Molecular mechanism of intracellular membrane biogenesis in Synechocystis sp. PCC6803

蓝藻(又称蓝藻)是一种通过光合作用生长的细菌，其生长方式与植物相似。叶绿体(植物细胞内的光合体)来自自由生活的蓝藻，这是蓝藻和叶绿体有许多相似之处的原因。蓝藻广泛存在于环境中。例如，它们在河流、湖泊和海洋中非常丰富，它们对地球的生态做出了重要贡献。蓝藻作为“生物燃料”的可能来源，现在正引起人们越来越大的兴趣。我们也许最终能够改造蓝藻来生产细胞工厂，利用太阳光的能量来生产氢气等燃料。蓝藻具有比大多数细菌更复杂的细胞结构。细胞内部是类囊体膜，这是一个复杂的内部膜系统，是光合作用“光反应”的场所。类囊体膜含有从阳光中吸收能量的色素，以及将太阳能转化为储存的化学能的第一步蛋白质。虽然我们现在知道了很多关于光合作用蛋白质的细节，但我们对类囊体膜是如何形成的知之甚少。我们建议以被认为对类囊体膜产生重要的基因作为起点来研究这个问题。目前还不可能产生完全缺乏这些基因的突变体。然而，当每个细胞的基因拷贝数减少时，类囊体膜的合成就会大大减少。虽然这些基因已经被鉴定，但我们不知道它们编码的蛋白质是如何参与类囊体膜的生成的。我们将使用一种可以很容易进行转基因的“模型”蓝藻来研究这个问题。我们将改造这种蓝藻，这样我们就可以控制这两个基因的表达：我们将能够打开和关闭蛋白质的生产。这将给我们提供一种控制类囊体膜产生的方法。我们将能够观察到当基因失活时类囊体膜的降解，以及当基因再次激活时重新组装。为了更详细地了解被确认为对膜合成重要的蛋白质的功能，我们将鉴定在细胞中与这些蛋白质相互作用的其他蛋白质，并产生用荧光标记‘标记’这些蛋白质的突变体。这将使我们能够在荧光显微镜中看到蛋白质的分布和行为。一种可能性是，蛋白质最初位于细胞周围的细胞膜中。在这里，它们可以帮助收集合成类囊体膜所需的其他膜成分，并将这些成分包装成‘囊泡’--然后可以将膜成分穿梭到类囊体膜上的小膜体。通过观察膜重组过程中荧光蛋白的分布，我们将能够了解它们是如何参与的。如果我们能够了解类囊体膜是如何组装的，我们就可以更好地改变类囊体膜的功能，例如从太阳能中生产氢气。从长远来看，我们甚至能够在不同种类的细菌中诱导产生类似的膜系统，这给了我们一种生产微生物“细胞工厂”的新工具。

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'.