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, 288 (5), 3632-40

Organization and Flexibility of Cyanobacterial Thylakoid Membranes Examined by Neutron Scattering

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Organization and Flexibility of Cyanobacterial Thylakoid Membranes Examined by Neutron Scattering

Michelle Liberton et al. J Biol Chem.

Abstract

Cyanobacteria are prokaryotes that can use photosynthesis to convert sunlight into cellular fuel. Knowledge of the organization of the membrane systems in cyanobacteria is critical to understanding the metabolic processes in these organisms. We examined the wild-type strain of Synechocystis sp. PCC 6803 and a series of mutants with altered light-harvesting phycobilisome antenna systems for changes in thylakoid membrane architecture under different conditions. Using small-angle neutron scattering, it was possible to resolve correlation distances of subcellular structures in live cells on the nanometer scale and capture dynamic light-induced changes to these distances. Measurements made from samples with varied scattering contrasts confirmed that these distances could be attributed to the thylakoid lamellar system. We found that the changes to the thylakoid system were reversible between light- and dark-adapted states, demonstrating a robust structural flexibility in the architecture of cyanobacterial cells. Chemical disruption of photosynthetic electron transfer diminished these changes, confirming the involvement of the photosynthetic apparatus. We have correlated these findings with electron microscopy data to understand the origin of the changes in the membranes and found that light induces an expansion in the center-to-center distances between the thylakoid membrane layers. These combined data lend a dynamic dimension to the intracellular organization in cyanobacterial cells.

Figures

FIGURE 1.
FIGURE 1.
Transmission electron micrographs of WT and phycobilisome antenna mutants. Whole cell images and enlargements illustrating thylakoid membrane spacing are shown for WT (A and B), CB (C and D), CK (E and F), and PAL (G and H). White bars depict the center-to-center thylakoid membrane spacing. T, thylakoid membranes; P, polyphosphate bodies. Scales bars = 500 nm (A, C, E, and G) and 50 nm (B, D, F, and H).
FIGURE 2.
FIGURE 2.
Contrast variation for WT in the light and dark. SANS intensity was measured for samples under both light and dark conditions for a range of ratios of D2O to H2O. A, scattering profiles for samples in the dark and equilibrated in 100% D2O (●) and 20% D2O (○). B, the square root of scattering intensity for the diffraction peak at the lowest q is plotted as a function of D2O concentration for samples in the light (○) and dark (●). The intensities from samples in 20% D2O were excluded from the linear regression fits (solid lines). The contrast match point for the major peak is 17.9% in the light and 16.5% in the dark, within the range of a peak that is composed primarily of lipids.
FIGURE 3.
FIGURE 3.
SANS data from cyanobacterial cells under light and dark conditions. Scattering intensities from WT (red), CB (blue), CK (green), and PAL (gold) in the light (A) and dark (B) over the entire experimental q range are shown. Peaks are labeled 1–5. In A, intensities for WT and PAL were vertically offset by −12 and 6 cm−1, respectively. In B, intensities for WT, CB, and PAL were vertically offset by −12, −3, and 6 cm−1, respectively.
FIGURE 4.
FIGURE 4.
Time course of dynamic changes in cyanobacterial strains during dark-to-light transition. A, WT during the transition from dark to light. B, PAL during the transition from dark to light, showing that peaks shift slightly in the q range but do not appear or disappear. C, CK during light-to-dark transition. D, CK during dark-to-light transition.
FIGURE 5.
FIGURE 5.
Inhibition of electron transport prevents light/dark-induced changes. Strains were preincubated in the dark in the presence of DCMU, and data were collected in the dark and upon exposure to light for WT (A), CB (B), CK (C), and PAL (D) strains. Untreated light (solid red circles) and dark (solid black circles) scattering profiles are reproduced from Fig. 2 for comparison with the DCMU-treated light (open red circles) and dark (open black circles) profiles. In A, intensities were vertically offset by 0.4 cm−1 for treated and untreated samples exposed to light. In B, intensities were vertically offset by 0.7 cm−1 for untreated samples and by 0.3 cm−1 for treated samples exposed to light. In C and D, intensities for treated and untreated samples exposed to light were vertically offset by 0.3 cm−1.
FIGURE 6.
FIGURE 6.
Quasi-elastic line widths and fits to jump diffusion model used to determine average water diffusion coefficients. The half-width at half-maximum (HWHM) values of the q-dependent and q-independent Lorentzian components of the quasi-elastic energy spectra are shown for WT Synechocystis in the dark (●), WT Synechocystis illuminated with white light (○), and BL21 E. coli (■). Fits of the WT dark, WT illuminated, and E. coli dark q-dependent line widths to the jump diffusion model are shown as solid, dashed, and dotted lines, respectively.
FIGURE 7.
FIGURE 7.
Schematic drawing of thylakoid membrane organization in cyanobacterial cells from SANS data and TEM analysis. Different membrane and phycobilisome arrangements are numbered 1–5 to correlate with SANS peaks. 1, center-to-center repeat distance between thylakoid membrane pairs, shown with WT phycobilisomes; 2, repeat distance between thylakoid membrane pairs insufficient for phycobilisomes; 3, repeat distance originating from closely appressed thylakoid membrane pairs; 4 and 5, repeat distance originating from a single thylakoid membrane layer with lumens of varying size.

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