Table of Contents

• Coverage
• Dataset Description
  • Methods & Sampling
  • Data Processing Description
• Related Publications
• Parameters
• Project Information
• Funding

This dataset includes two forms of the data, one as plain-text .csv (See "Data Files" section, and one in Microsoft Excel (.xlsx) format from MSDial software (See "Data Files" and "Supplemental Files" sections).

The first experiment was done using one strain of Vibrio vulnificus as prey. Equal volumes of the prey bacterial suspensions in artificial seawater were dispensed and tested against a single strain of protist, bacteriophages, and HBx. Predator-free control was also included in the analysis. Predator-prey dual cultures were filtered sequentially through 0.8µm, 0.45µm, 0.2 µm, and 0.01µm membrane filters to obtain extracellular metabolites. Filtrates were acidified and extracted by solid phase extraction (Dittmar et al. 2008) using the C18 cartridges, followed by reverse-phase liquid chromatography coupled with positive and negative electrospray ionization (ESI) FT-ICR MS.  

Instrument summary:

Liquid Chromatography
Reverse phase LC separations were performed online with a C18 custom packed (ReprosSil_Pur 120 Å C18-AQ, 5 μm., 360 μm o.d. X 75 μm i.d., 7.5 cm) outfitted on our nano-HPLC system (ACQUITY M-class, Waters, Milford, MA). An analytical gradient was modified from published gradients to fit our LC-MS/MS set-up (Soule et al. 2015). Analytical gradient going from 5% B to 100% B was carried out (A: 0.1% formic acid in water B: 0.1% formic acid in methanol). Samples were run in triplicates with 4 µL injections at a flow rate of 0.3 uL/min for the analytical separation.

Tandem Mass Spectrometry
Electrospray ionization (ESI) voltage was set to positive 3.5 kV with a source temperature of 300 ºC. MS analysis was performed on the NHMFL 14.5 T and 21 T FT-ICR MS mass spectrometer outfitted with a Velos Pro linear ion trap (Thermo Fisher Scientific) at the front end (Hendrickson et al. 2015; Schaub et al. 2008). Precursor spectra were obtained in the ICR cell with a 1E6 automatic gain control (AGC), sum 4 transient acquisition, and resolving power of 600k at 400 m/z. CID fragmentation spectra were collected from the Velos Pro Ion Trap on the top 3 most abundant ions in the precursor scan with sum 3 transient acquisition and collision energy of 40. Precursor and fragment spectra were collected in the 150-1500 m/z dynamic range. 

Location Summary:

Samples were generated in 2022-July by laboratory microcosm experiments, and the data were collected in 2022-07 and 2023-04.

In vitro microcosm experiments to isolate and characterize the prey cellular organic matter released as lysis products as a result of micropredators were conducted at PI Williams lab at Florida A&M University. 

FT-ICR MS data collection and analysis was conducted by co-PI Chen in the ICR user facility at the National High Magnetic Field Laboratory. 

Orbitrap MS data were collected at the Systems Mass Spectrometry Core at Georgia Institute of Technology in collaboration with Dr. Facundo Fernandez's lab. 

First, MSDial 4.90 (RIKEN), a freely available analysis software, was used for metabolite analysis loaded with a public library of tandem mass spectrometry experiments (13,303 compounds: positive mode) (Tsugawa et al. 2015). MSDial was used due to the ability to identify unique metabolites as well as compare relative abundance of metabolites within each sample. Data was organized by predator types for comparisons. Precursor parameters were set for accurate mass tolerance for MS and MS/MS (0.001 Da and 0.025 Da) and a minimum peak detection of 10k amp. Eight analyte adducts were selected, ([M+NH4]+, [M+Na]+ , [M+Na]+ , [2M+NH4]+ , [M+2H]2+, [M+H+NH4]2+, [M+H+Na]2+, [M+3H]3+) based on previous studies.

The identification score was set to 80% in MSDial for identifications and to limit false positives. A standard mixture was made with B12, ferrichrome, and ferrioxamine E, each at 5 mM. The standard mix runs were used for the alignment of all spectra for comparison. MSDial output includes metabolite retention times, m/z, compound name, and relative abundance within each sample set.

The dataset was also analyzed using Compound Discoverer (ThermoFisher Scientific, version 3.3 SP1).

After analysis, the data were exported from MSDial and saved as .xlsx. The pdf files (*_CompoundDiscovererReport.pdf) were exported from Compound Discoverer. See the "Supplemental Files" section for access to the .xslx and .pdf files.

NSF Award Abstract:

Microbes are the most abundant organisms on Earth and play an important role as degraders, cycling nutrients in the environment. Too many or too few bacteria may disrupt a sensitive ecological balance and proper functioning of environmental processes such as carbon, nitrogen and phosphorus cycles. The abundance of bacteria populations in any given environment is controlled by various biological, chemical and physical mechanisms. Among the biological agents are microscopic predators, or micropredators, of bacteria. The most studied of these are protists, viruses that infect bacteria, and a group of bacteria collectively known as the Bdellovibrio and like organisms (BALOs). These micropredators prey upon certain bacteria to obtain required nutrients or other cellular material for their replication. In the process, cellular products from the prey bacteria are released into the environment and utilized as nutrients by other microbes. Although the micropredators co-occur, and likely interact, in nature, most experimental studies have investigated their activities individually, rather than collectively. As a result, little is known about their collective role in controlling bacteria populations and the cycling of nutrients. The goal of the proposed research is to address this gap in knowledge by investigating all three as a collective group under simulated natural conditions representing a range of temperature, salinity and abundance of prey. This project is conducted at two Historically Black Universities (HBCUs) with strong records of training and mentoring students and postdocs from underrepresented populations in science. The project benefits up to 100 students by providing unique and meaningful educational and research training experiences at the undergraduate and graduate student levels and for early-career scientist. Specific activities include courses on scientific writing and presenting results at annual project workshops as well as national and international scientific meetings. Graduate students are being trained in modern advanced methodologies in chemistry and microbiology. There is an ongoing assessment module to document education and training outcomes.

Up to now, the two mainly accepted mechanisms of mortality in bacterial populations are heterotrophic protist grazing and viral infection. Increasingly, it has become evident that an understudied group of predatory bacteria, BALOs, can also contribute to bacterial mortality. Yet, the mechanisms underlying the dynamics of BALO-prey interactions are poorly understood, as are the interactions among the micropredators, BALOs, protists and bacterial viruses. Ultimately, these processes may have contrasting influences on the structure and functioning of the microbial loop, including impacting higher trophic levels and biogeochemical cycles. The investigators hypothesize that environmental factors significantly influence how mortality in bacterial populations is partitioned among the micropredators. To test this hypothesis researchers are (1) investigating the interactions amongst the micropredators, (2) examining the molecular-level composition and dynamics of dissolved organic matter as the result of the different mortality processes by the NMR/ FT-ICR mass spectrometry (MS) hybrid approach, and (3) modeling these tri-trophic dynamics. Intellectual Merit: Results from this research will define a new mechanistic understanding of mortality dynamics that influence the microbial loop and oceanic biogeochemical cycles.

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.