We designed a strategy to isolate and characterize response regulator genes from the cyanobacteri... more We designed a strategy to isolate and characterize response regulator genes from the cyanobacterium Synechococcus sp. strain PCC 7942 based on the premise that cyanobacterial response regulators would bear strong similarity to their counterparts from other eubacteria. Two response regulator genes, srrA and srrB, were isolated from Synechococcus and found to encode proteins similar to the OmpR subclass of response regulators. Disruption of either gene by insertional mutagenesis did not produce an obvious phenotype and did not affect the accumulation of psbAII mRNA under high-light conditions, indicating that these gene products are not involved in mediating the well characterized standard- to high-light transition response of photosystem II genes in this cyanobacterium. Analysis of the chromosomal region adjacent to srrA revealed the presence of another presumptive transcriptional activator gene. This gene, named lrrA, belongs to the lysR family. Attempts to disrupt lrrA or an adjacent ORF (orfG) were not successful, suggesting that these genes are important for the growth of Synechococcus.
Both light itself and excitation pressure have been implicated as the environmental signal that s... more Both light itself and excitation pressure have been implicated as the environmental signal that stimulates interchange of the two forms of the D1 protein of photosystem II (PS II) in Synechococcus sp. strain PCC 7942. We sought an explanation for conflicting reports regarding the role of photosynthetic electron transport in regulation of psbA expression and D1 interchange. Inhibitors that block at different points in the photosynthetic electron transport chain were administered and the effect on expression of psbAII, which encodes the high-light-induced form II of D1, was examined by measuring changes in transcript levels and in the activities of reporter enzymes. Both 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of PSII, and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), an inhibitor of the cytochrome b6/f complex, prevented high-light-induced increase in β-galactosidase activity from a psbAII::lacZ gene fusion when added at a concentration that completely inhibits photosynthetic electron transport (1 μM). The same effect was observed for luciferase activity from transcriptional and translational fusions of psbAII to the luxAB genes from Vibrio harveyi. DCMU (1 μM) arrested luciferase expression at low-light levels – thus eliminating the high light response – whereas a sublethal concentration (50 nM), which reduces electron transport by 50%, had intermediate effects on psbAII-driven luciferase activity. However, psbAII transcript levels, monitored by northern blot analysis, were not altered by electron transport inhibitors, either at low-light intensity or following a high-light exposure. The suppressive effect of DCMU on expression of reporter enzymes was not restricted to the high-light response of psbAII-driven reporter systems, but was also observed using an isopropyl-(-d)-thiogalactopyranoside (IPTG)-inducible trc promoter fused to luxAB. This construct only marginally responded to IPTG addition when DCMU was present. Thus, blocking electron transport in Synechococcus affects the translation machinery in a general way, and the use of electron transport inhibitors is of limited value when focusing on specific redox regulation of D1 protein synthesis or degradation.
We designed a strategy to isolate and characterize response regulator genes from the cyanobacteri... more We designed a strategy to isolate and characterize response regulator genes from the cyanobacterium Synechococcus sp. strain PCC 7942 based on the premise that cyanobacterial response regulators would bear strong similarity to their counterparts from other eubacteria. Two response regulator genes, srrA and srrB, were isolated from Synechococcus and found to encode proteins similar to the OmpR subclass of response regulators. Disruption of either gene by insertional mutagenesis did not produce an obvious phenotype and did not affect the accumulation of psbAII mRNA under high-light conditions, indicating that these gene products are not involved in mediating the well characterized standard- to high-light transition response of photosystem II genes in this cyanobacterium. Analysis of the chromosomal region adjacent to srrA revealed the presence of another presumptive transcriptional activator gene. This gene, named lrrA, belongs to the lysR family. Attempts to disrupt lrrA or an adjacent ORF (orfG) were not successful, suggesting that these genes are important for the growth of Synechococcus.
Both light itself and excitation pressure have been implicated as the environmental signal that s... more Both light itself and excitation pressure have been implicated as the environmental signal that stimulates interchange of the two forms of the D1 protein of photosystem II (PS II) in Synechococcus sp. strain PCC 7942. We sought an explanation for conflicting reports regarding the role of photosynthetic electron transport in regulation of psbA expression and D1 interchange. Inhibitors that block at different points in the photosynthetic electron transport chain were administered and the effect on expression of psbAII, which encodes the high-light-induced form II of D1, was examined by measuring changes in transcript levels and in the activities of reporter enzymes. Both 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of PSII, and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), an inhibitor of the cytochrome b6/f complex, prevented high-light-induced increase in β-galactosidase activity from a psbAII::lacZ gene fusion when added at a concentration that completely inhibits photosynthetic electron transport (1 μM). The same effect was observed for luciferase activity from transcriptional and translational fusions of psbAII to the luxAB genes from Vibrio harveyi. DCMU (1 μM) arrested luciferase expression at low-light levels – thus eliminating the high light response – whereas a sublethal concentration (50 nM), which reduces electron transport by 50%, had intermediate effects on psbAII-driven luciferase activity. However, psbAII transcript levels, monitored by northern blot analysis, were not altered by electron transport inhibitors, either at low-light intensity or following a high-light exposure. The suppressive effect of DCMU on expression of reporter enzymes was not restricted to the high-light response of psbAII-driven reporter systems, but was also observed using an isopropyl-(-d)-thiogalactopyranoside (IPTG)-inducible trc promoter fused to luxAB. This construct only marginally responded to IPTG addition when DCMU was present. Thus, blocking electron transport in Synechococcus affects the translation machinery in a general way, and the use of electron transport inhibitors is of limited value when focusing on specific redox regulation of D1 protein synthesis or degradation.
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