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     

Unique peptide identification of RNase A superfamily sequences based on reinforced merging algorithms

Human ribonuclease A (RNaseA) superfamily consists of eight RNases with high similarity in which RNase2 and RNase3 share 76.7% identity. The evolutionary variation of RNases results in differential structures and functions of the enzymes. To distinguish the characteristics of each RNase, we developed reinforced merging algorithms (RMA) to rapidly identify the unique peptide motifs for each member of the highly conserved human RNaseA superfamily. Many motifs in RNase3 identified by RMA correlated well with the antigenic regions predicted by DNAStar. Two unique peptide motifs were experimentally confirmed to contain epitopes for monoclonal antibodies (mAbs) specifically against RNase3. Further analysis of homologous RNases in different species revealed that the unique peptide motifs were located at the correspondent positions, and one of these motifs indeed matched the epitope for a specific anti-bovine pancreatic RNaseA (bpRNaseA) antibody. Our method provides a useful tool for identification of unique peptide motifs for further experimental design. The RMA system is available and free for academic use at http://bioinfo.life.nthu.edu.tw/rma/ and http://spider.cs.ntou.edu.tw/bioinformatics/RMA.html.     

Induction of a nitrogenase activity rhythm inSynechococcus and the protection of its nitrogenase against photosynthetic oxygen

All oxygen levels are detrimental to the nitrogenase activity ofSynechococcus RF-1 cells. In continuous light, cultures maintain a high dissolved oxygen concentration and a continuous but usually low rate of nitrogenase activity.Cultures adapted to a light-dark regimen will reduce acetylene almost exclusively during the dark periods. When switched to continuous light, they continue to exhibit a diurnal rhythm in nitrogenase activity. While in continuous light, each upsurge of nitrogenase activity coincides with a marked drop in the net oxygen production rate; this drop is due largely to a concomitant increase in the dark respiration rate of the culture.The endogenous nitrogenase activity rhythm can be induced in continuous light by periodically lowering the oxygen concentration of the culture by either bubbling nitrogen through it or by treating the culture with 3(3,4-dichlorophenol)-1,1-dimethylurea (DCMU or diuron).     

The aerobic nitrogen-fixingSynechococcus RF-1 containing uncommon polyglucan granules and multiple forms of α-amylase

The aerobic nitrogen-fixing unicellular cyanobacteriumSynechococcus RF-1 (pcc 8801) contains numerous irregular polyglucan granules ranging in size from 0.1 to 0.4 m. These morphologically unusual polyglucan granules are not found in the sheathed nitrogen-fixing unicellular cyanobacteriumGloeothece RF-6 (pcc 8803). Two forms of -amylase, ethyleneglycolbis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA)-sensitive and EGTA-insensitive, were found in theSynechococcus RF-1. In addition to their EGTA sensitivity, the two forms of -amylase also differed in their reactive pH range and in their zymogram disc gel electrophoresis. Each form of -amylase was stable and constant in concentration through a diurnal light/dark cycle.     

Calcium is required for the increase of dark respiration during diurnal nitrogen fixation by Synechococcus RF-1

Under 12/12 h light/dark cycles, 1 mM ethyleneglycol-bis-(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA, pH 8.0) added at the start of the dark period, inhibited the increase of dark respiration which was associated with nitrogen fixation in Synechococcus RF-1. Twenty-five millimolar NaNO₃ added 30 min before the start of dark period suppressed this respiratory increase. If 1.25 mM CaCl₂ was added to the EGTA-treated sample from 3 to at least 10 h later in the dark period, a quick rise in respiratory rate was observed. This rise was also reduced by 25 mM NaNO₃. Extracellular Ca²⁺ appears to be required for the increase in dark respiration associated with the rhythmic appearance of nitrogenase activity in the dark cycle.     

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