Even green pond scum can suffer from jet lag

December 02, 2002

Blue-green algae - better known as green pond scum - are the simplest organisms known that have an internal biological clock.

Biologists know that biological clocks play a critical role in most living organisms including humans, but, despite an abundance of theories, they don't really know why. Now, a research team from Vanderbilt, the University of Wisconsin-Madison and Harvard Medical School has taken a significant step toward answering this question. In an article published the week of Dec. 2 in the online edition of the Proceedings of the National Academy of Sciences, they report having successfully determined the structure of a biological clock protein for the first time.

The protein in question is called KaiC and it comes from a genus of blue-green algae named Synechococcus. Carl Johnson, the professor of biological sciences who headed up the study, directs one of the few labs in the world that studies biological clocks in prokaryotes, single-celled organisms that lack a nucleus.

In humans and other higher organisms, biological clocks control the wake-sleep cycle and have a profound influence on a number of different basic biological processes. When a person's internal clock is out of sync with the environment, jet lag is the result.

Not many scientists are studying biological clocks in prokaryotes because no one thought they had them until recently, Johnson says. That is because bacteria typically reproduce in less than 24 hours. Biologists had developed a general rule that a microorganism that divided in less than a day -becoming a whole new individual in the process - would gain no benefit from possessing an internal clock. So they had pretty much stopped looking for internal clocks in fast-growing bacteria.

Like many such rules of thumb, however, this neat formulation was disproved in the 1980s by a group of Taiwanese scientists who didn't know any better. They were investigating how blue-green algae (also known as cyanobacteria) found in rice paddies remove nitrogen from the air and fix it chemically so that plants can use it for food. It is quite a trick for a single-celled organism to combine photosynthesis and nitrogen fixation because the two processes are biochemically incompatible. The Taiwanese discovered that the tiny organisms had solved this problem by inventing the practice of moonlighting: They photosynthesize all day and then fix nitrogen after dark.

"When those of us who work in biological clocks saw their results, we realized that the algae must have a clock," Johnson says. This led Johnson, who studies biological clocks in a number of different organisms, to add blue-green algae to his repertoire. "The nice thing about bacteria is that you have a lot of genetic tools available and you can do analyses much faster than you can in higher organisms," he says.

Using these tools, Johnson and colleagues from Texas A&M University and the University of Nagoya, were able to sequence three genes - Kai A, B and C - that code for three critical clock proteins, called KaiA, KaiB and KaiC. Although other proteins are involved, the biological clock will not work without them. KaiC is the largest of the three. There were enough similarities between its primary genetic sequence and those of other proteins that act directly on the bacteria's chromosome to suggest that KaiC also had this ability, but there were enough differences so that the researchers were not certain.

Next the researchers purified the protein and studied its structure with electron microscopy.

"We were able to show that KaiC forms a ring-like, hexagonal structure and this structure gives us some important clues into how the biological clock regulates the expression of the entire bacterial genome," Johnson says.

Kai-C's structure is similar to that found in other proteins that are known to bind to bacterial DNA, so it strongly reinforces the genetic indications that this protein interacts directly with DNA. However, it lacks some of the characteristic genetic sequences of the other ring molecules, suggesting that its interaction may be novel in nature, Johnson says.

"Now, how does this protein work in terms of the Circadian clock? That's the $64,000 dollar question!" he says. The scientists know that the internal clock in the blue-green algae is significantly different from that even in single-celled eukaryotes, organisms with nuclei. For one thing, the clock in cyanobacteria controls the expression of virtually 100 percent of its genes, while in the clock in eukaryotes controls only 5 to 10 percent.

Johnson has an intriguing idea about how this bacterial clock might work. It takes advantage of the fact that blue-green algae's chromosome takes the form of a circle, rather than long linear molecules as is the case in higher organisms.

Double-stranded DNA can exist in two basic states: one is a relaxed state and the other is a state called super-coiling. DNA becomes super-coiled when it is subjected to torsional, or twisting stress. It is similar to the state that a length of hemp rope takes on when one end is held fixed and the other is rotated in the same direction that the rope is wound.

Whether DNA is a relaxed or a super-coiled state has a big influence on how well genes can operate. So Johnson speculates that KaiC might work by twisting the bacteria's DNA back and forth between the two states.

"We really don't know if that is the case," says Johnson, "but it is our working hypothesis."

The bacterial biological clock may have some biochemical similarities to those in animals and humans, but it is clear that it works much differently. Although the clocks in humans only affect a small subset of the entire genome, that still represents a tremendously large number of individual genes.

"We think we know a lot, but we are still really in the infancy of understanding how these clocks work," he says.

Co-authors of the paper are post-doctoral fellows Tetsuya Mori and Yao Xu from Vanderbilt; Sergei Saveliev, Michael M. Cox and Ross B. Inman from the University of Wisconsin-Madison; and Walter F. Stafford from Harvard Medical School.

The research was supported by funds from the National Science Foundation and the National Institutes of Health.

SIDEBAR: Why are biological clocks so important?

Ever since biological clocks were discovered, scientists have assumed that they must be beneficial. But it wasn't until 1998 that this general belief got some direct, experimental confirmation.

Johnson, working with Susan S. Golden from Texas A&M University and Takao Kondo from Nagoya University, performed a conceptually simple experiment that demonstrated that bacteria that had a biological clock that is in synch with the light/dark cycle have a competitive advantage over those who have biological clocks tuned to a different frequency.

First, they produced strains of cyanobacteria (blue-green algae) that had biological clocks that operated at frequencies of 22 hours and 30 hours. They inserted the gene that produces the luminescent enzyme luciferase into the bacteria's chromosome in a position where it was turned on and off by the bacteria's internal clock, allowing them to identify the different bacteria by timing the rhythm of light production. They also engineered some normal, wild-type bacteria which have a 25-hour clock so they would rhythmically luminesce as well. Next they created mixed colonies by combining the different strains in pairs: wild type and 22 hour; wild type and 30 hour; 22 hour and 30 hour. Then they put these mixed cultures into incubators with three different light-dark cycles - 22 hours, 24 hours and 30 hours - and monitored them for about a month.

They found that the strain whose internal biological clock most closely matched the light-dark cycle invariably outgrew the competing strain. In fact, they found that the selective advantage by having the correctly tuned biological clock was surprisingly strong, allowing the strain with the matching frequency to grow 20 to 30 percent faster than the strain with its internal clock tuned to the wrong frequency.

In a related series of experiments, Johnson's lab used a mutant strain of cyanobacteria that doesn't have a clock at all. They found that the "de-clocked" strain can compete perfectly well with the wild-type bacteria in a environment with constant light and temperature. As soon as the mixed culture is placed into a rhythmic environment, however, the wild-type organisms rapidly out-compete the bacteria without internal clocks.

"Our data show that having a properly tuned biological clock confers a major advantage in rhythmic environments," says Johnson. "We don't know why that is, but there are a lot of hypotheses." One proposal is that the internal clock allows the bacteria to anticipate when the light is going to arrive so they can get their photosynthetic system up and running in time to start capturing sunlight at the earliest possible moment.

Vanderbilt University

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