Sterol methyltransferases in uncultured bacteria complicate eukaryotic biomarker interpretations

Abstract

Sterane molecular fossils are broadly interpreted as eukaryotic biomarkers, although diverse bacteria also produce sterols. Steranes with side-chain methylations can act as more specific biomarkers if their sterol precursors are limited to particular eukaryotes and are absent in bacteria. One such sterane, 24-isopropylcholestane, has been attributed to demosponges and potentially represents the earliest evidence for animals on Earth, but enzymes that methylate sterols to give the 24-isopropyl side-chain remain undiscovered. Here, we show that sterol methyltransferases from both sponges and yet-uncultured bacteria function in vitro and identify three methyltransferases from symbiotic bacteria each capable of sequential methylations resulting in the 24-isopropyl sterol side-chain. We demonstrate that bacteria have the genomic capacity to synthesize side-chain alkylated sterols, and that bacterial symbionts may contribute to 24-isopropyl sterol biosynthesis in demosponges. Together, our results suggest bacteria should not be dismissed as potential contributing sources of side-chain alkylated sterane biomarkers in the rock record.

Fig. 1. Sterol methyltransferases from sponges and yet-uncultured bacteria perform multiple side-chain methylations in vitro.

a Structure of the sponge biomarker 24-isopropylcholestane. b Predominant pathway to 24-isopropyl sterols by bacterial sterol methyltransferases (SMTs). c Representative extracted ion chromatograms (m/z 456, 470, 484, 498) of total lipid extracts from in vitro reactions performed with desmosterol and E. coli lysates containing an empty plasmid, an SMT identified in a transcriptome of the demosponge Chondrilla nucula, a bacterial SMT identified in a metagenome from the demosponge Aplysina aerophoba, a bacterial SMT identified in a freshwater metagenome-assembled genome (MAG) assigned to the phylum Chlamydiae, and a myxobacterial SMT identified in a marine MAG assigned to the genus Sandaracinus. d Representative extracted ion chromatograms (m/z 456, 470, 484, 498) of total lipid extracts from in vitro reactions performed with the same lysates but with 24-methylenecholesterol as the substrate. e Side-chain structures of sterols identified in c and d. All lipids were derivatized to trimethylsilyls prior to GC-MS analysis. The raw data used to generate this figure are provided in Supplementary Data 2. Mass spectra of identified sterols are shown in Supplementary Fig. 3.

Fig. 2. Sterols identified as products of sponge and bacterial sterol methyltransferase in vitro reactions.

Sterols were detected using GC-MS. Products of in vitro reactions performed with both desmosterol and 24-methylenecholesterol as substrates are included. C₂₈ sterols include (epi)codisterol (II), 24-methylenecholesterol (III), and 24-methyldesmosterol (V). C₂₉ sterols include (epi)clerosterol (IV), fucosterol (VI), and isofucosterol (VII). C₃₀ sterols include 24S−24-isopropylcholesta-5,25-dienol (VIII) and 24-isopropylcholesta-5,24-dienol (IX). Metagenomic SMTs identified in gene clusters with at least two other sterol biosynthesis homologs are bolded. *NMR confirmed epiclerosterol and not clerosterol as products of the “Aplysina aerophoba JGIcombinedJ30088_10000835 1 of 2” and “Chlamydiae sp., drinking water 1 of 2” sterol methyltransferases. **24-isopropylcholesta-5,23-dienol was also detected as a product of the “Chlamydiae sp., drinking water 1 of 2” sterol methyltransferase by NMR, but was not detected by GC-MS.

Fig. 3. Gene clusters suggest de novo side-chain alkylated sterol biosynthesis in the bacterial domain.

a Gene clusters identified in metagenomes and metagenome-assembled genomes (MAGs) containing three or more sterol biosynthesis homologs and including functional sterol methyltransferases (bold). Genes are named for their Saccharomyces cerevisiae (erg) or Methylococcus capsulatus Bath (sdmAB) homologs. Dotted sterol methyltranferases did not methylate desmosterol or 24-methylenecholesterol in vitro. Striped patterns correspond to the number of methylations performed by each sterol methyltransferase in vitro. Gradients indicate partial sequences. b Schematic of a hypothetical bacterial side-chain alkylated sterol biosynthesis pathway. Arrow colors at each step correspond to the homologs identified in a. Detailed information on the source of each metagenomic gene cluster is available in Supplementary Table 4. Homology for each gene is described in Supplementary Data 1.

Fig. 4. Functional metagenomic SMTs are bacterial.

a Maximum likelihood tree generated from a concatenated alignment of the conserved methyltransferase and C-terminal domains of sterol methyltransferase (SMT) proteins. Metagenomic SMTs are in blue, sponge SMTs are in red, and other eukaryotic SMTs are in black. Bolded labels indicate SMTs tested in this study, and the number of circles corresponds to the number of carbons the SMT added to desmosterol at the C-24 position in vitro. The numbers in parentheses indicate the number of proteins in collapsed clades. b Maximum likelihood tree of oxidosqualene cyclases (OSCs). OSC groups are assigned as in ref. . Bolded labels indicate OSCs in metagenome gene clusters or metagenome-assembled genomes with SMTs tested in this study.