Dataset: Controlled laboratory study using model organisms Micromonas commoda RCC 299 and Ruegeria pomeroyi DSS-3

Name: Bacterial transcriptional response to picoeukaryote Micromonas commoda
Published: 2024-05-21
Keywords: phytoplankton-bacteria interactions, Micromonas commoda, transcriptome, Heterotrophic Bacteria, oceans

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Cite This Dataset

Spatial Extent: N:33.9519 E:-83.3576 S:33.9519 W:-83.3576

Temporal Extent: 2021 - 2021

Project:

Effects of Climate Change Variables on Microbial Autotroph-Heterotroph Carbon Flux (CC_Auto_Hetero_Fluxes)

Principal Investigator:

Mary Ann Moran (University of Georgia, UGA)

Scientist:

Maria Hamilton (University of Georgia, UGA)

Student:

Frank Xavier Ferrer-González (University of Georgia, UGA)

Technician:

Christa Smith (University of Georgia, UGA)

BCO-DMO Data Manager:

Karen Soenen (Woods Hole Oceanographic Institution, WHOI BCO-DMO)

Version:

Version Date:

2024-05-21

Restricted:

Validated:

Current State:

Data not available

Bacterial transcriptional response to picoeukaryote Micromonas commoda

Abstract:

Marine biogeochemical cycles are built on interactions between surface ocean microbes, particularly those connecting phytoplankton primary producers to heterotrophic bacteria. However, direct influences of bacteria on phytoplankton physiology are poorly known. In this study, three marine bacteria (Ruegeria pomeroyi DSS-3, Stenotrophomonas sp. SKA14, and Polaribacter dokdonensis MED152) were co-cultured with green alga Micromonas commoda, and the phytoplankter's transcriptome was studied by RNASeq. The presence of each bacterium invoked transcriptomic remodeling by M. commoda after 8 h in co-culture. Some aspects of the algal transcriptomic response were conserved across all three bacteria, while others were restricted to a single bacterium. M. commoda had both rapid and extensive responses to heterotrophic bacteria.

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Methods & Sampling

Axenic cultures of Micromonas commoda RCC299 (National Center for Marine Algae, NMCA) were grown in 1 L of organic-carbon free defined medium L1-Si [31] as modified by NCMA (https://ncma.bigelow.org/) at a salinity of 35 in 1900 mL vented polystyrene tissue culture flasks. Flasks were maintained at 18 oC under 16 h light at 160 μmol photons m−2s−1 and 8 h dark. Pre-cultures of Micromonas were sequentially upscaled (50 ml, 200 ml, 1 L) with transfers occurring during the exponential growth phase. After growing for 7 d (early stationary growth phase; ~2.7 × 106 cells ml−1), three marine bacteria pre-grown in YTSS medium (Ruegeria pomeroyi DSS-3, Stenotrophomonas sp. SKA14, and Polaribacter dokdonensis MED152 were washed 5 times in sterile L1 medium at 6000 RCF and inoculated into the axenic cultures at ~106 cells ml−1. Three or four replicate co-cultures were established for each bacterial strain and also for an axenic phytoplankton control. Three additional treatments were established with bacterial strains introduced individually into L1 medium with 400 μM C glucose as the sole carbon source (which supports all 3) at the same initial cell concentration as the co-cultures. As this treatment contained a single, known metabolite, it served as a control for co-culture transcriptome analysis. Bacterial contamination of the axenic phytoplankton cultures was ruled out based on lack of colony formation from culture aliquots spread onto YTSS plates and absence of bacterial-size particles in flow cytometry scattergrams.

Data Processing Description

Bacterial cell counts: Cell counts were periodically obtained by flow cytometry. Samples were fixed at a final concentration of 1% glutaraldehyde, incubated at 4°C for 20 min, and stored at −80°C. Just prior to analysis, an internal standard of 5 μm fluorescent particles (ACFP-50-5; Spherotech, Lake Forest, IL, USA) was added, followed by staining for 15 min with SYBR Green I (final concentration 0.75X; Life Technologies, Waltham, MA, USA). Samples were analyzed on an Agilent Quanteon flow cytometer (Acea, Biosciences Inc, San Diego CA) with a 405 nm laser using a 530/30 bandpass filter for SYBR Green.

Micromonas cell counts: Cell counts were periodically obtained by flow cytometry. Samples were fixed at a final concentration of 1% glutaraldehyde, incubated at 4°C for 20 min, and stored at −80°C. Just prior to analysis, an internal standard of 5 μm fluorescent particles (ACFP-50-5; Spherotech, Lake Forest, IL, USA) was added, followed by staining for 15 min with SYBR Green I (final concentration 0.75X; Life Technologies, Waltham, MA, USA). Samples were analyzed on an Agilent Quanteon flow cytometer (Acea, Biosciences Inc, San Diego CA) with a 405 nm laser using a 695/40 bandpass filter for chlorophyll a (phytoplankton).

Nutrient analysis: At the 8 h time point, 50 ml of each sample was used for nutrient analysis. Samples were filtered through 0.2 μm pore-size 47 mm Supor filters to remove cells. The filtrate was frozen and stored at -20°C. Nutrient analyses were performed by the University of Georgia Laboratory of Environmental Analysis. Concentrations of nitrate (NO3-), nitrite (NO2-), and phosphate (PO43-) were measured using ion chromatography on a DX500 Ion Chromatograph (Dionex Co.) with an initial cartridge treatment (OnGuard-Ag cartridge from Dionex) performed to remove chloride ions. Measurements for ammonium (NH4+) were done separately via the phenate method with spectrophotometric analysis on a Model Spectronic 21D (Spectronic Instrumentation).

More Information about this dataset

Funding Sources

Funding Source	Award Number
NSF Division of Ocean Sciences	OCE-1948104

Deployments

None available yet

Instruments

Flow Cytometer

Supplied Name: Agilent Quanteon flow cytometer (Acea, Biosciences Inc, San Diego CA)

Supplied Description:

Agilent Quanteon flow cytometer (Acea, Biosciences Inc, San Diego CA) with a 405 nm laser using a 530/30 bandpass filter for SYBR Green for bacterial cell counts. 405 nm laser using a 695/40 bandpass filter for chlorophyll a (phytoplankton) was used for micromonas cell counts.

Instrument Type

Generic Name: Flow Cytometer

Community Identifier: http://vocab.nerc.ac.uk/collection/L05/current/LAB37/

Generic Description:

Flow cytometers (FC or FCM) are automated instruments that quantitate properties of single cells, one cell at a time. They can measure cell size, cell granularity, the amounts of cell components such as total DNA, newly synthesized DNA, gene expression as the amount messenger RNA for a particular gene, amounts of specific surface receptors, amounts of intracellular proteins, or transient signalling events in living cells.
(from: http://www.bio.umass.edu/micro/immunology/facs542/facswhat.htm)

Ion Chromatograph

Supplied Name: DX500 Ion Chromatograph (Dionex Co.)

Supplied Description:

DX500 Ion Chromatograph (Dionex Co.) with an initial cartridge treatment (OnGuard-Ag cartridge from Dionex) performed to remove chloride ions.

Instrument Type

Generic Name: Ion Chromatograph

Generic Description:

Ion chromatography is a form of liquid chromatography that measures concentrations of ionic species by separating them based on their interaction with a resin. Ionic species separate differently depending on species type and size. Ion chromatographs are able to measure concentrations of major anions, such as fluoride, chloride, nitrate, nitrite, and sulfate, as well as major cations such as lithium, sodium, ammonium, potassium, calcium, and magnesium in the parts-per-billion (ppb) range. (from http://serc.carleton.edu/microbelife/research_methods/biogeochemical/ic.html)

Spectrophotometer

Supplied Name: Model Spectronic 21D (Spectronic Instrumentation)

Supplied Description:

Measurements for ammonium (NH4+) were done separately via the phenate method with spectrophotometric analysis on a Model Spectronic 21D (Spectronic Instrumentation).

Instrument Type

Generic Name: Spectrophotometer

Community Identifier: http://vocab.nerc.ac.uk/collection/L05/current/LAB20/

Generic Description:

An instrument used to measure the relative absorption of electromagnetic radiation of different wavelengths in the near infra-red, visible and ultraviolet wavebands by samples.

Parameters

Date and time formats are described in python strftime() syntax.

Supplied Name	Supplied description	Supplied Units	Standard Name
Treatment	Lab treatment description: DSS-3, MED152, SKA14, axenic	unitless	treatment
Time_h	Time after start of experiment	hour (h)	time_sample
Sample_ID	Identifier of lab sample	unitless	sample
Micromonas_cells_ml	Micormonas cell count	cells per milliliter (cell/ml)	cell_concentration
Bacteria_cells_ml	Bacteria cell count	cells per milliliter (cell/ml)	cell_concentration
NH4_uM	Ammonium (NH4+) concentration	micromolar (uM)	NH4
NO3_uM	Nitrate (NO3-) concentration	micromolar (uM)	NO3
PO4_uM	Phosphate (PO43-) concentration	micromolar (uM)	PO4

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Database

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Dataset: Controlled laboratory study using model organisms Micromonas commoda RCC 299 and Ruegeria pomeroyi DSS-3