Bacterial lipids are well-preserved in ancient rocks and certain ones have been used as indicators of specific bacterial metabolisms or environmental conditions existing at the time of rock deposition. Here we show that an anaerobic bacterium produces 3-methylhopanoids, pentacyclic lipids previously detected only in aerobic bacteria and widely used as biomarkers for methane-oxidizing bacteria. Both Rhodopila globiformis, a phototrophic purple nonsulfur bacterium isolated from an acidic warm spring in Yellowstone, and a newly isolated Rhodopila species from a geochemically similar spring in Lassen Volcanic National Park (USA), synthesized 3-methylhopanoids and a suite of related hopanoids and contained the genes encoding the necessary biosynthetic enzymes. Our results show that 3-methylhopanoids can be produced under anoxic conditions and challenges the use of 3-methylhopanoids as biomarkers of oxic conditions in ancient rocks and as prima facie evidence that methanotrophic bacteria were active when the rocks were deposited.

Eight species of heliobacteria have had their genomes sequenced. However, only two of these genomes have been analyzed in detail, those from the thermophilic Heliomicrobium (Hmi.) modesticaldum and the alkaliphilic Heliorestis (Hrs.) convoluta. Here we present analyses of the draft genome sequence of a species of heliobacterium that grows optimally at a moderate temperature and neutral pH. The organism, Heliophilum (Hph.) fasciatum, is phylogenetically unique among cultured heliobacteria and was isolated from rice soil, a common habitat for heliobacteria. The Hph. fasciatum genome contains 3.14 Mbp—similar to that of other reported heliobacteria—but has a G+C base ratio that lies between that of Hmi. modesticaldum and Hrs. convoluta. Many of the genomic features of Hmi. modesticaldum and Hrs. convoluta, such as the absence of genes encoding autotrophic pathways, the presence of a superoperonal cluster of photosynthesis-related genes, and genes encoding endospore-specific proteins, are also characteristic of the Hph. fasciatum genome. However, despite the fact that Hph. fasciatum is diazotrophic, classical nif genes encoding the alpha and beta subunits of dinitrogenase (nifDK) present in other heliobacteria could not be identified. Instead, genes encoding several highly divergent NifDK homologs were present, at least one of which likely encodes a functional dinitrogenase and another a methylthio-alkane reductase (MarDK) for sulfur assimilation. A classical NifH (dinitrogenase reductase) homolog was also absent in Hph. fasciatum, but a related protein was identified that likely carries out this function as well as electron delivery to MarDK. The N₂-fixing system of Hph. fasciatum is therefore distinct from that of other heliobacteria and may have unusual properties.

Bacterial lipids are well preserved in ancient rocks and certain ones have been used as indicators of specific bacterial metabolisms or environmental conditions existing at the time of rock deposition. Here we show that an anaerobic bacterium produces 3-methylbacteriohopanepolyols (3-MeBHPs), pentacyclic lipids previously detected only in aerobic bacteria and widely used as biomarkers for methane-oxidizing bacteria. Both Rhodopila globiformis, a phototrophic purple nonsulfur bacterium isolated from an acidic warm spring in Yellowstone, and a newly isolated Rhodopila species from a geochemically similar spring in Lassen Volcanic National Park (USA), synthesized 3-MeBHPs and a suite of related BHPs and contained the genes encoding the necessary biosynthetic enzymes. Our results show that 3-MeBHPs can be produced under anoxic conditions and challenges the use of 3-MeBHPs as biomarkers of oxic conditions in ancient rocks and as prima facie evidence that methanotrophic bacteria were active when the rocks were deposited.

Biomarkers are membrane lipids that can record microbial inputs to the sedimentary rock record and can provide a longstanding record of microbial life on Earth. Hopanoids, cyclic triterpenoid membrane lipids, are some of the most abundant bacterial lipid biomarkers on Earth. Previously, we identified the production of 3-methylhopanoids (3-MeBHPs) in Rhodopila sp. LVNP - a novel purple non-sulfur (PNS) anoxygenic phototroph we isolated from an acidic (pH 3.9) sulfidic spring in Lassen Volcanic National Park. We also detected 3-MeBHPs in a closely related microbe (Rhodopila globiformis) isolated from a similar spring in Yellowstone National Park (pH 3.4). We went back to the environments from which these cultures were isolated and performed lipid analyses on microbial consortia and sediments in situ to link the laboratory studies to source organisms in the natural environment. We used liquid chromatography mass spectrometry (LC-MS) analyses to characterize the intact bacteriohopanepolyol (BHP) profiles, 16S rRNA microbial community composition, and metagenomic evidence of methylated hopanoid production potential in the two acidic springs. Using the combination of these techniques, we detected a multitude of novel source organisms for both 3-MeBHPs and 2-methylhopanoids (2-MeBHPs). 3-MeBHPs are traditionally associated with oxygen requiring methanotrophic and acetic acid bacteria and have long been considered a signal for aerobic methanotrophy in the rock record. 2-MeBHPs are traditionally associated with cyanobacteria and the origins of oxygenic photosynthesis. Previous studies have found organisms that are exceptions to this but are considered not environmentally relevant outside of laboratory studies. Our work shows production of these geologically important lipid biomarkers in an anoxic natural environment where no cyanobacteria and no methanotrophs are present. This work combines laboratory studies and environmental analyses, which led to the discovery of new source organisms for methylated hopanoids. New evidence of non-traditional source organisms in these anoxic environments may have implications for re-interpretations of the sedimentary rock record.