In 1970, photobiology researcher J. Woodland Hastings and colleagues at Harvard University discovered that a marine bioluminescent bacteria — Vibrio fischeri, indigenous to the gut of deep-sea fish — only glowed when a high cell density was reached in culture (1). This discovery sparked a hunt for the signaling molecule that allowed the bacteria to coordinate their luminescence to such a degree that the colony lit up at the exact same moment.
Now, new tools and techniques are allowing researchers to not only understand why bacteria need to communicate with one another but also to manipulate these communication streams to improve antibiotics and develop clean energy technologies.
Central to quorum sensing is the ability of bacteria to monitor their surroundings in order to detect cell density so that when a population flourishes there can be a coming together of like “minds,” as it were, and a resulting coordination of “behaviors.” Yet, work by Rustem F. Ismagilov out of the University of Chicago has shown that a single cell confined to an extremely small space can activate quorum sensing.
“What matters in quorum sensing is the accumulation of the signaling molecule; if the signal concentration becomes high enough, even low numbers of cells can get the job done,” says Carey D. Nadell, a post-doc in the Princeton University lab of Bonnie Bassler, a pioneer in the field of quorum sensing.
Most of Bassler’s lab is knee-deep in working out the molecular mechanisms bacteria use to communicate. Nadell, who trained as an evolutionary biologist, is more interested in why bacteria bother to communicate at all, that is, what advantages quorum sensing confers on a bacterial cell or cells in the natural world. To this end, Nadell and colleagues examined why some bacteria exhibit opposing quorum-sensing phenotypes when placed under similar cell density conditions.
Prior work in the Bassler lab showed that the human pathogen Vibrio cholerae uses quorum sensing to up-regulate their production of biofilms at low cell densities and alternately, down-regulate biofilm production at high cell densities. This runs counter to the typical pattern of biofilm up-regulation at high cell densities observed in other human pathogens; for example Pseudomonas areuginosa, which causes infections in open wounds and the lungs of patients with cystic fibrosis, ramps up production of the materials needed to build biofilms (an extracellular mixture of polysaccharides, DNA, and proteins) only at high cell densities.
“We asked ourselves: What about the natural history of each of these pathogens would select for different types of quorum sensing?” says Nadell. Because V. cholerae is more of a roaming type of bacteria by nature, seeding quickly in the host to establish acute infection and then just as quickly dispersing, Nadell and his co-authors built the first evolutionary model of quorum sensing in biofilms to test their hypothesis that evolutionary pressures may determine quorum sensing phenotypes.
Biofilms give specific advantages to bacteria, such as protection from immune system challenges, predators, shear stress, and antibiotics; but because biofilms form sticky matrixes that act like glue, holding bacterial cells in place, biofilm formation can be a hindrance to cells trying to disperse in order to range from one environment to another.
Using computational models that tracked individual cell behavior in simulated space, Nadell and colleagues analyzed patterns of quorum sensing under varying conditions. In the case of V. cholerae, the team reported biofilm formation was associated with a positive ability of the bacteria to compete with native intestinal flora to establish residence (2). Once population density reached an apex, bacterial down-regulation of biofilm production allowed for a redirection of resources that enabled a short burst of biomass growth and rapid dispersal from the attachment site.
Now that the group has established a theoretical model for the evolution of quorum-sensing regulation of biofilm production, they are proceeding with live lab bacterial competition experiments. Ultimately, they hope that such research enhances the ability to manipulate quorum sensing to human advantage.
At the Albert Einstein College of Medicine of Yeshiva University in New York, manipulation of quorum sensing has enabled the Vern L. Schramm lab to develop novel antibiotic compounds that don’t trigger resistance.
“The number one health problem with bacterial infections today is due to the ability of antibiotics to kill bacteria and therefore select actively for resistant strains, and once you have a resistant strain it’s very difficult to get rid of that from health care facilities,” says Schramm.
For this reason, he hypothesized that antibiotics that could reduce the infective functions of bacteria — such as biofilm formation — but not kill them, would reduce the risk that resistance would later develop. And previous research has demonstrated that mutant bacterial strains defective in quorum sensing produce attenuated infections in host species. For example, mice infected intranasally with quorum-sensing deficient Streptococcus pneumoniae showed decreased lung infection rates.
So, the Schramm lab set about to block quorum sensing in two highly virulent bacterial species: Vibrio cholerae and Escherichia coli 0157: H7. To accomplish this, they targeted a bacterial enzyme, MTAN, known to be essential in the biosynthesis of autoinducer signaling molecules.
Because the Schramm lab has experience producing transition state analogue inhibitors of human enzymes (two are currently in clinical trials: one to treat gout, another, leukemia), they decided to develop an inhibitor of MTAN. The concept of a transition state inhibitor is to capture the exact moment in time —one-tenth of one trillionth of a second, to be exact — when the substrate, in this case MTA, is broken by the catalytic activity of the enzyme, here, MTAN.
The technology involved uses kinetic isotope effects, which measures the effect of substituting a heavy isotope at the position where bonds are being broken during a catalytic reaction. Each substitution of mass, e.g., carbon 13 to replace carbon 12, acts as a reporter on what is happening at the transition state. This information is used to create an analogue that binds to the enzyme more tightly than its natural substrate, in effect “locking up” the enzyme and preventing it from acting on its natural substrate.
Work published in Nature Chemical Biology showed that three synthetic small-molecule transition-state analogues developed by Schramm’s team against quorum sensing exhibited high potency in disrupting quorum sensing in both V. cholerae and E. coli, with biofilm production significantly reduced (3).
“There was always a question if MTAN was really involved in quorum sensing or not,” says Schramm. “The answer has been to knock out this enzyme with both our drug and gene depletion.”
To prove that no resistance developed in bacteria treated with their transition state analogue antibiotics, the lab grew the bacteria out for 26 successive generations, a 226 expansion of cell number, and found the final generation was just as sensitive to his antibiotic as the first. Now, Schramm is looking to move this work into animal testing in the near future and sees myriad applications in both industry and healthcare.
Manipulating quorum sensing is likely to have far-reaching applications in realms outside of medicine. For example, Thomas K. Wood out of Pennsylvania State University is particularly interested in clean energy production and bioreactors.
“The engineer in me wants to elucidate how to use biofilms for beneficial purposes,” says Wood. And because the bacteria used in bioreactors to make useful end products will be more robust in biofilms, less prone to demise in fluctuating conditions, it is important to be able to control biofilms at varying times.
In a recent Nature Communications paper (4), Wood and colleagues have shown proof-of-principle that biofilms can be controlled — his group was able to lay down biofilm and then disperse it as well, using a population-driven quorum-sensing switch coupled to genetically engineered biofilm disperser proteins.
To accomplish this, his group engineered a synthetic biofilm quorum-sensing circuit that utilized the homoserine lactone quorum-sensing circuit native to P. aeruginosa to induce a new form of communication between two genetically engineered E. coli cells — one froma biofilm colonizer cell, the other, a biofilm disperser cell.
In addition, Wood engineered two proteins involved in biofilm dispersal —BdcA and Hha — to enhance their ability to disperse biofilms and cloned those mutated genes into the E. coli colonizer cells and dispersal cells respectively. In a complex biofeedback loop, the disperser cell, churning out the quorum-sensing P. aeruginosa signal, induces the colonizer cell to express the BdcA gene, which acts as a biofilm disperser.
This, in essence, allowed Wood to control consortial biofilm production. In terms of medical applications, it may be feasible at some future point to remove biofilms in vivo using the same BdcA network. His lab is actively investigating this avenue.
Clearly listening in on bacterial conversations is presenting researchers with an expansive new vocabulary to mine.
- Nealson, K. H., T. Platt, and J. W. Hastings. 1970. Cellular control of the synthesis and activity of the bacterial luminescent system. Journal of bacteriology 104(1):313-322.
- Nadell, C. D., J. B. Xavier, S. A. Levin, and K. R. Foster. 2008. The evolution of quorum sensing in bacterial biofilms. PLoS Biol 6(1):e14+.
- Gutierrez, J. A., T. Crowder, A. Rinaldo-Matthis, M.-C. C. Ho, S. C. Almo, and V. L. Schramm. 2009. Transition state analogs of 5'-methylthioadenosine nucleosidase disrupt quorum sensing. Nature chemical biology 5(4):251-257.
- Hong, S. H., M. Hegde, J. Kim, X. Wang, A. Jayaraman, and T. K. Wood. 2012. Synthetic quorum-sensing circuit to control consortial biofilm formation and dispersal in a microfluidic device. Nature Communications 3(January):613+.