Non-Photosynthetic Bacteria Also Have a Circadian Rhythm

by Elio
 

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.
 

Fig. 1 Entrainment by light and a free-running rhythm in B. subtilis. Bioluminescence of P_ytvA::lux (A) and P_kinC::lux (D)under 5 days of entrainment with cycles of darkness and blue light (12-hour D/12-hour L) and after release to constant darkness conditions [DD; (B) and (E) for 5 days. The temperature was kept constant at 25.5°C. The detrended data are presented as means ± SD. The shading in (A) and (D) shows the timing of the LD cycle (yellow, light phase; gray, dark phase) relative to the bioluminescence. The horizontal bar in (B) and (E) (lower left) shows the time window of 48 hours selected for the analysis of period length. The calculated period length is plotted in (C) and (F); individual data points [(C), N = 16; (F), N = 7] are shown along with the median and interquartile range. Source. Frontispiece: Circadian rhythms. Source.

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.