Site-directed mutagenesis of the basic residues 321K to321 G in the CP 47 protein of photosystem II alters the chloride requirement for growth and oxygen-evolving activity in Synechocystis 6803

CP 47, a component of photosystem II (PSII) in higher plants, algae and cyanobacteria, is encoded by the psbB gene. Site-specific mutagenesis has been used to alter a portion of the psbB gene encoding the large extrinsic loop E of CP 47 in the cyanobacterium Synechocystis 6803. Alteration of a lysine residue occurring at position 321 to glycine produced a strain with altered PSII activity. This strain grew at wild-type rates in complete BG-11 media (480 µM chloride). However, oxygen evolution rates for this mutant in complete media were only 60% of the observed wild-type rates. Quantum yield measurements at low light intensities indicated that the mutant had 66% of the fully functional PSII centers contained in the control strain. The mutant proved to be extremely sensitive to photoinactivation at high light intensities, exhibiting a 3-fold increase in the rate of photoinactivation. When this mutant was grown in media depleted of chloride (30 µM chloride), it lost the ability to grow photoautotrophically while the control strain exhibited a normal rate of growth. The effect of chloride depletion on the growth rate of the mutant was reversed by the addition of 480 µM bromide to the chloride-depleted BG-11 media. In the presence of glucose, the mutant and control strains grew at comparable rates in either chloride-containing or chloride-depleted media. Oxygen evolution rates for the mutant were further depressed (28% of control rates) under chloride-limiting conditions. Addition of bromide restored these rates to those observed under chloride-sufficient conditions. Measurements of the variable fluorescence yield indicated that the mutant assembled fewer functional centers in the absence of chloride. These results indicate that the mutation K321G in CP 47 affects PSII stability and/or assembly under conditions where chloride is limiting.     

Unmasking a temperature-dependent effect of the P. anserina i-AAA protease on aging and development

Different molecular pathways involved in maintaining mitochondrial function are of fundamental importance to control cellular homeostasis. Mitochondrial i-AAA protease is part of such a surveillance system and PaIAP is the putative ortholog in the fungal aging model Podospora anserina. Here we investigated the role of PaIAP in aging and development. Deletion of the gene encoding PaIAP resulted in a specific phenotype. When incubated at 27°C, spore germination and fruiting body formation are not different from that of the corresponding wild-type strain. Unexpectedly, the lifespan of the deletion strain is strongly increased. In contrast, cultivation at an elevated temperature of 37°C leads to impairments in spore germination and fruiting body formation, and to a reduced lifespan. The higher PaIAP abundance in wild-type strains of the fungus grown at elevated temperature and the phenotype of the deletion strain unmasks a temperaturerelated role of the protein. The protease appears to be part of a molecular system that has evolved to allow survival under changing temperatures as they characteristically occur in nature.     

Targeted genetic inactivation of the photosystem I reaction center in the cyanobacterium Synechocystis sp. PCC 6803.

We describe the first complete segregation of a targeted inactivation of psaA encoding one of the P700-chlorophyll a apoproteins of photosystem (PS) I. A kanamycin resistance gene was used to interrupt the psaA gene in the unicellular cyanobacterium Synechocystis sp. PCC 6803. Selection of a fully segregated mutant, ADK9, was performed under light-activated heterotrophic growth (LAHG) conditions; complete darkness except for 5 min of light every 24 h and 5 mM glucose. Under these conditions, wild-type cells showed a 4-fold decrease in chlorophyll (chl) per cell, primarily due to a decrease of PS I reaction centers. Evidence for the absence of PS I in ADK9 includes: the lack of EPR (electron paramagnetic resonance) signal I, from P700+; undetectable P700-apoprotein; greatly reduced whole-chain photosynthesis rates; and greatly reduced chl per cell, resulting in a turquoise blue phenotype. The PS I peripheral proteins PSA-C and PSA-D were not detected in this mutant. ADK9 does assemble near wild-type levels of functional PS II per cell, evidenced by: EPR signal II from YD+; high rates of oxygen evolution with 2,6-dichloro-p-benzoquinone (DCBQ), an electron acceptor from PS II; and accumulation of D1, a PS II core polypeptide. The success of this transformation indicates that this cyanobacterium may be utilized for site-directed mutagenesis of the PS I core.     

Zeaxanthin accumulation in the absence of a functional xanthophyll cycle protects Chlamydomonas reinhardtii from photooxidative stress

Xanthophylls participate in light harvesting and are essential in protecting the chloroplast from photooxidative damage. To investigate the roles of xanthophylls in photoprotection, we isolated and characterized extragenic suppressors of the npq1 lor1 double mutant of Chlamydomonas reinhardtii, which lacks zeaxanthin and lutein and undergoes irreversible photooxidative bleaching and cell death at moderate to high light intensities. Here, we describe three suppressor strains that carry point mutations in the coding sequence of the zeaxanthin epoxidase gene, resulting in the constitutive accumulation of zeaxanthin in a range of concentrations. The presence of zeaxanthin in these strains was sufficient to prevent photooxidative damage in the npq1 lor1 background. The size of the light-harvesting antenna in the suppressors decreased in high light in a manner that was proportional to the relative content of zeaxanthin, with the strain having the most zeaxanthin showing a severe reduction in levels of the major light-harvesting complex II proteins in high light. We show that the effect of constitutive zeaxanthin on light harvesting is not the main cause of increased photoprotection, because in the absence of zeaxanthin, a strain with a smaller light-harvesting antenna showed only minor protection against photobleaching in high light. Furthermore, the zeaxanthin-accumulating suppressors were able to tolerate higher levels of exogenous reactive oxygen than their parental strain under conditions that did not affect light harvesting. Our results are consistent with an antioxidant role of zeaxanthin in the quenching of singlet oxygen and/or free radicals in the thylakoid membrane in vivo.     

Expression of the isiA gene is essential for the survival of the cyanobacterium Synechococcus sp. PCC 7942 by protecting photosystem II from excess light under iron limitation

Iron deficiency is known to suppress primary productivity in both marine and freshwater ecosystems. In response to iron deficiency, certain cyanobacteria induce a chlorophyll (Chl)-protein complex, CP43', which is encoded by the isiA gene. The deduced amino-acid sequence of CP43' predicts some structural similarity to the CP43 polypeptide of photosystem II, but the function of CP43' remains uncertain. In order to assess its physiological role, the isiA gene of a cyanobacterium, Synechococcus sp. PCC7942, was inactivated by insertion mutagenesis (giving isiA cells). Compared with isiA cells, under iron deprivation, wild-type cells showed both lower rates of photosystem II-mediated O2 evolution at limiting light irradiances and decreased yields of room temperature Chl fluorescence at various irradiances. These observations strongly suggest that the decreased photosystem II activity in wild-type cells with CP43' is attributable to increased non-radiative dissipation of light energy. In agreement with this hypothesis, isiA cells were more susceptible to photoinhibition of photosynthesis than wild-type cells, resulting in much slower growth rates under iron limitation. Based on these results, we suggest that CP43' functions as a non-radiative dissipator of light energy, thus protecting photosystem II from excessive excitation under iron-deficient conditions.     

Cells Lacking Pfh1, a Fission Yeast Homolog of Mammalian Frataxin Protein,Display Constitutive Activation of the Iron Starvation Response

Friedreich ataxia is a genetic disease caused by deficiencies in frataxin. This protein has homologs not only in higher eukaryotes but also in bacteria, fungi, and plants. The function of this protein is still controversial. We have identified a frataxin homolog in fission yeast, and we have analyzed whether its depletion leads to any of the phenotypes observed in other organisms. Cells deleted in pfh1 are sensitive to growth under aerobic conditions, display increased levels of total iron, hallmarks of oxidative stress such as protein carbonylation, decreased aconitase activity, and lower levels of oxygen consumption compared with wild-type cells. This mitochondrial protein seems to be important for iron and/or reactive oxygen species homeostasis. We have analyzed the proteome of cells devoid of Pfh1, and we determined that gene products up- and down-regulated upon iron depletion in wild-type cells are constitutively misregulated in this mutant. Because of the particular signaling pathway components governing the iron starvation response in fission yeast, our experiments suggest that cells lacking Pfh1 display a decrease of cytosolic available iron that triggers activation of Grx4, the common regulator of the iron starvation gene expression program. Our Schizosaccharomyces pombe Δpfh1 strain constitutes a new and useful model system to study Friedreich ataxia.