Diffusion of phycobilisomes on the thylakoid membranes of the cyanobacterium Synechococcus 7942. Effects of phycobilisome size, temperature, and membrane lipid composition.

A variant of fluorescence recovery after photobleaching allows us to observe the diffusion of photosynthetic complexes in cyanobacterial thylakoid membranes in vivo. The unicellular cyanobacterium Synechococcus sp. PCC7942 is a wonderful model organism for fluorescence recovery after photobleaching, because it has a favorable membrane geometry and is well characterized and transformable. In Synechococcus 7942 (as in other cyanobacteria) we find that photosystem II is immobile, but phycobilisomes diffuse rapidly on the membrane surface. The diffusion coefficient is 3 x 10(-10) cm(2) s(-1) at 30 degrees C. This shows that the association of phycobilisomes with reaction centers is dynamic; there are no stable phycobilisome-reaction center complexes in vivo. We report the effects of mutations that change the phycobilisome size and membrane lipid composition. 1) In a mutant with no phycobilisome rods, the phycobilisomes remain mobile with a slightly faster diffusion coefficient. This confirms that the diffusion we observe is of intact phycobilisomes rather than detached rod elements. The faster diffusion coefficient in the mutant indicates that the rate of diffusion is partly determined by the phycobilisome size. 2) The temperature dependence of the phycobilisome diffusion coefficient indicates that the phycobilisomes have no integral membrane domain. It is likely that association with the membrane is mediated by multiple weak interactions with lipid head groups. 3) Changing the lipid composition of the thylakoid membrane has a dramatic effect on phycobilisome mobility. The results cannot be explained in terms of changes in the fluidity of the membrane; they suggest that lipids play a role in controlling phycobilisome-reaction center interaction.

How do cyanobacteria sense and respond to light?

Cyanobacteria exhibit numerous responses to changes in the intensity and spectral quality of light. What sensors do cyanobacteria use to detect light and what are the mechanisms of signal transduction? The publication in 1996 of the complete genome sequence of the cyanobacterium Synechocystis 6803 provided a tremendous stimulus for research in this field, and many light-sensors and signal transducers have now been identified. However, our knowledge of cyanobacterial light-signal transduction remains fragmentary. This review summarizes what we know about the ways in which cyanobacteria perceive light, some of the ways which they respond to light signals and some recent achievements in elucidating the signal transduction mechanisms. Some problems in characterizing cyanobacterial signal transduction pathways are outlined and alternative experimental strategies are discussed.

Long-wave sensitivity in deep-sea stomiid dragonfish with far-red bioluminescence: evidence for a dietary origin of the chlorophyll-derived retinal photosensitizer of Malacosteus niger.

Both residual downwelling sunlight and bioluminescence, which are the two main sources of illumination available in the deep sea, have limited wavebands concentrated around 450-500 nm. Consequently, the wavelengths of maximum absorption (lambdamax) of the vast majority of deep-sea fish visual pigments also cluster in this part of the spectrum. Three genera of deep-sea loose-jawed dragonfish (Aristostomias, Pachystomias and Malacosteus), however, in addition to the blue bioluminescence typical of most deep-sea animals, also produce far-red light (maximum emission >700 nm) from suborbital photophores. All three genera are sensitive in this part of the spectrum, to which all other animals of the deep sea are blind, potentially affording them a private waveband for illuminating prey and for interspecific communication that is immune from detection by predators and prey. Aristostomias and Pachystomias enhance their long-wave visual sensitivity by the possession of at least three visual pigments that are long-wave shifted (lambdamax values ca. 515, 550 and 590 nm) compared with those of other deep-sea fishes. Malacosteus, on the other hand, although it does possess two of these red-shifted pigments (lambdamax values ca. 520 and 540 nm), lacks the most long-wave-sensitive pigments found in the other two genera. However, it further enhances its long-wave sensitivity with a chlorophyll-derived photosensitizer within its outer segments. The fluorescence emission and excitation spectra of this pigment are very similar to spectra obtained from mesopelagic copepods, which are an important component of diet of Malacosteus, suggesting a dietary origin for this pigment.

Effects of tubulin assembly inhibitors on cell division in prokaryotes in vivo.

The bacterial cell division protein FtsZ is a structural analogue of tubulin. Bacterial mutants in which the ftsZ gene is inactivated are unable to divide. Numerous inhibitors of tubulin assembly are known, some of which are used as fungicides. The strong structural homology between FtsZ and tubulin raises the possibility that some of these inhibitors could affect bacterial cell division. Here we report that the tubulin assembly inhibitors thiabendazole and 2-methylbenzimidazole cause cell elongation in Escherichia coli and cyanobacteria.

Involvement of an FtsH homologue in the assembly of functional photosystem I in the cyanobacterium Synechocystis sp. PCC 6803.

The Synechocystis sp. PCC 6803 genome encodes four putative homologues of the AAA protease FtsH, two of which (slr0228 and sll1463) have been subjected to insertional mutagenesis in this study. Disruption of sll1463 had no discernible effect but disruption of slr0228 caused a 60% reduction in the abundance of functional photosystem I, without affecting the cellular content of photosystem II or phycobilisomes. Fluorescence and immunoblotting analyses show reductions in PS I polypeptides and possible structural alterations in the residual PS I, indicating an important role for slr0228 in PS I biogenesis.

Bacterial orthologues indicate the malarial plastid gene ycf24 is essential.

ycf24 is a well conserved gene found in all major groups of bacteria, as well as on red algal plastid genomes and the vestigal plastid genome of apicomplexan pathogens like the malaria parasite Plasmodium falciparum (ORF470). Some database annotations describe Ycf24 as an ABC transporter subunit, but we find the level of significance is low. To investigate ycf24's function we disrupted it in the cyanobacterium Synechocystis sp., strain PCC6803 which has a multi-copy genome. This showed ycf24 is essential, partial loss producing a terminal phenotype of chlorosis, reduced cell size, loss of DNA, and a striking arrest in cytokinesis. Attempts to disrupt the single copy of ycf24 in E. coli failed to give stable transformants. When Ycf24 was over-expressed in E. coli as a soluble fusion protein, it localized mostly as a band on either side of the nucleoid and nucleoid partitioning was aberrant. We propose the relict plastid organelle of apicomplexans retains its capacity for protein synthesis because Ycf24 is essential.

Cyanobacterial ycf27 gene products regulate energy transfer from phycobilisomes to photosystems I and II.

Two open reading frames (slr0115 and slr0947) in the genome of the cyanobacterium Synechocystis sp. PCC 6803 are shown to be involved in the regulation of the coupling of phycobilisomes to photosynthetic reaction centres. Homologues of these genes, called ycf27, have been found in a range of phycobilin-containing organisms. The slr0115 and slr0947 gene products are OmpR-type DNA-binding response regulator proteins. Deletion of slr0115 results in increased efficiency of energy transfer from phycobilisomes to photosystem II relative to photosystem I. Reduction of the copy number of slr0947 has the opposite phenotypic effect. We have given the slr0115 and slr0947 genes the designations rpaA and rpaB respectively.

Enhanced retinal longwave sensitivity using a chlorophyll-derived photosensitiser in Malacosteus niger, a deep-sea dragon fish with far red bioluminescence.

Through partial bleaching of both visual pigment extracts and cell suspensions we show that the deep-sea stomiid Malacosteus niger, which produces far red bioluminescence, has two visual pigments within its retina which form a rhodopsin/porphyropsin pigment pair with lambda max values around 520 and 540 nm, but lacks the very longwave sensitive visual pigments (lambda max > 550 nm) observed in two other red light producing stomiids. The presence of only a single opsin gene in the M. niger genome was confirmed by molecular and cladistic analysis. To compensate for its apparently reduced longwave sensitivity compared to related species, the outer segments of M. niger contain additional pigments, which we identify as a mixture of defarnesylated and demetallated derivatives of bacteriochlorophylls c and d, that are used as a photosensitiser to enhance its sensitivity to longwave radiation.

A gene required for the regulation of photosynthetic light harvesting in the cyanobacterium Synechocystis 6803.

A gene required for the short-term regulation of photosynthetic light harvesting (the state transition) has been identified in the cyanobacterium Synechocystis sp. PCC6803. The open reading frame is designated sll1926 in the complete Synechocystis gene sequence. The deduced amino acid sequence has no homologues in current sequence databases and no recognizable sequence motifs. It encodes a putative integral membrane protein of 16 kDa, which we have designated RpaC (regulator of phycobilisome association C). Fluorescence measurements of an insertional inactivation mutant of rpaC (Deltasll1926) show that it is specifically unable to perform state transitions. Deltasll1926 has approximately wild-type levels of PS1, PS2 and phycobilisomes. Measurements of oxygen evolution and uptake show Deltasll1926 to have no deficiency in electron transport rates. In vitro [gamma-32P]-ATP labelling experiments suggest that RpaC is not the 15 kDa membrane phosphoprotein previously implicated in state transitions. Deltasll1926 grows more slowly than the wild type only at very low light intensities.

Inhibition by phosphate of light-state transitions in cyanobacterial cells.

Light-state transitions in cyanobacteria are a rapid physiological adaptation mechanism which changes the distribution of excitation energy absorbed by the light-harvesting complexes between Photosystem II and Photosystem I. State transitions in two cyanobacterial species are shown to be inhibited by buffers containing 0.2-0.4 M phosphate. Both the state 1 and the state 2 transition are inhibited, so that cells may be locked in the state to which they were adapted before the addition of phosphate. The inhibition of the state 1 transition is due to inhibition of photosynthetic electron transport. However, the inhibition of the state 2 transition is probably due to a direct effect on the biochemical signal transduction pathway. The implications for the biochemical mechanism of state transitions are discussed.

State 1-State 2 transitions in the cyanobacterium Synechococcus 6301 are controlled by the redox state of electron carriers between Photosystems I and II.

The mechanism by which state 1-state 2 transitions in the cyanobacterium Synechococcus 6301 are controlled was investigated by examining the effects of a variety of chemical and illumination treatments which modify the redox state of the plastoquinone pool. The extent to which these treatments modify excitation energy distribution was determined by 77K fluorescence emission spectroscopy. It was found that treatment which lead to the oxidation of the plastoquinone pool induce a shift towards state 1 whereas treatments which lead to the reduction of the plastoquinone pool induce a shift towards state 2. We therefore propose that state transitions in cyanobacteria are triggered by changes in the redox state of plastoquinone or a closely associated electron carrier. Alternative proposals have included control by the extent of cyclic electron transport around PS I and control by localised electrochemical gradients around PS I and PS II. Neither of these proposals is consistent with the results reported here.

State transitions, photosystem stoichiometry adjustment and non-photochemical quenching in cyanobacterial cells acclimated to light absorbed by photosystem I or photosystem II.

Cells of the cyanobacterium Synechococcus 6301 were grown in yellow light absorbed primarily by the phycobilisome (PBS) light-harvesting antenna of photosystem II (PS II), and in red light absorbed primarily by chlorophyll and, therefore, by photosystem I (PS I). Chromatic acclimation of the cells produced a higher phycocyanin/chlorophyll ratio and higher PBS-PS II/PS I ratio in cells grown under PS I-light. State 1-state 2 transitions were demonstrated as changes in the yield of chlorophyll fluorescence in both cell types. The amplitude of state transitions was substantially lower in the PS II-light grown cells, suggesting a specific attenuation of fluorescence yield by a superimposed non-photochemical quenching of excitation. 77 K fluorescence emission spectra of each cell type in state 1 and in state 2 suggested that state transitions regulate excitation energy transfer from the phycobilisome antenna to the reaction centre of PS II and are distinct from photosystem stoichiometry adjustments. The kinetics of photosystem stoichiometry adjustment and the kinetics of the appearance of the non-photochemical quenching process were measured upon switching PS I-light grown cells to PS II-light, and vice versa. Photosystem stoichiometry adjustment was complete within about 48 h, while the non-photochemical quenching occurred within about 25 h. It is proposed that there are at least three distinct phenomena exerting specific effects on the rate of light absorption and light utilization by the two photoreactions: state transitions; photosystem stoichiometry adjustment; and non-photochemical excitation quenching. The relationship between these three distinct processes is discussed.