small regulatory RNAs (sRNAs) control; traits, nutrient uptake, stress response, viral immunity and quorum sensing. Quorum regulated small RNAs (Qrr sRNAs), can repress mRNA translation by pairing with a ribosome binding site inhibiting ribosome access, resulting in mRNA degradation.

Figure 1. Model for Qrr sRNA regulation of aphA, LuxR/HapR and LuxO.

In Vibro harveyi quorum sensing, at low cell density in the absence of autoinducers,

      

1.The quorum response regulator protein Lux O is phosphorylated. Phospho-LuxO activated the expression of 5 genes. 
2.The 5 genes, encode 5 Qrr sRNAs which activate translation of the low cell density master regulator AphA

3.Simultaneously repressing the high cell density master regulator LuxR.

 High densities when autoinducers are present,

4.  LuxO is dephosphorylated and is inactive

5.  The production of Qrr sRNAs ceases without Qrr sRNA and AphA is not produced

6.  Only LuxR is translated.

Vibro chloerae, is closely related and differs slightly with only four genes expressed by LuxO-P (phosphorylated luxO)  and the LuxR counterpart is called HapR. Both are captured in Figure 1 (Shao et al 2012), The numbers added to the diagram refer to the processes outlined above. The Phosphorylation-dephosphorylation of luxO determines the expression of the structural genes for bioluminescence (Munn, 2011).

This study illustrates the production pattern of the newly identified quorum-sensing low cell density master regulator AphA in both Vibro harveyi and Vibro cholera. This study included several tests with both Vibro harveyi and Vibro cholerae to investigate into the factors that could affect the production of AphA in quorum sensing.

              1. How regulation affects AphA protein level

·         Wild type – high cell density mode

·         LuxOD47E - mimicking phosphor-LuxO, low cell density mode

·         ∆LuxR/∆HapR – high cell density mode, but luxR/HapR are independent

·         LuxOD47E ∆LuxR/∆HapR - low cell density mode, but luxR/HapR are independent

This test used a western blot of the four genetic backgrounds (above), and compared the results from each background. When comparing the Wild type and LuxOD47E it showed that the AphA protein is dramatically reduced once Vibro harveyi is in high density mode. Similar results were found when comparing LuxR deletion strains (∆LuxR with LuxOD47E ∆LuxR). The LuxOD47E ∆LuxR strain observed Qrr sRNAs responsible for inducing the high-level production of AphA, at low cell density. In the Vibro cholerae tests distinct of Vibro haveyi, at high cell density there remained detectable AphA proteins.

       2.Does Proper base pairing between Qrr sRNAs and aphA mRNA affect aphA protein production?

In this test, a plasmid encoding a Vibro harveyi AphA-GFP translational fusion was engineered into Esterichia coli. Esterichia coli was used to avoid interference from other Vibro harveyi that could alter AphA regulation. Another plasmid was added to the Esterichia coli strain this time the plasmid encodes Qrr4. AphA-GFP production increased when wild type Vibro harveyi Qrr 4 was expressed in Esterichia coli, this showed that the Qrr sRNAs act independently, and suggests base pairing.

The reason for this investigation was that a sequence comparison of Vibro harveyi and Vibro cholera Apha mRNAs with the Qrr sRNAs revealed a potential binding site (~130 upstream of the start codon in the 5’UTR of the AphA mRNA). The complementary sequence in the Qrr sRNAs contains two sections (region I and region II). The complementary pattern suggested that the Qrr sRNAs could control AphA production through base pairing between one or both regions.

The idea of base pairing was then examined again by making alterations to the sequence in region I and region II, these were called mutation I and mutation II. Both mutations eliminated the activation of AphA-GFP production. Another set of mutations were carried out called mutation A and B, these disrupted pairing in each region. These were introduced to the pervious mutations; combining non-complementary mutations did not restore regulation. This shows that Qrr 4 activated AphA production at low density through base pairing to the aphA mRNA 5’UTR. Similar results were obtained for Vibro cholerae AphA-GFP, except for a significant difference in basal levels of AphA.

                            3. Can Qrr sRNA discriminate between target mRNAs?

This test used the three targets, aphA, LuxR and luxO which were chosen because they are present in both Vibro harveyi and Vibro cholerae. An Esterichia coli strain containing an AphA-GFP, LuxR-GFP or LuxO-GFP translational fusion was also used. Qrr sRNAs employ distinct pairing regions to discriminate between different targets. The results showed that Region I is uniquely used for AphA activation however region II is used for all three targets (aphA, LuxR and LuxO).

4. Prediction: Qrr1 should work as well as Qrr4 in regulating LuxR and LuxO.

To test their prediction the authors used GFP reporters in Esterichia coli to compare the strength of Qrr4 ans Qrr1 regulation of AphA, LuxR and LuxO. Qrr1 is less effective activating AphA-GFP than Qrr4. Both Qrr1 and 4 repress LuxR-GFP and LuxO-GFP production to similar levels.

Overall the results suggest that evolution of multiple Qrr genes in Vibrios is linked to newly emerged targets that are under their control. In Vibro harveyi and Vibro cholerae Qrr 1 became dedicated to regulation of targets (LuxR and LuxO). The other Qrr sRNAs became available to control additional targets (AphA).

To conclude the use of Quorum sensing in bacteria, is dependent on density of populations. The genes expressed and eventual behaviour of the bacteria is then influenced by high or low population densities. In Low densities AphA is produced, however in high densities due to the absence of Qrr sRNAs there is a reduced production of AphA. This paper showed that the main influences to AphA production is base pairing, specific binding regions and the type of Qrr sRNA. These can all be subjected to mutation and affect the amount of AphA produced.

The importance of quorum sensing in bacteria means they are able to quickly change their behaviour in the presence of environmental change. Marine snow associated bacteria have recently been shown to produce AHLs (Munn, 2011). AHL is another signal molecule called acyl homoserine lactone. This means that using quorum sensing marine snow related bacteria can track the highest density of members of their species and therefore the highest/lowest concentrations of DOM.

This paper links to my previous post about bioluminescence  by explaining the intercellular communication system that is associated with some bioluminescent bacteria. Quorum Sensing is a mechanism that initiates the luminescence of some organisms like Vibro Fisheri when they reach an optimum population density. The paper has provided a greater insight into the genes and processes incorporated in quorum sensing, it would be interesting to determine if there are some clear behaviour patterns exhibited by the bacteria and how phylogeny of these bacteria may affect the Qrr genes, since this study showed that Qrr 1 did not activate AphA as well as Qrr4.

Shao Y, Bassier B.L, (2012) Quorum-sensing non-coding small RNAs use unique pairing regions to differentially control mRNA targets, Molecular Microbiology, issue 83, pages 599-611

Munn C. (2011) Marine Microbiology, ecology and applications, 2^nd edition, Taylor and Francis group LLC, pages 81- 82.