YeiE protein abundance, DNA binding, and influence of sulfite on growth and gene expression in Salmonella
Data files
Dec 03, 2025 version files 22.16 MB
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complemented_yeiE_GC_trial_1.csv
26.17 KB
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complemented_yeiE_GC_trial_2.csv
27.57 KB
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complemented_yeiE_GC_trial_3.csv
27.78 KB
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complemented_yeiH_GC_trial_1.csv
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complemented_yeiH_GC_trial_2.csv
20.84 KB
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complemented_yeiH_GC_trial_3.csv
26.18 KB
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complemented_yeiH_GC_trial_4.csv
23.60 KB
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GC_yeiE_sulfite_trial_1.csv
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GC_yeiE_sulfite_trial_2.csv
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GC_yeiE_sulfite_trial_3.csv
80.02 KB
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low_sulfite_growth_curve_trial_1.csv
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low_sulfite_growth_curve_trial_2.csv
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low_sulfite_growth_curve_trial_3.csv
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low_sulfite_growth_curve_trial_4.csv
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PyeiE_in_WT_and_∆yeiE_with_sulfite_trials1-6.csv
36.45 KB
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PyeiE_induction_with_sulfite_trials_1-3.csv
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PyeiH_in_WT_and_∆yeiE_with_sulfite_trials_1-3.csv
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PyeiH_induction_by_sulfite_trials_1-4.csv
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RawBlotsandCoomassies.tif
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RawEMSAs.tif
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RawYeiEProteinPurification.tif
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README.md
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sulfite_reductase_fold_growth_anaerobic_minimal_media_trials_1-3.csv
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sulfite_reductase_GC_minimal_and_rich_media_with_sulfite_trial_1.csv
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sulfite_reductase_GC_minimal_and_rich_media_with_sulfite_trial_2.csv
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sulfite_reductase_GC_minimal_and_rich_media_with_sulfite_trial_3.csv
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yeiE_and_yeiH_growth_curve_sulfite_trial_1.csv
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yeiE_and_yeiH_growth_curve_sulfite_trial_2.csv
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yeiE_and_yeiH_growth_curve_sulfite_trial_3.csv
20.96 KB
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YeiE_normalization_calculations_trials1-3.csv
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YeiE_promoter_activity_in_WT_and_del_yeiE_time_course_trial_1.csv
12.84 KB
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YeiE_promoter_activity_in_WT_and_del_yeiE_time_course_trial_2.csv
11.76 KB
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YeiE_promoter_activity_in_WT_and_del_yeiE_time_course_trial_3.csv
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Abstract
YeiE is a LysR-type transcriptional regulator that binds to sulfite to influence downstream gene expression. This dataset contains the raw data associated with the following experiments: growth curves of Salmonella enterica serotype Typhimurium (ATCC14028s) WT, ∆yeiE, ∆yeiH , ∆cysJ, ∆asrA, and ∆cysJ∆asrA mutants exposed to sulfite using optical density (OD600) to monitor bacterial growth over time; anaerobic growth of the WT, ∆yeiE, ∆cysJ, ∆asrA, and ∆cysJ∆asrA mutants in minimal media with sulfite or sulfate as sulfur source using colony forming units (CFU); influence of sulfite on the activity of promoters for yeiE (PyeiE) and yeiH (PyeiH) using transcriptional reporters in which PyeiE or PyeiH drives expression of luciferase genes (luxCDABE) to release light (relative luminescence units, RLU); raw data associated with western blotting and Coomassie blue-stained SDS-PAGE gels of YeiE-3XFLAG from Salmonella treated with sulfite (or control treatments) and associated protein normalization calculations; and protein purification and electrophoretic mobility shift assays (EMSAs) of YeiE-3xFLAG with DNA.
Dataset DOI: 10.5061/dryad.j0zpc86qq
Description of the data and file structure
Growth curves are optical density at 600 nm (OD600) collected on a Tecan MPlex plate reader. AVERAGE (or AVG) is averaged data from technical repeats. BLANK is the average value of uninoculated media subtracted from the average value of the indicated strain/condition. Metadata are included at the bottom of the sheet. Kinetic data from bacteria grown on the plate reader are presented with each independent trial in a separate .csv file.
Anaerobic fold growth was determined by serial diluting and plating cultures to determine colony forming units (CFU) per milliliter. Fold change was calculated using the following formula: CFU/mL at 24 hours divided by CFU/mL at 0 hours.
Gene expression data are OD600 and/or relative luminescence units (RLU) collected on a Tecan MPlex plate reader.
Western blot and Coomassie blue-stained gel images are from total proteins of 14028S with a yeiE-3xFLAG chromosomal fusion treated with 1 mM sulfate/sulfite/untreated conditions separated on 10% SDS-PAGE gels in duplicate for each sample. One gel was stained with Coomassie blue and the other was processed for western blotting to identify YeiE-3XFLAG (~36kDa). All blots and gels were imaged on ChemiDoc MP and raw images are organized on a single .tif with details of exposure settings listed.
YeiE-3XFLAG protein abundance was determined from densitometry values of total protein and YeiE-3XFLAG. Within an experiment, the total volume of all bands on Coomassie blue-stained gels was measured using Image Lab software and compared with the sample of lowest mean intensity within a gel to generate a normalization factor for each sample. The intensity of the ~36 kDa YeiE-3xFLAG band was multiplied by the total protein normalization factor for the sample to generate relative YeiE-3xFLAG. Relative YeiE-3xFLAG was normalized to time zero to calculate fold change in YeiE-3xFLAG expression.
Protein purification from E. coli BL21 carrying pBAD18Cm-yeiE-strII and induced with 0.2% arabinose for 2 hours. Recombinant YeiE was purified using a Strep-Tactin XT column (IBA Lifesciences GmbH). Fractions were separated on 10% SDS-PAGE gel stained with Coomassie blue, and imaged using ChemiDoc MP. Elution 2 (E2) indicated on annotated image was used as rYeiE for EMSA in this study.
Electrophoretic mobility shift assays of purified rYeiE ((YeiE-strII) at the indicated concentrations incubated with 40 ng DNA from the indicated regulatory regions. DNA-protein complexes were separated on a native 6% polyacrylamide gel with in-gel SYBR green staining and imaged on ChemiDoc MP. All raw images are organized on one single .tif and details of exposure settings are listed.
Files and variables
Files:
- complemented_yeiE_GC_trial_1.csv
- complemented_yeiE_GC_trial_2.csv
- complemented_yeiE_GC_trial_3.csv
Description: growth curve
Variables
- Growth (OD600) of the WT, ∆yeiE+pWSK29, or ∆yeiE+pYeiE in Lennox broth with 5mM sulfite, 5mM sulfate, or media alone.
Files:
- YeiE_promoter_activity_in_WT_and_del_yeiE_time_course_trial_1.csv
- YeiE_promoter_activity_in_WT_and_del_yeiE_time_course_trial_2.csv
- YeiE_promoter_activity_in_WT_and_del_yeiE_time_course_trial_3.csv
Description: gene expression
Variables
- Gene expression (RLU) from the WT+promoterless plasmid, ∆yeiE+promoterless plasmid, WT+PyeiE reporter, and ∆yeiE+PyeiE reporter grown in Lennox broth pH 7.0.
Files:
- complemented_yeiH_GC_trial_1.csv
- complemented_yeiH_GC_trial_2.csv
- complemented_yeiH_GC_trial_3.csv
- complemented_yeiH_GC_trial_4.csv
Description: growth curve
Variables
- Growth (OD600) of the WT, ∆yeiH+pWSK29, or ∆yeiH+pYeiH in Lennox broth with 5mM sulfite, 5mM sulfate, or media alone.
File: PyeiE_induction_with_sulfite_trials_1-3.csv
Description: gene expression
Variables
- Gene expression (RLU) from the WT with PyeiE transcriptional reporter treated with 1mM sulfite, 1mM sulfate, or 2mM NaCl at exponential growth.
File: PyeiE_in_WT_and_∆yeiE_with_sulfite_trials1-6.csv
Description: gene expression
Variables
- Gene expression on a per-cell basis (RLU/OD600) from the WT or ∆yeiE with PyeiE transcriptional reporter treated with 2mM sulfite, 2mM sulfate, or 4mM NaCl at exponential growth.
File: PyeiH_in_WT_and_∆yeiE_with_sulfite_trials_1-3.csv
Description: gene expression
Variables
- Gene expression on a per-cell basis (RLU/OD600) from the WT or ∆yeiE with PyeiH transcriptional reporter treated with 0.1mM sulfite, 0.1mM sulfate, or 0.2mM NaCl at exponential growth.
File: sulfite_reductase_fold_growth_anaerobic_minimal_media_trials_1-3.csv
Description: anaerobic fold growth
Variables
- CFU/mL from the WT, ∆yeiE, ∆cysJ, ∆asrA, or ∆cysJ∆asrA grown in M9-glucose minimal media with 1mM sulfite or 1mM sulfate as sulfur source.
File: PyeiH_induction_by_sulfite_trials_1-4.csv
Description: gene expression
Variables
- Gene expression on a per-cell basis (RLU/OD600) from the WT with PyeiH reporter treated with the indicated concentrations of sulfite or sulfate (mM) at exponential growth.
Files:
- sulfite_reductase_GC_minimal_and_rich_media_with_sulfite_trial_1.csv
- sulfite_reductase_GC_minimal_and_rich_media_with_sulfite_trial_2.csv
- sulfite_reductase_GC_minimal_and_rich_media_with_sulfite_trial_3.csv
Description: growth curve
Variables
- Growth (OD600) of the WT, ∆yeiE, ∆cysJ, ∆asrA, or ∆cysJ∆asrA grown in M9-glucose minimal media with 1mM sulfite or 1mM sulfate as sulfur source or Lennox broth with 10mM sulfite or 10mM sulfate.
Files:
- GC_yeiE_sulfite_trial_1.csv
- GC_yeiE_sulfite_trial_2.csv
- GC_yeiE_sulfite_trial_3.csv
Description: growth curves
Variables
- Growth (OD600) of the WT or ∆yeiE mutant in Lennox broth with the indicated concentrations of sulfite or sulfate (mM)
Files:
- yeiE_and_yeiH_growth_curve_sulfite_trial_1.csv
- yeiE_and_yeiH_growth_curve_sulfite_trial_2.csv
- yeiE_and_yeiH_growth_curve_sulfite_trial_3.csv
Description: growth curves
Variables
- Growth (OD600) of the WT, ∆yeiH+pWSK29, and ∆yeiH+pYeiH in Lennox broth with the indicated concentrations of sulfite or sulfate (mM)
Files:
- low_sulfite_growth_curve_trial_1.csv
- low_sulfite_growth_curve_trial_2.csv
- low_sulfite_growth_curve_trial_3.csv
- low_sulfite_growth_curve_trial_4.csv
Description: growth curves
Variables
- Growth (OD600) of the WT, ∆yeiH+pWSK29, and ∆yeiH+pYeiH in Lennox broth with the indicated concentrations of sulfite or sulfate (mM)
File: RawBlotsandCoomassies.tif
Description: Raw western blot and Coomassie blue stained gel images
Variables:
- Three biological replicates of ATCC14028s yeiE-3XFLAG treated with 1mM sulfite, sulfate, or not treated for the indicated times. One gel was stained with Coomassie-blue and the other gel was used for western blotting using mouse anti-FLAG IgG.
File: YeiE_normalization_calculations_trials1-3.csv
Description: YeiE-3XFLAG protein abundance
Variables:
- Raw and normalized densitometry values used for quantification of all YeiE-3XFLAG western blots and Coomassie blue stained gels used for quantification of rYeiE over time.
File: RawEMSAs.tif
Description: EMSA gel images
Variables:
- Two biological replicates of EMSA of purified rYeiE (YeiE-strII) at the indicated concentrations incubated with 40 ng DNA.
File: RawYeiEProteinPurification.tif
Description: Protein purification gels
Variables:
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YeiE-Strep-tag II isolated from E. coli BL21 carrying pBAD18Cm-yeiE-str using a Strep-Tactin XT column. Fractions were separated on a 10% SDS-PAGE gel and visualized with Coomassie blue staining. Elution 2 (E2) indicated on annotated image was used as rYeiE for EMSA in this study.
Growth curves:
Overnight cultures of the indicated bacterial strains were diluted 1:100 into Lennox broth or sulfur-free M9 glucose minimal media with the indicated concentrations (mM) of sodium sulfite (Na2SO3), sodium sulfate (Na2SO4), sodium chloride (NaCl), or no additive. Bacterial suspensions were plated into clear 96-well plates and incubated at 37 °C with agitation on a plate reader (Tecan MPlex). Optical density at 600 nm (OD600) data were collected using either iTecan or Magellan software and exported into Microsoft Excel for analysis. Data from duplicate or triplicate technical repeats were averaged, and then the average of the uninoculated media (also reported as BLANK or Lennox) was subtracted to generate blanked data for growth in Lennox broth. Data from growth in M9 minimal media was presented alongside uninoculated media to represent the lower limit of detection of the assay.
Anaerobic fold growth:
Washed overnight cultures of the indicated bacterial strains were pelleted and moved into an anaerobic chamber (Bactron EZ, Shel Lab) with internal atmosphere composed of 90 % N2, 5 % H2 and 5 % CO2. Cell pellets were re-suspended in sulfur-free M9 minimal media and diluted 1:100 into M9 glucose with 1mM sulfite or sulfate. An aliquot was removed, serially diluted, and plated to determine starting colony forming units (CFU) per milliliter. Cultures were incubated at 37 °C in the anaerobic chamber for 24 hours at which time the CFU/mL was determined. Fold growth was CFU/mL at 24 hours divided by starting CFU/mL.
Gene expression:
The WT and ∆yeiE mutant bacterial strains containing a transcriptional reporter plasmid in which the promoter of interest (PyeiE or PyeiH) drove expression of luciferase genes (luxCDABE) or promoter-less luciferase were used. Overnight cultures were diluted 1:100 into Lennox broth with the indicated concentrations (mM) of sodium sulfite, sodium sulfate, sodium chloride, or no additive. For determination of native PyeiE activity, bacterial suspensions were plated into white 96-well plates and incubated in a plate reader at 37 °C with agitation. Relative luminescence units (RLU) were collected using either iTecan or Magellan software and exported into Microsoft Excel for analysis. Data from duplicate or triplicate technical repeats were averaged and then the average of the uninoculated media (also reported as BLANK) was subtracted to generate blanked RLU data. To determine the influence of sulfite on gene expression, overnight cultures of bacterial strains containing the transcriptional reporter plasmids were diluted 1:100 into 50mL of Lennox broth. Bacteria were grown at 37 °C with agitation for 1.5 hours at which time the indicated concentrations of sodium sulfite, sodium sulfate, or sodium chloride (mM) were added. At the indicated times, bacteria were removed to determine the RLU and OD600. Duplicate measurements were averaged and the RLU was divided by the OD600.
Western blotting for YeiE-3XFLAG and protein quantification:
Three biological replicates of western blot and Coomassie blue stained gels of total protein lysates from 14028S with a yeiE-3xFLAG chromosomal fusion grown as for gene expression (above) treated with 1 mM sulfate/sulfite/untreated conditions, normalized by OD600, and proteins separated on 10 % SDS-PAGE gels. YeiE-3XFLAG was detected using mouse anti-FLAG IgG (1:1000 dilution, Life Technologies, Lot ZD393479A) as primary antibody and horse radish peroxidase conjugated goat anti-mouse IgG (1:10,000 dilution; Pierce, Lot 46-170-060115) as secondary antibody. Blots were developed with Clarity Max Western ECL Substrate following the manufacturer’s instructions (Bio-Rad). Band at ~36kDa represents YeiE. All blots and gels were imaged on ChemiDoc MP.
The total volume of all bands on Coomassie blue-stained gels was measured using Image Lab (BioRad v6.1.0) and compared with the sample of lowest mean intensity within a gel to generate a normalization factor for each sample. The intensity of the ~36 kDa YeiE-3xFLAG band was multiplied by the total protein normalization factor for the sample to generate relative YeiE-3xFLAG. Relative YeiE-3xFLAG was normalized to time zero to calculate fold change in YeiE-3xFLAG expression.
Protein purification and EMSA:
YeiE-Strep-tag II was expressed in E. coli BL21 carrying pBAD18Cm-yeiE-strII induced with 0.2 % arabinose for 2 hours. Recombinant YeiE was purified using a Strep-Tactin XT column (IBA Lifesciences GmbH). Fractions were separated on 10 % SDS-PAGE gel and visualized by Coomassie blue staining. Elution 2 (E2) indicated on annotated image was used as rYeiE for EMSA in this study.
Purified rYeiE (YeiE-strII) at the indicated concentrations was incubated with 40 ng DNA from the indicated regulatory regions. DNA-protein complexes were separated on a native 6 % polyacrylamide gel with in-gel SYBR green staining and imaged on ChemiDoc MP.
All raw images are organized on one single .tif and details of exposure settings are listed next to the images.