We have used a luciferase reporter gene and continuous automated monitoring of bioluminescence to demonstrate unequivocally that cyanobacteria exhibit circadian behaviors that are fundamentally the same as circadian rhythms of eukaryotes. We also show that these rhythms can be studied by molecular methods in Synechococcus sp. PCC7942, a strain for which genetic transformation is well established. A promoterless segment of the Vibrio harveyi luciferase structural genes (luxAB) was introduced downstream of the promoter for the Synechococcus psbAI gene, which encodes a photosystem H protein. This reporter construction was recombined into the Synechococcus chromosome, and bioluminescence was monitored under conditions of constant illumination following entrainment to light and dark cycles. The reporter strain, AMC149, expressed a rhythm of bioluminescence which satisfies the criteria of circadian rhythms: persistence in constant conditions, phase resetting by light/dark signals, and temperature compensation of the period. Rhythmic changes in levels of the native psbAl message following light/dark entrainment supported the reporter data.The behavior of this prokaryote disproves the dogma that circadian mechanisms must be based on eukaryotic cellular organization. Moreover, the cyanobacterial strain described here provides an efficient experimental system for molecular analysis of the circadian clock.Despite decades of study, the biochemical mechanism of circadian clocks remains a mystery. Circadian rhythms have been found in a wide spectrum of organisms (1) but, until recently, only in eukaryotes (2, 3). In the last few years circadian rhythms have been reported in the prokaryotic cyanobacteria (4-7). Unfortunately, these studies employed genetically intractable cyanobacterial strains and laborious assays to detect the rhythms. These difficulties have impeded the demonstration that the prokaryotic rhythms are equivalent to the circadian rhythms of eukaryotes.Proof that prokaryotes have circadian pacemakers has threefold significance. (i) With regard to the evolutionary emergence of circadian behavior: Can the simpler organization of prokaryotes support a circadian mechanism? Is circadian behavior adaptive for prokaryotic niches as well as for eukaryotic niches? (ii) The previous failure of attempts to discover circadian clocks in prokaryotes has led to a "eukaryotes-only" dogma which limited the types ofmodels that have been considered for the underlying clock mechanism (3). Now that prokaryotic cellular organization appears to be fully competent to generate circadian oscillations, a broader range of mechanisms can be seriously evaluated as candidates for the circadian pacemaker. (iii) The realization thatThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.prokaryotes express circadian behavior is significant from the perspective of designing an optimal s...

Eight stations in the main body of Chesapeake Bay and one on the continental shelf were sampled seven times over a period of 13 months to investigate the nitrogenous nutrition of the phytoplankton.The rates at which the phytoplankton were utilizing NO,, NO,, NHr+, and urea N were determined.The data demonstrate that for a large portion of the year there is inadequate N nutrient available to permit a single doubling of the particulate N. Over temperatures from 4" -28°C and salinities from 2-32&, there was a universally high phytoplankton preference for NH4+ and urea N over NO,-and NO,.A relative preference index indicated that NHa' concentrations in excess of O-5-1.0 pg-atom N liter-l almost totally suppressed NO, utilization.Urea N was used after NH,+ in order of prcfcrence, and when the sum of available NH4+ and urea N was insufficient to meet the phytoplankton N nutrient demand, NO; was used. When the sum of all available N nutrients was less than that required to satiate the phytoplankton demand, NIL', urea N, NO,-, and NO,-were all utilized at rates proportional to their availability. For the midbay region in October 1973, NO, was the dominant N nutrient present both in the water and in the diet of the phytoplankton.

The spring freshet increases density stratification in Chesapeake Bay and minimizes oxygen tr111nsfer from the surface to the deep layer so that waters below 10m depth experience oxygen depletion which may lead to anoxia during June to September. Respiration in the water of the deep layer is the major factor contributing to oxygen depletion. Benthic respiration seems secondary. Organic matter from the previous year which bas settled into the deep layer during winter provides moot of the oxygen demand but some new production in the surface layer may sink and thus supplement the organic matter accumulated in the deep layer.

The biochemistry and circadian regulation of luminescence in two Pyrocystis species, P. lunula Hulburt and P. noctiluca Murray et Haeckel, were compared with a wellstudied species, Gonyaulax polyedra Stein. All exhibit circadian rhythms and all have similar luciferins and luciferases. However, the Pyrocystis species lack a second protein involved in the reaction in Gonyaulax, the luciferin (substrate) binding protein, which sequesters the luciferin at the cytoplasmic pH and releases it upon acidification, thus controlling the characteristic flashing, which is similar in the three species. More striking is the difference in the circadian regulation of luminescence, which in Gonyaulax involves the daily synthesis and destruction of the two proteins, along with the luminous organelles (scintillons) from which light is emitted, and which are present in all species. In the Pyrocystis species, the amount of luciferase is the same in extracts made during the day and night phases; its circadian regulation in vivo may be attributed to a change in its localization from day to night phase.

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Phosphomonoester concentrations were 0 to 0.09 E.cg-atom liter-l in Chesapeake Bay from December 1972 to December 1973. Alkaline phosphatase activity associated with natural phytoplankton assemblages indicated the cells' potential to utilize the monoesters as a phosphorus source. However, ecological interpretation of alkaline phosphatase activity data is complicated by the necessity to increase the monoester concentration in order to measure enzyme activity fluorometrically.The half-saturation constant ( K, ) was 0.31 for 3-0-methyl fluorescein hydrolysis by a natural phytoplankton assemblage and 0.75 PM for glucose-6-POp by a nanoplankter in culture, and maximum velocities (V,) were 3.2 and 6.4 nm (pg Chl a h)".In one experiment with a natural phytoplankton assemblage, organisms in the 0.8-5-pm size range comprised 78% of the plant biomass and were responsible for 70% of the phosphorus uptake from glucose-6-POs when size fractionation preceded experimental incubations. Phosphomonocsters may contribute to phytoplankton phosphorus nutrition during much of the year, but are in greatest demand in spring in Chesapeake Bay.

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In the unicellular algae Pyrocystis lunula Schütt and Gonyaulax polyedra Stein, bioluminescence and its circadian regulation are similar in several respects, but there are also several important differences. As in G. polyedra, P. lunula emits light both as bright flashes and as a low intensity glow. At 20° C, the individual flashes are considerably brighter than in G. polyedra, and their durations are typically less than 500 ms. Both species show a circadian rhythm in the frequency of spontaneous flashes, which peaks in the night‐phase under light–dark cycles and continues in both continuous light and dark. However, compared to G. polyedra, the circadian system in P. lunula is more sensitive to light: 10 min exposures (500 μmol · m–2· s–1 white light) can shift the phase of the rhythm by more than 8 h, and rhythmicity is completely suppressed at an irradiance above 20 μmol · m–2· s–1, where the G. polyedra rhythym persists for weeks. Like G. polyedra, period length increases with increasing irradiance of continuous red light but decreases with increasing intensity of continuous blue light. The glow in P. lunula differs markedly from that in G. polyedra in that it occurs at about the same intensity at all times during the circadian cycle; thus, it is not under circadian control but may fluctuate 5–10‐fold in intensity within a time frame of seconds. This suggests that the glow may differ in its physiological basis in the two organisms. The results also indicate that the circadian regulation of luciferase activity differs in the two species. In G. polyedra, the organelle responsible for bioluminescence and luciferase is lost and then reformed on a daily basis; in P. lunula, the luciferase is conserved and localized elsewhere during the nonbioluminescent phase of the cycle.