Category Archives: Microorganisms

Yes, Archaea also have circadian clocks!

ResearchBlogging.orgIf you ever glanced at the circadian literature, you have probably encountered the statement that “circadian rhythms are ubiquitous in living systems”. In all of my formal and informal writing I qualified that statement somewhat, stating something along the lines of “most organisms living on or near the Earth’s surface have circadian rhythms”. Why?
In the earliest days of chronobiology, it made sense to do most of the work on readily available organisms: plants, insects, mammals and birds. During the 20th century, thousands of species of animals, fungi, protists and plants – all living on the planet’s surface – were tested for the possession of the circadian clock, and one was always found. Hence the “ubiquitous” statement seen in so many papers.
But, as it was later discovered, for some marine organisms moon cycles are more important than day-night cycles so they have evolved lunar clocks in addition or instead of circadian clocks (see sponges and cnidaria, for some examples). In the intertidal zone, the tides are more important for survival than the daily rhythms, so the organisms living there have evolved tidal clocks. Animals that live in caves have lost circadian rhythms, at least in behavioral output (a clock may still be operating underneath, driving metabolic or developmental rhythms). In the polar regions, rhythmicity may be seasonal. In subterranean animals, like Blind Naked mole-rats, most individuals are without rhythms, but young males that leave the colony in order to join another one develop rhythmicity during their adventurous journey. In social insects, only the individuals that go outside the hive to forage exhibit daily rhythms.
How does one figure out if an organism has a clock? You need to pick a good output and a way to continuously monitor it. Then you put the organism in constant conditions of light, temperature, air pressure, sound etc., and monitor the output for many days. If you do the statistics on the data at the end of the experiment and see that there is a periodicity in the data (for at least the first 2-3 days)that is reasonably close to 24 hours (between 16 and 32 hours is usually thought to be the limits), you know that your organism of choice has a circadian clock.
In a related experiment, you expose the organism to an environmental periodicity – usually a light-dark cycle, as it is usually the strongest cue, as the evolution of circadian clocks and light-detecting mechanisms is closely intertwined – to see if the rhythmicity of the organism can be synchronized (entrained) to the environmental cycle, indicating that it is a biological function and not the chance quirk in your data. Without these two experiments providing positive data it does not make sense to do any further investigations into mechanisms of entrainment, anatomical location of the clock or the cellular mechanism of the clock.
The trick is to find a good output to monitor. It is easy for rodents – they will run in running wheels (so will cockroaches). Songbirds will jump from perch to perch. Lizards will walk around the cage and tilt the floor from one side to another. And while behavioral output – the general locomotor activity – is not the most reliable (it is very prone to masking effects, so for instance mice will generally not run in wheels in bright light, while rats will), it is usually the easiest and cheapest to monitor and, in most cases (see an example where it failed, while monitoring body temperature worked) will be sufficient.
But what do you do when the organism does not have a measurable behavioral output, especially one that can be continuously monitored by machines? You start thinking very, very hard. And you come up with an alternative. You may be able to implant radiotransmitters and monitor body temperature. Or you may record vocalizations. Or you may take small blood samples several times per day and assay for something like melatonin.
The technological constrains limited our ability to discover circadian clocks in bacteria until the 1990s. Until then, the existence of such clocks was a mystery (one that everyone in the field was eager to see solved). I have written several posts about the discoveries of clocks in bacteria: Circadian Clocks in Microorganisms, Clocks in Bacteria I: Synechococcus elongatus, Clocks in Bacteria II: Adaptive Function of Clocks in Cyanobacteria, Clocks in Bacteria III: Evolution of Clocks in Cyanobacteria, Clocks in Bacteria IV: Clocks in other bacteria, Clocks in Bacteria V: How about E.coli? The understanding of the way bacterial clocks work (more like a relay or a switch than a clock) made us rethink the clock metaphor we have been using for almost a century.
So it appears that most Eukaryotes have clocks and at least some bacteria have them as well. But the other large group – the Third Domain: Archaea – eluded us thus far. After all, Archaea are notoriously difficult to culture in the laboratory and it took some time to figure out how to keep them alive outside of their natural extreme environments.
Do Archaea have clocks? We did not know. Until now. A couple of weeks ago, PLoS ONE published a paper that is the first to demonstrate the daily rhythms in an Archaeon: Diurnally Entrained Anticipatory Behavior in Archaea by Kenia Whitehead, Min Pan, Ken-ichi Masumura, Richard Bonneau and Nitin S. Baliga. Here is the text of the Abstract:

By sensing changes in one or few environmental factors biological systems can anticipate future changes in multiple factors over a wide range of time scales (daily to seasonal). This anticipatory behavior is important to the fitness of diverse species, and in context of the diurnal cycle it is overall typical of eukaryotes and some photoautotrophic bacteria but is yet to be observed in archaea. Here, we report the first observation of light-dark (LD)-entrained diurnal oscillatory transcription in up to 12% of all genes of a halophilic archaeon Halobacterium salinarum NRC-1. Significantly, the diurnally entrained transcription was observed under constant darkness after removal of the LD stimulus (free-running rhythms). The memory of diurnal entrainment was also associated with the synchronization of oxic and anoxic physiologies to the LD cycle. Our results suggest that under nutrient limited conditions halophilic archaea take advantage of the causal influence of sunlight (via temperature) on O2 diffusivity in a closed hypersaline environment to streamline their physiology and operate oxically during nighttime and anoxically during daytime.

What does that mean? What did they do?
First, they picked a good candidate species – Halobacterium salinarum. Why is it a good candidate? Because it lives near the Earth’s surface, in salty lakes and ponds (like this one, in Africa):
salinarium in a lake.gif
Many Archaea live in places where no light ever penetrates: deep inside the rock or ice or the oceanic floor. Some Archaea are exposed to light in cyclical fashion but not a 24-hour cycle – I have written somewhere before that I expect the Archaea living in the waters of the Old Faithful geiser in Yellowstone National Park to have a 45-minute clock instead. But Halobacterium salinarum is exposed to the natural periodicity of the day-night cycle on the surface and is thus a good candidate for an Archaeon that may have evolved a circadian clock. This is how the Halobacterium salinarum look like under the microscope:
salinarium micrograph.gif
There is another reason this is a good candidate. The light-dark cycle has a potential adaptive consequence to the critter. Water that is saturated with salt will have a high variation of its oxygen content and this variation is dependent on the environmental temperature: when it is colder outside, oxygen can more readily disolve in the salty water. When it is warm, it cannot.
The environment where Halobacterium salinarum lives is cold during the night and hot during the day. But the temperature changes are much more gradual and slow than changes in illumination (as well as less dependable: there are colder and warmer days), so being in tune with the light is a better way to synchronize one’s activities than measuring temperature (or oxygen content) directly. By entraining to a ligh-dark cycle, these organisms can make switches in their oxygen-dependent metabolism in a more timely (and thus more energy-efficient) fashion: by predicting instead of reacting to the changes in temperature over the course of 24 hours.
So, Whitehead et al placed some Halobacterium salinarum in light-dark cycles and subsequently released them into constant darkness. But what did they measure? Archaea are known to be lousy wheel-runners!
In bacteria, much of the work is done by measuring bioluminescence coming from the expression of the luciferase gene inserted next to one of the clock gene promoters. But here, we don’t know which if any gene is a clock gene and we do not have the technology ready yet. But, these days microarrays are cheaper and easier to use then some years ago when I started grad school. And remember that Everything Important Cycles!
So they took samples of the organism six times per day and ran them on microarrays, comparing the expression of all the genes between the sampling times, both during entrainment to LD cycles and in the subsequent DD (constant dark) environment:
archaea microarrays.JPG
What they discovered is that about 12% of the genes cycle with the period of 24 hours in LD cycles and continue to cycle in DD with a circadian period of around 21.6 hours:
archaea periods.JPG
What is most interesting is that the genes that cycle are the genes that are involved in oxygen (or oxygen-dependent) metabolism – exactly the kinds of genes that are expected to cycle in this organism. Some of these genes are also know to be directly regulated by oxygen. Now we know they can also be regulated – directly and/or indirectly through a clock – by light, inducing expression in preparation for the changes in oxygen concentration, not just in direct response. In this way, the cell is ready to use oxygen a little bit ahead of time. No time wasted.
I am very excited about this finding. This opens up a whole avenue of future research, something that the authors also realize:

Indeed, further detailed experimentation is necessary to ascertain precise phasing, temperature compensation, adaptability to different periods of entrainment etc. to ascertain the mechanistic underpinnings of this diurnal entrainment and its physiological implications.

Once we know there is a clock in Archaea – and now we do due to this paper – we can start studying it in detail.
Furthermore, this finding has big implications for the study of the evolutionary origins of the circadian clock (and light-reception associated with it). The molecular mechanism of the clock is very different between Bacteria and Eukaryotes, leading the field to conclude that the clock evolved independently in these two groups (and perhaps more – some people think that protist, plant, fungal and animal clocks evolved independently of each other as well). Now we can try to figure out how Archaea measure time. Is their mechanism similar to that in Bacteria? Or in Eukaryotes? Or something completely different, indicating another independent evolutionary origin? Or something in-between Bacteria and Eukaryotes, containing some elements of both, suggesting that perhaps there was only one evolutionary origin for clocks in all the life on Earth. The authors note that this last scenario is a strong contender:

Finally, the discovery of diurnal entrainment of gene expression in an archaeon also raises important questions regarding the origin of light-responsive clock mechanisms. This is because archaeal information processing machinery is assembled from components that share ancestry with eukaryotic (general transcription factors and RNA polymerase) and bacterial (sequence-specific transcription regulators) counterparts [44]. Furthermore, components of both bacterial [45,46] and eukaryotic [47] clocks are encoded in its genome [6,32].

Of course, since this is an Open Access article, you can and should read it yourself to get more details. And post ratings, notes and comments while you are there.
Whitehead, K., Pan, M., Masumura, K., Bonneau, R., & Baliga, N. (2009). Diurnally Entrained Anticipatory Behavior in Archaea PLoS ONE, 4 (5) DOI: 10.1371/journal.pone.0005485
Update: see comment thread for more. Unfortunately, scientists still at this day and age do not report everything and keep data secret. Apparently, this was the case in the question posed by this study. I hear from trusted sources that there is still not evidence for a clock in Archaea beyond the direct effects of light on gene expression and O2 metabolism.

New microbiology aggregator

New microbiology aggregator just went live in Belgrade, built (as always) by Vedran Vucic.

Scarlett Johansson – Bioterrorist?

You may have heard the story that Scarlett Johansson had a cold when she appeared on Jay Leno’s show the other day. And you may have heard that she got the cold from her ‘The Spirit’ co-star Samuel L. Jackson. And you may have heard that she had to blow her nose into a tissue during the show. And you may have heard that this particular tissue is now up for sale on eBay. And you may have heard that all proceeds of this sale will benefit USA Harvest, the charity of Scarlett Johansson’s choice.
What you may not know is that, due to the content of the tissue being regarded as biohazard (or even bioterrorism), you may not be able to have it shipped to you if you live outside of United States.
Update: sold for $5,300

When science bloggers publish, then blog about it ;-)

On Tuesday night, when I posted my personal picks from this week’s crop of articles published in PLoS ONE, I omitted (due to a technical glitch on the site), to point out that a blog-friend of mine John Logsdon published his first PLoS ONE paper on that day:

It’s a updated and detailed report on the ongoing work in my lab to generate and curate an “inventory” of genes involved in meiosis that are present across major eukaryotic lineages. This paper focuses on the protist, Trichomonas vaginalis, an organism not known to have a sexual phase in its life cycle.

Here is the paper (and check John’s post for his experiences publishing in PLoS ONE):
An Expanded Inventory of Conserved Meiotic Genes Provides Evidence for Sex in Trichomonas vaginalis:

Meiosis is a defining feature of eukaryotes but its phylogenetic distribution has not been broadly determined, especially among eukaryotic microorganisms (i.e. protists)–which represent the majority of eukaryotic ‘supergroups’. We surveyed genomes of animals, fungi, plants and protists for meiotic genes, focusing on the evolutionarily divergent parasitic protist Trichomonas vaginalis. We identified homologs of 29 components of the meiotic recombination machinery, as well as the synaptonemal and meiotic sister chromatid cohesion complexes. T. vaginalis has orthologs of 27 of 29 meiotic genes, including eight of nine genes that encode meiosis-specific proteins in model organisms. Although meiosis has not been observed in T. vaginalis, our findings suggest it is either currently sexual or a recent asexual, consistent with observed, albeit unusual, sexual cycles in their distant parabasalid relatives, the hypermastigotes. T. vaginalis may use meiotic gene homologs to mediate homologous recombination and genetic exchange. Overall, this expanded inventory of meiotic genes forms a useful “meiosis detection toolkit”. Our analyses indicate that these meiotic genes arose, or were already present, early in eukaryotic evolution; thus, the eukaryotic cenancestor contained most or all components of this set and was likely capable of performing meiotic recombination using near-universal meiotic machinery.

‘The Hairy Beast’ or ‘Super Virgin’?

Ha! Made you look! Which is exactly the point! Go and add your own ideas in the comments there….

A cellular riddle

It takes 38 minutes for the E.coli genome to replicate. Yet, E.coli can bo coaxed to divide in a much shorter time: 20 minutes. How is this possible?
Larry poses the riddle and provides the solution.
The key is that complex biochemical processes are taught sequentially, one by one, because that is how we think and process information. Yet, unless there is a need for precise timing (in which case there will be a timer triggering the starts and ends of cellular events), most processes occur all the time, simultaneously, in parallel. How do we teach that?

Microbial genomics in PLoS

Considering this I am kinda baffled by this. There is tons of microbial metagenomics and genomics in PLoS journals.