Methods for the isolation of functional photosystem II core particles from the cyanobacterium Synechocystis sp. PCC 6803.

This chapter contains the description of several methods used for the isolation of functional photosystem II (PS II) core particles from wild type, photosystem I-less, and CP47 histidine-tagged cells of the cyanobacterium Synechocystis sp. PCC 6803. The presented protocols cover cultivation of photosystem I-containing and photosystem I-less cells, isolation of thylakoid membranes, purification of PS II core particles using a weak cation exchange or metal affinity column chromatography, and characterization of the final preparation.

Small Cab-like proteins retard degradation of photosystem II-associated chlorophyll in Synechocystis sp. PCC 6803: kinetic analysis of pigment labeling with 15N and 13C.

Isotope (Na(15)NO(3), ((15)NH(4))SO(4) or [(13)C]glucose) labeling was used to analyze chlorophyll synthesis and degradation rates in a set of Synechocystis mutants that lacked single or multiple small Cab-like proteins (SCPs), as well as photosystem I or II. When all five small Cab-like proteins were inactivated in the wild-type background, chlorophyll stability was not affected unless the scpABCDE(-) strain was grown at a moderately high light intensity of 100-300 micromol photons m(-2) s(-1). However, the half-life time of chlorophyll was 5-fold shorter in the photosystem I-less/scpABCDE(-) strain than in the photosystem I-less strain even when grown at low light intensity (~3 micromol photons m(-2) s(-1)) (32 +/- 5 and 161 +/- 25 h, respectively).

Continuous chlorophyll degradation accompanied by chlorophyllide and phytol reutilization for chlorophyll synthesis in Synechocystis sp. PCC 6803.

Chlorophyll synthesis and degradation were analyzed in the cyanobacterium Synechocystis sp. PCC 6803 by incubating cells in the presence of 13C-labeled glucose or 15N-containing salts. Upon mass spectral analysis of chlorophyll isolated from cells grown in the presence of 13C-glucose for different time periods, four chlorophyll pools were detected that differed markedly in the amount of 13C incorporated into the porphyrin (Por) and phytol (Phy) moieties of the molecule.

Phycobilin/chlorophyll excitation equilibration upon carotenoid-induced non-photochemical fluorescence quenching in phycobilisomes of the cyanobacterium Synechocystis sp. PCC 6803.

To determine the mechanism of carotenoid-sensitized non-photochemical quenching in cyanobacteria, the kinetics of blue-light-induced quenching and fluorescence spectra were studied in the wild type and mutants of Synechocystis sp. PCC 6803 grown with or without iron. The blue-light-induced quenching was observed in the wild type as well as in mutants lacking PS II or IsiA confirming that neither IsiA nor PS II is required for carotenoid-triggered fluorescence quenching.

15N-labeling to determine chlorophyll synthesis and degradation in Synechocystis sp. PCC 6803 strains lacking one or both photosystems.

Rates of chlorophyll synthesis and degradation were analyzed in Synechocystis sp. PCC 6803 wild type and mutants lacking one or both photosystems by labeling cells with ((15)NH(4))(2)SO(4) and Na(15)NO(3). Pigments extracted from cells were separated by HPLC and incorporation of the (15)N label into porphyrins was subsequently examined by MALDI-TOF mass spectrometry.

Subtle modification of isotope ratio proteomics; an integrated strategy for expression proteomics.

Use of minor modification of isotope ratio to code samples for expression proteomics is being investigated. Alteration of (13)C abundance to approximately 2% yields a measurable effect on peptide isotopic distribution and inferred isotope ratio. Elevation of (13)C abundance to 4% leads to extension of isotopic distribution and background peaks across every unit of the mass range.

Isolation of functional photosystem II core particles from the Cyanobacterium synechocystis sp. PCC 6803.

This chapter contains the description of several methods used for the isolation of functional photosystem (PS)II core particles from wild-type (wt), PSI-less, and CP47 histidine-tagged cells of the cyanobacterium Synechocystis sp. PCC 6803. These protocols discuss the cultivation of PSI-containing and PSI-less cells, isolation of thylakoid membranes, purification of PSII core particles using a weak cation exchange or metal affinity column chromatography, and characterization of the final preparation.

Multiple deletions of small Cab-like proteins in the cyanobacterium Synechocystis sp. PCC 6803: consequences for pigment biosynthesis and accumulation.

Deletion of the genes for four or five small Cab-like proteins (SCPs) in photosystem (PS) I-less and PS I-less/PS II-less strains of Synechocystis sp. PCC 6803 caused a large decrease in the chlorophyll and carotenoid content of the cells without accumulation of early intermediates in the chlorophyll biosynthesis pathway, suggesting limited chlorophyll availability. The PS II/PS I ratio increased upon deletion of multiple SCPs in a wild type background, similar to what is observed in the presence of subsaturating concentrations of gabaculin, an inhibitor of an early step in the tetrapyrrole biosynthesis pathway.

Energy and electron transfer in photosystem II of a chlorophyll b-containing Synechocystis sp. PCC 6803 mutant.

Using a Synechocystis sp. PCC 6803 mutant strain that lacks photosystem (PS) I and that synthesizes chlorophyll (Chl) b, a pigment that is not naturally present in the wild-type cyanobacterium, the functional consequences of incorporation of this pigment into the PS II core complex were investigated. Despite substitution of up to 75% of the Chl a in the PS II core complex by Chl b, the modified PS II centers remained essentially functional and were able to oxidize water and reduce Q(A), even upon selective excitation of Chl b at 460 nm.

The presence of chlorophyll b in Synechocystis sp. PCC 6803 disturbs tetrapyrrole biosynthesis and enhances chlorophyll degradation.

Both chlorophyll (Chl) a and b accumulate in the light in a Synechocystis sp. PCC 6803 strain that expresses higher plant genes coding for a light-harvesting complex II protein and Chl a oxygenase. This cyanobacterial strain also lacks photosystem (PS) I and cannot synthesize Chl in darkness because of the lack of chlL.

Regulation of the tetrapyrrole biosynthetic pathway leading to heme and chlorophyll in plants and cyanobacteria.

Photosynthetic organisms synthesize chlorophylls, hemes, and bilin pigments via a common tetrapyrrole biosynthetic pathway. This review summarizes current knowledge about the regulation of this pathway in plants, algae, and cyanobacteria. Particular emphasis is placed on the regulation of glutamate-1-semialdehyde formation and on the channelling of protoporphyrin IX into the heme and chlorophyll branches.

Small Cab-like proteins regulating tetrapyrrole biosynthesis in the cyanobacterium Synechocystis sp. PCC 6803.

In the cyanobacterium Synechocystis sp. PCC 6803 five open reading frames (scpA-scpE) have been identified that code for single-helix proteins resembling helices I and III of chlorophyll a/b-binding (Cab) antenna proteins from higher plants. They have been named SCPs (small Cab-like proteins).