The chemist examined the role of activated oxygen molecules in biological processes.
Tools for tracking quorum-sensing signals in bacterial colonies
May 1, 2014|
COURTESY OF CHIEN-YI CHANG AND PAUL WILLIAMS
To infiltrate the defenses of a bacterial colony, a scientist needs to think a bit like a CIA agent. How are cells coordinating their activities? Which bacteria are sending messages to their neighbors, and what are they saying? Since the discovery more than 40 years ago that bacteria use chemical signals to communicate with each other and synchronize their behaviors, biologists have been trying to decode the molecular language of so-called quorum sensing.
Scientists now know that quorum sensing is a fundamental ability of many single-celled organisms. It allows bacteria to perceive the presence of neighbors, detect when they’ve reached a threshold number, and change their behavior in response—producing a toxin or altering their growth pattern, for instance. Pathogenic bacteria use quorum sensing to form hard-to-treat biofilm infections in the lungs; commensal bacteria in the gut and skin also communicate with their neighbors using quorum-sensing signals, as do bacteria in the oceans and soils.
Today, commercially available reporters can quickly identify known quorum-sensing molecules, and novel technologies developed by chemists and engineers are laying bare the consequences of quorum-sensing signaling for bacterial colonies. But the challenge with many quorum-sensing experiments is that researchers still don’t know what they’re missing. While some quorum-related messengers have been characterized for decades, others are still being discovered, and some experiments only detect the most common signals. And quorum-sensing findings made in the lab don’t always hold true in clinical trials or in model organisms, so researchers who have been in the field for decades caution novices to take their first results with a grain of salt.
The Scientist asked experts who study quorum sensing in bacteria for a run-down of their favorite techniques. Here’s what they said.
A few basic biochemistry experiments can reveal whether the bacteria you’re studying are using quorum sensing and hint at the molecules they are deploying for these purposes, says Paul Williams of the U.K.’s University of Nottingham. In the early 1990s, when Williams discovered the first quorum-sensing signal used by the plant pathogen Erwinia carotovora, mass spectrometry and microfluidics weren’t at his disposal, so he relied on more traditional techniques.
Williams was among the first to take advantage of a light-based reporter system to detect the presence of N-acyl homoserine lactones (AHLs), a class of quorum-sensing signal molecules common in gram-negative bacteria. The marine bacterium Vibrio fischeri had been discovered to light up when it sensed AHLs, and Williams’s Nottingham colleague, Gordon Stewart, constructed a reporter system using its genes. Today, researchers can purchase kits containing such reporters to determine when bacteria produce such signals. Alternatively, adding AHL molecules—which are also commercially available—can reveal whether bacteria respond by altering their growth, for example, or by producing more of a particular compound.
“There’s something to be said for going straight to mass spec these days,” Williams says. Basic chemical-fractionation methods can enrich for known quorum-sensing molecules or for the signaling molecule in question, and mass spec can then identify those molecules. The advantage to this method: it could turn up a messenger that hasn’t been studied before.
What you can learn: These basic experiments can indicate easily and cheaply how important quorum sensing is to a given organism or behavior. “Any standard microbiology lab can easily do these kinds of assays,” says Kendra Rumbaugh, a clinical microbiologist at Texas Tech University who uses such reporters to test whether quorum sensing exacerbates infections in patients’ surgical wounds.
Challenges: When used to detect quorum-sensing inhibitors, AHL reporter systems are prone to false positives. “A seeming inhibitor may be interacting with your reporter gene, but not your quorum-sensing gene,” Williams cautions. “It’s surprising how often people are tripped up by this.” Make sure you deploy careful controls for each part of the assay. If you use mass spec to analyze a supernatant, Williams says, take extra care to confirm that the molecules you find are really involved in quorum sensing. Not every molecule that alters bacterial growth, for instance, is a quorum-sensing signal.
What it takes: The protocols are relatively simple. ATCC (American Type Culture Collection) offers Vibrio harveyi-based reporter kits for around $300 per strain, or $2,000 for a panel of strains. Many labs have created their own reporter strains of bacteria and are often willing to share. The isolated AHL molecules cost less than $200 from Sigma-Aldrich. Mass spectrometry costs vary by institution.
COURTESY OF JENNA EUN AND DOUGLAS WEIBELOutside the lab, bacteria don’t generally live in environments that resemble shaking flasks of culture media. Instead, they most often grow in small, high-density colonies and are exposed to gradients of chemicals. When they’re monitoring their neighbors and responding to quorum-sensing signals, the physical structure of the bacterial community is vital. Over the past decade, scientists have developed new ways to mimic these tiny, dense bacterial communities by growing the microbes in confined environments.
At the University of Wisconsin–Madison, biomedical engineer Douglas Weibel creates droplets of bacteria to limit their growth. He constructs microfluidic devices that allow him to combine bacteria, growth media, and any chemical factors of interest into microliter-size droplets that line up inside channels in the device to keep them intact and separate (Angew Chem Int Ed Engl, 52:8908-11, 2013). “You can make a million droplets per hour,” Weibel says. “That’s like if you could set up a million petri dishes per hour.”
What you can learn: By altering the distance between droplets or by combining multiple droplets into larger communities of bacteria, Weibel and his colleagues can observe how the bacteria adapt to different densities or chemicals. Because droplet microfluidics allows the creation of so many samples at once, it can be used as a high-throughput way to screen genetic mutants for how they react to density changes and point to quorum-sensing genes.
Challenges: Not every variable can be tested on bacteria that are contained in tiny droplets inside a microfluidics device. The most testable properties are ones that can be observed under a microscope, such as cell density, shape, or clumping. Studying gene expression, on the other hand, would be harder.
What it takes: Weibel admits that microfluidics can scare away many biologists, but notes that in the context of droplets it’s relatively straightforward. Unlike microfluidic setups that require constantly redesigning the channels for every experiment, droplet devices are more of a one-size-fits-all approach. That means you can team up with a lab that has lithography expertise, create a device once, and go on your way. A single mold for a microfluidics device can usually be produced for around $200; the materials and equipment needed to cast silicone into the mold run closer to $1,000 (Nat Rev Microbiol, 11:337-48, 2013).
If a researcher wants to study how bacteria form dense biofilms in response to quorum sensing, but doesn’t have access to microfluidics to create controlled physical spaces for colonies, Williams recommends biofilm flow-cell systems. “It’s basically a bigger version of microfluidics,” he says, which makes it easier to set up. Rather than housing cells in channels that are microns wide, flow cell inlets are on the order of a few millimeters in width.
Researchers can control what substances are pushed through the insides of a flow cell—a series of chambers on a glass plate. Then, they can watch under a confocal or fluorescent microscope as the bacteria contained within the flow cell form biofilms. The combination of adding flow to the experiments—allowing you to quickly alter conditions—and being able to view changes in real time over a matter of days makes flow cells superior to petri dishes for studying biofilms.
What You Can Learn: Flow cells provide an easy way to compare the biofilm behavior of a few different bacterial strains, or to see how different molecules or growth mediums affect biofilm formation. “If you put in a quorum-sensing mutant or you run a medium lacking some signaling molecule through, you’ll be able to see a difference in the biofilms,” Williams says. In 2013, Williams and his colleagues used a flow-cell system to study how often Pseudomonas aeruginosa “cheat” at quorum sensing and don’t participate in normal cooperative behaviors, and how this affects the antibiotic resistance of P. aeruginosa biofilms.
Challenges: With this less-technically-advanced version of microfluidics, air bubbles can be a big problem. One study on flow cells found that bubbles ruined one in three experiments—flow cells containing air have to be discarded. Running the flow cells at lower temperatures can help limit bubble formation, but means that the experiment veers further from normal physiological conditions (Biofouling, 28:835-42, 2012).
What it Takes: Access to a confocal, fluorescent, or reflected light microscope and a flow-cell system. Biosurface Technologies charges $385 for a basic FC91 capillary flow cell, which contains four glass chambers; the reusable single-chamber FC81 polycarbonate transmission flow cell starts at $460.
COURTESY OF JODI CONNELL. ADAPTED FROM PNAS, DOI:10.1073/pnas.1309729110, 2013At the University of Texas at Austin, a team of scientists has been working to perfect its technique of trapping small numbers of bacteria into microcavities they call “lobster traps.” Each trap holds as little as one picoliter of liquid, and can contain only a few hundred bacteria. “We wanted to look at the minimum number of cells required for quorum sensing,” says Jodi Connell at the University of Texas at Austin, who is the first author of many of the papers on lobster traps. (See “Lobster-Pot Science,” The Scientist, May 2011.)
The latest advance from Connell and her colleagues is a method for completely sealing off these compartments, thus keeping bacteria from moving in or out. The team first suspends the bacteria in a gelatin-based substance that’s liquid at high temperatures and Jell-O–like at room temperature. Then, the researchers use laser-based lithography to cross-link photosensitive molecules contained in the gel, which creates semiporous dividers that enclose each bacterial community. (See “Building 3-D Microbial Communities,” The Scientist, October 7, 2013.) Chemicals such as quorum-sensing signals can cross between communities; the bacteria themselves cannot.
As with microfluidics droplets, some characteristics of the populations—those that can be visibly observed—can be studied while the bacteria remain in the compartments. But with the lobster traps, the growth of colonies can be stopped, samples removed, and other experiments run, such as gene-expression profiling of the cells.
What you can learn: “Allowing populations to grow in confined conditions like this lets us control their shape and density,” Connell says. In one recent experiment, she and UT Austin colleagues in Marvin Whiteley’s lab studied the interactions between confined colonies of Staphylococcus aureus and P. aeruginosa—pathogens that often coexist in wounds, catheters, and lung infections. They discovered that the proximity of a sufficient density of P. aeruginosa in an adjacent lobster trap helps S. aureus survive treatment with some antibiotics (PNAS,doi:10.1073/pnas.1309729110, 2013). In other studies, they’ve established that population size is as important as population density in mediating what quorum-sensing signals are sent between cells and how they’re interpreted.
Challenges: Lobster traps aren’t yet high throughput enough to run more than a few experiments at once, Connell says. So they can’t be used, for example, as a tool in large-scale mutagenesis screens to identify quorum-sensing genes. And the cost of the devices is still relatively high.
What it takes: These experiments require specific equipment and expertise, Connell says. Molds are shaped around specific bacterial colonies, so they aren’t made until the colonies are already established. This means that every new experiment requires access to microfabrication equipment, which is not standard in most microbiology labs. The machines used to create the molds around colonies—spin coaters or dip coaters—can cost as much as $10,000.
May 27, 2014
Re: "Outside the lab, bacteria don’t generally live in environments that resemble shaking flasks of culture media."
Inside and outside the lab, bacteria live in environments where ecological variation in nutrient availability determines what is metabolized to become the species-specific chemical signals that control the physiology of reproduction (e.g., in species from microbes to man via conserved molecular mechanisms). The species-specific chemicals that control the physiology of reproduction are commonly called pheromones.
Use of the term "pheromones" in this article would link nutrient-dependent pheromone-controlled ecological adaptations from natural selection of nutrients via chemical signals that are commonly called food odors. Thus, the link from ecological variation in the epigenetic landscape to the physical landscape of DNA in the organized genomes of species from microbes to man becomes clear. The link is food odors and pheromones via the epigenetic effects of olfactory/pheromonal input on cell type differentiation!
Researchers need only associate nutrient uptake with chemical signals (e.g., food odors) and associate the controlled physiology of reproduction with social odors (i.e., pheromones). They will then understand why the human ability to sniff out sublte differences in cell types of bacterial colonies growing on media in the lab arises via the conserved molecular mechanisms that enable the ability of individuals to chemically sense (i.e., sniff out) the differences between a nutrient source and a conspecific's pheromones.
Understanding how the conserved molecular mechanisms of sensing (e.g., food odors) and signaling (e.g., conspecifics) link ecological variation to ecological adaptations manifested in the morphological and behavioral phenotypes of species from microbes to man via cell type differentiation also helps eliminate ideas about mutations, natural selection, and evolution.
We can reevaluate what Darwin actually wrote and meant. In attempts to get others to understand his theory, Darwin pleaded for them to put 'conditions of life' before consideration of natural selection for anything else. We've since seen that some people understand that 'conditions of life are nutrient-dependent and pheromone-controlled. However, those conditions are not well-represented "...in environments that resemble shaking flasks of culture media." There's no mention of the pheromone-controlled physiology of reproduction or the seemingly futile cycles of thermodynamically controlled nutrient-dependent protein biosynthesis and degradation.
See for example: The Man Who Bottled Evolution, and see for comparison: Nutrient-dependent/pheromone-controlled adaptive evolution: a model. Quorum sensing is an example of how ecological variation results in ecological adaptations.
May 27, 2014
Here's Kohl commenting again to try to sell his perfumes. He's posting the usual baloney: "The species-specific chemicals that control the physiology of reproduction are commonly called pheromones." No they aren't.
Any good scientist will immediately see that this is false.