Thermoluminescence and flash-induced oxygen yield in herbicide resistant mutants of the D1 protein in Synechococcus PCC7942.

Several strains of Synechococcus PCC7942 carrying point mutations in the gene psbA were studied by thermoluminescence and polarographic measurement of flash-induced oxygen yield. The following results were obtained: (a) Replacement of Ser-264 in D1 by Ala (mutant Di1) or Gly (mutant G264) resulting in DCMU and atrazine resistance leads to a downshift of the thermoluminescence (TL) B-band peak temperature from 40 degrees C in wild-type thylakoids to about 30 degrees C. In dark adapted samples of both mutants the TL and oxygen yield pattern induced by a train of single turnover flashes were strongly damped indicative of a high miss factor. (b) In contrast to Ser-264 mutants, replacement of Phe-255 in D1 by Tyr (mutant Tyr5) induced strong resistance to atrazine but not to DCMU and did not affect the peak termperature of the B-band and the flash-induced TL and oxygen yield patterns. In this respect mutant Tyr5 resembles the wild type. (c) No significant differences have been found between strains with single site mutations in psbAI and normal psbAII/psbAIII genes, and strains with same mutations in psbAI but additional deletion of psbAII and psbAIII. Obviously in strains were psbAI is present, PS II complexes containing gene products of psbAII and psbAIII are not assembled in detectable amounts. (d) Strains with double mutations at positions 264 and 255 display a downshift of the B-band peak temperature. Their oscillatory patterns of B-band intensity and oxygen yield are highly damped. This behaviour is similar to strains D1 and G264 which are modified at position 264 only. We extend reports on additivity of mutation effects on herbicide binding to binding of QB. (e) Mutations at the QB site not only influence the binding of QB and herbicides but also change the thermoluminescence quantum yield and the lifetimes of the redox states S2 and S3 of the water oxidase. This finding might indicate long ranging effects on Photosystem II exerted by structural modifications of the QB site. From these data we conclude that Ser-264 is essential for binding of atrazine, DCMU and QB, whereas Phe-255 is involved in atrazine binding and its substitution by Tyr does not markedly affect QB or DCMU binding in Synechococcus PCC7942.     

cpmA, a Gene Involved in an Output Pathway of the Cyanobacterial Circadian System

We generated random mutations in Synechococcus sp. strain PCC 7942 to look for genes of output pathways in the cyanobacterial circadian system. A derivative of transposon Tn5 was introduced into the chromosomes of reporter strains in which cyanobacterial promoters drive the Vibrio harveyi luxAB genes and produce an oscillation of bioluminescence as a function of circadian gene expression. Among low-amplitude mutants, one mutant, tnp6, had an insertion in a 780-bp open reading frame. The tnp6 mutation produced an altered circadian phasing phenotype in the expression rhythms of psbAI::luxAB, psbAII::luxAB, and kaiA::luxAB but had no or little effect on those of psbAIII::luxAB, purF::luxAB, kaiB::luxAB, rpoD2::luxAB, ndhD::luxAB, and conII::luxAB. This suggests that the interrupted gene in tnp6, named cpmA (circadian phase modifier), is part of a circadian output pathway that regulates the expression rhythms of psbAI, psbAII, and kaiA.     

Light availability influences the ratio of two forms of D1 in cyanobacterial thylakoids

The psbA multigene family in Synechococcus sp. strain PCC 7942 encodes two forms of the D1 protein; Form I, the product of psbAI, differs from Form II, the product of psbAII and psbAIII, at 25 of 360 amino acid positions. D1 is essential for photosynthesis as a protein component of the photosystem II reaction center. Antisera were raised against purified hybrid proteins encoded by psbAI-lacZ and psbAIII-lacZ translational gene fusions that contain the unique amino termini of Form I and Form II, respectively. Form specificity of each antiserum was verified by Western analysis using thylakoid membranes from mutant strains containing only Form I or Form II. Western analysis of thylakoid membranes from wild-type cells cultured at different light intensities detected both forms of D1 in the membrane and showed changes in the ratio of the two forms. The D1 composition of the membrane matched predicted ratios of the forms based on differential gene expression: psbAI is expressed highest at low light, and both psbAII and psbAIII are expressed highest at high light. Along a gradient of light intensity from 5 microE. m-2.s-1 to 482 microE.m-2.s-1, the relative amount of Form I in thylakoid membranes decreased 58%, while the relative amount of Form II increased 60%. Maximum detection of Form I coupled with minimum detection of Form II in membranes from cells harvested at light intensities below 390 microE.m-2.s-1 suggests a central role for Form I in photosystem II. Increased incorporation of Form II into the thylakoid membrane occurred at light intensities reported by others to be photoinhibitory, suggesting that Form II serves a role in adaptation to high light.     

Translation of the psbA mRNA of Chlamydomonas reinhardtii requires a structured RNA element contained within the 5' untranslated region

Translational regulation is a key modulator of gene expression in chloroplasts of higher plants and algae. Genetic analysis has shown that translation of chloroplast mRNAs requires nuclear-encoded factors that interact with chloroplastic mRNAs in a message-specific manner. Using site-specific mutations of the chloroplastic psbA mRNA, we show that RNA elements contained within the 5' untranslated region of the mRNA are required for translation. One of these elements is a Shine- Dalgarno consensus sequence, which is necessary for ribosome association and psbA translation. A second element required for high levels of psbA translation is located adjacent to and upstream of the Shine-Dalgarno sequence, and maps to the location on the RNA previously identified as the site of message-specific protein binding. This second element appears to act as a translational attenuator that must be overcome to activate translation. Mutations that affect the secondary structure of these RNA elements greatly reduce the level of psbA translation, suggesting that secondary structure of these RNA elements plays a role in psbA translation. These data suggest a mechanism for translational activation of the chloroplast psbA mRNA in which an RNA element containing the ribosome-binding site is bound by message- specific RNA binding proteins allowing for increased ribosome association and translation initiation. These elements may be involved in the light-regulated translation of the psbA mRNA.