Molecular mechanism of intracellular membrane biogenesis in Synechocystis sp. PCC6803

Lead Research Organisation: Queen Mary, University of London
Department Name: Sch of Biological and Chemical Sciences


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'.

Technical Summary

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.
Description Cyanobacteria are bacteria that live by photosynthesis. They are extremely important for global ecology, they have potential for solar-powered production of biofuels and other products, and in addition they share many characteristics with plant chloroplasts, to which they are evolutionarily related. Therefore they are a useful model system for studying the many aspects of photosynthesis that are common to chloroplasts and cyanobacteria. Like chloroplasts, but unlike most other bacteria, cyanobacteria have a complex internal membrane system, the thylakoid membranes that are the site of the photosynthetic light reactions. There are many unresolved questions concerning the biogenesis of the thylakoid membranes and the photosynthetic complexes that they contain. A number of genes have been identified that seem to be important for thylakoid membrane biogenesis, since mutants lacking these genes are deficient in thylakoid membrane structure and/or function. We set out to understand how the proteins encoded by these genes function, using a new approach based on creating mutants in which the native gene is replaced by a gene that codes for the protein linked to an artificial fluorescent protein tag. If the tagged protein is still assembled and functional, we can then use fluorescence microscopy to observe the protein in action in live cells, allowing us to test different models for the way in which the protein acts. We focused in particular on a protein called Vipp1, which has been suggested to directly required for the biogenesis of the thylakoid membrane system in cyanobacteria and chloroplasts. We created strains in which Vipp1 is tagged with Green Fluorescent Protein (GFP) in two species of cyanobacteria. We used two different species to provide confirmation that the effects we saw were generally important, and also because the species we chose have different cell shapes. We found that, with fluorescence microscopy, some effects can be more easily observed in quantified in bacteria with a particular cell shape, and therefore it is useful to employ species with a range of cell shapes. In both species of cyanobacteria, we found that the Vipp1 protein could be tagged with GFP without significantly impairing protein function. Observation with the fluorescence microscope showed something unexpected: when we grow cells at low light intensities Vipp1 is dispersed in the cytoplasm of the cell. However, exposure of cells to high light triggers a dramatic change in the behaviour of Vipp1: over a time-scale of 5 to 30 minutes it forms concentrated clusters in the cytoplasm and close to the membrane. Vipp1 behaves in a
similar way when cells are exposed to other stresses, such as mineral deprivation. Vipp1 function appears to be particularly important under high light stress, which suggests that the formation of Vipp1 clusters is key to its role in the cell. Careful observation of the behaviour of the Vipp1 clusters ruled out one possibility, that they are involved in transport of material from the cytoplasmic membrane to the thylakoid membranes. To get a better idea of their role, we isolated the Vipp1 clusters from cells that had been exposed to high light. We then identified the other proteins found in the Vipp1 clusters. We found a large collection of proteins associated with Vipp1 under these conditions, including components of the photosynthetic apparatus, other stress-related proteins, and a diverse collection of proteins required for the protein synthesis and complex assembly.

These findings suggest a new model for Vipp1 action: that it is required to co-ordinate the formation of emergency protein synthesis centres, required for the rapid production of new proteins under stress conditions. Since proteins related to Vipp1 are found in many non-photosynthetic bacteria such as E. coli, we may have identified a stress defence mechanism that is widespread in bacteria. Future work will test this possibility.

A second major research output concerns the localisation and activity of the bidirectional hydrogenase in cyanobacteria. This enzyme has attracted biotechnogical interest as it potentially provides a route to efficient solar-powered biological hydrogen generation. Our work establishes a specific thylakoid membrane localisation of the hydrogenase, and shows that this localisation is important for physiological electron supply, and is under physiological control.
Exploitation Route Our work on Vipp1 and other thylakoid membrane biogenesis factors makes a significant contribution to understanding cyanobacterial thylakoid membrane generation, currently the subject of intense research.

If our current model of Vipp1 action proves to be correct, it will have considerable implications for the possible modes of action of related proteins such as PspA, which are widespread in non-photosynthetic bacteria and appear important for stress protection in many situations, including infection.

Our work on the localisation and activity of the cyanobacterial hydrogenase will be important for all those trying to optimise the efficiency of solar-powered biohydrogen production.
Sectors Energy,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

Description So far, one aspect of our work has attracted biotechnological attention: this is our work on electron transport switches in cyanobacteria that depend on the spatial distribution of electron transport complexes. We are currently in discussion with a bioenergy firm with a view to testing mutants for their potential for solar-powered biofuel production.
First Year Of Impact 2012
Sector Energy
Impact Types Economic

Description ScyCode
Amount € 2,500,000 (EUR)
Funding ID ScyCode 
Organisation German Research Foundation 
Sector Charity/Non Profit
Country Germany
Start 01/2019 
End 12/2021