Summary of Study ST004469

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002822. The data can be accessed directly via it's Project DOI: 10.21228/M8WG3X This work is supported by NIH grant, U2C- DK119886. See: https://www.metabolomicsworkbench.org/about/howtocite.php

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Study ID	ST004469
Study Title	Diel light dynamics regulate arsenic methylation in cyanobacteria and in microbial mats
Study Summary	The ability of microorganisms to biomethylate arsenic has ancient origins. The primary biomethylation product, trivalent monomethylarsonous acid (MMAIII), is highly toxic and oxidation to the less harmful pentavalent species requires molecular oxygen. As arsenic biomethylation evolved before the Great Oxidation Event, it has been hypothesized that early microorganisms may have used MMAIII as an antibiotic. To explore this in the context of early cyanobacterial mats creating localized oxygen oases, we assessed the light- and oxygen-dependent dynamics of arsenic methylation in cyanobacterial cultures and in an ancient ocean analog cyanobacterial mat (Laguna Pozo Bravo, Argentina). During dark and anoxic conditions Synechococcus sp. SAG2156 mainly produced MMAIII, whereas in the light dimethylarsinic acid (DMAV) was released. We propose that methylation levels are regulated by the availability of reduced thioredoxin, crucial for preparing the methyltransferase ArsM for the next methylation step of MMA to DMA. This availability is governed by photosynthesis. Accumulation of MMA under anoxic conditions at night and of DMA during the day was also observed in the porewater of the natural microbial mat. Metatranscriptomics revealed that mat inhabitants responded, for instance, by increased expression of arsH, facilitating oxidation of MMAIII during daytime oxic conditions. Our results overall show that cyanobacteria may employ MMAIII as an antibiotic, but only transiently at night. Beyond providing insights about environmental factors that have shaped the biosphere over geological time, the role of light and oxygen in the biogeochemistry of methylated arsenic compounds is critical for assessing their toxicity and potential health effects.
Institute	Universität Phillips-Marburg
Department	Biogeochemistry
Laboratory	AG Klatt
Last Name	Doherty
First Name	Daniel
Address	Hans-Meerwein-Straße 4, 35043 Marburg, Germany
Email	dohertyd@staff.uni-marburg.de
Phone	+4915204161820
Submit Date	2025-11-06
Raw Data Available	Yes
Raw Data File Type(s)	mzXML
Analysis Type Detail	LC-MS
Release Date	2026-01-12
Release Version	1

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Project:

Project ID:	PR002822
Project DOI:	doi: 10.21228/M8WG3X
Project Title:	Diel light dynamics regulate arsenic methylation in cyanobacteria and in microbial mats
Project Summary:	The ability of microorganisms to biomethylate arsenic has ancient origins. The primary biomethylation product, trivalent monomethylarsonous acid (MMAIII), is highly toxic and oxidation to the less harmful pentavalent species requires molecular oxygen. As arsenic biomethylation evolved before the Great Oxidation Event, it has been hypothesized that early microorganisms may have used MMAIII as an antibiotic. To explore this in the context of early cyanobacterial mats creating localized oxygen oases, we assessed the light- and oxygen-dependent dynamics of arsenic methylation in cyanobacterial cultures and in an ancient ocean analog cyanobacterial mat (Laguna Pozo Bravo, Argentina). During dark and anoxic conditions Synechococcus sp. SAG2156 mainly produced MMAIII, whereas in the light dimethylarsinic acid (DMAV) was released. We propose that methylation levels are regulated by the availability of reduced thioredoxin, crucial for preparing the methyltransferase ArsM for the next methylation step of MMA to DMA. This availability is governed by photosynthesis. Accumulation of MMA under anoxic conditions at night and of DMA during the day was also observed in the porewater of the natural microbial mat. Metatranscriptomics revealed that mat inhabitants responded, for instance, by increased expression of arsH, facilitating oxidation of MMAIII during daytime oxic conditions. Our results overall show that cyanobacteria may employ MMAIII as an antibiotic, but only transiently at night. Beyond providing insights about environmental factors that have shaped the biosphere over geological time, the role of light and oxygen in the biogeochemistry of methylated arsenic compounds is critical for assessing their toxicity and potential health effects.
Institute:	Universität Phillips-Marburg
Last Name:	Doherty
First Name:	Daniel
Address:	Hans-Meerwein-Straße 4, 35043 Marburg, Germany
Email:	dohertyd@staff.uni-marburg.de
Phone:	+4915204161820

Subject:

Subject ID:	SU004645
Subject Type:	Bacteria
Subject Species:	Synechococcus sp. SAG2156

Factors:

Subject type: Bacteria; Subject species: Synechococcus sp. SAG2156 (Factor headings shown in green)

mb_sample_id	local_sample_id	Sample source	Treatment
SA531913	Syn 4c	Cyanobacteria lipid extract	180uM PO4, 180uM As(V)
SA531912	Syn 5a	Cyanobacteria lipid extract	180uM PO4, 1.8mM As(V)
SA531914	Syn 3c	Cyanobacteria lipid extract	180uM PO4, 18uM As(V)
SA531915	Syn8a	Cyanobacteria lipid extract	Control

Showing results 1 to 4 of 4

Showing results 1 to 4 of 4

Collection:

Collection ID:	CO004638
Collection Summary:	Synechococcus sp. SAG2156 cultures were grown at 30 degrees Celsius in ASN(III) medium, modified by adding HEPES to achieve strong pH buffering at approximately 7.3. The light intensity used for cultivation was ~55 µmol photons m-2 s-1, as measured with a spherical light probe (Walz, Germany) connected to a light meter (Licor). At exponential growth phase, cultures where collected in a 0.2 um glass fiber filter, flash frozen and saved for lipid extraction.
Sample Type:	Bacterial cells
Collection Method:	Lipid extraction

Treatment:

Treatment ID:	TR004654
Treatment Summary:	To assess if Synechococcus sp. SAG2156 is capable of producing arsenolipids, it was grown in the absence and presence of 180µM PO4 (phosphate) and 18µM As(V), 180µM PO4 and 180µM As(V), 180µM PO4 and 1.8 mM As(V), and only 180µM PO4. The cyanobacterial cultures were grown in liquid ASN(III) medium (1), modified by adding HEPES to achieve strong pH buffering at approximately 7.3. The light intensity used for cultivation was ~55 µmol photons m-2 s-1, as measured with a spherical light probe (Walz, Germany) connected to a light meter (Licor). Growth was monitored non-invasively by measuring OD directly in the growth vials using a custom-made photometric setup. In the mid-exponential growth phase (OD750 ~0.8), 20 ml of cyanobacterial culture was filtered onto pre-combusted (400 ºC for 6 h) 0.2 µm Whatman GF/F filters (GE Healtchcare Life Sciences). To ensure that no unincorporated arsenic species were caught on the filters, they were also rinsed with washing buffer (at least 2 volumes of the volume of the culture filtered, 35‰ NaCl solution).

Treatment ID:

TR004654

Treatment Summary:

To assess if Synechococcus sp. SAG2156 is capable of producing arsenolipids, it was grown in the absence and presence of 180µM PO4 (phosphate) and 18µM As(V), 180µM PO4 and 180µM As(V), 180µM PO4 and 1.8 mM As(V), and only 180µM PO4. The cyanobacterial cultures were grown in liquid ASN(III) medium (1), modified by adding HEPES to achieve strong pH buffering at approximately 7.3. The light intensity used for cultivation was ~55 µmol photons m-2 s-1, as measured with a spherical light probe (Walz, Germany) connected to a light meter (Licor). Growth was monitored non-invasively by measuring OD directly in the growth vials using a custom-made photometric setup. In the mid-exponential growth phase (OD750 ~0.8), 20 ml of cyanobacterial culture was filtered onto pre-combusted (400 ºC for 6 h) 0.2 µm Whatman GF/F filters (GE Healtchcare Life Sciences). To ensure that no unincorporated arsenic species were caught on the filters, they were also rinsed with washing buffer (at least 2 volumes of the volume of the culture filtered, 35‰ NaCl solution).

Sample Preparation:

Sampleprep ID:	SP004651
Sampleprep Summary:	The lipid extraction protocol consisted of shredding the filters to smaller pieces and treated with 7 ml of methanol:methyl-tert-buthyl-ether:water (1:3:1) followed by 30min of shaking at 5 ºC, and sonification in ice cool water bath for 10 min. After sonification the addition of 4.8 ml of methanol:MQ water (1:3) solution leads to the separation of the upper organic and lower water phases, which must be properly separated and cleaned by centrifugation at 1000rpm for 10 min at 5 ºC. All of the upper, organic phase was separated from the rest of sample and the solvents were evaporated by evaporating the solution with N2 gas. The dried pellet was resuspended in 500 µL dichlorometane:methanol (1:9) solution and stored at -20 ºC. This was an adapted method of Giavalisco et al., 2011. Giavalisco P, Li Y, Matthes A, Eckhardt A, Hubberten HM, Hesse H, et al. Elemental formula annotation of polar and lipophilic metabolites using 13C, 15N and 34S isotope labelling, in combination with high-resolution mass spectrometry. Plant Journal. 2011. 68(2):364–76.

Sampleprep ID:

SP004651

Sampleprep Summary:

The lipid extraction protocol consisted of shredding the filters to smaller pieces and treated with 7 ml of methanol:methyl-tert-buthyl-ether:water (1:3:1) followed by 30min of shaking at 5 ºC, and sonification in ice cool water bath for 10 min. After sonification the addition of 4.8 ml of methanol:MQ water (1:3) solution leads to the separation of the upper organic and lower water phases, which must be properly separated and cleaned by centrifugation at 1000rpm for 10 min at 5 ºC. All of the upper, organic phase was separated from the rest of sample and the solvents were evaporated by evaporating the solution with N2 gas. The dried pellet was resuspended in 500 µL dichlorometane:methanol (1:9) solution and stored at -20 ºC. This was an adapted method of Giavalisco et al., 2011. Giavalisco P, Li Y, Matthes A, Eckhardt A, Hubberten HM, Hesse H, et al. Elemental formula annotation of polar and lipophilic metabolites using 13C, 15N and 34S isotope labelling, in combination with high-resolution mass spectrometry. Plant Journal. 2011. 68(2):364–76.

Combined analysis:

Analysis ID	AN007492
Chromatography ID	CH005681
MS ID	MS007188
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Waters Acquity
Column	Water Acquity UPLC BEH C18 (150 x 1.7mm, 2.1um)
MS Type	API
MS instrument type	QTOF
MS instrument name	Bruker maXis Impact qTOF
Ion Mode	POSITIVE
Units	Peak Area

Chromatography:

Chromatography ID:	CH005681
Instrument Name:	Waters Acquity
Column Name:	Water Acquity UPLC BEH C18 (150 x 1.7mm, 2.1um)
Column Temperature:	65 C
Flow Gradient:	0min:0%B, 2min:0%B, 2.1min:15%B, 20min:85%B, 20.5min: 100%B, 28min:100%B
Flow Rate:	0.4 ml/min
Solvent A:	85% methanol/15% waer; 0.04% formic acid; 0.1% ammonium hydroxide
Solvent B:	50% isopropanol/50% methanol; 0.04% formic acid; 0.1% ammonium hydroxide
Chromatography Type:	Reversed phase

MS:

MS ID:	MS007188
Analysis ID:	AN007492
Instrument Name:	Bruker maXis Impact qTOF
Instrument Type:	QTOF
MS Type:	API
MS Comments:	Used Compass DataAnalysis software 4.4, searched for compounds with m/z and MS2 fragments as per Glabonjat et al., 2019. Target arsenolipids (mainly arsenosugar phytols and arsenic hydrocarbons) were not found in our study. Glabonjat, Ronald & Raber, Georg & Jensen, Kenneth & Schubotz, Florence & Boyd, Eric & Francesconi, Kevin. (2019). Origin of arsenolipids in sediments from Great Salt Lake. Environmental Chemistry. 16. 303-311. 10.1071/EN19135.
Ion Mode:	POSITIVE