The prokaryotic Synechococcus sp. RF-1 exhibited a nitrogen fixation circadian rhythm with characteristics remarkably similar to the circadian rhythm of eukaryotes. The rhythm had a freerunning period of about 24 hours when the length of the preentrained cycle did not differ too much from 24 hours, and it was insensitive to changes in temperature from 220C to 330C. Because the endogenous rhythm of nitrogen fixation was not affected by a phase-shift of its previous cycles, the circadian rhythm in Synechococcus sp. RF-1 was not considered to be controlled simply by a feedback mechanism.medium (13)
In the prokaryote Synechococcus RF-1, circadian changes in the uptake of L-leucine and 2-amino isobutyric acid were observed. Uptake rates in the light period were higher than in the dark period for cultures entrained by 12/12 hour light/dark cycles. The periodic changes in L-leucine uptake persisted for at least 72 hours into continuous light (L/L). The rhythm had a freerunning period of about 24 hours in L/L at 290C. A single dark treatment of 12 hours could initiate rhythmic leucine uptake in an L/L culture. The phase of rhythm could be shifted by a pulse of low temperature (0°C). The free-running periodicity was "temperature-compensated" from 21 to 370C. A 24 hour depletion of extracellular Ca2+ before the free-running L/L condition reduced the variation in uptake rate but had little effect on the periodicity of the rhythm. The periodicity was also not affected by the introduction of 25 mm NaNO3. The uptake rates for 20 natural amino acids were studied at 12 hour intervals in cultures exposed to 12/12 hour light/dark cycles. For eight of these amino acids Circadian rhythms have been documented for organisms throughout the eukaryotic kingdoms (3). Circadian rhythmicity had not been detected in any prokaryote until recently, when endogenous circadian timing in nitrogen-fixation (5,8,12) and cell division (12,16) were reported in some strains of unicellular cyanobacteria belonging to the genus Svnechococcus.In eukaryotic organisms, endogenous circadian changes in the uptake rates of histidine and lysine have been observed in the yeast Saccharomyces cerevisiae (4).In this report, we investigated the uptake rates of natural amino acids in the prokaryotic Synechococcus RF-1 in an attempt to observe endogenous circadian variations. For comparison, we also investigated uptake rates of L-leucine in Svnechococcuis PCC 7942 and Synechocystis PCC 6803. MATERIALS AND METHODSSvnechococcus RF-1 (PCC 8801) was cultured as described previously (8 When cells of Synechococcus RF-1 were cultured under 12/ 12 h L/D cycles, the uptake rates of leucine (Fig. 1A) and non-metabolizable AIB (Fig. 2) fluctuated periodically and were several times higher during the light period than during the dark period. After the cultures were subsequently exposed to L/L, the periodic variations in leucine and AIB uptake persisted for at least 72 h (Fig. 1B) and 60 h ( Fig. 2A), respectively, without a noticeable change in periodicity. The average rhythm period under free-running conditions was about 24 h. The amino acid uptake rhythm was about 12 h out of phase with the nitrogen-fixation (5,8)
Sixteen nif and 'nif-associated genes (expressed only under conditions of nitrogen fixation) in Synechococcus sp. strain RF-1 have been cloned and sequenced. All of the nif and nif-associated genes identified in Synechococcus RF-1 were arranged in a continuous cluster spanning approximately 18 kb and containing seven operons. The nifH operon (nifH-nifD-nifK) has been reported previously. nifB, fdXN, nifS8 nifU and nifP were found to be located upstream of the nifH operon. nifB-WxN-nifS-nifU were expressed as an operon. A nifPlike gene was found to be located just upstream of nifB. nifE, nifN, nifX, nifW and the nif-associated hesA, hesB and 'fdx' were found to be located downstream from nifK. The genes located downstream from nifK are arranged nifE-nifN-nifX-orf-nifW-hesA-hesB-' fdx' and span approximately 7 kb. The function of the ORF situated between nifX and niNV is not known. However, it was identified as a counterpart of ORF-2 in Anabaena sp. strain PCC 7120 based on the deduced amino acid sequence. Northern hybridization and primer extension analysis indicated that the nif and nif-associated genes are organized in nifE-nifN, nifx-orf, niW-hesA-hesB and fdx'-containing operons, respectively. According to the results of this study and previous reports, the genes are expressed in a rhythmic pattern with peaks during the dark phase when the culture is grown in a 12 h IighVl2 h dark regimen. The rhythm persisted after the culture was transferred to continuous illumination.
Under continuous illumination this unicellular aerobic cyanobacterium fixes dinitrogen continuously at a variable and usually low rate. Exposure of the culture to diurnal light/dark cycles invariably results in the virtual restriction of nitrogenase activity to the dark periods. The rhythmic diurnal dinitrogen fixation pattern becomes a truely endogenous cycle which persists for at least 4 days with decreasing magnitude on exposing the culture to continuous illumination. The free running time of the rhythm appears to decrease from an initial 26 h to 22 h in the course of 4 days. This appears to be the first record of an endogenous rhythm in a prokaryote.
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