Demethylated hopanoids in ‘Ca. Methylomirabilis oxyfera’ as biomarkers for environmental nitrite-dependent methane oxidation

Nadine T. Smit; D. Rush; Diana X. Sahonero-Canavesi; Monique Verweij; Olivia Rasigraf; S. Guerrero Cruz; Mike S.M. Jetten; Jaap S. Sinninghe Damsté; Stefan Schouten

doi:10.1016/j.orggeochem.2019.07.008

Demethylated hopanoids in ‘Ca. Methylomirabilis oxyfera’ as biomarkers for environmental nitrite-dependent methane oxidation

Nadine T. Smit^*, D. Rush, Diana X. Sahonero-Canavesi, Monique Verweij, Olivia Rasigraf, S. Guerrero Cruz, Mike S.M. Jetten, Jaap S. Sinninghe Damsté, Stefan Schouten

^*Corresponding author for this work

Research output: Contribution to journal › Article › Academic › peer-review

Abstract

Hopanoids are lipids that are widespread in the bacterial domain and well established molecular biomarkers in modern and paleo environments. In particular, the occurrence of ¹³C-depleted 3-methylated hopanoids are characteristic of aerobic bacteria involved in methane oxidation. Previously the intra-aerobic methanotroph ‘Candidatus Methylomirabilis oxyfera’ (‘Ca. M. oxyfera’), which performs nitrite-dependent methane oxidation in anoxic environments, has been shown to synthesize bacteriohopanepolyols (BHPs) and their 3-methylated counterparts. However, since ‘Ca. M. oxyfera’ does not utilize methane as a carbon source, its biomass and lipids do not show the characteristic ¹³C-depletion. Therefore, the detection of ‘Ca. M. oxyfera’ in various environments is challenging, and still underexplored. Here, we re-investigated the hopanoid content of ‘Ca. M. oxyfera’ bacteria using enrichment cultures. We found the GC-amenable hopanoids of ‘Ca. M. oxyfera’ to be dominated by four demethylated hopanoids of which only one, 22,29,30-trisnorhopan-21-one, had been identified previously. The three novel hopanoids were tentatively identified as 22,29,30-trisnorhopan-21-ol, 3-methyl-22,29,30-trisnorhopan-21-one and 3-methyl-22,29,30-trisnorhopan-21-ol. These unique demethylated hopanoids are most likely biosynthesized directly by ‘Ca. M. oxyfera’ bacteria. Bioinformatical analysis of the ‘Ca. M. oxyfera’ genome revealed potential candidate genes responsible for the demethylation of hopanoids. For the sensitive detection of the four trisnorhopanoid biomarkers in environmental samples, a multiple reaction monitoring (MRM) method was developed and used to successfully detect the trisnorhopanoids in a peatland where the presence of ‘Ca. M. oxyfera’ had been confirmed previously by DNA-based analyses. These new biomarkers may be a novel tool to trace nitrite-dependent methane oxidation in various (past) environments.

Original language	English
Article number	103899
Journal	Organic Geochemistry
Volume	137
DOIs	https://doi.org/10.1016/j.orggeochem.2019.07.008
Publication status	Published - Nov 2019

Funding

We thank Irene Rijpstra for laboratory assistance and helpful comments in compound identification as well as Laura Villanueva for discussions about hopanoid biosynthesis pathways. We also thank Annika Vaksmaa for providing methanotrophic ANME-2D/‘ Ca . M. oxyfera’ biomass for comparison. We thank Martin Blumenberg and anonymous reviewer, as well as the Associate Editor for constructive comments. This study received funding from the Netherlands Earth System Science Center (NESSC) and Soehngen Institute for Anaerobic Microbiology (SIAM) through Gravitation grants (024.002.001 and 024.002.002) from the Dutch Ministry for Education, Culture and Science . DXSC received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement n◦ 694569 – MICROLIPIDS) and MSMJ by ERC AG ecomom 339880. Appendix A

Keywords

3-Methyl 22,29,30-trisnorhopan-21-one
Candidatus Methylomirabilis oxyfera
Demethylated hopanoids
Methylation
Nitrite-dependent methane oxidation
Trisnorhopanoids

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10.1016/j.orggeochem.2019.07.008

1-s2.0-S0146638019301299-mainFinal published version, 685 KBLicence: CC BY-NC-ND

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Smit, N. T., Rush, D., Sahonero-Canavesi, D. X., Verweij, M., Rasigraf, O., Guerrero Cruz, S., Jetten, M. S. M., Sinninghe Damsté, J. S., & Schouten, S. (2019). Demethylated hopanoids in ‘Ca. Methylomirabilis oxyfera’ as biomarkers for environmental nitrite-dependent methane oxidation. Organic Geochemistry, 137, Article 103899. https://doi.org/10.1016/j.orggeochem.2019.07.008

@article{ea95accd85294c83a7d8afebdeeb5a42,

title = "Demethylated hopanoids in {\textquoteleft}Ca. Methylomirabilis oxyfera{\textquoteright} as biomarkers for environmental nitrite-dependent methane oxidation",

abstract = "Hopanoids are lipids that are widespread in the bacterial domain and well established molecular biomarkers in modern and paleo environments. In particular, the occurrence of 13C-depleted 3-methylated hopanoids are characteristic of aerobic bacteria involved in methane oxidation. Previously the intra-aerobic methanotroph {\textquoteleft}Candidatus Methylomirabilis oxyfera{\textquoteright} ({\textquoteleft}Ca. M. oxyfera{\textquoteright}), which performs nitrite-dependent methane oxidation in anoxic environments, has been shown to synthesize bacteriohopanepolyols (BHPs) and their 3-methylated counterparts. However, since {\textquoteleft}Ca. M. oxyfera{\textquoteright} does not utilize methane as a carbon source, its biomass and lipids do not show the characteristic 13C-depletion. Therefore, the detection of {\textquoteleft}Ca. M. oxyfera{\textquoteright} in various environments is challenging, and still underexplored. Here, we re-investigated the hopanoid content of {\textquoteleft}Ca. M. oxyfera{\textquoteright} bacteria using enrichment cultures. We found the GC-amenable hopanoids of {\textquoteleft}Ca. M. oxyfera{\textquoteright} to be dominated by four demethylated hopanoids of which only one, 22,29,30-trisnorhopan-21-one, had been identified previously. The three novel hopanoids were tentatively identified as 22,29,30-trisnorhopan-21-ol, 3-methyl-22,29,30-trisnorhopan-21-one and 3-methyl-22,29,30-trisnorhopan-21-ol. These unique demethylated hopanoids are most likely biosynthesized directly by {\textquoteleft}Ca. M. oxyfera{\textquoteright} bacteria. Bioinformatical analysis of the {\textquoteleft}Ca. M. oxyfera{\textquoteright} genome revealed potential candidate genes responsible for the demethylation of hopanoids. For the sensitive detection of the four trisnorhopanoid biomarkers in environmental samples, a multiple reaction monitoring (MRM) method was developed and used to successfully detect the trisnorhopanoids in a peatland where the presence of {\textquoteleft}Ca. M. oxyfera{\textquoteright} had been confirmed previously by DNA-based analyses. These new biomarkers may be a novel tool to trace nitrite-dependent methane oxidation in various (past) environments.",

keywords = "3-Methyl 22,29,30-trisnorhopan-21-one, Candidatus Methylomirabilis oxyfera, Demethylated hopanoids, Methylation, Nitrite-dependent methane oxidation, Trisnorhopanoids",

author = "Smit, {Nadine T.} and D. Rush and Sahonero-Canavesi, {Diana X.} and Monique Verweij and Olivia Rasigraf and {Guerrero Cruz}, S. and Jetten, {Mike S.M.} and {Sinninghe Damst{\'e}}, {Jaap S.} and Stefan Schouten",

year = "2019",

month = nov,

doi = "10.1016/j.orggeochem.2019.07.008",

language = "English",

volume = "137",

journal = "Organic Geochemistry",

issn = "0146-6380",

publisher = "Elsevier Ltd",

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TY - JOUR

T1 - Demethylated hopanoids in ‘Ca. Methylomirabilis oxyfera’ as biomarkers for environmental nitrite-dependent methane oxidation

AU - Smit, Nadine T.

AU - Rush, D.

AU - Sahonero-Canavesi, Diana X.

AU - Verweij, Monique

AU - Rasigraf, Olivia

AU - Guerrero Cruz, S.

AU - Jetten, Mike S.M.

AU - Sinninghe Damsté, Jaap S.

AU - Schouten, Stefan

PY - 2019/11

Y1 - 2019/11

N2 - Hopanoids are lipids that are widespread in the bacterial domain and well established molecular biomarkers in modern and paleo environments. In particular, the occurrence of 13C-depleted 3-methylated hopanoids are characteristic of aerobic bacteria involved in methane oxidation. Previously the intra-aerobic methanotroph ‘Candidatus Methylomirabilis oxyfera’ (‘Ca. M. oxyfera’), which performs nitrite-dependent methane oxidation in anoxic environments, has been shown to synthesize bacteriohopanepolyols (BHPs) and their 3-methylated counterparts. However, since ‘Ca. M. oxyfera’ does not utilize methane as a carbon source, its biomass and lipids do not show the characteristic 13C-depletion. Therefore, the detection of ‘Ca. M. oxyfera’ in various environments is challenging, and still underexplored. Here, we re-investigated the hopanoid content of ‘Ca. M. oxyfera’ bacteria using enrichment cultures. We found the GC-amenable hopanoids of ‘Ca. M. oxyfera’ to be dominated by four demethylated hopanoids of which only one, 22,29,30-trisnorhopan-21-one, had been identified previously. The three novel hopanoids were tentatively identified as 22,29,30-trisnorhopan-21-ol, 3-methyl-22,29,30-trisnorhopan-21-one and 3-methyl-22,29,30-trisnorhopan-21-ol. These unique demethylated hopanoids are most likely biosynthesized directly by ‘Ca. M. oxyfera’ bacteria. Bioinformatical analysis of the ‘Ca. M. oxyfera’ genome revealed potential candidate genes responsible for the demethylation of hopanoids. For the sensitive detection of the four trisnorhopanoid biomarkers in environmental samples, a multiple reaction monitoring (MRM) method was developed and used to successfully detect the trisnorhopanoids in a peatland where the presence of ‘Ca. M. oxyfera’ had been confirmed previously by DNA-based analyses. These new biomarkers may be a novel tool to trace nitrite-dependent methane oxidation in various (past) environments.

AB - Hopanoids are lipids that are widespread in the bacterial domain and well established molecular biomarkers in modern and paleo environments. In particular, the occurrence of 13C-depleted 3-methylated hopanoids are characteristic of aerobic bacteria involved in methane oxidation. Previously the intra-aerobic methanotroph ‘Candidatus Methylomirabilis oxyfera’ (‘Ca. M. oxyfera’), which performs nitrite-dependent methane oxidation in anoxic environments, has been shown to synthesize bacteriohopanepolyols (BHPs) and their 3-methylated counterparts. However, since ‘Ca. M. oxyfera’ does not utilize methane as a carbon source, its biomass and lipids do not show the characteristic 13C-depletion. Therefore, the detection of ‘Ca. M. oxyfera’ in various environments is challenging, and still underexplored. Here, we re-investigated the hopanoid content of ‘Ca. M. oxyfera’ bacteria using enrichment cultures. We found the GC-amenable hopanoids of ‘Ca. M. oxyfera’ to be dominated by four demethylated hopanoids of which only one, 22,29,30-trisnorhopan-21-one, had been identified previously. The three novel hopanoids were tentatively identified as 22,29,30-trisnorhopan-21-ol, 3-methyl-22,29,30-trisnorhopan-21-one and 3-methyl-22,29,30-trisnorhopan-21-ol. These unique demethylated hopanoids are most likely biosynthesized directly by ‘Ca. M. oxyfera’ bacteria. Bioinformatical analysis of the ‘Ca. M. oxyfera’ genome revealed potential candidate genes responsible for the demethylation of hopanoids. For the sensitive detection of the four trisnorhopanoid biomarkers in environmental samples, a multiple reaction monitoring (MRM) method was developed and used to successfully detect the trisnorhopanoids in a peatland where the presence of ‘Ca. M. oxyfera’ had been confirmed previously by DNA-based analyses. These new biomarkers may be a novel tool to trace nitrite-dependent methane oxidation in various (past) environments.

KW - 3-Methyl 22,29,30-trisnorhopan-21-one

KW - Candidatus Methylomirabilis oxyfera

KW - Demethylated hopanoids

KW - Methylation

KW - Nitrite-dependent methane oxidation

KW - Trisnorhopanoids

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U2 - 10.1016/j.orggeochem.2019.07.008

DO - 10.1016/j.orggeochem.2019.07.008

M3 - Article

AN - SCOPUS:85070716031

SN - 0146-6380

VL - 137

JO - Organic Geochemistry

JF - Organic Geochemistry

M1 - 103899

ER -

Demethylated hopanoids in ‘Ca. Methylomirabilis oxyfera’ as biomarkers for environmental nitrite-dependent methane oxidation

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Keywords

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