25Winter 2011

The Primal ConversationIntercellular Communication in Bacteria

AAron koenig ‘14

Biology

Historically, conventional think-ing in evolutionary biology has drawn a bright line sepa-

rating multicellular from unicellular organisms. Only the cells of animals, plants, and fungi were thought capable of achieving the synchrony required to function as a collective. Bacteria, in contrast, were perceived as blind, deaf, and mute, with each cell single-mind-edly focusing on its own metabolism and reproduction. However, recent discoveries in the field of bacterial cell-cell signaling have exposed the ubiqui-ty of multicellular behavior in bacterial populations, dramatically altering our understanding of the microbial world.

Introduction to Quorum Sensing

the first complete descrip-tions of cell-cell signaling systems used by microbes to regulate gene ex-pression were obtained from studies of the marine bacteria Vibrio fischeri and Vibrio harveyi over 30 years ago (1). The genes studied encode light-producing luciferase enzymes and are up-regulated in response to increases in population density (1). Underlying this unusual behavior is a pheromone, or autoinducer, from the family of acyl-homoserine lactones (AHL) (2). Ev-ery Vibrio cell contains the gene luxI, whose protein product, known as a synthase, synthesizes AHL (2). As the cells divide, increasing their population density, the concentration of AHL also increases (2). An intracellular recep-tor, encoded by the gene luxR, binds AHL when the concentration of the pheromone reaches a specific threshold (2). The LuxR-AHL complex acts as a transcriptional activator, stimulating production of the luciferase enzymes and LuxI, which produces the original autoinducer (2). Further increases to the concentration of LuxI establish a positive feedback loop, leading to pop-ulation-wide production of light (2).

In some respects, the mecha-nism of cell-cell signaling used by Vi-brio cells to assess population den-sity resembles a democratic system of government. Each cell casts a vote through its contribution of phero-mones. When cells detect the presence of a sufficient concentration of phero-mone, analogous to the recognition of a quorum, the group takes action. A nonscientist friend of Stephen Win-ans, a researcher at Cornell University studying the role of bacterial signaling in plant crown gall disease, serendipi-tously devised the term “quorum sens-ing” to describe multicellular bacterial behavior (3). As described by microbi-ologists, quorum sensing refers to the ability of bacteria to detect population density and coordinate corresponding shifts in gene expression patterns (4).

Multicellular Bacterial Behaviors Regulated by Quorum Sensing

Transcriptional regulatory pro-teins activated by binding to phero-mones enhance gene expression in

genes whose promoters contain a specific sequence element recognized by the protein-pheromone complex. While the synthases of pheromones and their associated receptors origi-nated early on in bacterial evolution-ary history — the LuxI/R system of the vibrios has homologues in evolution-arily distant human and plant patho-gens — even closely related quorum sensing systems can be repurposed to control a variety of behaviors (5). Be-haviors activated in density-dependent response pathways include biolumi-nescence, symbiotic plant root nodule formation by nitrogen fixers, bacterial mating, antibiotic production, biofilm formation, and extracellular DNA up-take in response to harsh conditions (6). In all of these cases, bacteria only benefit from engaging in these activi-ties collectively at high cell densities.

Vibrio fischeri, for example, en-gages in a partnership with the Hawai-ian Bobtail Squid by producing light when the bacteria grow in the nutrient-rich environment of the squid’s spe-cialized light producing organs. This relationship provides direct benefits for the squid, which can escape the

Image retreved from http://upload.wikimedia.org/wikipedia/commons/c/cf/Quorum_sensing_diagram.png (Accessed 29 Jan 2011).

Schematic diagram of Quorum sensing.

Dartmouth unDergraDuate Journal of Science26

notice of predators on moonlit nights by counter-illuminating themselves. By using the light produced by their complement of V. fischeri, the squid can eliminate telltale shadows on the seabed. Light production only confers a selective advantage for bacteria when they are growing within the confines of the squid. Free-floating populations of V. fischeri do not express high levels of luciferase. Using the LuxI/R quorum sensing system, the bacteria are able to avoid the individual fitness cost of luciferase synthesis in the open ocean.

While many bacterial species, in-cluding V. fischeri, do no harm to the hosts they colonize, quorum sensing also aids bacterial pathogens. Highly developed immune systems, like those of humans, are adept at preventing in-fectious disease; however, bacteria are able to avoid a host immune response by suppressing expression of virulence factors until enough cells are present to overrun the host (7). Interestingly, not all pathogens conform to this model of virulence regulation by quorum sens-ing. In Vibrio cholerae, the etiologi-cal agent of cholera, a two-pheromone quorum-sensing network arranged in parallel represses virulence factor production at high population densi-ties, but permits their expression at low densities (2). More background

on the parthenogenesis of cholera re-veals the evolutionary rationale for this idiosyncrasy, as it is the diarrhea resulting from cholera that allows V. cholerae to spread (2). Concurrent in-hibition of biofilm formation permits bacteria to detach from their intesti-nal hideout and “go with the flow” (2).

Biofilm formation, involving the creation of multicellular aggregates, is an important intermediate step in the progression of bacterial infection. Al-though significant variation exists in the structure and composition of bio-films, most bacterial species create bio-films through the secretion of exopoly-saccharides, the primary constituent of the extracellular matrix that binds cells into a biofilm (8). Bacteria in biofilms are more resistant to antibiotics and the host immune system, properties useful for maintaining chronic infec-tions (9). it is estimated that biofilms participate in 65 percent of human bacterial infections, making biofilm formation and maintenance the focus of extensive research (8). Perhaps not unexpectedly, quorum-sensing signals have been shown to make important contributions to biofilm development in many species of microorganisms (8).

Disrupting Quorum Sensing Systems

By employing intercellular signal-ing, bacteria enjoy a seemingly limit-less ability to adapt to the vagaries of their environment. Our body’s defens-es are all too often unable to adequately respond to the challenges posed by a cooperative group of bacteria produc-ing virulence factors or encasing them-selves in protective biofilms. Fortu-nately, this model of host-bacterial interaction, in which bacteria always have the upper hand, is only partially correct. While fighting infections by Pseudonomas aeruginosa, a process dependent on quorum sensing, the hu-man body is able to use its paraoxonase family of organophosphate-hydrolyz-ing enzymes to degrade the acyl homo-serine lactone pheromone produced by the bacteria (6). Inhibition of quorum sensing, or “quorum quenching,” has also been observed in barley and fungi. This implies that quorum quenching is an evolutionarily successful strategy for combating bacterial infection (10).

In an era dominated by multi-drug resistant bacteria, alternatives to anti-biotics active on a broad class of micro-organisms are in high demand. It is a sobering thought that Penicillum fungi, from which the “miracle” drug penicillin was derived, produce small molecules that inhibit quorum sensing (6). These molecules mimic the pheromones of specific bacterial species, competitively binding to receptors of the true phero-mone to prohibit activation of the quo-rum-sensing pathway (6). Apparently, even the organisms responsible for sparking the mass production of anti-biotics cannot solely rely on that class of biomolecules for protection. Bio-technology may yet restore Penicillum fungi to the forefront of anti-microbi-al warfare due to recent advances in methods of disrupting quorum sensing.

The quorum sensing systems of gram-negative bacteria use acyl homos-erine lactones as primary pheromones. Despite the potential for cross-species signaling, most pheromone-receptor systems are used for intraspecies com-munication, specific to a single bacte-rial species or strain (11). Exceptions do exist, however. For example, the synthase of AI-2, a pheromone struc-

Image courtesy of Nick Hobgood.

Vibrio fischeri has a symbiotic relationship with the Bobtail Squid.

27Winter 2011

turally unrelated to the acyl homos-erine lactones, is encoded in the ge-nomes of nearly 50 percent of all fully sequenced bacteria (2). A recent study by Belgian researchers affiliated with Ghent University found that the ex-pression of genes regulated by quorum sensing in multiple vibrio species could be reduced by AI-2 quorum sensing in-hibitors that bind to the LuxPQ recep-tor of AI-2 (12). Libraries of candidate quorum-sensing inhibitors can be rap-idly screened using lethal genes linked to pheromone-dependent promoters, assaying the success of each inhibitor by locating bacteria that grow (13). A group of British researchers has taken a different approach to block bacterial communication, designing a chemical-ly inert polymer that traps the phero-mones of V. fisheri (14). The ultimate goal of quorum-inhibition research is to produce a broad-spectrum inhibitor, which could act on a single pheromone produced and recognized by multiple bacterial species, as in the case of AI-2.

Emerging ApplicationsIn the study of bacterial quorum

sensing, much emphasis has been placed on applying emerging findings on the structure of intercellular sig-naling pathways to the prevention and treatment of bacterial infections. The potential of quorum sensing, however, also stems from its unique combina-tion of bacteria with simple systems

for intercellular signaling. From a bioengineering perspective, quorum-sensing bacteria create tantalizing possibilities for gene circuit design, in which well-defined chemical in-puts are linked to desired outputs (6).

One astonishing multicellu-lar machine has already been devel-oped: a micro-scale clock driven by the oscillating fluorescence output of recombinant bacteria (15). The gene circuit driving this behavior contains components from the LuxI/R phero-mone-receptor pair of the vibrios, but links their activation to the expres-sion of Green Fluorescent Protein and an enzyme that quenches the LuxI/R pheromone signal (15). Coupling the transcriptional activator to its own re-pressor leads to the establishment of tunable, periodic fluorescence (15). Removal of excess extracellular AHL via a microfluidics system is required for the maintenance of oscillation, which demonstrates, as in the V. fisch-eri – squid symbiosis, the importance of environment to the long-term stabil-ity of quorum sensing behaviors (15).

ConclusionsFrom bacterial biofilms to net-

works of neurons, life depends on cel-lular communication as the basis of its continued survival. Although the methods of intercellular communica-tion employed by plants, animals, and fungi are far more complex than those of bacteria, the existence of quorum-sensing systems indicates that pre-cursors of multicellularity penetrate further into the tree of life than we previously expected. Moreover, the regulatory networks associated with quorum sensing are hardly archaic. Humans consist of roughly one trillion cells, far outnumbered by the ten tril-lion bacterial cells estimated to reside on or around our body (16). Quorum sensing systems mediate interactions between our body and its associated microbes that enrich our lives tremen-dously by aiding us in digestion, pro-tection against environmental hazards, and vitamin synthesis (16). Converse-ly, quorum sensing is used to maximize harm to our body by invasive patho-gens, often overshadowing the posi-tive contributions of our microbiome.

The challenge of fully elucidat-

ing quorum sensing will continue to stimulate research into bacterial com-munication in the near future. It is likely that bacteria have been commu-nicating using pheromones for billions of years. For the first time in history, when bacteria talk, the world listens.

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Image courtesy of MethoxyRoxy

Neurons are well known for communicating via synaptic connections.