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.     

Dinitrogen-fixing endogenous rhythm in Synechococcus RF-1

Abstract Using the Southern hybridization technique, 22 strains of archaebacteria were tested for the presence of DNA sequences homologous to the Anabaena nifH gene, which codes for the Fe protein component of nitrogenase. Total DNA from all the 14 methanogenic strains tested was shown to carry homology to nifH . With total DNA from 3 Halobacterium strains, hybridization was also detected, though unspecific binding cannot completely be excluded. No hybridization was detected with DNA from Thermoplasma acidophilum , nor with the DNA from 4 strains belonging to the sulfur-dependent branch of the archaebacteria. Results strongly support the hypothesis of an ancient origin of nif genes.     

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.     

Calcium requirement in nitrogen fixation in the cyanobacterium Synechococcus RF-1

Under diurnal 16/8-h light-dark cycles, ethyleneglycol-bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA) at 1 mM completely blocked the appearance of rhythmic N₂-fixing activity in Synechococcus RF-1. Ca²⁺ at 2 mM, when supplied either together with or several hours after the EGTA application, restored the nitrogenase activity, whereas, when Ca²⁺ was supplied several hours later, the peak of nitrogenase activity was shifted from the dark to the light period in which the activity is normally suppressed. Sr²⁺ also reversed the inhibition by EGTA, but only partially. When O₂ in the gas phase above the culture was below 1%, the inhibition of nitrogenase activity by EGTA was reduced to less than 20% of the control value without EGTA. Thus Ca²⁺ appears to be required by the cell to protect its nitrogenase from inactivation by O₂. In media without EGTA, a close correlation between nitrogenase activity and concentrations of Ca²⁺ was also observed.Abbreviation EGTA ethyleneglycol-bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid     

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.     

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.     

Purification of RuBisCO from the thermophilic cyanobacterium Synechococcus sp. strain a-1

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), the key enzyme of the Calvin Benson cycle, has been purified from a thermophilic cyanobacterium, Synechococcus sp. strain a-1 and characterized. The enzyme is an L₈S₈-type hexadecamer with a molecular mass of 530 kDa. The enzyme was stable against heat treatment up to 70°C, which is the highest value among the RuBisCOs so far purified. The K_m value for ribulose bisphosphate on the carboxylase activity was substantially higher than those observed for RuBisCOs obtained from mesophilic autotrophs. The N-terminal amino acid sequence for the large subunit of the enzyme was highly similar to those of the other cyanobacteria despite the significant differences in heat stability.     

Calcium carbonate formation by Synechococcus sp. strain PCC 8806 and Synechococcus sp. strain PCC 8807

Precipitation of CaCO3 catalyzed by the growth and physiology of cyanobacteria in the genus Synechococcus represents a potential mechanism for sequestration of atmospheric CO2 produced during the burning of coal for power generation. Synechococcus sp. strain PCC 8806 and Synechococcus sp. strain PCC 8807 were tested in microcosm experiments for their ability to calcify when exposed to a fixed calcium concentration of 3.4 mM and dissolved inorganic carbon concentrations of 0.5, 1.25 and 2.5 mM. Synechococcus sp. strain PCC 8806 removed calcium continuously over the duration of the experiment producing approximately 18.6 mg of solid phase calcium. Calcium removal occurred over a two-day time period when Synechococcus sp. strain PCC 8807 was tested and only 8.9 mg of solid phase calcium was produced. Creation of an alkaline growth environment catalyzed by the physiology of the cyanobacteria appeared to be the primary factor responsible for CaCO3 precipitation in these experiments.     

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.