Processing of cell surface lipoproteins in Streptomyces coelicolor. A new paradigm?

细菌可分为两大类：革兰氏阴性菌有两层膜包裹细胞，即细胞质膜和外膜。在这些膜之间也有一个叫做周质的空间。通过细胞膜输出的蛋白质通常最终进入周质。革兰氏阳性细菌只有一层膜，被厚厚的细胞壁包围。跨膜输出的蛋白质必须固定在膜的外表面或细胞壁上，以避免完全从细胞中丢失。这些细胞表面蛋白充当与外部世界的接触点，参与许多基本过程，包括感知环境变化、蛋白质折叠、呼吸和营养物质清除。它们在表面附着中也起着重要作用，对于宿主成功感染致病细菌是必不可少的。细菌将蛋白质附着到膜上的主要方式之一是将脂分子附着在蛋白质的N端。被广泛接受的模型认为，这些‘脂蛋白’首先通过蛋白质N-末端的一个信号序列靶向SEC(分泌)途径。SEC将它们穿过细胞质膜运输到细胞外，在那里一种名为LGT的酶连接脂质分子。然后，信号序列被一种名为LSP的酶切割。在革兰氏阳性细菌中，这条途径在这里结束，蛋白质附着在膜的外表面。在革兰氏阴性细菌中，这些蛋白质有另一种脂分子，被一种名为LNT的酶附着，这使得它们能够被运输到外膜。只有一小部分保留在内膜中。在革兰氏阴性细菌中，所有参与脂蛋白生物合成的酶都是必不可少的，可能是因为去除其中任何一种酶都会导致脂蛋白在内膜积累并破坏稳定。在革兰氏阳性细菌中，它们对许多致病微生物的毒力是必不可少的。因此，它们是新抗菌药物的极佳靶点，如LSP的强力抑制剂球霉素。我们一直在研究革兰氏阳性菌天蓝色链霉菌的脂蛋白生物合成，并发现了一些令人惊讶的差异，这些差异可能会挑战广泛接受的模型。以前，人们认为所有的脂蛋白都是由SEC输出的，但天蓝色链霉菌的情况并非如此。我们有很好的证据表明，一些天蓝色链霉菌脂蛋白可以通过TAT分泌途径输出。SEC运输线性蛋白质，而TAT通常用于运输在细胞内组装的完全折叠的蛋白质。这对目前的模型提出了挑战，它还表明LGT酶可以将脂类附着到线状和完全折叠的蛋白质上。天蓝色乳杆菌的不同寻常之处在于它含有两种LGT酶，其中一种可能作用于SEC-，另一种作用于TAT依赖的脂蛋白。最后，也许最令人费解的是，天蓝色链霉菌含有两种LNT酶。这种酶执行一个以前被认为是革兰氏阴性细菌独有的步骤。由于革兰氏阳性菌没有外膜，天蓝色葡萄球菌中这些酶的功能尚不清楚。在这项研究中，我们将研究TAT和SEC依赖的脂蛋白在输出和修饰方面的差异。我们将研究这两种LGT酶在这些加工途径中的功能。我们还将测试LNT酶的活性，以确定它们在革兰氏阴性细菌中是否具有与LNT相同的功能。我们预计，这项研究的结果将导致我们对细菌脂蛋白生物合成的理解发生范式转变。

Bacteria can be divided into two main groups; Gram negative bacteria have two membranes surrounding the cell, the cytoplasmic membrane and the outer membrane. They also have a space between these membranes called the periplasm. Proteins which are exported across the cytoplasmic membrane typically end up in the periplasm. Gram positive bacteria have only a single membrane, surrounded by a thick cell wall. Proteins which are exported across the membrane must be anchored to the outer surface of the membrane, or the cell wall, to avoid being lost entirely from the cell. These cell surface proteins act as a point of contact with the outside world and are involved in a number of essential processes including sensing environmental changes, protein folding, respiration and nutrient scavenging. They also have an important role in surface attachment and are essential for successful host infection in disease-causing bacteria. One of the main ways in which bacteria attach proteins to their membranes is by attaching a lipid molecule to the N-terminal end of the protein. The widely accepted model suggests that these 'lipoproteins' are first targeted to the Sec (secretion) pathway by a signal sequence at the N-terminus of the protein. Sec transports them across the cytoplasmic membrane to the outside of the cell where an enzyme called Lgt attaches the lipid molecule. The signal sequence is then cleaved by an enzyme called Lsp. In Gram positive bacteria the pathway ends here and the proteins are attached to the outer face of the membrane. In Gram negative bacteria these proteins have another lipid molecule attached by an enzyme called Lnt and this allows them to be transported to the outer membrane. Only a small proportion is retained in the inner membrane. In Gram negative bacteria all the enzymes involved in lipoprotein biosynthesis are essential, probably because removing any one of them results in lipoproteins accumulating in, and destabilising, the inner membrane. In Gram positive bacteria they are essential for the virulence of many disease-causing organisms. They therefore represent an excellent target for new antibacterial drugs such as globomycin, a strong inhibitor of Lsp. We have been studying lipoprotein biosynthesis in the Gram positive bacterium Streptomyces coelicolor, and have discovered some surprising differences which could challenge the widely accepted model. Previously it was assumed that all lipoproteins were exported by Sec but this is not the case in S. coelicolor. We have good evidence that some S. coelicolor lipoproteins can be exported by the Tat secretion pathway. While Sec transports linear proteins, Tat is typically used to transport fully folded proteins which are assembled inside the cell. This challenges the current model and it also suggests that the Lgt enzyme can attach lipids to both linear and fully folded proteins. S. coelicolor is unusual in that it contains two Lgt enzymes and it is possible that one acts on Sec-, and the other on Tat-dependent lipoproteins. Finally, and perhaps most puzzlingly of all, S. coelicolor contains two Lnt enzymes. This enzyme performs a step previously thought to be unique to Gram negative bacteria. Since Gram positive bacteria do not have an outer membrane the function of these enzymes in S. coelicolor are unclear. In this study we will investigate the differences in the export and modification of Tat- and Sec-dependent lipoproteins. We will examine the functions of the two Lgt enzymes in these processing pathways. We will also test the activities of the Lnt enzymes to see if they have the same function as Lnt in Gram negative bacteria. We anticipate that the results of this study will lead to a paradigm shift in our understanding of bacterial lipoprotein biosynthesis.