Signs Of Intelligent Life:Bacteria Start Small, But Grow Smart

It's All Part Of The Growing Field Of "Sociomicrobiology,” The Study Of Microbe Social Activity

By Elizabeth Sharpe

Bacteria are tiny. Generally around a few micrometers long, they're only visible through a microscope. And they're everywhere. This may explain why the market is flooded with disinfectants and antibiotics aimed at killing the multitude of bacteria that share our daily lives, lest they get out of hand and cause nasty infections.

But these microbe-killers often don't work. They were designed to kill individual organisms, which are indeed vulnerable when alone. But bacteria don't like single life much. Instead, they thrive better in communities called "biofilms.” When bacteria attach to a surface like an artificial heart valve, they start to replicate and form a small colony of bacteria, releasing and surrounding themselves with a slimy extracellular matrix structure from which a biofilm gets its name.

And when bacteria get together, the party creates a lot of changes.

These changes are of increasing interest to scientists. In a paper published January 2005 in Trends in Microbiology, E. Peter Greenberg calls the new and growing field "sociomicrobiology,” the study of microbe social activity. Greenberg is chair of the department of microbiology at the University of Washington (UW) in Seattle, Wash.

There is growing evidence that as a collective force, the bacterial community exerts a sort of peer pressure on the solitary bacteria, so that the bacterial cells begin to act and respond differently than they would on their own. Think of bacteria in a factory assembly line: Each bacterium performs a special kind of work for the biofilm community. And in order to be effective at their jobs, the bacteria use a special communication system to "talk” to each other.

Sound surprising? It's only recently that scientists recognized that bacteria in a community operate more like a multi-cellular organism. It's a far cry from what scientists used to believe thirty years ago. In the past, many biologists insisted the smaller the organism, the less it needed social activity, citing bacteria as an example. In fact, bacteria in biofilms were thought to retain their individual characteristics and functions, like individual people in a crowd. It seemed illogical that a single-celled bacterium could manage something as sophisticated as say, a division of labor. More "highly evolved” species like humans take this division of labor for granted. Cells in the body have unique duties: blood cells act like blood cells and skin cells like skin cells. The cell types don't get their roles mixed up.

But bacteria in a biofilm may have more diversified roles than we think. Greenberg studies how a species of the bacteria Pseudomonas aeruginosa forms a mushroom-shaped colony on a piece of glass. A single bacterium of this species is 2 microns in length, and the biofilm it forms is about 120 microns tall in lab set-ups. What Greenberg finds most curious is that there seems to be a strong selective process for the bacteria to exhibit different traits based upon where they reside in the colony. Bacteria on the stalk of the mushroom-shaped biofilm are starved for oxygen and express genes that make a surfactant–a slippery substance. They also are more likely than their counterparts in the cap to swim away, perhaps to attach elsewhere. In contrast, bacteria in the cap have plenty of oxygen, grow faster, and don't make the surfactant.

Diversification

Bacteria in biofilms, Greenberg says, seem to be adapting to a unique condition created by their position in the biofilm. Perhaps their diversification may increase their capability as a group to defend themselves against disinfectants, antibiotics, or the body's immune system.

Pradeep Singh thinks so. He calls it "biological insurance,” and draws a comparison to a forest. In an ecosystem of a forest, a mono-species forest is going to be more susceptible to a limited amount of nutrients than a multi-species forest when there is a drought. Singh says bacteria may work the same way. Biofilms that are diverse are better able to survive. Singh is an assistant professor of medicine and microbiology at the University of Iowa in Iowa City, Ia.

Singh also studies Pseudomonas aeruginosa, bacteria that invade the lungs of many cystic fibrosis patients. He grew identical clones of the bacterium. When he harvested bacteria from the biofilm that formed, he found they had changed some of functional traits they expressed in their original state: their ability to swim or the production of secreted products like toxins and detergents. He published his findings in the Nov. 15, 2004 issue of the Proceedings of the National Academy of Sciences.

We've known for a long time that in the lungs of people with cystic fibrosis, bacteria change genetically over time, says Greenberg. Pseudomonas aeruginosa has been studied at a genomic level by Maynard Olson in the department of medicine at the UW. Greenberg has collaborated with Singh and says Singh's work proves Olson's work on a mechanistic level. If there are different regions in these biofilms that have different conditions, there is going to be selection; if any mutant arises, it's going to be selected.

Yet, both Greenberg and Singh's conclusions come from examining bacteria outside the human body. There are a number of difficulties that prevent scientists from being able to accurately investigate biofilms that form inside the body, says Greenberg. Singh, too, warns against assuming that the same process he found in laboratory experiments is occurring in the lungs of cystic fibrosis patients. Many strains of bacteria may be harbored in a patient's lungs simultaneously, so it is difficult to know at what stage each of the bacteria diversify and what functional traits change.

Still, Singh speculates that one way to make biofilms more vulnerable may be to develop a way to block diversification. And that may be possible, Greenberg says, by using existing substances that can block the bacteria's genetic change.

Bacteria communicate

Bacteria interact to support the biofilm in another way as well. The theory that some bacterial cells like Vibrio fischeri communicate through a specialized signal system was proposed by Woody Hastings back in the 1970s. While it seemed incredulous then, the signal system called "quorum sensing” coined by Greenberg and colleagues Stephen Winans and Clay Fuqua is widely accepted today. Bacteria in a biofilm sense their own density or "quorum,” so they send a chemical signal out to the others of the same species in the colony, and the others sense and respond to what they "hear.”

It was V. fischeri that illuminated the process. This is a species of marine bacteria that lives in the light organ of a squid the size of a pencil eraser. When enough of the bacteria get together, they recognize that there are a lot of the other V. fischeri around releasing the same chemical signal from the "autoinducer” gene. So, in essence, their signal induces all the bacteria to change their behavior: to become luminescent. V. fischeri swimming around in the ocean don't waste the energy to light up, because what's the point? They are so small. It's only when they reach a critical mass that they know it's time to light up.

But the ability to communicate may be key to explaining how bacteria can turn deadly. Take, for example, bacteria like Pseudomonas aeruginosa. People with cystic fibrosis have a genetic defect that compromises the lungs' ability to fight off bacterial infection. P. aeruginosa emits a chemical signal when the bacteria recognize there are enough of them bound together in the lungs of the cystic fibrosis patient to release so-called virulence factors that allow them to cause an infection. Greenberg compares it to an amassing army. When there are enough troops present, they show their weapons. In the case of P. aeruginosa, these weapons are toxins that break down proteins and lipids. The chronic lung infection eventually leads to respiratory failure and fatal pneumonia, claiming the lives of at least 5 out of 10 cystic fibrosis patients by the age of 30.

But it may not be just members of the same species that communicate with each other. Vibrio harveyi, for example, uses multiple signals to communicate, according to Bonnie Bassler, a professor of molecular biology at Princeton University in Princeton, N.J. She says it doesn't do bacteria much good to only be aware of their own same-species number. Usually, a biofilm is made up of mixed species. On your teeth, for example, there are 600 species of bacteria. So, bacteria can't rely on just one set of information. V. harveyi makes a nonspecific or "generic” signal that other bacteria understand and cause them to respond. This species is not alone. Of the 200 bacterial genomes that have been mapped, more than 50 percent of them have been found to have the gene that makes the molecule called AI-2, or autoinducer-2, that other bacterial species recognize.

It's a communication system Bassler calls "bacterial esperanto.” AI-2 signals all the bacteria in a mixed community that there a lot of them, so now is the time to turn on a set of genes that will be good for the biofilm, like those that code for virulence factors. Bassler says researchers are still looking at different species of bacteria to find out exactly what genes the AI-2 turns on.

"We think this is the molecule that says 'other.' It says 'I'm not alone,'” says Bassler of AI-2. "But there have to be molecules that say who the other guy is and we don't know anything about those yet.” But she and many other researchers are trying to find those molecules. "It's going to be a very rich chemical language with a lot of words in it.” Right now researchers only know two.

But these two are powerful. In P. aeruginosa, Greenberg knows that quorum sensing is an Achilles' heel. Messing up communication is an effective point of attack. He wants to develop a strategic missile that blocks P. aeruginosa'sability to hurt patients. But that's easier said than done.

How to stop ‘em

In fact, many researchers are looking at ways to stop bacteria from forming biofilms.

One strategy may involve how organic materials and proteins collect on the surface of an implant in the body, says James Bryers, professor of bioengineering and a lead investigator at UW Engineered Biomaterials, a National Science Foundation Engineering Research Center at the UW.

Despite attempts by biomaterials engineers to create an anti-biofilm surface, the surface remains largely ineffective. The surface that a bacterium sees is already "hidden” by the organic compounds that have adhered to it. In terms of an artificial heart, there are two species of bacteria that pose a threat: Staphylococcus epidermidis, relatively benign, exisiting all over your skin, and Staphylococcus aureus, the same bacteria that causes food poisoning. Bryers says these two species can stick to an artificial device because they have receptors that link to fibronectin, a natural protein found in the body. The bacteria can't attach if he generates molecules that block the sticky arm of the bacteria from attaching to fibronectin. He says this can be done by containing the anti-adhesion molecules in the device.

But there may be another way as well, says Bryers. Since fibronectin is oriented up from the device, why not turn the protein on its head. If there is no fibronectin out there waving about, attracting bacteria, then there may not be any way for bacteria to stick to the surface. His idea comes in response to engineers who have introduced stealth surfaces, that is, engineered surfaces of implanted devices that promote healing in the body by attracting mammalian cells. This is often done by tethering fibronectin to the surface. But if bacteria recognize only one end of it, then why not specifically orient the protein so the bacteria don't see it there?

Still other methods are being considered. Researchers at the Center for Biofilm Engineering at Montana State University (MSU) in Bozeman, Mont. are developing surface coatings for implant devices that could potentially inhibit biofilms from attaching and forming, says Phil Stewart, chair of the Center. They are working on understanding cell-to-cell chemical signals in order to jam the bacterial communication system, and they are even screening extracts of plants that may prevent biofilms.

Plants are often successful in ways the human body is not. In the mid-1990s, researchers Staffan Kjelleberg and Peter Steinberg in Australia found a seaweed, indigenous to Australia, New Zealand, and sub-Antarctic islands, which naturally produced compounds called furanones that prevented bacteria from attaching. The furanones resemble some of quorum sensing molecules. The researchers formed a company called Biosignal Ltd. to develop technology based on these naturally-derived compounds. The company went public in April 2004.

But not all biofilms are bad. In fact, biofilms are being used to clean up pollutants in water, soil, and at industrial sites. Those found in soil particles, in surface waters like streams and lakes, and in decaying organic materials, like leaves, are mostly beneficial to humans because they help cycle nutrients, says Gill Geesey, professor of microbiology at MSU. "They're helping us survive.” These biofilms make oxygen, nitrogen, and phosphorous, which get incorporated into plant material that becomes our food.

Elizabeth Sharpe is a science writer and editor at University of Washington Engineered Biomaterials (UWEB), a National Science Foundation Engineering Research Center.

Images

Top: Biofilms have wide-ranging effects. Chart: Courtesy of the Center for Biofilm Engineering, Montana State University and Peg Dirckx.

Middle: Biofilm formed on a natural hot spring in Yellowstone National Park. In this 1ft x 1ft square picture, there are millions of bacteria. Photo: Gill Geesey, Montana State University

Bottom: Cross-section of a stainless steel tube coated with thick, thriving biofilm. Photo: Center for Biofilm Engineering, Montana State University, and N. Zelver

For more information:

The Center for Biofilm Engineering http://www.erc.montana.edu/

BiofilmsOnline.com http://www.biofilmsonline.com/

The University of Washington Engineered Biomaterials Summer Symposium, co-sponsored by the Center for Biofilm Engineering: Bugs and Biomaterials http://www.uweb.engr.washington.edu/about/ news/summersymposium.html