Molecular mechanism of intracellular membrane biogenesis in Synechocystis sp. PCC6803

Cyanobacteria (otherwise known as blue-green algae) are bacteria that grow by photosynthesis in a similar way to plants. Chloroplasts (the photosynthetic bodies within plant cells) are descended from free-living cyanobacteria, accounting for the many similarities between cyanobacteria and chloroplasts. Cyanobacteria are widespread in the environment. For example they are very abundant in rivers, lakes, and the oceans, where they make an important contribution to the ecology of the planet. Cyanobacteria are now attracting increasing interest as possible sources of 'biofuels'. We may eventually be able to modify cyanobacteria to produce cell factories using the energy of sunlight to produce fuels such as hydrogen. Cyanobacteria have a more complex cell structure than most bacteria. Inside the cells are the thylakoid membranes, a complex internal membrane system which is the site of the 'light reactions' of photosynthesis. The thylakoid membranes contain the pigments that absorb energy from sunlight, and the proteins that carry out the first steps in converting solar energy to stored chemical energy. Although we now know a lot of detail about the photosynthetic proteins, we know rather little about how the thylakoid membranes are made. We propose to investigate this question using as a starting point genes which are believed to be important for thylakoid membrane production. It has not yet been possible to produce mutants completely lacking these genes. However, when the number of gene copies per cell is reduced, thylakoid membrane synthesis is greatly decreased. Although the genes have been identified, we do not know how the proteins that they encode are involved in thylakoid membrane generation. We will investigate this question using a 'model' cyanobacterium that can easily be genetically modified. We will modify this cyanobacterium so we can control the expression of both genes: we will be able to switch the production of the proteins on and off. This should give us a way to control thylakoid membrane generation. We will be able to watch thylakoid membrane degradation when the genes are inactivated, and reassembly when the genes are activated again. To get more detail on the function of proteins identified as being important for membrane synthesis, we will identify other proteins that interact with these proteins in the cell and we will produce mutants in which these proteins are 'tagged' with fluorescent labels. This will enable us to see the distribution and behaviour of the proteins in a fluorescence microscope. One possibility is that the proteins are initially located in the cytoplasmic membrane surrounding the cells. Here they may help to collect together other membrane components required for thylakoid membrane synthesis, and package these components into 'vesicles' - small membrane bodies which could then shuttle the membrane components to the thylakoids. By observing the distribution of the fluorescent proteins during membrane reassembly we will be able to see how they are involved. If we can understand how thylakoid membranes are assembled we will be in a better position to modify thylakoid membrane function, for example to produce hydrogen from solar energy. In the long-term we may even be able to induce the production of similar membrane systems in different kinds of bacteria, giving us a new tool for the production of microbial 'cell factories'.

Cyanobacteria contain thylakoid membranes, a complex internal membrane system that is the site of the light-reactions of photosynthesis. We know rather little about how the thylakoid membranes are assembled. They may originate as invaginations of the cytoplasmic membrane, they may be a completely independent membrane system, or they may be assembled from vesicles originating from the plasma membrane. We do not know if thylakoid membranes can be assembled de novo, or if some pre-existing thylakoid membrane is always required ('membrane inheritance'). Genetic studies have identified VIPP1 as a protein likely to be specifically involved in thylakoid biogenesis in cyanobacteria and chloroplasts. The researcher co-investigator has identified other candidate proteins, including a prohibitin-like protein which is the product of the slr1768 open reading-frame in the cyanobacterium Synechocystis 6803. In the cases of both VIPP1 and slr1768, the cyanobacterial null mutants will not segregate (ie viable cells always retain at least one copy of the wild-type gene). We will generate Synechocystis mutants in which VIPP1, slr1768 and othe candidates are under the control of inducible promoters, allowing us to switch off and re-activate thylakoid biogenesis and thus enabling us to observe the degradation and reassembly of the thylakoid system. We will use biochemical approaches to identify interaction partners for canadidate thylakoid biogenesis proteins and we will generate mutants in which these proteins are GFP-tagged. Using confocal and TIRF microscopy we will be able to visualise the distribution and dynamics of these proteins at the onset of membrane biogenesis. We will be able to test the hypothesis that they are involved in microdomain formation in the cytoplasmic membrane, followed by vesicle formation. These studies will provide important background information for 'bioengineering' of thylakoid membranes for the production of biofuels, for example.