As oxygen filled the world, life’s universal clock began to tick

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The Earth’s earliest days were largely free of oxygen. Then, around 2.5 billion years ago, primitive bacteria started to flood the atmosphere with this vital gas. They produced it in the process of harnessing the sun’s energy to make their own nutrients, just as plants do today. The building oxygen levels reddened the planet, as black iron minerals oxidised into rusty hues. They also killed off most of the world’s microbes, which were unable to cope with this new destructive gas. And in the survivors of this planetary upheaval, life’s first clock began to tick and tock.

Today, all life on Earth runs on internal clocks. These ‘circadian rhythms’ are the reason we feel sleepy at night, and why our hormones, temperature and hunger levels rise and fall with a 24-hour cycle. They’re molecular metronomes that keep the events inside our bodies ticking in time with the world around us.

Until now, it seemed that the major branches of the tree of life each had their own timekeeping systems, evolved independently of the others. But Akhilesh Reddy and John O’Neill from the University of Cambridge have disproved that idea, by finding a universal clock that ticks in all kingdoms of life. “It’s exciting because it shows that circadian rhythms are likely as primitive as life on Earth,” says Erik Herzog from Washington University.

The universal clock involves proteins called peroxiredoxins (PRX), which help cells to cope with oxygen. This element, though crucial for life, also produces dangerous molecules called free radicals that trigger destructive chemical reactions when they bump into the other components of our cells. PRX proteins protect our cells by absorbing the damage. They’re bodyguards that take the hits that other components cannot. “These proteins had been around ever since cells have used oxygen to make energy,” says Reddy.

In mopping up free radicals, PRXs are transformed into an inactive ‘oxidised’ state, before being recycled back into their natural form. These flips and flops take place over a 24-hour cycle, and thought the details are still elusive, it’s easy to imagine how this could drive a daily rhythm.

Everything old is new again

The PRX clock that Reddy and O’Neill found is unusual in that it involves single proteins that are chemically changed, and then changed back. By contrast, most other clocks depend on genes being switched on (transcription) and proteins being made (translation). In general, a set of clock genes activates and produces proteins that turn off those same genes. This “transcription-translation feedback loop” produces levels of proteins that rise and fall throughout the day. They then control the activation of other genes, and they can be tweaked by external factors like light.

All the kingdoms of life have these loop-based clocks but oddly, each one seemed to use different cogs. Humans rely on clock genes that are unrelated to those used by plants, or fungi, or bacteria. “There’s no common logic other than they happen to work in a feedback loop,” says Reddy, “and even that’s sometimes forced.”

The PRX discovery clearly shows that clocks don’t need to run on feedback loops. But some scientists argue that we’ve known about that for decades. Patricia Lakin-Thomas, a circadian researcher at York University in Canada, says that the idea of these alternative clocks actually pre-dates the more familiar loop-based ones. They’ve just been buried or ignored by scientists who have focused on the feedback loops.

For example, since the 1960s, scientists have shown that a type of alga shows a daily cycle in its photosynthesis, even without a nucleus. That’s where gene activity and protein manufacture occurs. If clocks were only based on feedback loops, a nucleus-free cell should lose its rhythm. The alga showed that this isn’t true.

There’s other evidence, but the most startling demonstration came in 2005. Takao Kondo created a clock using just three bacterial clock proteins, floating in a test tube. There were no genes being switched on or off, and no new proteins being made. There were just three proteins, modifying each other in a perpetual loop. “That was a complete shift in the field and pretty unanticipated,” says Reddy.

O’Neill and Reddy followed Kondo’s experiments by showing that red blood cells, which contain no nucleus or DNA, also show circadian rhythms. So does Ostreococcus tauri, a microbe that doesn’t produce proteins in the dark. Neither cycle involved transcription or translation; instead, they were driven by PRX.

Now, the duo have found that PRX drives daily rhythms in all living things. By using antibodies that recognise the oxidised form of PRX, O’Neill and Reddy found that this state rises and falls with a daily rhythm in mice, flies, flowers, fungi, bacteria, and archaea (another group of microbes that makes up an entire domain of life). In mouse livers, for example, almost all PRX proteins were oxidised at noon only to return to their normal state by midnight.

Lakin-Thomas says that the study “could be the most important advance in the field” since Kondo’s three-protein trick from seven years ago. “This is the first rhythm of any kind that has been demonstrated in so many organisms across kingdoms,” she says.

Cogs or hands?

O’Neill and Reddy think that these clocks may have evolved around 2.5 billion years ago to help primordial cells cope with a sudden influx of oxygen. During this aptly-named Great Oxidation Event, microbes that lacked the right defensive molecules were torn apart by free radicals. And the survivors gained an advantage if their defences were tuned to the daily rhythms of oxygen production.

Back then, photosynthetic microbes gave off more oxygen during daylight hours, just as their modern counterparts do today (although each day was just 18 hours long since the Earth was spinning faster). Since oxygen levels rose and fell with a daily cycle, defences against free radicals worked best if they followed suit. And so, the first clocks arose from proteins that protected us from oxygen – a universal timepiece crafted by a blind watchmaker.

This chain of events is still a hypothesis but like all good ones, it provides some testable predictions. For example, if a species hasn’t evolved defences against free radicals, it shouldn’t have any circadian rhythms either. One such species exists – a methane-eating archaeon called Methanopyri that lives in hot undersea vents. Does it have a clock? Finding out will be tricky. This isn’t a microbe that can be easily grown in a lab, and even if it lacks any of the known clocks, the PRX discovery shows that there may well we ones that we don’t know about.

O’Neill and Reddy are also trying to work out how the PRX clocks interact with those based on feedback loops. Both sets are probably coordinated, but they can also work independently. When the duo knocked out the other clock genes in flies, fungi or bacteria, the PRX cycles still rose and fell (although their timing was slightly off). And when they removed the PRX-clocks from bacteria and a plant, the loop-based ones still worked.

Perhaps the PRX proteins are indeed universal cogs of a primitive fundamental clock. As living things diversified, they added other feedback-loop clocks on top of this ancestral one. Alternatively, PRX could just be the hands of a clock, driven by other cogs that may differ from kingdom to kingdom. To Lakin-Thomas, that’s the big remaining question – we now know that PRX cycles are universal, but how fundamental are they? And are their other biochemical clocks just waiting to be discovered?

To address these issues, O’Neill and Reddy are developing techniques that will allow them to study clocks in any species. Reddy says that in the past, “you’d have your mammalian, and fungal and fly people working on individual systems, none of them with the tools to talk to each other.” They’re now developing such tools, to reveal even more about how universal clocks might work.

Reference: Edgar, Green, Zhao, van Oojien, Olmedo, Qin, Xu, Pan, Valekunja, Feeney, Maywood, Hastings, Baliga, Merrow, Millar, Johnson, Kyriacou, O’Neill & Reddy. 2012. Peroxiredoxins are conserved markers of circadian rhythms. Nature http://dx.doi.org/10.1038/nature11088

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