Bacterial sterol methylation confounds eukaryotic biomarker interpretations

Sterol lipids are required by most eukaryotes and are readily preserved as sterane molecular fossils. These geologic steranes are broadly interpreted as biomarkers for ancient eukaryotes1,2 although diverse bacteria also produce sterols3. Steranes with side-chain methylations can act as more specific biomarkers4 if their sterol precursors are limited to particular extant eukaryotes and are absent in bacteria. An abundance of one such sterane, 24-isopropylcholestane, in late Neoproterozoic rocks has been attributed to marine demosponges and potentially represents the earliest evidence for animals on Earth5. However, debates over this interpretation6–14 continue given the potential for alternative sources of 24-isopropylcholestane and the lack of experimental evidence demonstrating the function of enzymes that methylate sterols to give the 24-isopropyl side-chain. Here we show that sterol methyltransferases from both sponges and bacteria are functional and identify three bacterial methyltransferases each capable of sequential methylations resulting in the 24-isopropyl sterol side-chain. We identified two of these propylating enzymes in a demosponge metagenome suggesting bacterial symbionts contribute to 24-isopropyl sterol biosynthesis in demosponges. Our results demonstrate yet-uncultured bacteria have the genomic capacity to synthesize side-chain alkylated sterols and should therefore be considered when interpreting side-chain alkylated sterane biomarkers in the rock record.