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    • br Materials and methods br Results and discussion

    2021-12-09


    Materials and methods
    Results and discussion In order to identify genes required for photoautotrophic growth, we screened a random insertion mutant library of Synechocystis 6803 and obtained many mutants with impaired photoautotrophic growth. About 6000 random insertion colonies of the mutant library were maintained in BG11 with glucose using 96-well or Petri-dish plates. The mutants were tested in BG11 without glucose for axl inhibitor growth capability. Mutants showing no or little growth under photoautotrophic conditions were further screened. Interrupted genes in confirmed mutants, including slr0906 (psbB) and slr2094 (fbp1), were identified using inverse PCR and sequencing. slr2094 encodes the fructose-1,6-/sedoheptulose-1,7-bisphosphatase bifunctional enzyme (F-I) [6]. In mutant 691-72, C.K2 was inserted at the HaeIII site 763bp away from the 5′ end of slr2094 (Fig. 1). To exclude the possibility that the phenotype of mutant 691-72 might be caused by a second mutation, we independently constructed another slr2094 mutant by targeted insertion. Synechocystis 6803 was transformed with pHB1485, in which the cloned slr2094 was inserted at the KpnI site by C.K2, resulting in mutant DRHB1485 (Fig. 1). The complete segregation of the mutant was confirmed by PCR using primers slr2094-1 and slr2094-2 (Fig. 1). We assayed the fructose-1,6-bisphosphatase activity in the wild-type strain and the mutant DRHB1485. The mutant showed little fructose-1,6-bisphosphatase activity (1.33±0.29nm NADPH mgprotein−1min−1), relative to the wild-type (62.27±1.47nm NADPH mgprotein−1min−1). The targeted insertion mutant DRHB1485 showed a similar phenotype to the mutant 691-72. Under photoautotrophic conditions, DRHB1485 grew at 0.04±0.01 doublings day−1, while the wild-type at 0.89±0.06 doublings day−1. Under photoheterotrophic conditions, the mutant and the wild-type grew at similar rates (1.03±0.01 and 1.11±0.01 doublings day−1, respectively). We also interrupted slr2094 with C.CE2 at the KpnI site by transforming Synechocystis 6803 with pHB1486. The resulted mutant DRHB1486 showed the same phenotype as 691-72 and DRHB1485 (data not shown). Because three independent mutants showed an identical phenotype, we conclude that the impaired phototrophic growth must be attributed to the null mutation of slr2094. The two genes slr2095 and slr2096 downstream of slr2094, both encoding putative transposases, are unlikely required for the photoautotrophic growth. Polar effects could be excluded. The products of reactions catalyzed by fructose-1,6-bisphosphatase or sedoheptulose-1,7-bisphosphatase in the Calvin cycle are precursors of many metabolites. Consequently, the activities of these two enzymes affect the partitioning of carbon sources among end products. Miyagawa et al. overexpressed the FBPase/SBPase gene fbpI from Synechococcus PCC 7942 in tobacco chloroplasts [18]. Compared with the wild-type, the transgenic plants showed higher photosynthetic CO2 fixation, accelerated growth rate, increased levels of intermediates of the Calvin cycle and carbohydrates. The results reflected the role of FBPase/SBPase in the regulation of carbon flux in plant cells. Consistent with reports concerning higher plants, our results directly showed the role of FBPase/SBPase in photosynthesis in a cyanobacterium: inactivation of slr2094 led to loss of photoautotrophic growth. Previously, it was proposed that F-I rather than F-II was expressed in Synechocystis 6803 [6]. We found that a very low or virtually no activity of FBPase remained in the F-I mutant DRHB1485. This finding indicated that the gene encoding F-II was silenced no matter with or without active F-I in Synechocystis 6803. In higher plants, the expression of FBPase could be regulated by light [19], [20]. In Synechocystis 6803, cells grown under light-activated heterotrophic growth conditions showed greatly reduced FBPase activity [6]. We assayed the FBPase activity of the wild-type strain grown mixotrophically under different light intensities and found that the activity remained basically unchanged in the range of 2–150μEm−2s−1 (Fig. 2). Therefore, the FBPase activity is not regulated by light in the cyanobacterium.