Circadian Rhythm in Amino Acid Uptake by Synechococcus RF-1

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 free-running period of about 24 hours in L/L at 29°C. 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 37°C. 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 (l-Val, l-Leu, l-Ile, l-Pro, l-Phe, l-Trp, l-Met, and l-Tyr), the light/dark uptake rate ratios had values greater than 3 and the rhythm persisted in L/L.     

Setting of the Circadian N(2)-Fixing Rhythm of the Prokaryotic Synechococcus sp. RF-1 while Its nif Gene Is Repressed

The N₂-fixing activity of the prokaryotic Synechococcus sp. RF-1 was repressed in the presence of nitrate. When the cultures in nitrate-containing medium were exposed to diurnal light-dark cycles, an endogenous circadian N₂-fixing rhythm developed after the cells were transferred to nitrate-free medium and incubated in continuous light. The N₂-fixing phase of the rhythm coincided with the dark phase of the light-dark cycles that were imposed when the cells were in nitrate-containing medium. The results indicate that after the endogenous N₂-fixing rhythm has been set, it can be kept latent for at least 38 hours before first manifesting itself.     

Circadian rhythm mutants of the prokaryoticSynechococcus RF-1

Synechococcus RF-1 established circadian rhythms in nitrogen fixation and leucine uptake when growing in a diurnal light/dark regimen. The rhythms persisted in subsequent uniform light/light conditions. In order to analyze the circadian rhythm at the genetic level, mutants were induced by N-methyl-N-nitro-N-nitrosoguanidine and then isolated by procedures with the circadian nitrogen-fixing rhythm as a selecton marker. Characterization of the mutants with respect to the circadian rhythm indicated that some mutants were abnormal only in the nitrogen-fixing rhythm, while some simultaneously lost the ability to establish the nitrogen-fixing and leucine-uptake rhythms. The physiological properties of the circadian rhythm were compared. The genetic potential of the mutants that were abnormal in both rhythms is emphasized.     

Circadian Rhythm in the Locomotor Activity of a Surface-Dwelling Millipede Syngalobolus sp.

Locomotor activity of the surface-dwelling millipede Syngalobolus sp. was recorded under laboratory conditions. Infra-red diodes were used to detect the locomotor activity in an oval shaped chamber, which was connected with an event recorder. The results of 11 individuals showed that the millipedes entrained to light/dark (LD12:12 h) conditions with negative phase angle difference (-83.2 ± 24.72 min). The millipedes showed a clear-cut free-running rhythm with a period (t) of 23.8 ± 1.0 h (n = 9) in constant darkness (DD). The period in continuous light (LL) was relatively greater (25.2 ± 0.1 h; n = 3) than that in DD.     

Relationship between the nitrogenase activity and dark respiration rate of Synechococcus RF-1

Growth of three different anaerobic rumen fungi Neocallimastix frontalis, Piromonas communis and Sphaeromonas communis was assessed in vitro at regular intervals by measurements of protein and chitin content and of chitin synthase activity of the cell free extracts. Similar trends and a comparable amount of protein and chitin were observed in the three species. However, chitin synthase activity was higher in S. communis and contrary to the activity of the other two strains did not decrease after maximum enzyme activity was reached. There were positive correlations between chitin content, protein content and chitin synthase activity during the active growth phase of the fungi indicating that they could be confidently used to determine in vitro growth phase and biomass concentration.     

Circadian Rhythm in the Locomotor Activity of a Surface-Dwelling Millipede Syngalobolus sp.

Locomotor activity of the surface-dwelling millipede Syngalobolus sp. was recorded under laboratory conditions. Infra-red diodes were used to detect the locomotor activity in an oval shaped chamber, which was connected with an event recorder. The results of 11 individuals showed that the millipedes entrained to light/dark (LD12:12 h) conditions with negative phase angle difference (–83.2 ± 24.72 min). The millipedes showed a clear-cut free-running rhythm with a period (t) of 23.8 ± 1.0 h (n = 9) in constant darkness (DD). The period in continuous light (LL) was relatively greater (25.2 ± 0.1 h; n = 3) than that in DD.     

Priority of light/dark entrainment over temperature in setting the circadian rhythms of the prokaryote Synechococcus RF-1

Light/dark (L/D) and temperature are two major factors in the entrainment of circadian rhythms. The input pathways of these two environmental factors for the entrainment of circadian rhythms in Synechococcus RF-1 are different since the overt rhythms in mutant CR-1, one of the circadian-rhythm mutants of Synechococcus RF-1, could be established by temperature cycles but not by L/D. Therefore, it was of interest to investigate the phases of Synechococcus RF-1 cells entrained simultaneously by L/D and temperature. The circadian rhythms of nitrogenase activity and protein synthesis in RF-1 cells entrained by L/D, and by lowered or raised temperatures differed in their peaks of activity. Comparison of the phases of RF-1 cells entrained by L/D and temperature independently, and by L/D and temperature simultaneously indicated that L/D entrainment has priority over the temperature effect. Received: 8 February 1999 / Accepted: 1 April 1999     

Cloning of the cpcE and cpcF genes from Synechococcus sp. PCC 6301 and their inactivation in Synechococcus sp. PCC 7942

Two open reading frames denoted as cpcE and cpcF were cloned and sequenced from Synechococcus sp. PCC 6301. The cpcE and cpcF genes are located downstream of the cpcB2A2 gene cluster in the phycobilisome rod operon and can be transcribed independently of the upstream cpcB2A2 gene cluster. The cpcE and cpcF genes were separately inactivated by insertion of a kanamycin resistance cassette in Synechococcus sp. PCC 7942 to generate mutants R2EKM and R2FKM, respectively, both of which display a substantial reduction in spectroscopically detectable phycocyanin. The levels of - and -phycocyanin polypeptides were reduced in the R2EKM and R2FKM mutants although the phycocyanin and linker genes are transcribed at normal levels in the mutants as in the wild type indicating the requirement of the functional cpcE and cpcF genes for normal accumulation of phycocyanin. Two biliprotein fractions were isolated on sucrose density gradient from the R2EKM/R2FKM mutants. The faster sedimenting fraction consisted of intact phycobilisomes. The slower sedimenting biliprotein fraction was found to lack phycocyanin polypeptides, thus no free phycocyanin was detected in the mutants. Characterization of the phycocyanin from the mutants revealed that it was chromophorylated, had a _max similar to that from the wild type and could be assembled into the phycobilisome rods. Thus, although phycocyanin levels are reduced in the R2EKM and R2FKM mutants, the remaining phycocyanin seems to be chromophorylated and similar to that in the wild type with respect to phycobilisome rod assembly and energy transfer to the core.     

Cloning of the phycobilisome rod linker genes from the cyanobacterium Synechococcus sp. PCC 6301 and their inactivation in Synechococcus sp. PCC 7942

Coexpression of pairs of nonhaemolytic H1yA mutants in the recombination-deficient (recA) strain Escherichia coli HB101 resulted in a partial reconstitution of haemolytic activity, indicating that the mutation in one H1yA molecule can be complemented by the corresponding wild-type sequence in the other mutant HlyA molecule and vice versa. This suggests that two or more HlyA molecules aggregate prior to pore formation. Partial reconstitution of the haemolytic activity was obtained by the combined expression of a nonhaemolytic HlyA derivative containing a deletion of five repeat units in the repeat domain and several nonhaemolytic HlyA mutants affected in the pore-forming hydrophobic region. The simultaneous expression of two inactive mutant HlyA proteins affected in the region at which HlyA is covalently modified by HlyC and the repeat domain, respectively, resulted in a haemolytic phenotype on blood agar plates comparable to that of wild-type haemolysin. However, complementation was not possible between pairs of HlyA molecules containing site-directed mutations in the hydrophobic region and the modification region, respectively. In addition, no complementation was observed between HlyA mutants with specific mutations at different sites of the same functional domain, i.e. within the hydrophobic region, the modification region or the repeat domain. The aggregation of the HlyA molecules appears to take place after secretion, since no extracellular haemolytic activity was detected when a truncated but active HlyA lacking the C-terminal secretion sequence was expressed together with a non-haemolytic but transport-competent HlyA mutant containing a deletion in the repeat domain.     

Nucleotide Sequence of Plasmid pAQ1 of Marine Cyanobacterium Synechococcus sp. PCC7002

We have determined the complete nucleotide sequence of pAQ1,the smallest plasmid of the unicellular marine cyanobacteriumSynechococcus sp. PCC7002. The plasmid consists of 4,809 bpand has at least four open reading frames that potentially encodepolypeptides of 50 or more amino acids. We found that a palindromicelement, the core sequence of which is G(G/A)CGATCGCC, is over-representednot only in plasmid pAQ1 but also in the accumulated cyanobacterialgenomic sequences from Synechococcus sp. PCC6301, PCC7002, PCC7942,vulcanus and Synechocystis sp. PCC6803 within GenBank and EMBLdatabases. It suggests that this sequence might mediate generearrangement, thus increasing genetic diversity, since recombinationevents are frequent in cyanobacteria.     

Cyanobacterial phycobilisomes. Characterization of the phycobilisomes of Synechococcus sp. 6301.

A procedure is described for the preparation of stable phycobilisomes from the unicellular cyanobacterium Synechococcus sp. 6301 (also known as Anacystis nidulans). Excitation of the phycocyanin in these particles at 580 nm leads to maximum fluorescence emission, from allophycocyanin and allophycocyanin B, at 673 nm. Electron microscopy shows that the phycobilisomes are clusters of rods. The rods are made up of stacks of discs which exhibit the dimensions of short stacks made up primarily of phycocyanin (Eiserling, F. A., and Glazer, A. N. (1974) J. Ultrastruct. Res. 47, 16-25). Loss of the clusters, by dissociation into rods under suitable conditions, is associated with loss of energy transfer as shown by a shift in fluorescence emission maximum to 652 nm. Synechococcus sp. 6301 phycobilisomes were shown to contain five nonpigmented polypeptides in addition to the colored subunits (which carry the covalently bound tetrapyrrole prosthetic groups) of the phycobiliproteins. Evidence is presented to demonstrate that these colorless polypeptides are genuine components of the phycobilisome. The nonpigmented polypeptides represent approximately 12% of the protein of the phycobilisomes; phycocyanin, approximately 75%, and allophycocyanin, approximately 12%. Spectroscopic studies that phycocyanin is in the hexamer form, (alpha beta)6, in intact phycobilisomes, and that the circular dichroism and absorbance of this aggregate are little affected by incorporation into the phycobilisome structure.     

Circadian Rhythm of Irinotecan Tolerability in Mice

The toxicity of irinotecan (CPT-11), a topoisomerase-I inhibitor largely used in cancer patients, was investigated as a function of the circadian time of its administration in mice, with mortality, body weight loss, leukopenia, neutropenia, intestinal lesions, and bone marrow cell cycle phase distribution as end points. Four experiments were performed on a total of 773 male mice standardized with 12 h light/12 h darkness. Irinotecan was administered daily for 4 or 10 consecutive days (D1-4 and D1-10, respectively, in different experiments) at one of six circadian stages expressed in hours after light onset (HALO). The survival curves differed significantly as a function of the dosage and circadian time of drug administration by the D1-10 schedule, with 70% survival at 7 or 11 HALO and 51% at 19 or 23 HALO ( p = 0.039 from log rank test). CPT-11 administration at 19 or 23 HALO resulted in (1) greatest mean body weight loss at nadir; (2) most severe colic and bone marrow lesions and/or slowest recovery; and (3) deepest neutropenia nadir and/or slowest hematologic recovery. These circadian treatment time-related differences were statistically validated. The bone marrow cell cycle data revealed a four to eight-fold larger G2-M phase arrest following irinotecan administration at 19 or 23 HALO in comparison to the other times of drug administration, apparently representative of the repair of more extensive DNA damage ( p < 0.001 from ANOVA) when the medication was given at these circadian times. Overall, CPT-11 was better tolerated by mice treated during the light (animals’ rest) span. The results support the administration of CPT-11 to cancer patients in the second half of the night, during sleep, in order to improve drug tolerability.     

Functional subunit structure of photosystem 1 reaction center in Synechococcus sp

Photochemical activities of six different P700-chlorophyll a-proteins (CP1-a, -b₁, -b₂, -c, -d, and -e) separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis from digitonin particles of a thermophilic cyanobacterium Synechococcus sp. were examined. CP1-a, -b₁, -b₂, and -c contain the competent reaction center of photosystem 1: They were highly active in photooxidation of cytochrome c-553, the physiological electron donor to P700 in the organism, with methyl viologen as electron acceptor and showed flash-induced absorption changes indicating the charge separation between P700 and the secondary electron acceptors, P430 and A₂. The cytochrome photooxidation and P430 and A₂ photoresponses were significantly suppressed in CP1-d. CP1-e which lacks P430 and A₂ was least active in the cytochrome photooxidation. A₁, the primary electron acceptor of P700, is present in CP1-e as well as in other CP1 complexes. Comparison of the results with the polypeptide composition of CP1 complexes (Y. Takahashi, H. Koike, and S. Katoh, 1982, Arch. Biochem. Biophys.219, 209–218). indicates that CP1-c which contains four polypeptides with molecular weights of 62,000, 60,000, 14,000, and 10,000 represents the functional core of the photosystem 1 reaction center. P700, A₁, and antenna chlorophyll are associated with 62,000- and 60,000-dalton polypeptides, whereas 14,000- and 10,000-dalton polypeptides are assumed to carry P430 and A₂. The 13,000-dalton polypeptide which is associated with CP1-a, -b₁, and -b₂ is not required for the functioning of the reaction center.