Thursday, February 23, 2006

Circadian Clocks in Microorganisms

Many papers in chronobiology state that circadian clocks are ubiqutous. That has been a mantra since at least 1960. This suggests that most or all organisms on Earth possess biological clocks.

In the pioneering days of chronobiology, it was a common practice to go out in the woods and collect as many species as possible and document the existence of circadian rhythms. Technical limitations certainly influenced what kinds of organisms were usually tested.

Rhythms of locomotor activity are the easiest to measure. Rodents, as well as large walking insects like cockroaches, will turn running wheels, each revolution triggering a switch that sends a signal to the computer. Songbirds will jump from one perch to another, each perch flipping a switch connected to a computer. Lizards, while walking around the cage will tilt the cage from left to right around an axis - a metal bar on the bottom - which will turn a switch. Plants that exhibit leaf movements (closing at night, opening during the day) were the prime experimental models for a while (e.g., Kalanchoe, mimosa, tobacco).

Monitoring rhythms in other organisms is much harder: it is mighty difficult to make a fish run in a running wheel, or build hopping perches sensitive enough to be triggered by the landing of a butterfly. That was even harder back in the late 1940s and early 1950s when most of this work was done.

It is no suprise that nobody looked at microorganisms back then - it was just technically too hard. The fact is that most of the pioneers in the field came in from vertebrate physiology, ethology or ecology. It is easy for us, large mammals, to forget that we are not among the dominant life-forms on the planet - that title goes to bacteria, in terms of numbers of individuals, in terms of biodiversity, and in terms of total biomass. See if you can find mammals, or even all animals on the Tree of Life (click to enlarge):
Some old papers, mostly parts of Conference Proceedings of various kinds, mention as fact that Bacteria do not have clocks but do not provide any citations. It took me years to dig out three papers (Rogers and Greenbank, 1930, Halberg and Connor, 1961; and Sturtevant, 1973) with relevance to this question and all three are ambiguous about the final verdict. Why is nobody revisiting this with modern molecular techniques?

Being unicellular does not preclude one from having a clock, though, as single-cell Protista and Fungi all have circadian rhythms, which have been studied quite extensively since the 1970s or so (I intend to delve some more in that literature and write some posts on them in the future).

One group of bacteria does have a clock - the unicellular Cyanobacteria (if you are above a certain age, you may remember them under their old name: blue-green algae), in particular those species that do not form chains, e.g., Synechococcus and Nostoc. This was discovered very recently - only ten years ago (Mori et al. 1996). I was two years into my Masters when that paper appeared and I remember the excitement. I will certainly write a post or two on those soon:
There has not yet been a single study of any kind of rhythmicity in Archaea. Most of those microorganisms live in strange places - miles deep under the surface of the earth, in rocks, in ice, on the ocean floors and in the hydrothermal vents. They mostly do not inhabit rhythmic environments, so perhaps they do not need to have clocks - but it would be really nice to know if that is really the case:
Old Faithful, the famous geyser in Yellowstone park contains Archea. As the geyser erupts every 45 minutes or so, the microbes are suddenly exposed to very different environment: light, turbulence, lower temperature. Should we expect them to evolve a 45-minute clock that will help them predict the eruption so they can limit some sensitive biochemical reactions to the quiet periods and switch on the defenses agains light and cold every 45 minutes?

In The Geometry of Biological Time, Arthur T. Winfree suggested an experiment (on Page 580) that it
"... should be possible to demonstrate the effect by bacterial selection experiments in a chemostat. By alternating the nutrient influx from glucose without oxygen, to oxygen without glucose, to alanine and oxygen, cells would be forced into a three-point metabolic cycle." and "... reversing the order of the driving cycle, it should be possible also to select cells whose clocks run backward."
In a later edition (after we learned that cyanobacteria have clocks) he suggested, instead, to use
"one of the species of cyanobacteria that revealed no circadian rhythms in surveys before Mori et al. (1996), and use light as the alternative nutrient".
As of today, nobody has performed such an experiment, although Elowitz and Leibler (2000) came pretty close with a study in which they produced oscillations in Escherichia coli with periods of 3-4 hours, which are slower than the cell-division cycle:
So, if most of Life on Earth is Prokaryotic (Eubacteria and Archaea), and those groups do not have clocks, then clocks are not ubiqutous, are they? In my papers and in my Dissertation I try to hedge a bit by stating that they are found in "organisms that live on or close to the surface of the Earth", thus at least avoiding the deep-oceanic, deep-soil, and parasitic microorganisms (as well as burrowing and cave organisms that may have secondarily lost their clock).


Blogger Impatient Patient said...

Can one organism with a circadian rhythm affect another organism's circadian rhythm?

5:45 PM  
Blogger Bora Zivkovic said...

This is a great question! The answer is Yes.

For instance in a rodent called Octogon degus, olfactory signals from one individual to another mutually entrain their rhtyhms (I think it has to be one male and one female, though). Other rodents may synchronize their rhtyhms when kept in group conditions without any rhyhtmic clues from the environment.

There is also a model (I do not know how well it is empirically supported) in which there is a feedback between the clock of dinoflagellate Gonyolax polyedra (I think it has been recently reclassified and renamed, but all teh literature used this name) and its environment, i.e., the little protists affect their environment which in turn entrains their clock which in turn makes teh environment rhythmmic (salinity, or pH or something), and so on.

Those are just some examples at the time-scales of day-to-day activity of organisms. At an evolutionary scale, many organisms evolve the rhythms in ways that synchronize with other species (usually trying to avoid each other). There are a number of cases of evolutionary arms-races around the circadian clock, as well as cases of competitive exlusion in time (as opposed to space). Some golden spiny mice in Israel - two closely related species - are sympatric (live in the same place) in one canyon. One of the species remains nocturnal, while the other one is forced to turn diurnal (only in that geographical area), with con-commitant changes in the anatomy and physiology of the eyes, etc.

10:09 PM  
Blogger Impatient Patient said...

Very cool.

My next question is about glia pro-inflammatory cytokines and the SCN.

The SCN regulates circadian rhythms- I think.

How do viruses and bacteria and inflammatory processes mess up circadian rhythms?

Does this happen in animals as well as humans?

If the circadian rhythm is affected by light, (and I am serious here) could sunglasses affect the circadian rhythm of people.

I have a million more questions, I was at Aetiology and read Tara's blog called Are Female Night Owls Screwed? I have been looking at glial cytokine effects on the maintenance of chronic pain, and I am pretty sure the SCN was mentioned in one of the papers or articles I came across. The hypothalamus is involved .... Very long and boring story...

Can one reset a real person's clock by going outside in the sunshine daily and dimming lights at night?

Is it better on the circadian rhythm to live close to the equator as opposed to somewhere where the day/night changes? Obviously not such a factor in 24/7 society. What genetic variations, if any , have been found in people's SCN's that live in different places - have their bodies adapted to different light levels. I think of the Artic and the Inuit. WHat happens when you move suddenly and mess with it?

I could go on all nite-

Thanks for answering the first question.


1:51 AM  
Blogger Bora Zivkovic said...

Lots of questions - some do not have answers yet. Others - I have touched upon in a number of previous posts - I sure hope you are browsing the blog some more.

Yes, there is genetic variation.

Exposure to bright light during the day and dark during the night entrains the clock.

Glia appear to have a big role in the workings of the SCN.

Cytokines? Dunno.

Sunglasses should not have a big effect (except to make it harder to reset the clock if you consciously try to expose yourself to bringht light).

Disease generally do not affect the clock - the clock tends to work perfectly well (though aging makes it a little sloppy over time) until the moment of death.

2:26 AM  
Blogger Impatient Patient said...

I have looked through your blog- I am a visitor at least once every couple of days. Usually via Aetiology.

Your name strikes me as very cool. I have no idea of your actual nationality, but I worked in a ukrainian school last year- the only English only girl- and have a new appreciation for names from different countries.

I will snoop some more and see what you have that will help me understand my bunches and bunches of questions.

And then- FOUND SOME MORE- actually only one. In your opinionion, can a person mess up their circadian rhythm by working straight nights for 18 years, if they started at a fairly young age? If you have a link to a previous post, I would love to read it, if you have answered this already. I need sleep, so thank you for your patience with everything. I will check back tomorrow.

2:47 AM  
Blogger Bora Zivkovic said...

Working one night can mess up your rhythms. Working nights for 18 years can be deadly - but, there is always that human variation....

7:16 AM  
Blogger Bora Zivkovic said...

There have been some papers out recently showing that night-shift nurses have a much greater incidence of certain diseases (cancers, ulcer) than day-shift nurses. It's all around the internet and easy to find.

Weather in North Carolina is as usual: we go through all four seasons within each week.

6:55 PM  
Blogger Impatient Patient said...

Can be deadly....Do you have any information that says that in a lot of words? That is exactly the answer I was looking for, and dreading at the same time.

I will keep digging on my own as well.

Have a great weekend. What is North Carolina like this time of year?

6:58 PM  
Blogger Impatient Patient said...

I have found that literature- what I am looking for specifically is if autoimmune conditions are affected by circadian rhythm disruptions. Poking and poking around....

1:16 PM  
Blogger Impatient Patient said...

Hello- I am back with another question. DO the bacteria and viruses in people have a 24/25 hour rhythm? DO we know that yet? I have been going thru your blog and a bunch of other stuff- I feel like I am in the land of the smart without my brain as I try to make sure I am not screwing things up as I try to understand.

Your patience is much appreciated!

12:37 AM  
Blogger Bora Zivkovic said...

Nobody has looked yet. I'd love to know if they do. I'll have a better look at those E.coli old papers I cited above and perhaps post more on it at a later date.

12:46 AM  
Blogger Impatient Patient said...

You wrote in a linked post:
XVI. CRs have so far proved surprisingly intractable to chemical perturbation [True. Heavy water, lithium, melatonin and a couple of other hormones are the only substances that affect the clock].

May I also ask what other hormones affect the Circadian Rhythm?

Also- How do glia affect Circadian Rhythm and is it microglia or astrocytes or both?

Pro-inflammatory cytokines are activated by glia and astrocytes. Think chain reaction/vicious cycle- A leads to B leads to C etc... They are a pain in the ass when they are part of auto-immune disorders. Auto immune disorders tend to have a lot of sleep disturbance. So wher are the SCN and Circadian Rhythm all in this recipe for disaster?

I think I will just sign myself as Pain in the Butt-LOL!! Thank you again.

1:01 AM  
Blogger Impatient Patient said...

About the hormones- I read the sex hormones influence the Circadian rhythm. Does Vitamin D as well? Sorry- I just wanted to clarify if it was anything other than sex hormones. The pineal gland is involved in testosterone synthesis, though, is it not?

Pain in the Butt Again- Thanks!!!

1:05 AM  
Blogger Bora Zivkovic said...

Don't know much about cytokines.

Vitamin D? No.

Estrogen and testosterone - yes.

Pineal does not have a direct effect on testosterone synthesis, but in some species may play an indirect role (via photoperiodism).

Astrocytes, last I heard.

1:28 AM  
Blogger Impatient Patient said...

Aah- the astrocytes are the ones I was hoping not to hear about.

This is how it goes with the glia thing in chronic pain/some immune disorders- just so you won't think I am nuts.

When the microglia are activated, they send out signals that basically call up some of the pro-inflammatory cytokines. TNF, interleukin 1 and interleukin 6.
When the astocytes get involved, is when the pain/sleep cycle really seems to have a problem and it becomes set.

Cytokines also send signals to the brain that an injury has happened, which tells the rat or the person to slow down and rest and be depressed. There is a very basic news article called SLACKER OR SICK that describes this. Barr is one of the authors of the study the article is based on, it can be googled.

My interest in the whole circadian rhythm thing is many-fold. I am a light worshipper. I cannot sleep in the basement without being MISERABLE. I need light to wake me. WInter up North is agony. Now that we have light earlier, and later, I am finally feeling human. It has taken a while!

I also have a lot of friends/family/acquaintances that have had difficulty "resetting" or even setting their clock after an illness- whether it be short term or prolonged. Some people have chronic illness. SOme are night workers. I am also curious as to how some people who work nights still stay connected to the daytime. They can switch back and forth easily. Others are NOT able to. All nice people, just different abilities in their body's coping mechanism.

How does short and long term disease affect brain function in regards to Circadian Rhythms. Does CR have anything to do with the staggering rise of depression and mental illness. Is that too simple. What happens to a continent in which the people are never really outside during the day? We are in office buildings and our vehicles, rather than our gardens or other natural settings. Do fluorescent lights mess us up?

You don't have to answer, I will keep snooping around, but these are some of the questions I think of now. I have hundreds of others, related somehow, written down.....

Maybe....Only the Shadow knows!!!??

1:48 AM  

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