Discovery of a cryptic sphingolipid pathway in E.coli - structural and functional analysis.

A very important, large family of biological molecules are called lipids. They include fats and steroids such as cholesterol. Another important sub-family are known as sphingolipids (SLs) and ceramides (which are like SLs with two tails). All these lipids are found in the cell membrane - scientists have found that animal, plants and bacterial cells have a protective, water-resistant outer shell that is composed of molecules with a water-loving (hydrophilic) head group and a long, water-hating (hydrophobic) tail. It is these molecules that provide that layer. However, they don't just have a structural role - they have been shown to be important when cells divide and when cells communicate with each other. There is a high turnover of lipids in the every cell, they are constantly being made and broken down. This is tightly controlled. In particular, changes in SL levels are strongly linked with old age and diseases such as Alzheimer's, Parkinson's Disease, diabetes, asthma, cancer and nerve-wasting diseases. It is rare to find molecules made by both plants, animals and bacteria; SLs and ceramides are exactly that - they are very large family of 100s of molecules, each slightly different - they contain amino acids, fatty acids and sugars. However, the core structures are the same. An exciting area of research with direct implications for human health is the discovery that humans are hosts for many different types of bacteria - collectively these are known as the microbiota/microbiome. These bacteria live in our mouths, on our skin and in our gut and help us metabolise our food and are also thought to play protective roles. They keep us healthy; so we have to understand when is a bacteria good and when is a bacteria bad - pathogenic? What are the chemical triggers?
Every cell make SLs by a multi-step pathway using simple building blocks - the steps are catalysed (sped up) by molecular machines called enzymes. In recent years, research has focussed on the enzymes involved in human SL biosynthesis but very little is known about how microbes make them. We made a breakthrough when we teamed up with American scientists to reveal that a simple, safe Caulobacter bacterium that lives in fresh water can make the same core SLs as we can, but it makes them through a different route - that's called convergent evolution. We then used genetics to look at the DNA of other bacteria - what we thought to present in a small number of microbes is more much more widespread. We have even found them in E. coli - a very common bacteria that can be good and bad. Scientists have used E. coli for many years because they are safe and easy to grow, easy to engineer and we have a blue-print of how they work. Now we have made an exciting discovery that E. coli make SLs we want to understand the molecular details of the process - we will study the enzymes involved. We will determine the 3D structure of the key SPT enzyme and how it engages with a lipid carrier. We will explore how the two lipids chains of SLs are installed. We will also grow E.coli in specially marked building blocks and that will reveal how the core molecules are made. This is a team effort with UK and USA scientists each bringing their own expertise to this project. We will use our skills as chemists, microbiologists and molecular biologists to uncover the secrets that have been hidden in E. coli until now. Our results will be of interest to academic microbiologists and chemists as well as those interested how molecules evolved. We are building a inventory called Lipid Maps of all the important lipid molecules in Nature. Because E. coli has been a model microbe for >50 years, it is rare to find something new - so it is exciting to work in this area.

Sphingolipids (SLs) and ceramides play essential roles in membrane structure and cell signalling in yeast, plants, mammals. Levels of these lipids are tightly controlled and unbalanced SL levels are now linked to many diseases such as Alzeimer's. Parkinson's Disease, diabetes and asthma. It was though that only a certain small group bacteria have the ability to make SLs but breakthrough studies have shown that microbes from the human microbiota (Bacteroides, P. gingivalis) have been shown to produce SLs that mediate interactions with host cells. In contrast to higher eukaryotes, very little is known about microbial SL biosynthesis, regulation and transport. To fully understand the molecular details we need model microbial systems to lay down the road map of the genes and enzymes involved. In collaboration with groups in the USA we recently showed that strains of the fresh water environmental organism Caulobacter crescentus can make SLs and ceramide - we found that they make these through a different route with novel enzymes - an example of convergent evolution. Using bioinformatics we discovered the same SL core genes are conserved in many microbes including the classic molecular biology workhorse E. coli. In this new project we will study the core enzymes involved in making SLs in E. coli. Despite being the bacterium used by 1000s of academic and industrial labs for >50 years, approximately 25% of the genes (~1000) in E.coli have still no annotated function. We found the cryptic SL genes by sequence homology and we'll use chemical, biochemical and structural tools to determine the molecular details of the pathway in E. coli. We will use enzyme assay, analytical chemistry, chemical cross-linking, X-ray crystallography, labelling, mutagenesis to deliver our aim to map bacterial SL biosynthesis. Future studies in pathogenic E.coli (still a cause of many deaths) will uncover their roles in cellular function. We expect our work to have impact in academia and industry.