Regulation of Superoxide Radicals in Escherichia coli Sara H. Schilling 2007

University of St. Thomas

To learn more about the regulatory systems that protect E. coli bacteria cells from harmful superoxide radicals Overall Goal

Why? Information about protective systems in E. coli can be applied to understand similar systems in humans

Superoxide Radicals in E. coli Fe 2+ + O 2 

Superoxide Radicals in E. coli Fe 2+ + O 2  Fe 3+ + O 2 Radicals damage DNA, creating mutations

Breakdown of Superoxide Radicals SOD 2O 2 + 2H + 

Breakdown of Superoxide Radicals SOD 2O 2 + 2H +  H 2 O 2 + O 2

Gene Expression DNA sodA

Gene Expression DNA  mRNA Transcription sodA

Gene Expression DNA   Protein mRNA TranscriptionTranslation sodASOD

Protein Regulation sodA gene  SOD protein

Protein Regulation Fur sodA gene  SOD protein

Previous Research Fur activates sodA transcription (Schaeffer, 2006)

Previous Research Fur activates sodA transcription (Schaeffer, 2006) Fur sodA gene  MORE SOD protein

Previous Research Fur activates sodA transcription (Schaeffer, 2006) Fur sodA gene  MORE SOD protein Fur regulates sodA transcription when there are Fe +2 and many superoxide radicals present (Rollefson, et al. 2004)

Forms of Fur Description Zn 2 FurFur with zinc ions at each binding site Zn 1 FurFur with one zinc ion and one open binding site Fe 3+ FurFur with a zinc ion and a ferric ion at the binding sites Fe 2+ FurFur with a zinc ion and a ferrous ion at the binding sites

Forms of Fur Description Zn 2 FurFur with zinc ions at each binding site Zn 1 FurFur with one zinc ion and one open binding site Fe 3+ FurFur with a zinc ion and a ferric ion at the binding sites Fe 2+ FurFur with a zinc ion and a ferrous ion at the binding sites

Forms of Fur Description Zn 2 FurFur with zinc ions at each binding site Zn 1 FurFur with one zinc ion and one open binding site Fe 3+ FurFur with a zinc ion and a ferric ion at the binding sites Fe 2+ FurFur with a zinc ion and a ferrous ion at the binding sites

Forms of Fur Description Zn 2 FurFur with zinc ions at each binding site Zn 1 FurFur with one zinc ion and one open binding site Fe 3+ FurFur with a zinc ion and a ferric ion at the binding sites Fe 2+ FurFur with a zinc ion and a ferrous ion at the binding sites

Forms of Fur Description Zn 2 FurFur with zinc ions at each binding site Zn 1 FurFur with one zinc ion and one open binding site Fe 3+ FurFur with a zinc ion and a ferric ion at the binding sites Fe 2+ FurFur with a zinc ion and a ferrous ion at the binding sites

First Goal To compare activation of sodA transcription in the presence of the three metal-ion complexes of Fur: Zn 1 Fur Zn 2 Fur Fe 3+ Fur

First Hypothesis Based on the research by Rollefson, et al. (2004), I hypothesized that Zn 2 Fur would be the metal-ion complex of Fur that most activates sodA transcription

Second Goal To determine the effect of Fur concentration on activation of sodA transcription: 0 nM 50 nM 100 nM 150 nM 200 nM

Second Hypothesis Based on research by Shaeffer (2006), I hypothesized that increased Fur concentration would increase activation of sodA transcription

Third Goal To determine the root of and eliminate the negative control signaling that was present in the Schaeffer study

Third Goal To determine the root of and eliminate the negative control signaling that was present in the Schaeffer study Fourth Goal To optimize DNA band signaling by modifying the Schaeffer Protocols

Methods—PCR Polymerase Chain Reaction Diagramed used by permission from K. Shaeffer

Methods—Transcription DNA PCR Purification Transcription in Presence of the Three forms of Fur at Increasing Concentration Negative Controls Constructed mRNA

Methods—Reverse Transcription mRNA Reverse Transcription Negative Controls Constructed cDNA PCR Amplified cDNA

Methods—Gel Electrophoresis Photo by Author

Methods—Visualization VersaDoc Camera Photo by K. Shaeffer used with permission

Results — sodA transcription of Zn 1 Fur Lane 1-2: sodA transcribed in absence of Zn 1 Fur, Lane 3-4: sodA transcribed in presence of 50 nM Zn 1 Fur; Lane 5-6: sodA transcribed in presence of 100 nM Zn 1 Fur, Lane 7-8: sodA transcribed in presence of 150 nM Zn 1 Fur, Lane 9-10: sodA transcribed in presence of 0 nM Zn 1 Fur

Results — sodA transcription of Zn 1 Fur Lane 1-2: sodA transcribed in absence of Zn 1 Fur, Lane 3-4: sodA transcribed in presence of 50 nM Zn 1 Fur; Lane 5-6: sodA transcribed in presence of 100 nM Zn 1 Fur, Lane 7-8: sodA transcribed in presence of 150 nM Zn 1 Fur, Lane 9-10: sodA transcribed in presence of 0 nM Zn 1 Fur

Results—sodA transcription with Fe +3 Fur Lane 1-2: sodA transcribed in absence of Fe 3+ Fur, Lane 3-4: sodA transcribed in presence of 50 nM Fe 3+ Fur; Lane 5-6: sodA transcribed in presence of 100 nM Fe 3+ Fur, Lane 7-8: sodA transcribed in presence of 150 nM Fe 3+ Fur, Lane 9-10: sodA transcribed in presence of 0 nM Fe 3+ Fur

Results—sodA transcription with Fe +3 Fur Lane 1-2: sodA transcribed in absence of Fe 3+ Fur, Lane 3-4: sodA transcribed in presence of 50 nM Fe 3+ Fur; Lane 5-6: sodA transcribed in presence of 100 nM Fe 3+ Fur, Lane 7-8: sodA transcribed in presence of 150 nM Fe 3+ Fur, Lane 9-10: sodA transcribed in presence of 0 nM Fe 3+ Fur

Results — sodA Transcription with Zn 2 Fur Lane 1-2: sodA transcribed in absence of Zn 2 Fur, Lane 3-4: sodA transcribed in presence of 50 nM Zn 2 Fur; Lane 5-6: sodA transcribed in presence of 100 nM Zn 2 Fur, Lane 7-8: sodA transcribed in presence of 150 nM Zn 2 Fur, Lane 9-10: sodA transcribed in presence of 0 nM Zn 2 Fur

Results—Negative Controls Initial Trial Lanes 1-3: positive controls, Lane 4: negative control (without Master Mix), Lane 5: negative control (without RT primers), Lane 6: empty, Lane 7: negative control (without cDNA), Lanes 8-10: positive controls

Results—Negative Controls Initial Trial Lanes 1-3: positive controls, Lane 4: negative control (without Master Mix), Lane 5: negative control (without RT primers), Lane 6: empty, Lane 7: negative control (without cDNA), Lanes 8-10: positive controls No cDNA

Results—Negative Controls Transcription Assay Components Lane 1: NTP-initiator mixture, Lane 2: RT primer #2, Lane 3: RT primer #3, Lane 4: negative control (without NTP- initiator mixture), Lane 5: negative control (without mRNA), Lane 6: negative control (without DNase), Lane 7: dNTP mixture, Lane 8: positive control Lane 1-2: empty, Lane 3: DNase, Lane 4: RNA polymerase, Lane 5: negative control (without DNA), Lane 6: RNase inhibitor, Lane 7: empty, Lane 8: negative control (without cDNA)

Results—Negative Controls Signaling Components Run with DNase Lane 1: positive control, Lane 2: empty, Lane 3: RNase inhibitor incubated with DNase, Lane 4: NTP-initiator mixture incubated with DNase, Lane 5: 0.5  L RNA polymerase incubated with DNase, Lane 6: 2.0 RNA polymerase incubated with DNase, Lane 7: RNase inhibitor, NTP- initiator mixture, and RNA polymerase incubated with DNase, Lane 8: DNA incubated with DNase

Results—Negative Controls Signaling Components Run with DNase Lane 1: positive control, Lane 2: empty, Lane 3: RNase inhibitor incubated with DNase, Lane 4: NTP-initiator mixture incubated with DNase, Lane 5: 0.5  L RNA polymerase incubated with DNase, Lane 6: 2.0 RNA polymerase incubated with DNase, Lane 7: RNase inhibitor, NTP- initiator mixture, and RNA polymerase incubated with DNase, Lane 8: DNA incubated with DNase Positive Control

Results—Negative Controls Constructed during RT-PCR Lane 1: positive control used in the negative controls (originally run in Figure 9, Lane 1), Lane 2: positive control (originally run in Figure 4, Lane 2), Lane 3: negative control (without mRNA, RT primers 2 and 3, reverse transcriptase, and dNTP mixture), Lane 4: negative control (without RT primers 2 and 3), Lane 5: negative control (without reverse transcriptase), Lane 6: negative control (without mRNA), Lane 7: negative control (without dNTP mixture), Lane 8: negative control (without cDNA), Lane 9: negative control (without Master Mix), Lane 10: negative control (without cDNA or RT primers)

Results—Protocol Optimization PCR Products with Different Concentrations of Primers Lane 4: PCR product containing 4  L of sodA primers; Lane 6: PCR product containing 1  L of sodA primers; Lane 8: PCR product containing 8  L sodA primers

Results—Protocol Optimization PCR Products with Different Concentrations of Primers Lane 4: PCR product containing 4  L of sodA primers; Lane 6: PCR product containing 1  L of sodA primers; Lane 8: PCR product containing 8  L sodA primers 4  L

Results—Protocol Optimization PCR Products with Different Concentrations of Primers Lane 4: PCR product containing 4  L of sodA primers; Lane 6: PCR product containing 1  L of sodA primers; Lane 8: PCR product containing 8  L sodA primers 8  L

Results—Protocol Optimization PCR Products with Different Concentrations of Primers Lane 4: PCR product containing 4  L of sodA primers; Lane 6: PCR product containing 1  L of sodA primers; Lane 8: PCR product containing 8  L sodA primers 1  L

Discussion—First Goal Hypothesis neither supported nor refuted -sodA transcription in presence of Zn 2 Fur unsuccessful Zn 1 Fur most activated sodA transcription To determine what form of Fur most activates sodA transcription

Future Work—First Goal Repeat sodA transcription in presence of Zn 2 Fur Perform sodA transcription in the presence of other metal-ion complexes of Fur

Discussion—Second Goal Hypothesis correct -Activation of sodA transcription did increase with Fur concentration To determine the effect of Fur concentration on sodA transcription

Discussion—Third Goal Partially successful -Negative control signaling present -Cause of signaling determined to originate during process of RT-PCR To eliminate and determine the cause of negative control signaling

Future Work—Third Goal Determine what in RT-PCR is causing the signaling - Examine each component of the RT-PCR assay

Discussion—Fourth Goal PCR product with 1  L of each sodA primer produced the best signaling –Amplification protocol was modified to reflect the optimization To optimize the Shaeffer PCR Protocol

Applications of Research Break down more harmful superoxide radicals

Applications of Research Break down more harmful superoxide radicals Fur – sodA interaction may serve as model in human systems

Applications of Research Break down more harmful superoxide radicals Fur – sodA interaction may serve as model in human systems May lead to synthesis of drugs that model regulatory proteins and modify expression of genes

Acknowledgements Dr. Kathy Olson University of St. Thomas Chemistry and Biology Departments Mrs. Lois Fruen Dr. Jacob Miller Team Research

Regulation of Superoxide Radicals in Escherichia coli Sara H. Schilling 2007