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| Dataset Title: | [Fe-Labeling Expt: E.coli and phage T4 or T5] - Iron concentrations of phage from experiments of iron-labelled E. coli infected with T4 and T5 bacteriophage, 2018 and 2019. (EAGER: Iron-Virus Interactions in the Ocean) 
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| Institution: | BCO-DMO (Dataset ID: bcodmo_dataset_757485) |
| Information: | Summary
| License
| ISO 19115
| Metadata
| Background
| Data Access Form
| Files
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![[The graph you specified. Please be patient.]](https://erddap.bco-dmo.org/erddap/tabledap/bcodmo_dataset_757485.png?expt_round,Cu_63_nmol&.draw=markers&.marker=5%7C5&.color=0x000000&.colorBar=%7C%7C%7C%7C%7C&.bgColor=0xffccccff)
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Attributes {
s {
date_utc {
String bcodmo_name "date_utc";
String description "UTC date when media was filtered and the experiment initiated";
String long_name "Date Utc";
String source_name "date_utc";
String time_precision "1970-01-01";
String units "unitless";
}
expt_round {
Float32 _FillValue NaN;
Float32 actual_range 1.0, 5.0;
String bcodmo_name "exp_id";
String description "round of experiment and sample analysis on the XR ICP-MS";
String long_name "Expt Round";
String units "unitless";
}
sample {
String bcodmo_name "sample";
String description "sample identifier: 1 for Fe-less (un-spiked) 0.02 um filtered M9 Minimal Media; 2 for 10 uM 57FeSO4 spiked 0.02 um filtered M9 minimal media; or 3 for 0.02 um filtered SM Buffer. Each stage of the experiment was designated sequentially as follows: 20’s for pelleted E. coli cultures grown to mid-logarithmic phase and rinsed three times in Fe-less media (no 57Fe spike) by centrifugation and re-suspension; 30’s for supernatant of pelleted bacteria (20’s); 40’s for unfiltered centrifuged supernatant of bacterial culture infected with phage overnight; 50’s for 0.22 um filtered dissolved fraction; 60’s for 0.02 um filtered soluble fraction; 70’s for bacterial samples treated with chloroform; 80’s for the supernatant (sample layer) above the sucrose cushion following ultracentrifugation; 90’s for sucrose cushion pellet re-suspended in 0.02 um filtered SM buffer; DIA for dialyzed samples post-dialysis over 6 buffer tank changes; and B for dialysis buffer: B1 pre-dialysis; B1-2 post-initial dialysis; B3-2 post-third dialysis buffer tank change; and B6-2 post- final dialysis buffer tank change. Samples were treated in triplicate: A; B; C for E. coli phage T4 samples; D; E; F for chloroform-lysed bacterial control samples; and G; H; I for Blanks; and M; N; O for E. coli phage T5 samples.";
String long_name "Sample";
String nerc_identifier "https://vocab.nerc.ac.uk/collection/P02/current/ACYC/";
String units "unitless";
}
description {
String bcodmo_name "brief_desc";
String description "description of phage purification step and type of sample";
String long_name "Description";
String units "unitless";
}
Fe_56_nM {
String bcodmo_name "Fe";
String description "Concentration of 56Fe as determined by XR ICP-MS";
String long_name "Fe 56 N M";
String units "nanoMolar";
}
Fe_57_nM {
String bcodmo_name "Fe";
String description "Concentration of 57Fe as determined by XR ICP-MS";
String long_name "Fe 57 N M";
String units "nanoMolar";
}
volume {
String bcodmo_name "volume";
String description "sample volume";
String long_name "Volume";
String units "liters";
}
Fe_56_nmol {
String bcodmo_name "Fe";
String description "56Fe concentration as determined by XR ICP-MS";
String long_name "Fe 56 Nmol";
String units "nanomoles";
}
Fe_57_nmol {
String bcodmo_name "Fe";
String description "57Fe concentration as determined by XR ICP-MS";
String long_name "Fe 57 Nmol";
String units "nanomoles";
}
Cu_63_nmol {
Float32 _FillValue NaN;
Float32 actual_range 6.03e-4, 265.0;
String bcodmo_name "Cu";
String description "63Cu concentration as determined by XR ICP-MS";
String long_name "Cu 63 Nmol";
String units "nanomoles";
}
Zn_66_nmol {
Float32 _FillValue NaN;
Float32 actual_range 0.0, 13800.0;
String bcodmo_name "Zn";
String description "66Zn concentration as determined by XR ICP-MS";
String long_name "Zn 66 Nmol";
String units "nanomoles";
}
Ni_60_nmol {
Float32 _FillValue NaN;
Float32 actual_range 2.19e-4, 1820.0;
String bcodmo_name "trace_metal_conc";
String description "60Ni concentration as determined by XR ICP-MS";
String long_name "Ni 60 Nmol";
String nerc_identifier "https://vocab.nerc.ac.uk/collection/P03/current/C035/";
String units "nanomoles";
}
Pb_208_nmol {
Float32 _FillValue NaN;
Float32 actual_range 5.05e-5, 340.0;
String bcodmo_name "trace_metal_conc";
String description "208Pb concentration as determined by XR ICP-MS";
String long_name "Pb 208 Nmol";
String nerc_identifier "https://vocab.nerc.ac.uk/collection/P03/current/C035/";
String units "nanomoles";
}
bact_cells_ml {
Float32 _FillValue NaN;
Float32 actual_range 0.344, 4.55e+8;
String bcodmo_name "bact_abundance";
String description "SYBR epifluorescence bacterial counts";
String long_name "Bact Cells Ml";
String nerc_identifier "https://vocab.nerc.ac.uk/collection/P02/current/BNTX";
String units "cells/milliliter";
}
phage_VPL_ml {
Float64 _FillValue NaN;
Float64 actual_range 4.49e+9, 4.62e+11;
String bcodmo_name "cell_concentration";
String description "phage cell concentration";
String long_name "Phage VPL Ml";
String units "virus-like particles/milliliter";
}
Fe_57_atoms_per_phage {
Float32 _FillValue NaN;
Float32 actual_range 74.3, 3200.0;
String bcodmo_name "Fe";
String description "57Fe content measured by XR ICP- MS; converted to moles; multiplied by Avogadro’s constant (6.022 x 1023 atoms/mol); and divided by number of phage in the sample";
String long_name "Fe 57 Atoms Per Phage";
String units "atoms";
}
notes_expt {
String bcodmo_name "comment";
String description "comments about samples";
String long_name "Notes Expt";
String units "unitless";
}
}
NC_GLOBAL {
String access_formats ".htmlTable,.csv,.json,.mat,.nc,.tsv";
String acquisition_description
"All materials were cleaned by soaking overnight with heating (1.5%
Citrad\\u00ae, by Decon Labs, Inc) in deionized water, rinsed in RO water, and
soaked in 10% TMG HCl (Fisher) in ultrapure water for 30 days, then rinsed
with ultrapure water, let dry in an AirClean 400 work station overnight, and
double-bagged in polyethylene bags (Mellett et al. 2018). M9 minimal media for
bacterial cultures was made using ultrapure water (18.2 M\\u03a9 cm),
containing final concentrations of 33.7 mM Na2HPO4\\u00b72H2O (Sigma-Aldrich
\\u226599.0% Titration), 22 mM KH2PO4 (ACS Reagent \\u226599% purity), 8.56 mM
NaCl (Certified ACS \\u226599.0% purity), 18.7 mM NH4Cl (Fisher Scientific
\\u226599.0% purity FCC), 0.1 M MgSO4 (Sigma-Aldrich \\u226599.99% trace metal
purity), 0.1 M CaCl2 (Alfa Aesar from Fisher Scientific 99.99% metals basis),
1 \\u00b5g/ml Thiamine HCl (Fisher Scientific 99% purity), and 0.5% Glucose
(Fisher Scientific 99% purity) in 1 L of Milli-Q (Kutter and Sulakvelidze
2004, Table 1;\\u00a0see Supplemental Documents below).
M9 minimal media was spiked with 57FeSO4 (final concentration: 10 \\u00b5M),
then filtered through a 0.02 \\u00b5m Whatman Anotop syringe filter that had
been rinsed with Milli-Q; the first few drops of media were discarded (Sample
2). Some un-spiked media was reserved for bacterial pellet rinses (Sample 1).
A volume of 20 mL media was inoculated with a frozen culture of the E. coli
strain ZK126 (Betty Kutter, Evergreen State College) which was grown over
eight generations exclusively on M9 minimal media with 57FeSO4. The culture
was placed into a polyethylene bag and vented to avoid contamination, then
incubated while shaking overnight at 37 \\u00b0C to reach late-logarithmic
growth. The following morning, three 45 mL aliquots of 10 \\u00b5M 57FeSO4
spiked and 0.02 \\u00b5m filtered media samples (one 45 mL blank sample
remained un-inoculated with E. coli) were weighed into 250 mL acid-cleaned
polycarbonate flasks. Each of the three aliquots were inoculated with 10% of
the overnight bacterial culture. The three bacterial cultures as well as the
blank samples were placed in a vented polyethylene bag and incubated while
shaking at 37 \\u00b0C. Once the culture reached mid-logarithmic growth, as
indicated by an absorbance (OD600) \\u00a0measured on a spectrophotometer
between 0.200-0.500 (Figure 1), the cultures were divided into 20 mL aliquots
(Figure 2, see Supplemental Documents, below).
To rinse excess 57Fe label from the surface of the bacterial cells, the
aliquoted cultures were transferred to a 50 mL falcon tube and centrifuged at
a speed of 6500 x g. The bacterial cells pelleted to the base of the tube, and
the supernatant was discarded. A volume of 10 mL of fresh Fe-less (not spiked
with 57FeSO4) 0.02 \\u00b5m filtered media was added and vortexed for 1 minute.
The rinsed bacterial cells were again centrifuged 6500 x g for 5 minutes to
pellet, and the cell rinsing was repeated three times. The final bacterial
pellet was re-suspended in 20 mL of Fe-less media. Samples designated A, B,
and C were T4 phage lysates, samples D, E, and F remained uninfected by phage
and served as bacterial control cells that were burst open by treatment with
chloroform, and samples G, H, and I were blanks of Fe-less media. For all
rounds (Round 1, 2, 3, 4, and 5 samples A, B, and C (Figure 3) were infected
with 5 \\u00b5l of T4 bacteriophage (Betty Kutter, Evergreen State College) at
a titer of 1.3 x 1011 phage/ml. For Rounds 2, 3, 4, and 5, samples M, N, and O
were infected with 5 \\u00b5l of T5 bacteriophage (ATCC\\u00ae 11303-B5\\u2122)
at a titer of 8.2 x 1011 phage/ml. Samples D, E, and F (bacterial controls) as
well as G, H, and I (blanks) remained uninfected and instead were stored at 4
\\u00b0C overnight to be used as phage-free lysis controls. Phage-infected
cultures A, B, and C were left shaking at 37 \\u00b0C overnight.
On the next day of the experiment, the uninfected bacterial controls D, E, and
F were treated with 20% chloroform (Fisher Scientific, Mobile phase for HPLC
applications \\u226599.8% purity) and vortexed for one minute to burst open the
bacterial cells without viral lysis. The necessity for this viral lysis-free
control is to account for colloids from within the bacterial cells that
contain 57Fe and would purify with T4 phage. For Round 5 all phages (A, B, C,
M, N, O) and blanks (G, H, I) were also treated with 20% chloroform and
vortexed for one minute. All the samples were then centrifuged in 50 mL Falcon
tubes at a speed of 9500 x g for 5 minutes to pellet the remaining bacterial
debris. The supernatant, which contained the T4 phage progeny for samples A,
B, and C and the T5 phage progeny for samples M, N, and O, was then filtered
using a 0.22 \\u00b5m Sterivex PVDF syringe filter (EMD Millipore), with a
Milli-Q pre-rinse and the first few drops of sample discarded. The filtrate
was the fraction containing phage and any soluble or colloidal 57Fe within the
dissolved size fraction (<0.22 \\u00b5m). The subsequent filtration of a
subsample of the dissolved fraction through a 0.02 \\u00b5m Whatman Anotop
syringe filter (with a Milli-Q pre-rinse and the first few drops of sample
discarded) was collected for the soluble fraction (<0.02 \\u00b5m). The
difference between the dissolved and the soluble fractions are used to
calculate iron within the colloidal fraction (0.2 \\u00b5m-0.02 \\u00b5m).
The phage within the dissolved fraction were further purified using a sucrose
cushion, which is a density-dependent technique used to concentrate and purify
phage by precipitating viral particles below a dense layer of sucrose (Hurwitz
et al. 2013). To do so, a 2.5 ml layer of 38% sucrose (Fisher Scientific) in
SM buffer (100 mM NaCl, 8 mM MgSO4, 50 mM Tris-HCl in Milli-Q, pH 7.5 and 0.02
\\u00b5m filtered) was added to the bottom of the ultracentrifuge tube (Beckman
Coulter), followed by 1 ml of sample and 10.5 ml of SM Buffer by carefully
tilting the tube so as not to disturb the dense sucrose layer. The samples
were spun in a Beckman Coulter SW40Ti swinging bucket ultracentrifuge, for 3
hours and 15 minutes at 175,000 x g (37,200 rpm). The sucrose and SM buffer
layers were then discarded, and the tubes were dried in a laminar flow clean
hood (Air Clean) for 20 minutes. The pelleted phages from samples A-C and M-O,
including any potential bacterial colloids of the same density as the phage as
accounted for in samples D-F, were then resuspended in 1 mL of SM Buffer.
All the samples were dialyzed using Float-A-Lyzer 100 kDa dialysis devices
(Fisher Scientific) in 1 L of dialysis buffer (10 mM NaCl, 50 mM Tris-Cl pH
8.0, 10 mM MgCl2) for a total of 6 buffer changes over 4 days. Bacterial and
viral counts were performed throughout for samples using SYBR nucleic acid
stain under epifluorescence microscopy (Noble & Furman 1998).
Metal concentrations were quantified using Element XR ICP-MS (Thermo) after
50-fold dilution with 5% nitric (Fisher Scientific, Optima) containing 10 ppb
rhodium as internal standard, and using external standard calibration curves.
Blank values after rhodium correction are listed in Table 2 (see Supplemental
Documents below).";
String awards_0_award_nid "713366";
String awards_0_award_number "OCE-1722761";
String awards_0_data_url "http://www.nsf.gov/awardsearch/showAward.do?AwardNumber=1722761";
String awards_0_funder_name "NSF Division of Ocean Sciences";
String awards_0_funding_acronym "NSF OCE";
String awards_0_funding_source_nid "355";
String awards_0_program_manager "Michael E. Sieracki";
String awards_0_program_manager_nid "50446";
String cdm_data_type "Other";
String comment
"Fe-Labeling Experiment: E.coli and T4, T5
PI's: K. Buck
version date: 2019-10-10";
String Conventions "COARDS, CF-1.6, ACDD-1.3";
String creator_email "info@bco-dmo.org";
String creator_name "BCO-DMO";
String creator_type "institution";
String creator_url "https://www.bco-dmo.org/";
String data_source "extract_data_as_tsv version 2.3 19 Dec 2019";
String date_created "2019-03-04T16:24:51Z";
String date_modified "2020-01-27T21:15:48Z";
String defaultDataQuery "&time<now";
String doi "10.1575/1912/bco-dmo.757485.1";
String history
"2024-11-08T05:46:41Z (local files)
2024-11-08T05:46:41Z https://erddap.bco-dmo.org/tabledap/bcodmo_dataset_757485.das";
String infoUrl "https://www.bco-dmo.org/dataset/757485";
String institution "BCO-DMO";
String instruments_0_acronym "ICP Mass Spec";
String instruments_0_dataset_instrument_description "Used to measure metal concentrations.";
String instruments_0_dataset_instrument_nid "757493";
String instruments_0_description "An ICP Mass Spec is an instrument that passes nebulized samples into an inductively-coupled gas plasma (8-10000 K) where they are atomized and ionized. Ions of specific mass-to-charge ratios are quantified in a quadrupole mass spectrometer.";
String instruments_0_instrument_external_identifier "https://vocab.nerc.ac.uk/collection/L05/current/LAB15/";
String instruments_0_instrument_name "Inductively Coupled Plasma Mass Spectrometer";
String instruments_0_instrument_nid "530";
String instruments_0_supplied_name "ELEMENT XR High Resolution Inductively Coupled Plasma Mass Spectrometer";
String instruments_1_acronym "Spectrophotometer";
String instruments_1_dataset_instrument_description "Used to measure bacterial cell concentrations.";
String instruments_1_dataset_instrument_nid "762163";
String instruments_1_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.";
String instruments_1_instrument_external_identifier "https://vocab.nerc.ac.uk/collection/L05/current/LAB20/";
String instruments_1_instrument_name "Spectrophotometer";
String instruments_1_instrument_nid "707";
String instruments_2_dataset_instrument_description "Used to concentrate cells and separate bacteria from phage.";
String instruments_2_dataset_instrument_nid "762164";
String instruments_2_description "A machine with a rapidly rotating container that applies centrifugal force to its contents, typically to separate fluids of different densities (e.g., cream from milk) or liquids from solids.";
String instruments_2_instrument_name "Centrifuge";
String instruments_2_instrument_nid "629890";
String instruments_2_supplied_name "Beckman Coulter SW40Ti swinging bucket ultracentrifuge";
String keywords "atoms, bact, bact_cells_ml, bco, bco-dmo, biological, cells, chemical, Cu_63_nmol, data, dataset, date, description, dmo, erddap, expt, expt_round, Fe_56_nM, Fe_56_nmol, Fe_57_atoms_per_phage, Fe_57_nM, Fe_57_nmol, management, Ni_60_nmol, nmol, notes, notes_expt, oceanography, office, Pb_208_nmol, per, phage, phage_VPL_ml, preliminary, round, sample, time, volume, vpl, Zn_66_nmol";
String license "https://www.bco-dmo.org/dataset/757485/license";
String metadata_source "https://www.bco-dmo.org/api/dataset/757485";
String param_mapping "{'757485': {}}";
String parameter_source "https://www.bco-dmo.org/mapserver/dataset/757485/parameters";
String people_0_affiliation "University of South Florida";
String people_0_affiliation_acronym "USF";
String people_0_person_name "Mya Breitbart";
String people_0_person_nid "51740";
String people_0_role "Principal Investigator";
String people_0_role_type "originator";
String people_1_affiliation "University of South Florida";
String people_1_affiliation_acronym "USF";
String people_1_person_name "Kristen N. Buck";
String people_1_person_nid "51624";
String people_1_role "Principal Investigator";
String people_1_role_type "originator";
String people_2_affiliation "University of South Florida";
String people_2_affiliation_acronym "USF";
String people_2_person_name "Chelsea Bonnain";
String people_2_person_nid "732872";
String people_2_role "Co-Principal Investigator";
String people_2_role_type "originator";
String people_3_affiliation "University of South Florida";
String people_3_affiliation_acronym "USF";
String people_3_person_name "Salvatore Caprara";
String people_3_person_nid "732874";
String people_3_role "Co-Principal Investigator";
String people_3_role_type "originator";
String people_4_affiliation "Woods Hole Oceanographic Institution";
String people_4_affiliation_acronym "WHOI BCO-DMO";
String people_4_person_name "Nancy Copley";
String people_4_person_nid "50396";
String people_4_role "BCO-DMO Data Manager";
String people_4_role_type "related";
String project "Fe-Virus";
String projects_0_acronym "Fe-Virus";
String projects_0_description
"Iron is an essential micronutrient for phytoplankton that is required for photosynthesis and respiration. Insufficient iron has been shown to limit phytoplankton growth in large regions of the surface ocean, and correspondingly, iron cycling is directly linked to carbon cycling in much of the marine environment. Nearly all iron in seawater (>99%) exists as complexes with organic molecules called ligands, which govern the concentration of iron dissolved in the water and the bioavailability of that iron to phytoplankton. However, despite the importance of iron-binding organic ligands, their sources and identities are largely unknown. Viruses, the majority of which are phages (viruses that infect bacteria), are extremely abundant in seawater and are in the same size fraction as dissolved iron. Recent evidence that non-marine phages contain iron as part of their structures has led to the proposal that marine phages may represent a previously overlooked class of organic iron-binding ligands. This project is determining the contribution of marine phages to dissolved iron pools and culture phage-host systems in the laboratory to determine if phages utilize bacterial iron-uptake receptors for infection in the manner of a Trojan horse. As the first study to examine the biogeochemical impact of trace elements contained within the structure of highly abundant marine phage particles, successful completion of the proposed research will be transformative for biological and chemical oceanography and have far-reaching implications for other fields, including human health where iron availability plays an important role in microbial pathogenesis. This project contributes to the multidisciplinary training of a graduate student and postdoctoral researcher. Research results will be disseminated through scientific publications and presentations, and the public will be educated about linkages between viruses and ocean chemistry via a hands-on exhibit for the annual St. Petersburg Science Festival.
Building upon evidence from non-marine model systems demonstrating the presence of iron ions in phage tail proteins and phage utilization of cell surface receptors for siderophore-bound iron, this project combines field and laboratory-based experiments to test the following three hypotheses regarding iron-virus interactions in the oceans: (1) Iron incorporated into phage tails originates from bacterial cell reserves, reducing the amount of iron available for remineralization upon lysis; (2) Phages constitute important iron-binding ligands in the oceans, accounting for a substantial portion of organically complexed colloidal dissolved iron; (3) Marine phages compete with siderophore-bound iron for uptake receptors on the bacterial cell surface and use iron in their tails as a Trojan horse for infection. Initial calculations predict that phages could account for up to 70% of the colloidal fraction of organically complexed dissolved iron in the surface ocean; therefore, this project is critical for advancing knowledge of trace-metal cycling as well as phage-host interactions. Additionally, if a portion of the cellular iron thought to be released from bacterial cells for remineralization following lysis is already incorporated into phage tails, then these findings will have significant implications for oceanic biogeochemical models. Through a combination of laboratory-based culture experiments and field sample measurements, this project could reveal the identity of a ubiquitous component of colloidal organic iron-binding ligands, modify the estimates of iron concentrations and species released through viral lysis, and potentially identify a novel receptor type for marine phage that may compete with the acquisition of siderophore-bound iron by host bacteria.";
String projects_0_end_date "2019-01";
String projects_0_name "EAGER: Iron-Virus Interactions in the Ocean";
String projects_0_project_nid "713367";
String projects_0_start_date "2017-02";
String publisher_name "Biological and Chemical Oceanographic Data Management Office (BCO-DMO)";
String publisher_type "institution";
String sourceUrl "(local files)";
String standard_name_vocabulary "CF Standard Name Table v55";
String summary "This data was collected as part of a study investigating the source of iron to bacteriophage (phage for short, or viruses that infect and kill bacteria) progeny. Evidence from a phage that infects E. coli shows iron incorporated into the tail fiber structure. This study aims at identifying whether the source of the iron is environmental or bacterially derived. E. coli bacterial cultures were grown in minimal media spiked with 10 \\u00b5M 57FeSO4 then infected with phage T4 or T5. The phages were purified by methods of centrifugation, filtration, density-dependent ultracentrifugation, and dialyzing. The resulting phage fractions were quantified by SYBR epifluorescence microscopy and metal concentrations were measured on an ELEMENT XR ICP-MS.";
String title "[Fe-Labeling Expt: E.coli and phage T4 or T5] - Iron concentrations of phage from experiments of iron-labelled E. coli infected with T4 and T5 bacteriophage, 2018 and 2019. (EAGER: Iron-Virus Interactions in the Ocean)";
String version "1";
String xml_source "osprey2erddap.update_xml() v1.3";
}
}
Data Access Protocol (DAP) and its
selection constraints.
The URL specifies what you want: the dataset, a description of the graph or the subset of the data, and the file type for the response.
Tabledap request URLs must be in the form
https://coastwatch.pfeg.noaa.gov/erddap/tabledap/datasetID.fileType{?query}
For example,
https://coastwatch.pfeg.noaa.gov/erddap/tabledap/pmelTaoDySst.htmlTable?longitude,latitude,time,station,wmo_platform_code,T_25&time>=2015-05-23T12:00:00Z&time<=2015-05-31T12:00:00Z
Thus, the query is often a comma-separated list of desired variable names,
followed by a collection of
constraints (e.g., variable<value),
each preceded by '&' (which is interpreted as "AND").
For details, see the tabledap Documentation.