DOI: 10.5061/dryad.j0zpc86qq
title: YeiE protein abundance, DNA binding, and influence of sulfite on growth and gene expression in <em>Salmonella</em>

description: 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.
description: <strong>Growth curves:</strong>
 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
 (Na<sub>2</sub>SO<sub>3</sub>), sodium sulfate
 (Na<sub>2</sub>SO<sub>4</sub>), 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 (OD<sub>600</sub>)
 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.
 <strong>Anaerobic fold growth:</strong>
 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 % N<sub>2</sub>, 5 %
 H<sub>2</sub> and 5 % CO<sub>2</sub>. 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.
 <strong>Gene expression:</strong>
 The WT and ∆<em>yeiE</em> mutant bacterial strains
 containing a transcriptional reporter plasmid in which the promoter of
 interest (P<em><sub>yeiE</sub></em> or
 P<em><sub>yeiH</sub></em>) drove expression of
 luciferase genes (<em>luxCDABE</em>) 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 OD<sub>600</sub>. Duplicate
 measurements were averaged and the RLU was divided by the
 OD<sub>600</sub>. <strong>Western
 blotting for YeiE-3XFLAG and protein
 quantification:</strong> Three biological
 replicates of western blot and Coomassie blue stained gels of total
 protein lysates from 14028S with a <em>yeiE-3xFLAG</em>
 chromosomal fusion grown as for gene expression (above) treated with 1 mM
 sulfate/sulfite/untreated conditions, normalized by
 OD<sub>600</sub>, 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. <strong>Protein
 purification and EMSA:</strong> YeiE-Strep-tag II
 was expressed in <em>E. coli</em> BL21 carrying
 pBAD18Cm-<em>yeiE-strII</em> 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.
description: # YeiE protein abundance, DNA binding, and influence of sulfite on growth
 and gene expression in *Salmonella* Dataset DOI:
 [10.5061/dryad.j0zpc86qq](10.5061/dryad.j0zpc86qq) ## Description of the
 data and file structure Growth curves are optical density at 600 nm
 (OD~600~) 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 OD~600~ 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 (OD~600~) 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+P*~yeiE~* reporter, and ∆*yeiE*+P*~yeiE~* 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 (OD~600~) 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/OD~600~) from the WT or ∆*yeiE* with P*~yeiE~* 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/OD~600~) from the WT or ∆*yeiE* with P*~yeiH~*
 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/OD~600~) 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 (OD~600~) 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 (OD~600~) 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 (OD~600~) 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 (OD~600~) 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**: * 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.