Over-production of the D1 protein of photosystem II reaction centre in the cyanobacterium Synechococcus sp. PCC 7942

Abstract

The unicellular cyanobacterium Synechococcus sp. PCC 7942 has three psbA genes encoding two different forms of the photosystem II reaction centre protein D1 (D1:1 and D1:2). The level of expression of these psbA genes and the synthesis of D1:1 and D1:2 are strongly regulated under varying light conditions. In order to better understand the regulatory mechanisms underlying these processes, we have constructed a strain of Synechococcus sp. PCC 7942 capable of over-producing psbA mRNA and D1 protein. In this study, we describe the over-expression of D1:1 using a tac-hybrid promoter in front of the psbAI gene in combination with lacIQ repressor system. Over-production of D1:1 was induced by growing cells for 12 h at 50 mumol photons m-2 s-1 in the presence of 40 or 80 micrograms/ml IPTG. The amount of psbAI mRNA and that of D1:1 protein in cells grown with IPTG was three times and two times higher, respectively. A higher concentration of IPTG (i.e., 150 micrograms/ml) did not further increase the production of the psbAI message or D1:1. The over-production of D1:1 caused a decrease in the level of D1:2 synthesised, resulting in most PSII reaction centres containing D1:1. However, the over-production of D1:1 had no effect on the pigment composition (chlorophyll a or phycocyanin/number of cells) or the light-saturated rate of photosynthesis. This and the fact that the total amounts of D1 and D2 proteins were not affected by IPTG suggest that the number of PSII centres within the membranes remained unchanged. From these results, we conclude that expression of psbAI can be regulated by using the tac promoter and lacIQ system. However, the accumulation of D1:1 protein into the membrane is regulated by the number of PSII centres.

Over-production of the D1:2 protein makes Synechococcus cells more tolerant to photoinhibition of photosystem II

“…Northern blotting was performed as described in Soitamo et al [29]. Five different probes were used to identify different m R N A species.…”

“…Five different probes were used to identify different m R N A species. Oligonucleotide probes specific for the 5' end were used to identify psbAI and psbAII/III messages [29]. tac-psbA messages were probed using a single-stranded 21 bp oligonucleotide originating directly upstream from the translation initiation codon ATG.…”

“…Western blotting was performed as described in Soitamo et al [29], except that the solubilisation of membrane proteins was carried out on ice for 3 h. The specificity of the antibodies used against D 1:1 and D 1:2 proteins has earlier been presented in Soitamo et al [29].…”

D1 polypeptide degradation may regulate psbA gene expression at transcriptional and translational levels in Synechocystis sp. PCC 6803

“…Many other factors such as the age of plant (Deng and Gruissem 1987;Klaff and Gruissem 1991), the maturation state of plastid (Kawaguchi et al 1992;Baumgartner et al 1993), and diurnal light rhythms (Salvador et al 1993) affect the expression of the psbA gene in plants. Cyanobacteria, in turn, have more than onepsbA gene, and these genes may be regulated by light in different ways (Schaefer and Golden 1989a,b;Bustos et al 1990; Kulkarni et al 1992;Soitamo et al 1994).…”

Dynamic responses of photosystem II and phycobilisomes to changing light in the cyanobacterium Synechococcus sp. PCC 7942

“…Protein samples containing equivalent amounts of Chl (0.5-1.5 gg) were separated on linear 15% LiDS-polyacrylamide gels, transferred electrophoretically to nitrocellulose followed by immuno-blot analysis according to Clarke and Critchley (1992) with detection using the ECL chemiluminescent kit (Amersham Braunschweig, Germany). The polyclonal antibodies against DI:I and Dl:2 are completely form-specific and show no detectable crossreactivity, as shown by Clarke et al (1993a) and Soitamo et al (1994) using inactivation mutant strains which express only D I:I or D1:2. The specificities of the polyclonal antibodies against total D 1 and phycobilisome have been characterized by Tyystj~irvi et al (1994) and Bhalerao et al (1994), respectively.…”