Bacteria's Key Innovation Helps Understand Evolution

Genomic analysis of E. coli shows multiple steps to evolve new trait

Several years ago researchers at Michigan State University (MSU) reported discovering a novel, evolutionary trait in a long-studied population of Escherichia coli, a rod-shaped bacterium commonly found in the lower intestine of mammals. The E. coli added a helping of citrate to its traditional diet of glucose, even though other E. coli can't consume citrate in the presence of oxygen.

These same biologists have now analyzed this new trait's genetic origins and found that in multiple cases, the evolving E. coli population needed more than one mutational step before the key innovation took hold.  Complex traits, like using a new food source, are thought to be difficult and arise rarely, making the research of broad interest to both evolutionary biologists and public health scientists.

The findings, reported in this week's journal Nature, document the step-by-step process by which organisms evolve new functions. The study also highlights the importance of evolutionary changes that alter the physical arrangement of genes, leading to new patterns of gene regulation.

E. coli normally can't digest citrate when oxygen is present because they don't express the right protein to absorb citrate molecules. Citrate is a salt or class of citric acid commonly found in fruit such as lemons. So how did this mutation occur?

To find the answer, postdoctoral researcher Zachary Blount and MSU Hannah Distinguished Professor of Microbiology and Molecular Genetics Richard Lenski analyzed dozens of complete genome sequences from bacteria that had evolved this new trait and had been sampled and stored at different time points in the history of the lineage.

The National Science Foundation's Division of Environmental Biology partly funded the research, as did the NSF-supported BEACON Center for the Study of Evolution in Action.

The team used samples from Lenski's long-term E. coli experiment that started in February 1988 and has been ongoing for more than 24 years. The experiment allows Lenski and his students and colleagues to study more than 56,000 generations of bacterial evolution. In terms of generations, it is the longest running evolution experiment in history.

Twelve populations of E. coli live in an incubator in Lenski's laboratory producing about seven new generations every 24 hours. Each day, scientists take one percent of each population and transfer it into a new food source, a flask containing fresh glucose, which the bacteria readily eat, and citrate, which one population discovered how to eat after more than 30,000 generations. The researchers also take samples every 500 generations, and freeze them for later study.

Because they freeze the samples, when something new emerges the scientists can go back to earlier generations to look for the steps that happened along the way, which is what occurred in this case.

The researchers found that at least three mutations were required for the bacteria to effectively use citrate when oxygen is present. One or more mutations were necessary to set the physiological stage for the two later events. Then a critical gene duplication occurred that effectively re-wired the expression of a previously silent gene.

"These bacteria have evolved to consume a food resource--citrate--that no wild E. coli uses. Three mutations are required for this to happen, and they must occur in a specific order," said George Gilchrist, NSF program manager for the BEACON Science and Technology Center. "This study shows that the first mutation is required to set the stage for the next two, but surprisingly, this turns out to occur repeatedly and independently in different populations. What this suggests is that complex traits, at least in the microbial world, can evolve quickly and repeatedly."

Additional co-authors include Jeff Barrick, University of Texas and Carla Davidson, University of Calgary.

To learn more about this research, see the journal article in Nature magazine.

 -NSF-

New Research Suggests Bacteria Are Social Microorganisms

MIT scientists: Bacteria plays different social roles, including attacking and defending other bacteria

New research from the Massachusetts Institute of Technology reveals that some unlikely subjects--bacteria--can have social structures similar to plants and animals.

The research shows that a few individuals in groups of closely related bacteria have the ability to produce chemical compounds that kill or slow the growth of other populations of bacteria in the environment, but not harm their own.

Published in the September 7 issue of the journal Science, the finding suggests that bacteria in the environment can play different social roles and that competition occurs not only among individual bacteria, but also among coexisting ecological populations.

The National Science Foundation, an independent federal agency that supports fundamental research and education across all fields of science and engineering, funded the research.

"Bacteria typically have been considered purely selfish organisms and bacterial populations as groups of clones," said Otto Cordero, a theoretical biologist and lead researcher on the paper. "This result contrasts with what we know about animal and plant populations, in which individuals can divide labors, perform different complementary roles and act synergistically."

Cordero and colleagues from MIT, along with researchers from the French Research Institute for Exploitation of the Sea and Woods Hole Oceanographic Institution in Massachusetts, studied whether population-level organization exists for bacteria in the wild.

They reasoned social structure can reduce conflict within populations of plants and animals and determine aggression towards competing biological populations. "Think of a population of lions in the Serengeti or a population of fish in a lake," said Cordero. But could the same be true for populations of bacteria?

"It is difficult to know what the environmental interactions really are, because microbes are too small for us to observe them in action," said Martin Polz, an organismic and evolutionary biologist at MIT and principal investigator for the Polz Microbial Ecology and Evolution Lab. "But our research provides strong evidence that antibiotics play a role in fending off competitors."

The researchers found evidence by looking at direct, aggressive competition between ecological populations of bacteria. They reconstructed a large network of bacterial fights--or antibiotic-mediated interactions--between bacteria from the ocean.

The scientists analyzed interactions called interference competitions, wherein bacteria produce antibiotics as a means of chemical warfare, to gain a competitive edge by directly hindering the survival of potential competitors.

This typically occurs when bacteria compete for the same portion of habitat.

The researchers assembled an all-against-all battleground for 185 closely-related, but distinct, members of an ocean-based family of bacteria called Vibrionaceae. They measured bacterial compounds produced by Vibrio isolates that directly antagonized other Vibrio isolates.

The framework provided Cordero and colleagues an opportunity to examine about 35,000 possible antibiotic-mediated interactions.

The researchers found that ecologically delineated bacterial populations act as socially cohesive units. "In these populations, a few individuals produced antibiotics to which closely related individuals in the population were resistant, whereas individuals in other populations were sensitive," said Cordero.

Thus, aggressive chemical reactions occur between, rather than within natural populations.

"It appears to be a group effort where individuals assume the role of antibiotic producers and hence defenders," said Polz. "Of course, competing groups could also produce antibiotics. It's a potential arms race out there."

"Those individuals that don't produce antibiotics can benefit from association with the producers, because they are resistant," added Cordero. "In other words, antibiotics have a social effect, because they can benefit the population as a whole."

The findings may help scientists answer questions about the natural role of antibiotics in human contexts.

"The research has the potential to bridge gaps in our understanding of the relationships between plants and humans and their non-disease- and disease-causing bacterial flora," said Robert Fleischmann, a program director in the Division of Biological Infrastructure for the National Science Foundation.

"We use antibiotics to kill pathogenic microbes, which cause harm to humans and animals," said Polz. "As an unfortunate side effect, this has lead to the widespread buildup of resistance, particularly in hospitals where pathogens and humans encounter each other often."

In addition, the results help scientists make sense of why closely related bacteria are so diverse in their gene content. Part of the answer, they say, is that the diversity allows the bacteria to play different social roles.

Social differentiation, for example, could mitigate the negative effects of two species competing for the same limiting resource--food or habitat, for instance--and generate population level behavior that emerges from the interaction between close relatives.

"Microbiology builds on the study of pure cultures," said Cordero, "that is genotypes isolated from their population. Our work shows that we need to start focusing on population based phenomena to better understand what these organisms are doing in the wild."

 -NSF-

When Viruses Infect Bacteria

Viruses are the most abundant parasites on Earth. Well known viruses, such as the flu virus, attack human hosts, while viruses such as the tobacco mosaic virus infect plant hosts.

More common, but less understood, are cases of viruses infecting bacteria known as bacteriophages, or phages. In part, this is due to the difficulty of culturing bacteria and viruses that have been cut off from their usual biological surroundings in a process called in vitro.

Researchers from the California Institute of Technology, funded in part by the National Science Foundation, were the first to use a clever technique to look at virus-bacterium interactions in vivo, that is, within an organism's normal state. The researchers report their results in the July 1st issue of the journal, Science.

As a test case, Rob Phillips and his team considered the interaction between viruses and bacteria in the hindgut, or posterior part, of a termite. Using new microfluidic technology, they were able to isolate single bacterial cells from the termite hindgut in six-nanoliter chambers on an array containing 765 such chambers.

They were then able to determine whether the chambers contained bacterial DNA, viral DNA or both. In the latter case, the researchers were able to statistically deduce whether the virus was specifically associated with the host--for example, by attaching to the host, shooting its DNA into the host, being incorporated into the host as a prophage (a viral genome inserted and integrated into the bacterial DNA), riding on a plasmid or by assembling new viruses within the host. And through this snapshot, the group recorded virus-bacterium associations.

Frequently there was a one-to-one virus-bacterium correspondence. However in some cases, the host was associated with a viral gene exhibiting marked diversity, suggesting possibly a more ancient infection, a more susceptible host or a phage replicating at a lower fidelity. By analyzing the bacteria and viruses based on their evolutionary development they were able to deduce that horizontal gene transfer, while it may be occurring, is not occurring at a rate high enough to randomize host-virus associations.

This study was by no means exhaustive. Many similar associations may still be found in the termite hindgut. And further inquiry may lead to a better understanding of the coevolution of a virus and its host. However, this was the first in vivo exercise, and it opens the doors of the field much wider than previously possible through in vitro culture alone.

-NSF-

Spacebound Bacteria Inspire Earthbound Remedies

J.D. Harrington

Headquarters, Washington     

 

Joe Caspermeyer

Arizona State University, Tempe

 

WASHINGTON -- Recent research aboard the space shuttle is giving scientists a better understanding of how infectious disease occurs in space and could someday improve astronaut health and provide novel treatments for people on Earth.

"With our space-based research efforts, including the International Space Station, we are not only continuing our human presence in space, but we are engaged in science that can make a real difference in people's lives here on Earth," said NASA Administrator Charles Bolden. "NASA's leadership in human spaceflight allows us to conduct innovative and ground-breaking science that reveals the unknown and unlocks the mysteries of how disease-causing agents work."

The research involves an opportunistic pathogen known as Pseudomonas aeruginosa, the same bacterium that caused astronaut Fred Haise to become sick during the Apollo 13 mission to the moon in 1970.

Scientists studying the bacterium aboard the shuttle hope to unlock the mysteries of how disease-causing agents work. They believe the research can lead to advanced vaccines and therapies to better fight infections. The findings are based on flight experiments with microbial pathogens on NASA shuttle missions to the International Space Station and appear in a recent edition of the journal Applied and Environmental Microbiology.

"For the first time, we're able to see that two very different species of bacteria - Salmonella and Pseudomonas - share the same basic regulating mechanism, or master control switch, that micro-manages many of the microbes' responses to the spaceflight environment," said Cheryl Nickerson, associate professor at the Center for Infectious Diseases and Vaccinology, the Biodesign Institute at Arizona State University (ASU) in Tempe. "We have shown that spaceflight affects common regulators in both bacteria that invariably cause disease in healthy individuals [Salmonella] and those that cause disease only in people with compromised immune systems [Pseudomonas]."

By studying the global gene expression patterns in bacterial pathogens like Pseudomonas and Salmonella, Nickerson's team learned more about how they react to reduced gravity.

Pseudomonas aeruginosa can coexist as a benign microbe in healthy individuals, but poses a serious threat to people with compromised immune systems. It is the leading cause of death for those suffering from cystic fibrosis and is a serious risk to burn victims. However, a high enough dosage of Salmonella typhimurium always will cause disease, even in healthy individuals.

During the initial study in 2006, two bacterial pathogens, Salmonella typhimurium and Pseudomonas aeruginosa, and one fungal pathogen, Candida albicans, were launched to the station aboard shuttles. They were allowed to grow in appropriately contained vessels for several days. Nickerson's team was the first to evaluate global gene and protein expression (how the bacteria react at the molecular level) and virulence changes in microbes in response to reduced gravity.

"We discovered that aspects of the environment that microbes encountered during spaceflight appeared to mimic key conditions that pathogens normally encounter in our bodies during the natural course of infection, particularly in the respiratory system, gastrointestinal system and urogenital tract," Nickerson said. NASA's Advanced Capabilities Division Director, Benjamin Neumann added that, "This means that in addition to safeguarding future space travelers, such research may aid the quest for better therapeutics against pathogens here on Earth."

The initial study and follow-on space experiments show that spaceflight creates a low fluid shear environment, where liquids exert little force as they flow over the surface of cells. The low fluid shear environment of spaceflight affects the molecular genetic regulators that can make microbes more infectious. These same regulators might function in a similar way to regulate microbial virulence during the course of infection in the human body.

"We have now shown that spaceflight conditions modified molecular pathways that are known to be involved in the virulence of Pseudomonas aeruginosa," said Aurelie Crabbe, a researcher in Dr. Nickerson's lab at ASU and the lead author of the paper. "Future work will establish whether Pseudomonas also exhibits increased virulence following spaceflight as did Salmonella."

NASA's Fundamental Space Biology Program sponsored and funded the research conducted by Crabbe and Nickerson along with their colleagues at the Biodesign Institute at ASU. They collaborated with the University of Colorado School of Medicine, University of Arizona, Belgian Nuclear Research Center, Villanova University, Tulane University, Affymetrix Inc, and NASA scientists.

For an abstract of the journal article on this research, visit http://www.ncbi.nlm.nih.gov/pubmed/21169425.

For more information about NASA programs, visit http://www.nasa.gov.

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