Uncovering the biosynthetic pathway and function of unusual aminolipids found in the marine heterotrophic bacterium Ruegeria Pomeroyi

Aminolipids are a widespread class of lipids present in several different bacterial taxa, but our understanding of their structures and role in microbial physiology is rather limited. The first evidence of the presence of aminolipids in bacteria dates back to the 1960s and 1970s, where some non-phosphorus (P) containing lipids, lacking a diacylglycerol backbone, were identified. More recent studies showed that aminolipids are widespread in different environments and sometimes they can constitute a strategy for bacteria to deal with nutrient deficiency, changes in temperature or pH. In marine environments, previous studies identified a few examples of aminolipids in Ruegeria pomeroyi DSS-3 including glutamine and ornithine aminolipids both of which were abundant when the bacterium was limited for key nutrients e.g. P. A comprehensive analysis of the lipid profile was conducted in this study (from both Ruegeria pomeroyi and Phaeobacter inhibens DMS17395) revealing that some unknown aminolipids appeared to be constitutively produced regardless of P concentrations. My PhD project thus aimed to elucidate the structure of these aminolipids, to identify the genes involved in their biosynthesis and to study their physiological roles and their regulation. The first aim (Chapter 3) was achieved using liquid chromatography coupled with high-resolution mass spectrometry. The new lipids were identified as a new class of sulfur-containing aminolipids (SALs), where the polar head group has either an amine or a sulfonate group. Several approaches were then used to elucidate the biosynthetic pathway for these novel lipids (Chapter 4) and one of the enzymes involved in the biosynthesis of this lipid – an acyltransferase, involved in the addition of the fatty acid chain to the polar head group was identified. Possible candidate genes involved in SAL synthesis were identified through comparative genomics between strains where these lipids were found (‘producers’) and the ones where they were absent (‘non-producers’). Subsequent knock-out mutagenesis and screening of a mutant library in P. inhibens were the key to identify the salA gene which was essential in SAL synthesis. SalA contains a LPAAT (lysophosphatidic acid acyltransferase) domain and appears to be common in bacteria of the Rhodobacteraceae family. Chapter 5 investigated how the production of SALs and its hydroxylation are affected by key parameters, including growth stage, nutrient limitation, temperature, and pH. In Chapter 6, the physiological and ecological role of SAL was investigated by analysing the distribution of SALs between the inner and outer membrane. The role of SAL in biofilm formation and interactions with protist grazers was also investigated using Phaeobacter inhibens and a salA mutant as the model. To summarise, this work presents structural elucidation of a new sulfurcontaining aminolipid and provides new insights into the physiological and ecological role of SALs in marine roseobacter clades