Ladderane Lipids in Anammox Bacteria: Occurence, Biosynthesis and Application as Environmental Markers

Abstract

Ladderane lipids are unusual membrane lipids of anammox bacteria, which contain either three or five cyclobutane rings, and are so far unique in nature. Anammox bacteria are recently identified members of the nitrogen cycle, with the ability to combine ammonium with nitrite to form dinitrogen gas, in waters or sediments containing little or no oxygen. In the natural environment they have been found in many different oxygen poor environments. For the first time, an anammox bacterium from the natural environment was successfully cultured from inoculum sediment from a Swedish fjord. The enrichment culture of “Candidatus Scalindua spp.” was shown to contain key features of anammox bacteria, including a compartmentalised cellular ultrastructure and ladderane lipids. The versatile metabolism of these anammox bacteria is thought to be a factor determining the widespread global distribution of the Scalindua genera in marine anoxic ecosystems. Ladderane lipids were applied to establish the presence of anammox bacteria and that the anammox reaction is a major process in nitrogen cycling in the Peruvian oxygen minimum zone (OMZ), supporting the idea that the anammox reaction plays a major role in the global cycling of nitrogen. Using novel high performance liquid chromatography (HPLC)/mass spectrometry (MS/MS) techniques, the ladderane core lipid and phospholipid composition of four different species of anammox bacteria was analysed. Ladderane phospholipids contained different combinations of hydrophobic tail types in combination with different types of polar headgroup. It was also found that ladderane lipids are not solely exclusive membrane lipids of the anammoxosome, and must be incorporated into other membranes in the anammox cell. It was also demonstrated that anammox bacteria produce shorter chained ladderane lipids under colder conditions, and vice versa in anammox biomass grown in enrichment cultures, in particulate organic matter and in surface sediments from a wide range of marine environments. To quantify the relationship between the C18 and C20 ladderane lipids containing five cyclobutane rings, the index of ladderane lipids with five cyclobutane rings (NL5) was calculated. When plotting the NL5 against in situ temperature a significant relationship was observed, best approximated by a 4th order sigmoidal curve (R2= 0.92, p=<0.0001, n=158). The NL5 can be used to discriminate between the origins of ladderane lipids in marine surface sediments and may have further application as a paleo-temperature proxy. The biosynthetic route of ladderane lipids was investigated using a comparative genomics analysis of K. stuttgartiensis and a wide range of bacterial and archaeal genomes. Genes were found to encode a new pathway for the anaerobic biosynthesis of polyunsaturated hydrocarbons, which could be subsequently folded into ladderane lipids. However, subsequent results of a 2_13C acetate labelling experiments on biomass of “Candidatus Brocadia fulgida”, indicated that the labelling pattern of the n-octyl chain used the type II fatty acid biosynthetic route. However, the labelling pattern of carbon atoms in the cyclobutane and cyclohexane moieties of the ladderane group did not correspond to known patterns of fatty acid synthesis. This suggests that a novel but as yet unknown route of biosynthesis is used for ladderane synthesis.