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Reading_QuorumSensing_1 - Quorum Sensing How Microbes Talk...

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Unformatted text preview: Quorum Sensing: How Microbes Talk to Each Other One of our most significant advances in understanding how bacterial cells function is the discovery over the last 20 years that bacterial cells communicate with each other. Different functions are regulated by bacterial communication, such as virulence in plant and animal pathogens, antibiotic production, horizontal gene transfer, differentiation or spore formation, and cell death. Types of Cell-Cell Signaling There are two general mechanisms through which cell-cell communication can occur: signaling through direct cell-cell contact or through extracellular signaling molecules. Communication through extracellular signaling molecules can be further subdivided into two general types: paracrine signaling and autocrine signaling. Paracrine signaling is when the cell type secreting the signaling molecule is a different than the cell that senses and responds to the secreted signaling molecule. Autocrine signaling is when the cell type secreting the signaling molecule is the same as the cell type that can sense and respond to the signaling molecule. Bacteria use all of these mechanisms of communication, but communication through autocrine signaling is more common. Quorum Sensing The function that is mediated by autocrine signaling is sensing population density. When the density of cells producing a signaling molecule in an environment is low, due to diffusion, the environmental concentration of the secreted signaling molecule will be low. However, if the density of the cells producing a signaling molecule in the environment is high, the environmental concentration of the secreted signaling molecule will be high enough to elicit a cellular response. The process of monitoring cellular population density is referred to as quorum sensing. Why do bacterial cells monitor their population density? The processes controlled by quorum sensing are those that are more efficiently carried out by a population of cells that single cells. For example, a single cell of a bacterium that causes disease may be unable to elicit disease symptoms when it expresses virulence functions. In contrast, many cells of this bacterium can elicit disease symptoms when they express virulence functions. Expressing virulence functions when they cannot be effective is an energetic burden on the cell. Textbook Reading Pages 374-376, “13.7 Quorum Sensing” The points to be remembered from this text: All the points to be remembered from this text are also presented in the text of this document. Discovery of the Mechanism of Quorum Sensing Quorum sensing was first discovered in the bioluminescent, marine bacterium Vibrio fischeri. Light production by this bacterium was monitored during growth of the cells in a batch culture. What they observed was that the intensity of light produced by the culture was not proportional 1 to the density of cells in the culture. The amount of light produced per cell was constantly changing during the period of exponential growth. Discovery of the Acyl-Homoserine Lactone There are two general hypotheses to explain this unusual form of gene expression observed in Vibrio fischeri. One hypothesis is that light production is regulated by an extracellular signal whose levels are constantly changing during growth of the culture, such as removal of an inhibitor or production of an inducer during growth. The other hypothesis is that there is an intracellular signal regulating light production. Scientists sought to distinguish whether light production was regulated through an external versus internal signal. They reasoned that, if light production was regulated through an external signal, they could transfer the extracellular medium from cells producing light to cells that were not and then observe the stimulation of light production in these cells. In contrast, if light production were stimulated by an internal signal, then transferring of the extracellular medium from cells producing light to cells that were not, would not result in stimulation of light production. Thus, cell free culture supernatants were harvested from V. fischeri cells that were grown to a high cell density when the levels of light production were high. To these supernatants, V. fischeri cells were added, and the level of light production per cell was measured at various times during the growth of the cells. As a control, V. fischeri cells were added to fresh media, and the level of light production per cell was measured at various times during growth of the cells. Scientists observed that light production was induced immediately after transfer of the V. fischeri cells to the culture supernatants, whereas in fresh media, light production did not occur until cells had reached a higher cell density. From these results, it was concluded that light production by V. fischeri was induced by an extracellular signal. Scientists then wanted to know whether light production by V. fischeri was induced by removal of an extracellular inhibitor that was present in the growth medium or by production and accumulation of an extracellular inducer molecule. Scientists reasoned that if an inducer was present in the extracellular medium of cells that produce light then they should be able to purify the compound, add it to fresh media, and induce light production. In contrast, if the extracellular medium of cells that produce light simply lacked an inhibitor, there would not be a compound that could be purified and transferred to fresh medium to induce light production. Thus, scientist fractionated culture supernatants, from cells producing light, into different compounds until they had a pure compound that could induce light production. The structure of the purified compound was determined to be an acyl-homoserine lactone (acyl-HSL). This compound has also been referred to as an autoinducer, as it is a compound produced by cells to induce themselves. Discovery of the Genes for the Acyl-HSL Regulatory System Scientists wanted to answer the questions of how the acyl-HSL induced light production, how the acyl-HSL was produced, and was the acyl-HSL a metabolic waste product or a dedicated signaling molecule. A breakthrough in answering these questions came when scientist cloned the genes from light production from V. fischeri into E. coli. 2 Cloning the genes for light production was accomplished by randomly fragmenting the chromosomal DNA of V. fischeri, ligating these fragments of DNA into a plasmid vector that can replicate in E. coli, and transforming the ligated DNA into E. coli. The transformed E. coli cells were then screed for those that produced light. The E. coli clones that produced light also showed cell density dependent regulation of light production as the V. fischeri cells do. Scientists could further show that these transformed E. coli cells now produced the acyl-HSL made by the V. fischeri cells. From the E. coli clone that produced light, the V. fischeri genes that were cloned into the plasmid were sequenced. Seven genes were identified, which are now known as luxA, luxB, luxC, luxD, luxE, luxI, and luxR. To determine which of these genes were directly involved in producing light and which were involved in producing and responding to the acyl-HSL, each gene was individually removed from the plasmid in such as was as to not affect expression of the neighboring genes. Each of the mutant E. coli clones was then assayed by the following three assays. 1. Test maximal level of light production. 2. Determine the level of acyl-HSL produced. 3. Measure the level of light production in cells at low density before and after addition of purified acyl-HSL. The following table summarizes the results obtained: Gene deleted Light Production Acyl-HSL Production Response to Acyl-HSL luxI low none normal luxR low low none luxCDABE* none normal none * Deletions of any one of the luxCDABE genes resulted in an identical phenotype. Think about what you can conclude regarding the function of each gene from the phenotypes listed in the above table. Quorum Sensing Through Peptides A similar phenomenon to how light production is regulated in V. fischeri was observed for expression of the genes required for surfactant production in B. subtilis. The expression of these surfactant production genes was constantly changing during growth of a B. subtilis culture, with the higher levels of expression at higher cell densities. Furthermore, culture supernatants from cells at high density could induce expression of surfactant genes in cells at low cell density. From these culture supernatants, a peptide was purified that induced expression of the surfactant genes. The gene that encoded the peptide-signaling molecule was immediately upstream of genes for a two-component regulatory system. This two-component regulatory system was required for cells to sense the signaling peptide and induce expression of the surfactant genes. Immediately upstream of the gene for the peptide signaling molecule was a gene that was required for production of the signaling peptide. Similar operons that encode a secreted peptide and a twocomponent regulatory system have been identified in a number of Gram-positive bacterial species. 3 ...
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