SlideShare a Scribd company logo

Final poster project microbiology

•Download as PPTX, PDF•
•
A
Andrew Arner

1. The study investigates the role of the Hfq protein in the stress responses and biochemical processes of Escherichia albertii, a recently discovered bacterial strain. 2. Catalase tests, sugar utilization tests, and assays exposing the bacteria to varying temperatures and pH levels were performed using wild type and Hfq-mutant strains. 3. The results showed that under the test conditions, the Hfq-mutant and wild type strains exhibited similar responses in oxidative stress tolerance, sugar metabolism, heat shock response, and acid stress response, suggesting Hfq does not play a role in these processes in E. albertii.

1 of 1
Download to read offline
Future Studies Hfq is an RNA chaperone capable of binding both sRNA and mRNA, which in turn effects the stability and translation of the mRNA transcript. This regulatory protein is highly conserved throughout many virulent bacterial strains. Research suggests that elimination of the Hfq gene from the bacterial genome results in a change in the virulence of the pathogen. Escherichia albertii is a newly discovered bacterial strain in the same genus as Escherichia coli. Using BLAST analysis, the Hfq gene was concluded to be present in E. albertii. Therefore we hypothesize that the removal of the Hfq gene from E. albertii will result in phenotypic changes that will alter the virulence of the pathogen. Biochemical assays, such as catalase tests and double sugar iron agar tests were performed to determine if Hfq played a role in the oxidative stress response as well as how the strain utilizes glucose and lactose. Further, the wild type and mutant strains were exposed to varying pH and temperature levels to determine if Hfq played a role in these responses, respectively. The results obtained concluded that under the given conditions, Hfq does not play a role in the oxidative stress response, glucose and lactose utilization, the pH stress response or in the response to heat shock. The Role of Hfq Protein in Heat Shock, Acid Stress and Biochemical Processes in Escherichia albertii Andrew Arner, Jim Kavulich, Nicholas Bastian Department of Biology, Saint Joseph’s University, Philadelphia, PA. 19131 Acknowledgements Mechanism of Action of Hfq References Proximal (Top) Distal (Bottom) sRNA/mRNA mRNA/sRNA sRNA binds mRNA The binding of sRNA and mRNA facilitated by Hfq ultimately affects the mRNA stability and its ability to undergo translation Wild Type Hfq- Round 1 Wild Type Hfq- 1 + + 2 + + Round 2 Wild Type Hfq- 1 + + 2 + + Note the similar degree of effervescence outlined by the boxes, suggesting Hfq does not play a role the oxidative stress response - A catalase assay was run by emulsifying a smear of both Hfq- and the wild type strains into 60 microliters of sterile water. The reagent was 3% H2O2. Catalase Assay - A DSI agar assay was run by stab streaking the same number of cells on the DSI agar - The results between the wild type and Hfq- strains suggest that Hfq does not play a role under these conditions - Both strains can utilize glucose but not lactose The red indicates the use of the peptone with alkaline products, increasing the pH Wild Type Hfq- The yellow indicates glucose use with acid production, dropping the pH Double Sugar Iron Agar Assay 1.00E+07 1.00E+08 1.00E+09 WT 7 Hfq- 7 Wt 5 Hfq- 5 cfu/mL Strain & pH Condition pH Stress Response in cfu/mL WT 7 Hfq- 7 Wt 5 Hfq- 5 - The pH stress response was determined by exposing the same number of cells of each strain to LB broth at pH 5 for 10 minutes. - Control strains were exposed to pH 7 for 10 minutes The Response to an Environment with pH 5 1.00E+08 1.50E+08 2.00E+08 2.50E+08 3.00E+08 3.50E+08 4.00E+08 4.50E+08 5.00E+08 5.50E+08 6.00E+08 Wt 35 Hfq- 35 Wt 50 Hfq- 50 cfu/mL Strain and Temperature in °C Heat Shock Response in cfu/mL Wt 35 Hfq- 35 Wt 50 Hfq- 50 - The response to heat was determined by exposing the same number of cells of each strain to similar conditions for a period of one hour - 50 °C (Wild type and Hfq-) - 35 °C (Wild type and Hfq-) - The bacteria were diluted in sterile saline by a factor of 10-6 The Response to Heat Shock at 50 °C Heat Shock Results Competitive Index: (Hfq-50:Wt50) ÷ (Hfq-35:Wt35) = (1.93 x 108/5.08 x 108) ÷ (2.48 x 108/5.63 x 108) = 0.860 Acid Stress Response pH 5 Competitive Index: (Hfq-5:Wt5) ÷ (Hfq-7:Wt7) = (7.93 x 107/6.2 x 108) ÷ (1.36 x 108/7.93 x 108) = 0.744 • Dr. Bhatt for his assistance and supplying of reagents for the assays Guang, Yang, Wang Ligui, and Wang Yong. "Hfq Regulates Acid Tolerance and Virulence by Responding to Acid Stress in Shigella Flexneri." Research in Microbiology 166.6 (2015): 476-85. Web. Shakhnovich, Elizabeth, Brigid Davis, and Mathew Waldor. "Hfq Negatively Regulates Type III Secretion in EHEC and Several Other Pathogens." Molecular Microbiology 74.2 (2009). Web. • Increase the temperature and alter the length of incubation in the heat shock assay to better express the effects of heat on the Hfq- mutant – Increase temperature to 60 °C • Alter the pH and the length of exposure to that environment to better determine how the Hfq- mutant responds to an acidic environment in relation to the wild type – Decrease the pH to 3 and 4 Abstract Dilutions, Averages and Standard Deviation Acid Stress Replicates Heat Shock Replicates NB: The figures depicted are a representation of average colony counts. For each strain at each condition, two rounds, each with two replicates, were plated to best represent the cell count in cfu/mL. Wt pH7 9.91 x 107 Hfq- pH7 4.95 x 107 Wt pH 5 2.45 x 107 Hfq- pH 5 1.62 x 107 Wt 35 °C 6.5 x 107 Hfq-3 35 °C 6.99 x 107 Wt 50 °C 6.9 x 107 Hfq- 50 °C 2.99 x 107 Condition SD NB: Any value at or close to 1 suggests no role in the response to that condition Summary and Conclusions - The similar degree of effervescence in all of the replicates of the catalase test suggests that Hfq does not play a role in the response to oxidative stress under these conditions - The results of the double sugar iron agar test depict that both the wild type and mutant strains can utilize glucose, but not lactose. This suggests that Hfq does not play a role in the utilization of glucose or lactose. - The comparative index for the heat shock assay (0.860) and acid stress assay (0.744) suggest that Hfq does not play a role in responding to either of these stresses under the given conditions.

More Related Content

Final poster project microbiology

  • 1. Future Studies Hfq is an RNA chaperone capable of binding both sRNA and mRNA, which in turn effects the stability and translation of the mRNA transcript. This regulatory protein is highly conserved throughout many virulent bacterial strains. Research suggests that elimination of the Hfq gene from the bacterial genome results in a change in the virulence of the pathogen. Escherichia albertii is a newly discovered bacterial strain in the same genus as Escherichia coli. Using BLAST analysis, the Hfq gene was concluded to be present in E. albertii. Therefore we hypothesize that the removal of the Hfq gene from E. albertii will result in phenotypic changes that will alter the virulence of the pathogen. Biochemical assays, such as catalase tests and double sugar iron agar tests were performed to determine if Hfq played a role in the oxidative stress response as well as how the strain utilizes glucose and lactose. Further, the wild type and mutant strains were exposed to varying pH and temperature levels to determine if Hfq played a role in these responses, respectively. The results obtained concluded that under the given conditions, Hfq does not play a role in the oxidative stress response, glucose and lactose utilization, the pH stress response or in the response to heat shock. The Role of Hfq Protein in Heat Shock, Acid Stress and Biochemical Processes in Escherichia albertii Andrew Arner, Jim Kavulich, Nicholas Bastian Department of Biology, Saint Joseph’s University, Philadelphia, PA. 19131 Acknowledgements Mechanism of Action of Hfq References Proximal (Top) Distal (Bottom) sRNA/mRNA mRNA/sRNA sRNA binds mRNA The binding of sRNA and mRNA facilitated by Hfq ultimately affects the mRNA stability and its ability to undergo translation Wild Type Hfq- Round 1 Wild Type Hfq- 1 + + 2 + + Round 2 Wild Type Hfq- 1 + + 2 + + Note the similar degree of effervescence outlined by the boxes, suggesting Hfq does not play a role the oxidative stress response - A catalase assay was run by emulsifying a smear of both Hfq- and the wild type strains into 60 microliters of sterile water. The reagent was 3% H2O2. Catalase Assay - A DSI agar assay was run by stab streaking the same number of cells on the DSI agar - The results between the wild type and Hfq- strains suggest that Hfq does not play a role under these conditions - Both strains can utilize glucose but not lactose The red indicates the use of the peptone with alkaline products, increasing the pH Wild Type Hfq- The yellow indicates glucose use with acid production, dropping the pH Double Sugar Iron Agar Assay 1.00E+07 1.00E+08 1.00E+09 WT 7 Hfq- 7 Wt 5 Hfq- 5 cfu/mL Strain & pH Condition pH Stress Response in cfu/mL WT 7 Hfq- 7 Wt 5 Hfq- 5 - The pH stress response was determined by exposing the same number of cells of each strain to LB broth at pH 5 for 10 minutes. - Control strains were exposed to pH 7 for 10 minutes The Response to an Environment with pH 5 1.00E+08 1.50E+08 2.00E+08 2.50E+08 3.00E+08 3.50E+08 4.00E+08 4.50E+08 5.00E+08 5.50E+08 6.00E+08 Wt 35 Hfq- 35 Wt 50 Hfq- 50 cfu/mL Strain and Temperature in °C Heat Shock Response in cfu/mL Wt 35 Hfq- 35 Wt 50 Hfq- 50 - The response to heat was determined by exposing the same number of cells of each strain to similar conditions for a period of one hour - 50 °C (Wild type and Hfq-) - 35 °C (Wild type and Hfq-) - The bacteria were diluted in sterile saline by a factor of 10-6 The Response to Heat Shock at 50 °C Heat Shock Results Competitive Index: (Hfq-50:Wt50) ÷ (Hfq-35:Wt35) = (1.93 x 108/5.08 x 108) ÷ (2.48 x 108/5.63 x 108) = 0.860 Acid Stress Response pH 5 Competitive Index: (Hfq-5:Wt5) ÷ (Hfq-7:Wt7) = (7.93 x 107/6.2 x 108) ÷ (1.36 x 108/7.93 x 108) = 0.744 • Dr. Bhatt for his assistance and supplying of reagents for the assays Guang, Yang, Wang Ligui, and Wang Yong. "Hfq Regulates Acid Tolerance and Virulence by Responding to Acid Stress in Shigella Flexneri." Research in Microbiology 166.6 (2015): 476-85. Web. Shakhnovich, Elizabeth, Brigid Davis, and Mathew Waldor. "Hfq Negatively Regulates Type III Secretion in EHEC and Several Other Pathogens." Molecular Microbiology 74.2 (2009). Web. • Increase the temperature and alter the length of incubation in the heat shock assay to better express the effects of heat on the Hfq- mutant – Increase temperature to 60 °C • Alter the pH and the length of exposure to that environment to better determine how the Hfq- mutant responds to an acidic environment in relation to the wild type – Decrease the pH to 3 and 4 Abstract Dilutions, Averages and Standard Deviation Acid Stress Replicates Heat Shock Replicates NB: The figures depicted are a representation of average colony counts. For each strain at each condition, two rounds, each with two replicates, were plated to best represent the cell count in cfu/mL. Wt pH7 9.91 x 107 Hfq- pH7 4.95 x 107 Wt pH 5 2.45 x 107 Hfq- pH 5 1.62 x 107 Wt 35 °C 6.5 x 107 Hfq-3 35 °C 6.99 x 107 Wt 50 °C 6.9 x 107 Hfq- 50 °C 2.99 x 107 Condition SD NB: Any value at or close to 1 suggests no role in the response to that condition Summary and Conclusions - The similar degree of effervescence in all of the replicates of the catalase test suggests that Hfq does not play a role in the response to oxidative stress under these conditions - The results of the double sugar iron agar test depict that both the wild type and mutant strains can utilize glucose, but not lactose. This suggests that Hfq does not play a role in the utilization of glucose or lactose. - The comparative index for the heat shock assay (0.860) and acid stress assay (0.744) suggest that Hfq does not play a role in responding to either of these stresses under the given conditions.