The very first entry in this blog, in December 2006, was about the amazing fact that some photosynthetic cyanobacteria have a circadian rhythm. I entitled it "It Don't Mean a Thing If It Ain't Got That Swing." Now, a group of German investigators report this is also true for an ordinary non-photosynthetic lab rat, Bacillus subtilis.
I was going to introduce the subject anew, when it occurred to me that my ancient text was still quite serviceable now, So, by your leave, I will copy parts of it here:
It’s not that many years ago that most people, me included, thought that bacteria lacked the ability to sense chemical gradients, communicate with one another, make fancy multicellular structures (with a few notable exceptions), and do some of the other things attributed to "higher" organisms. How things have changed! The number of complex processes that bacteria can do grows by the month, leading me to wonder at times if eukaryotes invented anything!…
To the list of phenomena that made this jump we can add circadian rhythms... It’s been known since about 1985 that such rhythms exist in some cyanobacteria. In these organisms, many processes exhibit daily fluctuations, including global gene expression, oxygen uptake, and carbohydrate synthesis. One gratifying reason for diurnal/nocturnal periodicity is that nitrogenase (the enzyme responsible for nitrogen fixation) is highly sensitive to the oxygen generated during photosynthesis. Unlike organisms, e.g. Anabaena, where photosynthesis and nitrogen fixation are carried out in different cells, unicellular cyanobacteria such as Synechococcus do both in the same cell. Here, the two processes are not separated in space but by time. During the day, they photosynthesize, at night they fix nitrogen.
Surprises abound. The genes involved in photosynthesis and nitrogen fixation are far from being the only ones showing circadian behavior. Most of the Synechococcus' promoters show such a rhythm, a global behavior that may be dictated by changes in DNA compaction (LINK). Note also that the cell division cycle in Synechococcus varies with light intensity and can be as short as about 6 hours, so there is nothing circadian about it (circadian, for those that need reminding, comes from the Latin for circa for about, and dies for day). Periodicity is not dependent on external stimuli such as light or dark periods, but that’s implied in the definition of circadian rhythms. In the words of Golden et al, "Unlike a metronome that merely taps out the beat, the circadian clock that times the events in a cell knows the score by heart." So, these organisms got that swing!
It gets better. The whole of the circadian rhythms has been attributed to a lowly number of proteins, four. SasA, a sensory kinase is the primary transducer, and three others are central timing mechanism. They are called Kai A, B, C ("kai" means cycle in Japanese). There is, of course more to it and for the whole cell physiology to behave cyclically, many other proteins are involved as well.
Just as you thought that it couldn’t get any more interesting, there comes a startling discovery: the three Kai proteins act rhythmically in vitro. Mix the three, add ATP, and you see phosphorylation of KaiC undergoing a 21 hour cycle at 30° (LINK). What is startling is that Kai proteins from mutants with altered periodicity show the same periodicity in vitro as the cells from which they are derived. And these are not just flimsy oscillations. They are totally convincing and maintained for at least four robust cycles. This finding is in keeping with the fact that the proteins do not have to be made anew and that the circadian rhythm does not depend on transcription and translation.
Now the big question is, what kind of biochemistry operates over such a long time? The Kai proteins interact with one another and one makes hexamers from monomers that have two potential phosphorylation sites. This may lead to a complex choreography of autophosphorylation and dephosphorylation, which may lead to such unusual kinetics. I find especially puzzling that the same kinetics is seen in vivo and in vitro, despite differences in concentration of the relevant proteins and in chemical and physical milieu. There may be exciting ways of explaining all this, but for now, to me it seems like a mystery.
Let me now return to the present. Cyanobacteria, being photosynthetic, are well equipped to sense light, but what about non-photosynthetic bacteria? A new report from the lab of Prof. Martha Merrow in Munich and others reveals that B. subtilis can also tell time. They constructed strains of this organism with the gene for the light-emitting enzyme luciferase fused to two genes. One, ytvA, codes for a blue light photoreceptor and the other one for an enzyme called KinC that is involved in biofilm formation and sporulation. Light emission correlated with the level of gene activity. They found that when the organisms were exposed to 12-hour cycles of light and dark for several days, levels of the ytvA promoter activity increased in the dark and decreased in the light. When these conditions were inverted, the pattern was reversed. This is in keeping with a circadian rhythm being present.
Strains whose rhythms had been synchronized with the environment were more fit; that is, when grown together under the conditions that impose periodicity, they outgrew the untrained ones. This fact may not seem surprising, but so far it has only been shown using bacterial systems (good for our side!). B. subtilis does not have the miraculous Kai proteins of Synechococcus, but they have something analogous, the so called PAS domains, which are structural motifs present in all eukaryotic circadian clocks.
It turns out that establishing that an organism has a circadian rhythm is not as simple as it may sound. It seems to be generally agreed that three criteria need to be met. First, under constant conditions, the oscillation should have a period close to 24 hours (but not exactly. Note the "circa" in circadian). Second, the rhythm must be "temperature compensated," that is, it maintains its rhythm over a range of temperatures. Third, the rhythm is "entrained" to an appropriate environmental cycle. When the timing of light and dark exposure is changed, so does the peaks in rhythms of gene expression. The authors conclude that these findings conclusively prove the existence of a circadian rhythm in this organism. They go on to say: "Our work opens the field of circadian clocks in the free-living, nonphotosynthetic prokaryotes, bringing considerable potential for impact upon biomedicine, ecology, and industrial processes." I agree that their work sheds light on novel but exciting aspects of bacterial physiology.
After writing this piece, it occurred to me to consult with a B. subtilis expert. None better than my old colleague and friend, A. L. (Linc) Sonenshein. This was his response:
Thinking about circadian rhythms in bacteria, it seems reasonable for them to accommodate to the variation in light and atmospheric temperature during the day. Since animals of virtually all types must be able to sleep at night and stay awake during the day, the bacteria that come in contact with them could have developed similar responses. B. subtilis does not infect humans or animals but just eating, growing, and surviving are its big issues. And since plants respond to light and temperature, bacteria that inhabit gardens and forests presumably have to "know" when is the best time to look for food. Was this ability developed by bacteria before eukaryotes arose? If so, the reason lies elsewhere.