Dataset: Synechococcus DOP Hydrolysis Experiment - cell counts and IVF

Name: In vivo fluorescence and flow cytometry cell counts from dissolved organic phosphorus (DOP) hydrolysis experiments with marine cyanobacterium Synechococcus laboratory cultures (WH8102 and WH5701) from 2018-2023
Published: 2024-06-06
Keywords: dissolved organic phosphorus, synechococcus, polyphosphate, phosphoester, in vivo fluorescence, flow cytometry, oceans

Cite This Dataset

DOI:10.26008/1912/bco-dmo.929471.1

Laboratory experiments at the University of Arizona, Tucson, Arizona, US

Temporal Extent: 2018 - 2023

Project:

Collaborative Research: Assessing the role of compound-specific phosphorus hydrolase transformations in the marine phosphorus cycle (P-hydrolase)

Principal Investigator:

Solange Duhamel (University of Arizona, UA)

Co-Principal Investigator:

Julia Diaz (University of California-San Diego, UCSD-SIO)

Scientist:

Kahina Djaoudi (University of Arizona, UA)

Emily Waggoner (University of Arizona, UA)

BCO-DMO Data Manager:

Amber D. York (Woods Hole Oceanographic Institution, WHOI BCO-DMO)

Version:

Version Date:

2024-06-06

Restricted:

Validated:

Yes

Current State:

Final no updates expected

In vivo fluorescence and flow cytometry cell counts from dissolved organic phosphorus (DOP) hydrolysis experiments with marine cyanobacterium Synechococcus laboratory cultures (WH8102 and WH5701) from 2018-2023

Abstract:

Marine cyanobacterium Synechococcus (WH8102 and WH5701) laboratory culture in vivo fluorescence and flow cytometry cell counts following growth in high and low phosphate media, as part of the DOP hydrolysis experiments in a study of "Dissolved organic phosphorus bond-class utilization by Synechococcus" (Waggoner et al. 2024). Study Abstract: Dissolved organic phosphorus (DOP) contains compounds with phosphoester (P-O-C), phosphoanhydride (P-O-P), and phosphorus-carbon (P-C) bonds. Despite DOP’s importance as a nutritional source for marine microorganisms, the bioavailability of each bond-class to the widespread cyanobacterium Synechococcus remains largely unknown. This study evaluates bond-class specific DOP utilization by cultures of an open ocean and a coastal ocean Synechococcus strain. Both strains exhibited comparable growth rates when provided phosphate, short-chain and long-chain polyphosphate (P-O-P), adenosine 5’-triphosphate (P-O-C and P-O-P), and glucose-6-phosphate (P-O-C) as the phosphorus source. However, growth rates on phosphomonoester adenosine 5’-monophosphate (P-O-C) and phosphodiester bis(4-methylumbelliferyl) phosphate (C-O-P-O-C) varied between strains, and neither strain grew on selected phosphonates. Consistent with the growth measurements, both strains preferentially hydrolyzed 3-polyphosphate, followed by adenosine 5’-triphosphate, and then adenosine 5’-monophosphate. The strains’ exoproteome contained phosphorus hydrolases, which combined with enhanced cell-free hydrolysis of 3-polyphosphate and adenosine 5’-triphosphate under phosphate deficiency, suggests active mineralization of short-chain polyphosphate by Synechococcus’ exoproteins. Synechococcus alkaline phosphatases presented broad substrate specificities, including activity towards short-chain polyphosphate, with varying affinities between the two strains. Collectively, these findings underscore the potentially significant role of compounds with phosphoanhydride bonds in Synechococcus phosphorus nutrition, thereby expanding our understanding of microbially-mediated DOP cycling in marine ecosystems.

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Archival Copy

Version Date	Archive	Persistent Identifier (PID)	Date PID Issued
2024-06-06	Marine Biological Laboratory/Woods Hole Oceanographic Institution Library (MBLWHOI DLA)	10.26008/1912/bco-dmo.929471.1	2024-07-15

Description

This dataset was utilized for Waggoner et al. (2024). See "Related Datasets" section on this page for other closely-related data from this study published in Waggoner et al. (2024). They are also listed under the BCO-DMO Project Page: https://www.bco-dmo.org/project/747715.

Methods & Sampling

This data is part of the DOP Hydrolysis Experiments:

Synechococcus Growth– Axenic Synechococcus WH8102 (open ocean strain) and WH5701 (coastal strain) were obtained from the National Center for Marine Algae and Microbiota (NCMA, Bigelow Laboratories, East Boothbay, Maine). Both strains were grown in batch culture using SN media (Waterbury et al. 1986) made with aged, filtered (0.2 µm), and autoclaved (120°C, 30 minutes) seawater from station ALOHA (A Long-term Oligotrophic Habitat Assessment). At the late-exponential phase, cultures were transferred in triplicate to one of two SN media: (1) +Pi (45 µmol L^-1 KH₂PO₄, following Waterbury et al. (1986)) and (2) -Pi (no KH₂PO₄ added; Pi below detection limit). All cultures were incubated at 25°C on a 12h:12h light cycle at 130 µmol m^-1 s^-1 in sterile culture flasks with a vent cap (0.22 µm hydrophobic membrane).

IVF and Flow Cytometry Cell Counts– For each triplicate culture flask and media type (+P and -P), in vivo fluorescence (IVF) was measured (AquaFluor®, Turner Designs) as a proxy for Synechococcus biomass. In parallel, over the growth curve, 2-mL Synechococcus culture aliquots were collected, fixed (final concentration of 0.2% paraformaldehyde), and stored at -80°C until cell abundance analysis using the Guava® EasyCyte flow cytometer (Millipore). Briefly, Synechococcus was enumerated in unstained samples based on red fluorescence (i.e., chlorophyll) and forward scatter signals using a low flow rate of 0.24 µL s^-1 for 1 minute. Instrument-specific beads (Guava® Check Kit, Luminex) were used to calibrate the instrument.

Organism identifiers (Life Science Identifier, LSID):
Synechococcus, urn:lsid:marinespecies.org:taxname:160572

Problems/Issues

This dataset is part of the DOP Hydrolysis Experiments. As such, IVF was measured consistently to track the culture growth curve and flow cytometry cell count samples were taken only on the days when DOP hydrolysis was measured (at each growth phase). As a result, there are more values of IVF than cell counts in the dataset.

Data Processing Description

DOP hydrolysis rates were normalized to flow cytometry cell counts to account for biomass differences between strains and treatments. The DOP hydrolysis rates can be found in the 'Synechococcus DOP Hydrolysis Experiment - hydrolysis rates' dataset (see "Related Datasets" section).

BCO-DMO Processing

* Sheet 1 of submitted file "Synechococcus_DOPHydrolysisExperiment_IVF_CellCounts.xlsx" was imported into the BCO-DMO data system for this dataset. Values "NaN" imported as missing data values.
** Missing data values are displayed differently based on the file format you download. They are blank in csv files, "NaN" in MatLab files, etc.

* Column names adjusted to conform to BCO-DMO naming conventions designed to support broad re-use by a variety of research tools and scripting languages. [Only numbers, letters, and underscores. Can not start with a number]

Data Files

File
929471_v1_syn-dop-exp-cellcounts-ivf.csv (Comma Separated Values (.csv), 3.04 KB) MD5:4e12eb5fffeb17e2f34fc48a76f895df Primary data file for dataset ID 929471, version 1

More Information about this dataset

Funding Sources

Funding Source	Award Number
NSF Division of Ocean Sciences	OCE-1737083
NSF Division of Ocean Sciences	OCE-2001212
NSF Division of Ocean Sciences	OCE-1736967
NSF Division of Ocean Sciences	OCE-1948042

Deployments

None available yet

Instruments

plate reader

Supplied Name: Molecular Devices M2 multimode plate reader (Spectra Max)

Supplied Description:

Instrument Type

Generic Name: plate reader

Generic Description:

Plate readers (also known as microplate readers) are laboratory instruments designed to detect biological, chemical or physical events of samples in microtiter plates. They are widely used in research, drug discovery, bioassay validation, quality control and manufacturing processes in the pharmaceutical and biotechnological industry and academic organizations. Sample reactions can be assayed in 6-1536 well format microtiter plates. The most common microplate format used in academic research laboratories or clinical diagnostic laboratories is 96-well (8 by 12 matrix) with a typical reaction volume between 100 and 200 uL per well. Higher density microplates (384- or 1536-well microplates) are typically used for screening applications, when throughput (number of samples per day processed) and assay cost per sample become critical parameters, with a typical assay volume between 5 and 50 µL per well. Common detection modes for microplate assays are absorbance, fluorescence intensity, luminescence, time-resolved fluorescence, and fluorescence polarization. From: http://en.wikipedia.org/wiki/Plate_reader, 2014-09-0-23.

Parameters

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

Supplied Name	Supplied description	Supplied Units	Standard Name
synechococcus_strain	Synechococcus strain. Two were tested, WH8102 and WH5701	unitless	sample_descrip
time_day	The day a culture aliquot was taken. Days since T0 of the experiment when the culture was transferred to new media and marked the start of the experiment.	days	time_elapsed
media_and_phosphate_level	culture was grown in SN media either with phosphate (+P) or without (-P). See methods.	unitless	treatment
in_vivo_fluor_trip1	in vivo fluorescence for triplicate flask #1	relative fluorescence units (RFU)	fluor
in_vivo_fluor_trip2	in vivo fluorescence for triplicate flask #2	relative fluorescence units (RFU)	fluor
in_vivo_fluor_trip3	in vivo fluorescence for triplicate flask #3	relative fluorescence units (RFU)	fluor
cell_counts_trip1	Flow cytometry cell counts for triplicate flask #1	cells per milliliter (cells mL-1)	cell_concentration
cell_counts_trip2	Flow cytometry cell counts for triplicate flask #2	cells per milliliter (cells mL-1)	cell_concentration
cell_counts_trip3	Flow cytometry cell counts for triplicate flask #3	cells per milliliter (cells mL-1)	cell_concentration