The History, Biochemistry, and Potential of Quorum Sensing

Abstract

Cellular Society

The History, Biochemistry, and Potential of Quorum Sensing

Greg Steinberg
The Pennsylvania State University
State College, Pennsylvania 16801

Abstract

Until the 1970s, bacteria have always been considered to act independently of a population. They were thought only to respond to chemical and physical signals such as concentrations and temperatures. Until recently, cell-to-cell communication was never thought plausible in simple prokaryotes, and was strictly a mechanism of higher level organisms. However, in 1972 it was shown that the marine bacteria Photobacterium fischeri exhibited bioluminescence only when in high cell concentrations (2). It was deduced that the cells are able to sense the overall cell population, and the bioluminescent phenotype was expressed once a necessary cell density was reached. Originally called auto-induction, quorum sensing (QS) is the term used to describe cell-to-cell communication in bacteria. Many bacteria have been show to “see and socialize” within a population (and even across species!). By recognizing cells around them, bacteria are able to coordinate phenotype expression under density-dependent conditions. The studies of these systems continually show that QS is widespread among most, if not all bacteria. QS seems to be an important control factor for antibiotic resistance, virulence factors, and metabolite production, and is now being targeted for prophylactic use in medicine, as well as an efficiency module for numerous industries.

Introduction

Quorum sensing is the ability of cells to sense cell density in a population. Cells use molecules called Auto-Inducers as transcriptional activators for density-dependent genes. As cells grow, they produce small concentrations of auto-inducers. In low concentrations, the auto-inducers do not have any activity. Once a certain cell-density is reached, enough auto-inducer is produced to trigger the expression of density dependent phenotypes. These new phenotypes contribute to factors that only help survival of the bacteria when large cell numbers are present. If these genes were expressed in small cell density, the cells would waste energy without exhibiting a use for the phenotype.

The first evidence of quorum sensing came from Myxobacterium in the 1960s. This experiment suggested that a secondary metabolite [substrate] was produced that triggered the differentiation of cells (8). In 1972, the classical QS system came from bioluminescence in the marine bacteria Photobacterium fischeria (now reclassified as Vibrio fischeri) (2). Until this research, cell-to-cell signaling was believed only to be a trait of multicellular organisms; these studies were initially taken with much skepticism. However, since the study of V. fischeri, many more density-dependent phenotypes have been identified in a wide range of bacteria.

QS is capable of controlling a large variety of phenotypes, many of which are of great interest in medicine and pathology. For example, some virulence factors in Streptococcus and Staphylococcus are only expressed once the population of bacteria is large enough to defeat the host immune system.

In Streptomyces species, mupirocin (a potent antibiotic) is only produced in late exponential and stationary phases (6). QS regulates the antibiotic production (15), so that they are only produced when surrounding cell count is high. By killing the cells around it, Streptomyces increases its own available nutrients.

A phenomenon known as swarming, the coordinated movement across a substrate by a population is also controlled by QS (7). Swarming motility is a virulence factor for some species, and also provides antibiotic resistance to the swarming cells (10). By inhibiting the QS of swarming cells, these virulence factors may be repressible.

It's believed that most prokaryotes have some sort of signaling mechanism for cell-to-cell communication. As experiments that attempt to elude QS mechanisms increasingly become more popular, so are the promises of QS targeting molecules for industry and medicine.


Biochemistry of Quorum Sensing

One of the most understood signaling mechanisms for prokaryotes is that of the bioluminescent bacteria Vibrio fisheri. V. fischeri is a Gram negative bacteria that lives in salt water either as non-luminous free plankton, or inside the light organs in marine fish where they are luminous. In 1972, it was shown that luminescence is a density-dependent phenotype; it is only expressed once a high enough density of cells is reached (2). In the experiments, it was shown that there is both an inhibitor and inducer (as the gene regulation mechanism was not investigated, the terms "repressor" and "activator" were not used) that are responsible for the regulation of the luminescent enzyme Luciferase. Cells were non-luminescent when first placed in fresh medium, where the inhibitor was present. The optical density of cells increased as expected; however luminescence did not change significantly until the threshold density was reached. This occurred when the bacteria were enough to consume the inhibitor and produce an activator, triggering luminescence. K.  Nealson described the activator molecule responsible for activation as an "Auto-inducer"; a bacterial signaling molecule (13). In 1977, Nealson grew V. fischeri cells in medium with a contained cell density of 107 CFU/ml. At this density in normal growth medium, the cells produced no significant luminescence. However, when the presumed auto-inducer was artificially added, the cells did show considerable luminescence; showing that the auto-inducer is responsible for luminescence (14). When compared to the in situ life of V. fischeri, the results correspond with the evolutionary benefit for sensing neighboring cells.    A QS system prevents single cells from producing the energy-consuming luminescence while not in the presence of a population; when the luminescence can not be seen. When the cells grow in symbioses with marine fish, nutrients are rich, and the cell density can trigger bioluminescence. New quorum sensing mechanisms are often compared to that of V. fischeri, and are often named "lux operon-like", after the auto-inducer regulatory mechanism of Luciferase.

Cells can use a variety of signaling molecules for QS systems; however some are much more common than others. Short peptides and amino acids, coded by gene sequences previously thought to be insignificant due to their short length, can be used as signaling molecules. These oligopeptides are used by Gram-positive bacteria by triggering phosphocascades. However, Homoserine Lactones (HSLs) are used by a wide variety of organisms. Bacteria also use a class of acylated HSLs (Acyl-Homoserine Lactone, or AHL) as their signaling molecule. HSLs and AHLs are found in Gram-negative bacteria. AHLs are hydrophobic, and able to diffuse through cell membranes to affect gene expressions. AHLs have a carbon side-chain with a variable length which is responsible for its effector selectivity.

Some signal molecules use transduction cascades to activate their target genes. And peptides generally use transport machinery in the membrane to interact with an intracellular target. Cells can even use more than one type of signal molecule to target gene expression. Pseudomonas aeruginus is a bacterial pathogen which causes severe infections in cystic fibrosis patients, often leading to death. P. aeruginus uses 2 AHLs (C4-HSL and C12-HSL) to trigger the formation of bio-film (11). These 2 AHLs are packaged into vesicles to convey their signal to neighboring cells. The packaging of the molecules is controlled by a molecule 2-heptyl-3-hydroxy-4-quinoline (Pseudomonas quinolone signal), which is also controlled by a quorum sensory molecule. Studies like that of Nakamura in P. aeruginus have shown how widespread quorum sensing is, and how important it is to the survival of a species.

Not only can cells respond to cells of the same species, but some experiments have shown cells to be able to respond to cells of other species (20). One of the most common signaling molecules is auto-inducer 2 (AI-2). AI-2 is highly conserved throughout bacteria; Gram-negative and Gram-positive bacteria both contain AI-2 producing genes. Many of these organisms use AI-2 as a growth regulatory molecule. The conservation of AI-2 suggests that cells can use it as an inter-species communication molecule. It's been hypothesized that different species in a population can use AI-2, but have reciprocal effects. A cell of one species can induce itself to enter logarithmic growth, while sending neighboring cells of different species into stationary phase.

QS systems are also capable of inter-kingdom communication. Agrobacterium tumefaciens is a bacteria that produces tumors in plants by transferring of a virulent Ti plasmid. A. tumefaciens can also transfer the Ti plasmid to other bacteria via conjugation. Conjugation requires high cell density. The Ti plasmid codes for conjugation genes which are expressed by a lux-like QS system activated by an AHL. The Ti plasmid forces tumor cells in the plant to produce opines, which trigger the conjugation to occur. (19)


Virulent Factors, Morphology, Antibiotic Susceptibility

One of the most important prospects of studying QS mechanisms is the idea of containing virulent factors in pathogens. There continues to be more studies being done on auto-inducer targeting molecules to fight against antibiotic resistance in industry and medicine. AI-2 mediated quorum sensing is common for activating virulent factors in enteric species. Cinnamaldehyde is one known molecule that disrupts AI-2 activity (1). While Cinnamaldehyde does not inhibit bacterial growth, it is able to inhibit AI-2 induced virulence factors in Vibrio spp., a common marine pathogen. Cinnamaldehyde inhibits the bio-film formation of a population, which makes the bacteria considerably more susceptible to stress and antibiotics. This suggests that molecules that target QS systems can be used themselves as a prophylactic, or in combination with an antibiotic to more efficiently kill pathogens. This is good news for the aquaculture industry, where Vibrio spp. infection is a large part of the economic loss. After years of overuse, Vibrio spp. has become resistant to common antibiotics used to prevent disease.

Molecules that mimic AHLs are also capable of inhibiting QS mediated phenotypes. It has been shown that N-nonanoyl-cyclopentylamide, an AHL-like molecule, is also capable of inhibiting virulence factors in Serratia marcescens (9). These new studies allude to a broad spectrum of molecules being used to inhibit microbial virulence. It may be possible to custom-make these molecules, targeting only specific systems and species. QS targeting molecules can be used in conjunction with antibiotics, rather than contributing to pathogen resistance by increasing the antibiotics being used.

Many pathogenic bacteria require the formation of bio-film colony morphology before they become virulent. These bacteria can often be beneficial to the host in a symbiotic relationship. However, bio-film can interfere with the host, starving it from oxygen and nutrients. Some marine plants have evolved to produce compounds that target QS systems responsible for bio-filming. The marine seaweed Delisea pulchra is one such host. D. pulchra has been shown to produce a halogenated furanone that interferes with bio-film formation on the level of quorum sensing (16).

The study of bio-filming bacteria is important in realizing how widespread quorum sensing systems are in bacteria. By studying marine sponges and invertebrates it is evident that most, if not all bacteria produce auto-inducers at some point in their life cycle (17). These studies also show that there is a sort of bio-cycle between growth stages in different species. That is to say, different bacteria species produce their auto-inducers at different life stages on the same host, which is indicative of the density-dependent nature of quorum sensing. Marine sponges appear to produce diketopiperazines (17) a molecule also shown to be produced by P. aeruginosa as an AHL system antagonist towards some bacteria (4). These molecules target the swarming motility in Serratia liquefaciens.

Swarming motility is a coordinated movement of bacteria across a substrate. Another example of multicellular activity in single-celled organisms, swarming motility is coordinated by quorum sensing. Reduced virulence was shown in Erwinia chrysanthemi when a null mutation was introduced to genes encoding for the AHLs responsible for swarming (10). Not much is known as to why some bacteria swarm, however the benefit has been demonstrated; antibiotic resistance. It's been suggested that alteration of LPS repels cationic antibiotics such as polymyxin and kanamycin in swarming Salmonella enterica. The proposed mechanism for resistance is addition of 4-amino-4-deoxy-L-arabinose to the lipid A of LPS, causing a more positively charged outer membrane. L-AraN is controlled under the QS pmrHFIJKLM operon, which is up-regulated in swarming cells (7).

By targeting QS systems, it may be possible to prevent microbial infection without the negative side effects of antibiotics. It has been shown that virulence can decrease a number of ways; mimic auto-inducers, or molecules that inhibit auto-inducer synthesis or deletion of genes that code for auto-inducers. Hopefully by controlling QS involved with population and morphology, virulent and parasitic phenotypes can be repressed for medical and industrial purposes.

References

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Industrial Manipulation of Quorum Sensing Systems

Quorum sensing has immediate obvious uses in medicine and improving human quality of life. But QS systems may also be manipulated for industrial uses. While QS antagonists can be used in industries such as aquaculture as an antibacterial agent, QS can also be manipulated for increased metabolite production for industrial purposes. While strain selection and genetic cloning are already techniques used in industry, QS may become easier, cheaper, and even more efficient than existing practices.

Acetic acid is a simple carboxylic acid, best known as the main ingredient in vinegar. Besides being a popular salad dressing, acetic acid is the precursor to many other products including Polyethylene Terephthalate (often called PET), a partially recyclable thermoplastic used in food and drink packaging. Acetic acid is normally produced synthetically from petrol products. Historically, humans have used bacteria to produce acetic acid, and acetic acid for vinegar is still produced biologically today. Oxidation of ethanol by Acetobacter produces acetic acid in aerobic conditions. Some anaerobes such as Clostridium can metabolize sugars straight to acetic acid, without the ethanol intermediate, making biologically produced ethanol much more efficient. It's very difficult to increase acetic acid yield in biological production because the product becomes toxic to cells, and will inhibit growth. The best available method to increase yield is to select strains that have a high resistance to acetic acid. Strains can also be genetically modified to produce high activity enzymes and growth; but it seems that a negative QS system is the biggest limiting factor in overall production.

Gluconacetobacter intermedius is an obligate aerobe, and produces acetic acid oxidatively from an alcohol precursor. By genetically manipulating cells to overexpress aldH from Gluconacetobacter polyoxogenes, cellsshow an increase in overall acetic acid production (3). aldH encodes a high activity aldehyde dehydrogenase - the enzyme responsible for acetic acid synthesis. aco from Acetobacter aceti, which encodes an aconitase can also be cloned to improve acetic acid production. Aconitase synthesizes isocitrate, an eventual precursor molecule for acetic acid.

Recently, it has been shown that genes that limit the overall acetic acid production in     G. intermedius are controlled by a quorum sensing system (5). The quorum sensing system affects fermentation negatively, decreasing fermentation, and therefore energy yield and growth rate. Biologically, decreasing growth rate decreases production of acetic acid before it becomes toxic to the cells. This control mechanism helps in preventing a toxic level of acetic acid. A higher product yield can also be achieved by inhibiting the negative QS regulating cell growth. Inhibition of the QS system gives a higher product yield than genetic manipulates such as aldH cloning (1.4 fold compared to 2.4 fold, respectively)(5).

By combining existing genetic techniques with new QS-level manipulation product yield could be even higher. While these techniques would most certainly be popularized in the vinegar industry, the plastic industry may stick to common petrol chemistry. Ideally, with further enhancement and on a larger scale, biological production may be a possible "green" solution to petrol reaction synthesis. Many industrial products have been known to be regulated by cell growth and density. QS may provide a way to molecularly trick cells into producing a desired product.

Abbreviations

QS – Quorum Sensing

HSL – Homoserine Lactone

ASL – Acyl-Homoserine Lacton

AI-2 – Autoinducer-2

LPS – Lipopolysaccharide


Conclusion

Although only discovered just 3 decades ago and previously taken with skepticism, it has been shown that quorum sensing is an integral part of bacterial survival and life cycles. The importance of quorum sensing has not gone unnoticed; new studies are popularly being done from both medical and industrial sides of microbiology. By studying the biochemistry of  quorum sensing, a better understanding of how bacteria become virulent can be obtained. Drugs that inhibit quorum sensing systems can help in containing virulent factors and keep patients, like those with Cystic Fibrosis, healthy. New drugs that target auto-inducers can provide a powerful solution to antibiotic resistance, which can save lives as well as industrial live stock. Manipulation of cell-to-cell communication can also result in a higher product yield in microbiological industries, such as acetic acid. By combining QS manipulation with genetic engineering and strain selection, a much higher product yield can be reached. Quorum sensing provides the perfect explanation for how industrial-products are regulated. It has been over 300 years since man first saw bacteria through a microscope, and although science has increased knowledge immeasurably since then, there still remains much to be seen.


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