Table of Contents

• Coverage
• Dataset Description
  • Methods & Sampling
  • Data Processing Description
• Data Files
• Supplemental Files
• Related Publications
• Related Datasets
• Parameters
• Instruments
• Project Information
• Funding

This dataset is part of a larger study on the impact of different physiological states/responses of Prochlorococcus on nitrite production potential in the presence of nitrate. The different perturbations yielding the different physiological states/responses and the respective analyses are listed below. Links to data for each perturbation can be found in the Related Datasets section.

The analyses include:

1. Cell concentration (Nitrogen limitation dataset, Light perturbation dataset, Temperature perturbation dataset)
2. Nitrite concentration (Nitrogen limitation dataset, Light perturbation dataset, Temperature perturbation dataset)
3. Nitrate + nitrite concentration (Nitrogen limitation dataset)
4. Bead standard normalized forward scatter (Nitrogen limitation dataset)
5. Bead standard normalized red (chlorophyll) fluorescence (Nitrogen limitation dataset)
6. FIRe fluorometry (Light perturbation dataset, Temperature perturbation dataset)

See Related Datasets section below for links to above mentioned datasets.

Cultures: The strains used in this study included Prochlorococcus MIT0915 and Prochlorococcus MIT0917. Before each experiment, all strains were  assayed for heterotrophic contaminants by staining cells with SYBR green and assessing the fluorescence and light scattering properties of both stained and unstained cells using a Guava easyCyte 12HT Flow Cytometer (MilliporeSigma, Burlington, MA, USA) – cultures that did not exhibit the presence of non-photosynthetic cells in the stained samples and had a single cyanobacteria population were presumed axenic and unialgal. Before each experiment, all presumed axenic cultures were  assessed for purity by confirming a lack of turbidity after inoculation into a panel of purity test broths as described previously (Berube et al., 2015).

Culture medium: The cultures were grown using AMP1-Mo-NO3 medium which is a variant of AMP1 artificial medium (Moore et al., 2007) with the following modifications: 400 µM ammonium sulfate was replaced with 800 µM sodium nitrate; the sodium molybdate concentration was increased from 0.3 nM to 100 nM; 1 mM HEPES pH=7.5 was replaced with 5 mM TAPS pH=8.2; and the concentration of sodium bicarbonate was decreased from 6 mM to 2.5 mM. The medium was filter sterilized using a 0.2 µm polyethersulfone (PES) membrane filter instead of using steam sterilization in an autoclave. A nitrogen-free variant (AMP1-Mo) was also prepared by omitting all inorganic nitrogen sources. 

Methodology and sampling for light and temperature perturbations: Batch cultures were grown as triplicates in 35 mL of medium in borosilicate glass culture tubes at a temperature of 24^oC and a photon flux of 20 µmol photons m^-2 s^-1 of blue light. Cultures were acclimated for at least 10 generations prior to starting the experiment. Each biological replicate was diluted to 1 x 10⁸ cells mL¹ in a total of 50 mL of nitrogen-free AMP1-Mo supplemented with 2.5 mM sodium bicarbonate in duplicate, one to serve as a control and one to be subjected to the perturbation. Shock experiments were conducted in light- and temperature-controlled water baths. In both experiments, the control box was maintained at 23–25^oC and 21–23 µmol photons m^-2 s^-1 of blue light. In the light shock experiment, experimental cultures were exposed to 220 µmol photons m^-2 s^-1 of broad spectrum light at 23-25^oC. In the cold shock experiment, the cultures were exposed to 21–23 µmol photons m^-2 s^-1 of blue light at 14–16^oC. Prior to initiating the cold shock, nitrogen-free AMP1-Mo medium was pre-chilled to 15^oC before inoculation with the harvested biomass. Prior to spiking with nitrate, an initial set of samples were collected for flow cytometry, nitrite concentration determination, and fluorescence induction and relaxation (FIRe) fluorometry. Nitrate was then added to each tube at a final concentration of 80 µM sodium nitrate. Samples for nitrite concentration determination were collected every 30 min for 150 min by syringe filtration of 4 mL of culture through a 0.2 µm PES membrane into a 14 mL polystyrene tube and stored at -20^oC. Endpoint flow cytometry and FIRe fluormetry samples were also collect at the final time point. Media-only control tubes were prepared using the same batch of nitrogen-free AMP1-Mo medium to facilitate assessment of background signal in nitrite concentration determination; the mean background signal was equivalent to 0.050 µM nitrite (+/- 0.004) for the light shock experiment and 0.076 µM nitrite (+/- 0.012) for the cold shock experiment.

Nitrate and nitrate + nitrite concentration assay: Nitrite was analyzed using an AA3 HR Autoanalyzer (Seal Analytical, Milwaukee, WI, USA). In this continuous segmented flow analyzer, nitrite is reacted with sulfanilamide and N-(1-naphthyl)ethylenediamine (NED) to produce a pink diazo dye. For determination of total nitrate and nitrite, any nitrate in the sample was first reduced to nitrite using a copper-cadmium coil. Absorbance was measured in a 1 cm flow cell using a 550 nm bandpass filter (SEAL Analytical method number G-384-08 for a multitest MT19B manifold). For each analysis, sodium nitrite and sodium nitrate standards were freshly prepared and serially diluted in nitrogen-free AMP1 medium to create a calibration curve at the following concentrations: 1.25, 0.5, 0.25, 0.08, 0.04, 0.03, and 0.015 µM. A linear calibration curve was applied to the data and the instrument’s response factor, represented by the slope of the calibration curve, remained consistent and reproducible throughout the nitrite analysis period (r² >0.99 in all runs). For samples in nitrogen-free AMP1 medium (AMP1-Mo) as a background matrix, the limit of detection (LOD) for nitrite was 0.010 µM and the limit of blank was 0.003 µM.

Cell concentration determination: Cell concentrations were obtained by flow cytometry using a Guava easyCyte 12HT Flow Cytometer (MilliporeSigma, Burlington, MA, USA). Prochlorococcus cells were detected based on the fluorescence of cellular pigments excited by a 488 nm laser. Cultures were first diluted to between 50 and 200 cells/µL and data were acquired for up to 6 min at a flow rate of 0.024 µL/s. Bead standards (Guava easyCheck beads; MilliporeSigma, Burlington, MA, USA), were run daily to confirm that the instrument was operating within normal parameters and within predefined tolerances for concentrations, scatter, and emission intensity of the beads. For each flow cytometry run, a diluted sample of Guava easyCheck beads was additionally run using the same parameters as the cultures to serve as a standard for the normalization of forward scatter and fluorescence data.

Fluorescence induction and relaxation fluorometry: The efficiency for energy conversion at photosystem II (Fv/Fm) for cultures subjected to light or cold shocks was measured using a FIRe Fluorometer System (Satlantic, Halifax, NS, Canada). A sample of each culture was diluted 10 fold in nitrogen-free AMP1 medium, incubated in the dark for 10 minutes in order to allow photosystems to open, and then transferred to a cylindrical quartz cuvette. A total of 10 profiles were collected for each sample. Background profiles for cell-free AMP1-Mo medium at 21 different gain values were collected to facilitate data processing; these files are named in format "AMP1NON.000" where "000" is a 3 digit counter from 000 through 020.

Flow cytometry data were exported as FSC 3.0 formatted files and analyzed using FlowJo version 10.6.1 (BD Biosciences, Ashland, OR, USA)

Fluorescence data were processed using fireworx version 0.9.1 (https://sourceforge.net/projects/fireworx/)

Continuous segmented flow nutrient autoanalyzer data were analyzed using AACE version 7.12 software (Seal Analytical, Mequon, WI, USA)

NSF Award Abstract:
The marine bacterium, Prochlorococcus, is a central part of the food web in the subtropical open ocean, one of the largest biomes on the planet. Like plants on land, Prochlorococcus and other phytoplankton are capable of photosynthesis, harnessing light to convert carbon dioxide into sugars and other organic matter. This matter feeds all the life in the sea. Akin to terrestrial plants, Prochlorococcus requires additional nutrients or "fertilizer" to grow and photosynthesize. Among these nutrients, nitrogen is often scarce across much of the sunlit ocean. Thus, understanding the means through which nitrogen is obtained and used by Prochlorococcus has important consequences for understanding how the nitrogen and carbon cycles are coupled in the ocean. Not all Prochlorococcus cells have the genetic capacity to use all sources of inorganic nitrogen, i.e. nitrate, nitrite, and ammonium. Some can use all three, some the last two, and some only ammonium. Cells that can use nitrate must sequentially transform it to nitrite and then to ammonium before they can make the building blocks for proteins. The genesis of this project derives from the observation that some Prochlorococcus cells release nitrite into the seawater during growth on nitrate. This project examines this feature of the physiology of these cell lines, and asks whether cells that release nitrite can support the growth of other cells than cannot use nitrate, in effect creating a cross-feeding situation that could make the system more robust. Understanding the drivers behind the coexistence of cells with different ways of obtaining nitrogen, a key currency in the ocean, will provide important insights on the flow of nitrogen in marine ecosystems. This project also sheds light on the structure of interactions between microbes and provides the broader scientific community (for instance, those studying diverse microbiomes related to human health and disease or agriculture) a new perspective on how microbes form beneficial partnerships. This project supports immersive laboratory-based research experiences for undergraduate students, who design and execute experiments directly related to the overall project goals. The project further supports the work of the investigators to engage with the general public on topics related to phytoplankton, photosynthesis, and the ecosystem services provided by these marine organisms.

In the low-light adapted LLI clade of Prochlorococcus, the focus of this project, nearly all cells possess the downstream half of the nitrate assimilation pathway (for the assimilation of nitrite). Only a fraction of LLI cells, however, have the complete nitrate assimilation pathway. Incomplete assimilatory nitrate reduction, with concomitant nitrite release, has been observed for LLI cells during growth on nitrate as the sole nitrogen source. Further, the nitrite released by cells growing on nitrate can support the growth of Prochlorococcus that can use nitrite but not the more oxidized nitrate. Overall, within a group of closely-related Prochlorococcus, there is genotypic and phenotypic diversity related to the production and consumption of nitrite, a central intermediate in the nitrogen cycle. The investigators propose to further develop Prochlorococcusas a model system to explore nitrite cycling within populations and provide new insights on how trait variability and the selection of complementary functions facilitates robustness and/or resiliency in microbial populations. The overarching hypothesis is that the population-level assembly of distinct functional types of Prochlorococcus emerges through interactions that are mediated, in part, by cross-feeding of nitrite. To address this broad hypothesis, the investigators are focusing on the following objectives: 1) assessing the physiological underpinnings of incomplete assimilatory nitrate reduction and nitrite release through in-vitro biochemical characterization of nitrite reductase enzymes and transcription profiling of cells subjected to light and temperature stress, 2) examining the nitrite production and consumption rates of Prochlorococcus strains across environmental gradients such as light, temperature, and nutrient availability in order to constrain the environmental parameters that modulate nitrite cycling, and 3) determining the frequencies and activities of nitrogen assimilation genotypes within laboratory and field populations, under varying environmental conditions and perturbations. Outcomes from objectives 1 and 2 help to constrain the tradeoffs associated with incomplete nitrate reduction and the release of nitrite (a valuable commodity to nitrogen limited cells) to facilitate modelling and interpretation of how partnerships between nitrogen assimilation genotypes are structured. These insights help to direct experiments in Objective 3, which aims to examine controlled laboratory co-cultures and field populations in order to produce quantitative data on the emergent features of Prochlorococcus populations where interactions are mediated by the cross-feeding of nitrite. These data are being used to develop an improved understanding of how interactions mediated by a common public good might give rise to emergent properties of populations, including resilience to perturbation and greater population-wide efficiency in nitrogen assimilation.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.