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Vitamin B12-Dependent Biosynthesis Ties Amplified 2-Methylhopanoid

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Vitamin B12-Dependent Biosynthesis Ties Amplified 2-Methylhopanoid Vitamin B12-dependent biosynthesis ties amplified 2- methylhopanoid production during oceanic anoxic events to nitrification Felix J. Ellinga,1, Jordon D. Hemingwaya, Thomas W. Evansb, Jenan J. Kharbusha,c, Eva Spieckd, Roger E. Summonsb, and Ann Pearsona aDepartment of Earth and Planetary Sciences, Harvard University, Cambridge, MA 02138; bDepartment of Earth, Atmospheric, and Planetary Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139; cDepartment of Earth and Environmental Science, University of Michigan, Ann Arbor, MI 48109; and dDepartment of Microbiology and Biotechnology, University of Hamburg, 22609 Hamburg, Germany Edited by Donald E. Canfield, Institute of Biology and Nordic Center for Earth Evolution, University of Southern Denmark, Odense M., Denmark, and approved November 11, 2020 (received for review June 15, 2020) Bacterial hopanoid lipids are ubiquitous in the geologic record and anoxygenic phototroph Rhodopseudomonas palustris (16) and serve as biomarkers for reconstructing Earth’s climatic and biogeochem- other freshwater and soil bacteria (17–21), illustrates that ical evolution. Specifically, the abundance of 2-methylhopanoids depos- 2-methylhopanoids are not exclusive to cyanobacteria. Although ited during Mesozoic ocean anoxic events (OAEs) and other intervals 2-methylhopanoids predominantly originate from cyanobacteria has been interpreted to reflect proliferation of nitrogen-fixing marine in environments such as freshwater and lagoonal microbial mats cyanobacteria. However, there currently is no conclusive evidence for (22, 23), it is plausible that other bacteria could have contributed 2-methylhopanoid production by extant marine cyanobacteria. As an to the geologic record of 2-methylhopanoids. However, it is alternative explanation, here we report 2-methylhopanoid production unlikely that freshwater cyanobacteria were primary contributors by bacteria of the genus Nitrobacter, cosmopolitan nitrite oxidizers that inhabit nutrient-rich freshwater, brackish, and marine environments. to the accumulation of 2-methylhopanoids in offshore marine The model organism Nitrobacter vulgaris produced only trace amounts environments during Mesozoic OAEs. Thus, numerous uncer- of 2-methylhopanoids when grown in minimal medium or with added tainties surround the origin of 2-methylhopanoids in the geo- methionine, the presumed biosynthetic methyl donor. Supplementa- logical record and the reasons behind their prevalence during tion of cultures with cobalamin (vitamin B12) increased nitrite oxidation episodes of ocean anoxia. rates and stimulated a 33-fold increase of 2-methylhopanoid abun- Screening of genomes and metagenomes for the hpnP gene dance, indicating that the biosynthetic reaction mechanism is cobalamin encoding a hopanoid C-2 methyltransferase (24) has led to the dependent. Because Nitrobacter spp. cannot synthesize cobala- identification of a small subset of freshwater and soil bacteria as min, we postulate that they acquire it from organisms inhabiting putative 2-methylhopanoid producers but has not revealed in- a shared ecological niche—for example, ammonia-oxidizing archaea. stances in marine cyanobacteria (24, 25). A more recent gene We propose that during nutrient-rich conditions, cobalamin-based mu- homology analysis suggested the presence of the hpnP gene in tualism intensifies upper water column nitrification, thus promot- diverse marine cyanobacteria (26). However, 2-methylhopanoids ing 2-methylhopanoid deposition. In contrast, anoxia underlying have not been detected in any of these cyanobacteria (15), oligotrophic surface ocean conditions in restricted basins would prompt shoaling of anaerobic ammonium oxidation, leading to low observed 2-methylhopanoid abundances. The first scenario Significance is consistent with hypotheses of enhanced nutrient loading during OAEs, while the second is consistent with the sedimentary record Hopanoids are bacterial membrane lipids that are abundant in of Pliocene–Pleistocene Mediterranean sapropel events. We thus the geologic record. Hopanoids methylated at the C-2 position hypothesize that nitrogen cycling in the Pliocene–Pleistocene Med- (2-methylhopanoids) are particularly abundant during Meso- iterranean resembled modern, highly stratified basins, whereas no zoic episodes of ocean anoxia, yet the sources and factors that modern analog exists for OAEs. contribute to accumulation of these biomarkers remain poorly understood. Here, we report 2-methylhopanoid production in nitrification | hopanoids | biomarker | cobalamin | oceanic anoxic event Nitrobacter, a ubiquitous group of nitrite-oxidizing bacteria that are favored under nutrient-rich growth environments, and we demonstrate that 2-methylhopanoid biosynthesis depends opanoids are a structurally diverse class of isoprenoid lipids on cobalamin (vitamin B ). We propose that Nitrobacter spp. that are involved in bacterial membrane homeostasis by 12 H acquire their cobalamin from ammonia-oxidizing archaea in a mediating membrane organization and stress response (1–4). As mutualistic relationship that promotes nitrification. Therefore, chemical fossils, hopanoids and their diagenetic products are accumulation of 2-methylhopanoids during the Mesozoic could ubiquitous in the geologic record where they serve as important ’ have resulted from intensification of the oxidative steps of the biomarkers for our planet s biogeochemical and microbial evolution marine nitrogen cycle in a nutrient-rich ocean. from the Proterozoic onward (5–8). Specifically, a subgroup of hopanoids methylated at the C-2 position (2-methylhopanoids) has Author contributions: F.J.E., J.D.H., J.J.K., and A.P. designed research; F.J.E., J.D.H., and been used as biomarkers for cyanobacteria (9) and invoked as evi- J.J.K. performed research; T.W.E., E.S., and R.E.S. contributed new reagents/analytic tools; dence for the proliferation of nitrogen-fixing cyanobacteria during F.J.E., J.D.H., T.W.E., and J.J.K. analyzed data; and F.J.E. and A.P. wrote the paper. intervals such as Mesozoic ocean anoxic events (OAEs) (10, 11) and The authors declare no competing interest. the Paleocene–Eocene Thermal Maximum (12). This article is a PNAS Direct Submission. The importance of hopanoids as biomarkers has sustained Published under the PNAS license. interest in understanding their sources and their role in bacterial 1To whom correspondence may be addressed. Email: [email protected]. physiology, leading to multiple recent studies that challenge This article contains supporting information online at https://www.pnas.org/lookup/suppl/ prior assumptions (2, 3, 13–15). Specifically, the occurrence of doi:10.1073/pnas.2012357117/-/DCSupplemental. 2-methylhopanoids in diverse alphaproteobacteria, including the First published December 14, 2020. 32996–33004 | PNAS | December 29, 2020 | vol. 117 | no. 52 www.pnas.org/cgi/doi/10.1073/pnas.2012357117 Downloaded by guest on September 26, 2021 suggesting this recent study (26) may have detected related genes ABRelative abundance (%) Fold-change of different function. 0 20 40 60 80 100 0 20406080 Identification of common source organisms and elucidation of the underlying biochemistry of 2-methylhopanoid biosynthesis Autotrophic (mid growth) could help constrain the factors controlling their geologic record. Autotrophic (early stationary) Based on previous detection of the hpnP gene in a species of the Autotrophic + Met. alphaproteobacterial genus Nitrobacter (24), we hypothesized Autotrophic + Cbl that Nitrobacter spp. could be an important but previously overlooked source of 2-methylhopanoids. Nitrobacter spp. are Autotrophic + Light + Cbl + Met. nitrite-oxidizing bacteria (NOB) that are abundant in soil, fresh Mixotrophic water, and the oceans where they share an ecological niche with Heterotrophic aerobic other NOB (Nitrospina, Nitrospira, Nitrococcus spp.), ammonia- Heterotrophic anaerobic oxidizing archaea (AOA), and ammonia-oxidizing bacteria - NO2 -limited + Cbl + Met. (27–29). O -limited + Cbl + Met. Here, we use a combination of genomic analyses and culture 2 experiments with the model organism Nitrobacter vulgaris AB1 to BHPs 2-methyl BHPs Total 2-methyl elucidate the factors driving 2-methylhopanoid biosynthesis in Diploptene 2-methyl Diploptene hopanoids this taxon. Our results suggest that the reaction mechanism is not Fig. 1. Hopanoid composition of N. vulgaris at different growth conditions only dependent on a radical S-adenosylmethionine (SAM) en- (average of triplicate cultures). (A) Relative abundances of polyfunctionalized zyme (23) but also on the enzymatic cofactor cobalamin (vitamin (BHPs) and nonfunctionalized (diploptene) hopanoids and their C-2–methylated B12). Because Nitrobacter spp. are cobalamin auxotrophs, we derivatives (2-methyl). (B) Fold change in total 2-methylhopanoid abundance (2- hypothesize that synergistic interaction between Nitrobacter and methyl BHPs + 2-methyl diploptene) relative to autotrophic early stationary-phase cobalamin-producing nitrifying archaea could be an important growth. Fold change values are given as averages of triplicate cultures with errors control on 2-methylhopanoid production. This hypothesis is bars representing propagated uncertainties (one SD). SI Appendix,TableS2has detailed information of fold change of individual hopanoid classes. Tested growth consistent with enhanced production of 2-methylhopanoids un- − der conditions of intensified oxidative nitrogen cycling in past conditions: autotrophic (NO2 as electron donor),
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