Elucidation of the bacterial sphingolipid biosynthetic pathway in Sphingomonas wittichii

细胞是由膜组成的，这些膜由称为“脂质”的化学物质组成-这些包含疏水（讨厌水）和亲水（喜欢水）部分。细胞膜必须很坚固，以保持细胞内容物，但也能够让分子（营养物质，金属，盐）-以及保持有毒物质和排出废物。它们还必须阻止水通过增加渗透压而涌入和破坏细胞。细胞已经进化出具有不同化学成分的不同膜。哺乳动物有复杂的膜，它们产生100多种不同的脂质。同样，酵母、植物和病毒也有物种特异性的脂质。细菌也有独特和不寻常的脂质-它们在免疫反应和炎症中也起着重要作用。哺乳动物已经进化到将自己的脂质识别为“自我”，但可以熟练地检测来自致病细菌，真菌和病毒的外来脂质。一旦被检测到，哺乳动物细胞可以发动有效的免疫反应来杀死入侵者。这就引出了一个问题，如果一种细菌进化到拥有与人类相似的脂质，我们如何区分它们？更仔细地观察脂质本身，我们的项目将集中在一个特殊的分支有趣的脂质称为“鞘脂”。早在100多年前，约翰·图姆就在人类大脑中发现了它们，他知道它们在大脑化学中起着重要作用。直到20世纪30年代，赫伯特卡特才发现鞘脂的化学性质--一个极性的水溶性头部和一个非极性的脂肪酸尾部。它们被发现由常见的氨基酸L-丝氨酸和长碳链（>C16）组成。长期以来，科学家们一直想知道鞘脂是如何在细胞内从普通的构建模块中产生，然后运输到外部的-当细胞快速生长和分裂时，这必须非常迅速地发生。此外，鞘脂是危险的-太多或太少的一个细胞可以是致命的，所以数量是微妙的控制方式，我们仍然不完全了解。为了揭示化学细节并探索所涉及的酶，我们和其他科学家正在研究人类，植物，酵母和细菌中的鞘脂生物合成。我们选择了一种有趣的细菌鞘氨醇单胞菌，因为它对人类无害-事实上它可以将毒素降解为无害分子。这些鞘氨醇单胞菌是非常不寻常的，因为它们制造的鞘脂在某种程度上类似于我们自己的鞘脂。我们将探索鞘氨醇单胞菌如何通过仔细表征编码进行丝氨酸和脂肪酸初始转化的酶的基因，通过复杂的第2和第3步，以及其他步骤来制造鞘脂。我们得到了帮助，因为美国能源部已经对鞘氨醇单胞菌的基因组进行了测序，并预测它有超过5000个基因。然而，我们不知道哪些参与了鞘脂的生物合成。我们将使用化学，生物化学，遗传和分子生物学方法来帮助我们理解每一步。我们已经有了一个开始，发现了一种不寻常的小蛋白质（约80个氨基酸长），我们认为它与鞘脂和脂肪酸的生物合成有关。大部分工作将在爱丁堡进行，但我们也将与圣安德鲁斯的吉姆奈史密斯合作，他可以确定蛋白质的三维结构，以及美国的遗传学专家特蕾莎邓恩。我们的团队合作将使我们领先于竞争对手。到资助结束时，我们将确定细菌鞘脂生物合成的基本路线图，并能够开始将其与人类，植物和酵母的路线图进行比较。我们将深入了解这些物种是如何进化到制造相同的鞘脂的，并开始了解每个物种如何控制每个细胞中的数量。在我们开展这项工作的同时，我们将确保向专家和公众举办研讨会，告诉他们我们的发现，并将在高评级的国际期刊上发表，这将有利于英国科学。

Cells are made of membranes which are composed of chemicals called 'lipids' - these contain hydrophobic (water hating) and hydrophilic (water loving) parts. Membranes have to be strong to keep cell contents in but also be able to let molecules in (nutrients, metals, salts) - as well as keeping toxic materials out and expelling waste. They must also stop water flooding in and bursting the cell by increased osmotic pressure. Cells have evolved different membranes with different chemical composition. Mammals have complicated membranes and they generate 100s of different lipids. Similarly, yeast, plants and viruses have species-specific lipids. Bacteria too have unique and unusual lipids - they also play important roles in the immune response and inflammation. Mammals have evolved to recognise their own lipids as 'self' but can expertly detect foreign lipids from pathogenic bacteria, fungi and viruses. Once detected, the mammalian cell can mount an effective immune response to kill the invader. This then begs the question, if a bacterium has evolved to have lipids similar to a human's - how do we tell them apart? Looking more closely at the lipids themselves our project will focus on a special branch of interesting lipids called 'sphingolipids'. They were discovered >100 years ago in human brains by John Thudichum who knew that they played an important role in brain chemistry. It took until the 1930s for Herbert Carter to work out the chemistry of the sphingolipids - a polar, water soluble head and a fatty acid non-polar tail. They were found to be made from the common amino acid L-serine and a long carbon (>C16) chain. Scientists have long wondered about how sphingolipids are made inside the cell from common building blocks and then transported to the outside - this must happen very quickly when the cells are rapidly growing and dividing. Also, sphingolipids are dangerous - too many or too little in one cell can be lethal so the amounts are delicately controlled in a way we still don't fully understand. To uncover the chemical details and explore the enzymes involved we and other scientists are studying sphingolipid biosynthesis in humans, plants, yeast and bacteria. We have chosen an interesting bacterium Sphingomonas wittichii because it is not harmful to man - in fact it can degrade toxins to harmless molecules. These Sphingomonas are highly unusual because they make sphingolipids that resemble our own to some extent. We will explore how Sphingomonas makes sphingolipids by carefully characterising the genes that encode the enzymes that carry out the initial conversion of serine and the fatty acid, through the complex 2nd and 3rd steps, and beyond. We are helped because the Department of Energy (USA) have already sequenced the Sphingomonas wittichii genome and predict it to have >5000 genes. However, we do not know which ones are involved in sphingolipid biosynthesis. We will use chemical, biochemical, genetic and molecular biology methods to help us understand each step. We have already made a start and found an unusual small protein (~80 amino acids long) that we think links sphingolipid and fatty acid biosynthesis. Most of the work will be carried out in Edinburgh but we will also work with Jim Naismith in St.Andrews who can determine the 3D structure of a protein, as well as a genetics expert in the USA, Teresa Dunn. Our teamwork will put us ahead of our competitors. By the end of the grant we will have determined the basic roadmap of bacterial sphingolipid biosynthesis and be able to begin to compare it with the map in humans, plants and yeast. We'll obtain insight into how these species evolved to make the same sphingolipid and begin to understand how each controls the amount in each cell. Whilst we carry out the work we will make sure we give seminars to experts and the general public telling them what we've found out and will also publish in highly-rated international journals that will benefit UK science.