How worms’ noses sense oxygen

Organisms ranging from bacteria to humans navigate environments that can contain dangerously too little or too much oxygen. Yet, scientists know little about how animals sense oxygen levels around them.

Researchers from the Berkeley and San Francisco campuses of the University of California have now discovered how the nematode C. elegans senses oxygen levels in order to steer clear of surrounding areas that are too low or too high in oxygen.

In the process, the researchers also discovered that the worm doesn’t like as much fresh air as people thought. While nematodes grown in laboratory Petri dishes are kept at the same oxygen concentration humans breathe in ambient air – 21 percent – nematodes appear to prefer only 6 percent oxygen.

“It was totally unexpected that they would actually prefer 6 percent. We don’t know why, though it probably gives them some survival advantage,” said Michael A. Marletta, professor of chemistry and of molecular and cell biology at UC Berkeley, and a faculty scientist at Lawrence Berkeley National Laboratory (LBNL). “And the bordering and clumping that worm experts refer to as social behavior is really the worms, in an artificial setting like a Petri dish, trying to get to an area of 6 percent oxygen, which they like. It’s a laboratory phenomenon.”

Bordering and clumping is a peculiar behavior in which worms cluster around the border of the Petri dish instead of spreading evenly around the surface. Marletta and his colleagues, members of the California Institute for Quantitative Biomedical Research (QB3), determined that the bacteria the worms feed on are at a higher density around the border of the dish, consuming oxygen along with the worms. Apparently, when oxygen levels are high, the worms pile onto the densest clumps of bacteria, because that’s where oxygen levels are lowest.

“The swarm of worms and density of bacteria together lower the oxygen concentration in that immediate environment,” he said. “We found that when we lower the oxygen concentration to six percent, the worms disperse in three minutes.”

At high concentrations, oxygen is toxic and corrosive. Worms avoid high oxygen presumably because the oxygen creates highly reactive chemicals that damage cells, though oxygen sensors also may help them find food, Marletta said.

Marletta, Cori Bargmann, a UC San Francisco professor of anatomy and biochemistry and biophysics, and their colleagues reported their results on June 27 in the Early Online Edition of Nature. The paper will be published in the July 15 issue of Nature.

Surprisingly, the worm’s oxygen sensors, which are actually enzymes that bind oxygen, are similar to enzymes used in humans and other animals to detect the signaling molecule nitric oxide, or NO. NO plays a major role in the cardiovascular system, activating an enzyme that triggers dilation of blood vessels and thereby controls blood pressure.

It was work on this NO-sensing enzyme that led Marletta into C. elegans research. The enzyme, guanylate cyclase, is found in smooth muscle, like that encircling blood vessels. NO binds and activates guanylate cyclase, triggering a cascade of chemical reactions that make the muscle relax, opening up the vessel and lowering blood pressure. NO also activates guanylate cyclase in the brain, where it is involved in learning in memory.

“NO is important in maintaining blood vessel homeostasis, and so is critical to cardiovascular function, gut motility and penile erection, among other things,” Marletta said.

Marletta’s students have painstakingly picked apart the guanylate cyclase protein, in particular the exact binding site for NO. It turns out to be a heme molecule nearly identical to the heme that binds oxygen in hemoglobin to carry it through the blood stream to muscle. The heme in hemoglobin cannot discriminate between oxygen, carbon monoxide (CO) or NO, which is why CO is toxic: it replaces oxygen.

The puzzle has been why the heme in guanylate cyclase is able to exclude oxygen from its binding site and reserve it only for NO. This molecular question – the key to understanding how NO works – has been a focus of the Marletta lab, and pursuit of the answer led directly to oxygen sensing in worms.

The discovery of a similar enzyme in the nematode, but one that binds oxygen instead of NO, will help Marletta and his colleagues discover the tricks used by the enzyme to let in or screen out oxygen from the heme binding site to selectively detect one or the other.

“This experiment helps us understand how NO receptors in muscle and brain are able to bind selectively to NO in low concentrations even when oxygen is present in far greater concentrations,” he said. “This could have implications across a wide swath of biology, in cases where organisms need to bind NO in low concentrations and very selectively.”

In order to understand how guanylate cyclase is put together, Marletta and his students went in search of similar enzymes in the genomes of other organisms. Patricia Pellicena, a UC Berkeley postdoctoral fellow with collaborator John Kuriyan, a UC Berkeley professor of chemistry, found homologues not only in the nematode, but also in more primitive organisms called bacterial prokaryotes. One group of these, the obligate anaerobes, die in the presence of oxygen, so they evidently require a sensitive oxygen detector.

This insight helped graduate students David S. Karow of UC Berkeley and Jesse M. Gray of UCSF make sense of puzzling data they were obtaining about C. elegans’ response to NO. Perhaps this enzyme was serving as an oxygen detector, not as a NO detector, in C. elegans?

By manipulating oxygen levels in Petri dishes filled with worms feeding on a lawn of bacteria, the researchers were able to show that bordering and clumping was actually a response to high oxygen levels. Karow and Gray employed a custom MEMS (microelectromechanical systems) chamber – only 100 microns (1/10 millimeter or 4 thousandths of an inch) thick – to study worm reactions to oxygen levels. Built at UC Berkeley by UCSF postdoctoral fellow Hang Lu, it has speeded behavioral studies of these denizens of the soil and served as a prototype for further nematode studies.

By knocking out several genes coding for parts of the guanylate cyclase enzyme, they were able to show that this enzyme was acting as an oxygen detector, primarily steering worms away from too much oxygen. The enzyme is found in three separate neurons that innervate the worm’s nose.

Bargmann speculates that the oxygen-sensing system used by C. elegans may be used by other animals who must avoid low-oxygen environments, including fish. Humans may also have such a detector to trigger hyperventilation during exercise or exposure to anoxic environments.

“We are immersed in a 21 percent oxygen atmosphere all the time, and our blood stream and lungs maintain the optimum oxygen levels in our tissues. So, we take oxygen levels for granted,” Bargmann said. “But most other animals on the planet live in water or the soil, such as C. elegans. And since oxygen diffuses much more slowly in those environments, they must evolve ways to sense oxygen and react to changes in oxygen levels.”

Marletta’s students continue to take apart the guanylate cyclase enzyme and, working with Kuriyan, are trying to crystallize the pieces in order to get X-ray diffraction data to determine the 3-D structure.

“Biology has learned to use NO in cell-to-cell signaling, evolving a system to generate and use it at very low concentrations. But what kind of receptors can work at very low concentrations of NO?” Marletta said. “We want to find out how nature engineered a guanylate cyclase protein that doesn’t bind oxygen but still binds NO.”

“It’s surprising,” he added, “that 225 years after Lavoisier discovered oxygen, we’re still finding out how organisms sense and use it, and little did we imagine that studying NO would lead us closer to understanding the fundamentals of oxygen utilization.”

The work was supported by the Howard Hughes Medical Institute and LBNL. Other coauthors of the paper are Andy J. Chang of UCSF, Jennifer S. Chang of the University of Michigan and Ronald E. Ellis of the University of Medicine and Dentistry of New Jersey.