Michelle Liberton

Ultrastructure of the membrane systems in the unicellular cyanobacterium Synechocystis sp. strain PCC 6803

Summary.Among prokaryotes, cyanobacteria are unique in having highly differentiated internal membrane systems. Like other Gram-negative bacteria, cyanobacteria such as Synechocystis sp. strain PCC 6803 have a cell envelope consisting of a plasma membrane, peptidoglycan layer, and outer membrane. In addition, these organisms have an internal system of thylakoid membranes where the electron transfer reactions of photosynthesis and respiration occur. A long-standing controversy concerning the cellular ultrastructures of these organisms has been whether the thylakoid membranes exist inside the cell as separate compartments, or if they have physical continuity with the plasma membrane. Advances in cellular preservation protocols as well as in image acquisition and manipulation techniques have facilitated a new examination of this topic. We have used a combination of electron microscopy techniques, including freeze-etched as well as freeze-substituted preparations, in conjunction with computer-aided image processing to generate highly detailed images of the membrane systems in Synechocystis cells. We show that the thylakoid membranes are in fact physically discontinuous from the plasma membrane in this cyanobacterium. Thylakoid membranes in Synechocystis sp. strain PCC 6803 thus represent bona fide intracellular organelles, the first example of such compartments in prokaryotic cells.

Global Proteomic Analysis Reveals an Exclusive Role of Thylakoid Membranes in Bioenergetics of a Model Cyanobacterium

Cyanobacteria are photosynthetic microbes with highly differentiated membrane systems. These organisms contain an outer membrane, plasma membrane, and an internal system of thylakoid membranes where the photosynthetic and respiratory machinery are found. This existence of compartmentalization and differentiation of membrane systems poses a number of challenges for cyanobacterial cells in terms of organization and distribution of proteins to the correct membrane system. Proteomics studies have long sought to identify the components of the different membrane systems in cyanobacteria, and to date about 450 different proteins have been attributed to either the plasma membrane or thylakoid membrane. Given the complexity of these membranes, many more proteins remain to be identified, and a comprehensive catalogue of plasma membrane and thylakoid membrane proteins is needed. Here we describe the identification of 635 differentially localized proteins in Synechocystis sp. PCC 6803 by quantitative iTRAQ isobaric labeling; of these, 459 proteins were localized to the plasma membrane and 176 were localized to the thylakoid membrane. Surprisingly, we found over 2.5 times the number of unique proteins identified in the plasma membrane compared with the thylakoid membrane. This suggests that the protein composition of the thylakoid membrane is more homogeneous than the plasma membrane, consistent with the role of the plasma membrane in diverse cellular processes including protein trafficking and nutrient import, compared with a more specialized role for the thylakoid membrane in cellular energetics. Thus, our data clearly define the two membrane systems with distinct functions. Overall, the protein compositions of the Synechocystis 6803 plasma membrane and thylakoid membrane are quite similar to that of the plasma membrane of Escherichia coli and thylakoid membrane of Arabidopsis chloroplasts, respectively. Synechocystis 6803 can therefore be described as a Gram-negative bacterium with an additional internal membrane system that fulfills the energetic requirements of the cell.

Population-level coordination of pigment response in individual cyanobacterial cells under altered nitrogen levels

Cyanobacterial phycobilisome (PBS) pigment-protein complexes harvest light and transfer the energy to reaction centers. Previous ensemble studies have shown that cyanobacteria respond to changes in nutrient availability by modifying the structure of PBS complexes, but this process has not been visualized for individual pigments at the single-cell level due to spectral overlap. We characterized the response of four key photosynthetic pigments to nitrogen depletion and repletion at the subcellular level in individual, live Synechocystis sp. PCC 6803 cells using hyperspectral confocal fluorescence microscopy and multivariate image analysis. Our results revealed that PBS degradation and re-synthesis comprise a rapid response to nitrogen fluctuations, with coordinated populations of cells undergoing pigment modifications. Chlorophyll fluorescence originating from photosystem I and II decreased during nitrogen starvation, but no alteration in subcellular chlorophyll localization was found. We observed differential rod and core pigment responses to nitrogen deprivation, suggesting that PBS complexes undergo a stepwise degradation process.

Phycobilisome Antenna Deletion in a Cyanobacterium does Not Improve Photosynthetic Energy Conversion Efficiency or Productivity in a Bench-Scale Photobioreactor System

Light harvesting in cyanobacteria is performed by large peripheral phycobilisome antenna complexes that absorb light and transfer it to membrane integral antenna closely associated with the photosynthetic reaction center. In eukaryotic microalgae exposed to high light, truncation of the chlorophyll light harvesting antenna system results in an overall increase in cell growth and photosynthetic efficiency by reducing excess light absorption and subsequent energy dissipation on an individual cell level. In order to test this model in cyanobacteria, we used an optimized photobioreactor system for precise regulation of growth parameters and collected data over a wide range of culture conditions, including different CO2 and light regimes. Wild-type Synechocystis 6803 and a PAL mutant that lacks phycobilisomes were grown in batch-mode in these bioreactors. Our data show that lack of phycobilisome antenna do not provide an advantage to Synechocystis 6803 cells under any of the conditions tested.

Three-Dimensional Arrangement of Thylakoid Membranes in Cyanothece sp. ATCC 51142, a Unicellular Cyanobacterium

In cyanobacteria and the chloroplasts of plants and algae, thylakoids are the complex internal membrane system where the light reactions of oxygenic photosynthesis occur. An early cyanobacterium, or cyanobacterial ancestor, was almost certainly the evolutionary precursor of chloroplasts via an endosymbiotic event. Despite this common ancestry, over evolutionary time the thylakoid membrane systems in photosynthetic organisms have diverged into architecturally dissimilar forms, so that a link between thylakoid membrane organization in chloroplasts and that of the cyanobacterial ancestor has been absent. In particular, conspicuously lacking has been evidence of a 3-dimensional organization that could be considered ancestral or intermediate to the present-day arrangement of grana and stromal thylakoids in chloroplasts.