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My Master thesis presentation

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Involvement of SOS response and C-di-GMP signaling in biofilm formation of Pseudomonas aeruginosa via DNA and Protein release. Supervising Professors Dr. Tao Wei, Chair Dr. Neal Guentzel Dr. Jilani Chaudry Nagarjun Kasaraneni, B.Tech
Pseudomonas aeruginosa ,[object Object],[object Object],[object Object],[object Object]
[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],Significance of the P.aeruginosa infections
Biofilm Importance in Present Day Antimicrobial Therapy ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Biofilm Matrix-enclosed bacterial populations adherent to each other and/or to surfaces Singh et al . (2002) Nature 417: 552
Developmental cycle of a biofilm J.W.Costerton et al., Annu. Rev. Microbiol, 1995
Strategies to prevent biofilm formation Rickard et al. (2003) TIM 11: 94 .
Published Data ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Riboncleiotide reductase & Hydroxyurea Riboncleiotide reductase (RNR) Cysteine thiyl radical attacks OH-C3’ leading to removal of hydroxide ion of C-2 ’ NTPs dNTPs
Hypothesis based on published data ,[object Object],[object Object],[object Object],[object Object],[object Object]
Mechanism of SOS response Bianco and Kowalczykowski, 1998
Cyclic-di-GMP signaling ,[object Object],[object Object],[object Object]
Cyclic-di-GMP signaling proteins in different bacteria Simm etal., Mol.Microbiol, 2004
Aminoglycoside response regulator (Arr) Hoffman et al., Nature 436, 1171, 2005
Outline of the work ,[object Object],[object Object],[object Object],[object Object],[object Object]
Biofilm stimulation is RecA dependent Hydroxyurea –inducible biofilm formation appears RecA-dependent .
SOS involvement in biofilm stimulation
Conclusion ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
DNA release in response to hydroxyurea Whether the release of DNA is under SOS control? [DNA μ g/ml]/cell
SOS controlled DNA release
Conclusion ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Protein release in response to hydroxyurea 60 kDa : Chaperonin GroEL 30 kDa : Elastase elsB 47 kDa : Elongation factor-TU 1: Cell lysate proteins HU- 2: Cell lysate proteins HU+ 3: Wt -HU Supernatent proteins 4: Wt+HU Supernatent proteins 5: RecA –HU supernatent proteins 6: RecA +HU Supernatent proteins 7: RecA Comp –HU Supernatent proteins 8: RecA Comp +HU Supernatent proteins.
Conclusion ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Correlation of macromolecule release with biofilm stimulation. ,[object Object],[object Object],[object Object],[object Object]
Correlation of protein release with biofilm stimulation
Conclusion ,[object Object],[object Object],[object Object],[object Object]
Complementation of the recA mutant biofilm with wild type proteins
Conclusion ,[object Object],[object Object],[object Object],[object Object],[object Object]
Protein release is regulated by Arr Summary From the Protein concentrations and SDS gel profiles suggests the Protein release in response to hydroxyurea appears to be regulated by Arr Wt arr 1 : Wt –HU Supernatent proteins 2 : Wt+HU Supernatent proteins 3 : arr –HU Supernatent proteins 4 : arr +HU Supernatent proteins Extracted at 8 hr time period.
Hypothesis based on results ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Evidence for biofilm rehabilitation ,[object Object],[object Object],[object Object],1:planktonic cells –HU 8hr 2:Planktonic cells +HU 8hr 3:Biofilm cells –HU 8hr 4:Biofilm cells +HU 8hr 5:Biofilm cells -HU 24hr 6:Biofilm cells +HU 24hr 1:planktonic cells –HU 8hr 2:Planktonic cells +HU 8hr 3:Planktonic cells +AboCbl 8hr 4:Planktonic cells +HU + AboCbl 8hr
Acknowledgements ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Thank you
Cynthia j.Kenyon. , Biomedical press, 1983 Cartoon of the SOS response

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Thesis ppt

  • 1. Involvement of SOS response and C-di-GMP signaling in biofilm formation of Pseudomonas aeruginosa via DNA and Protein release. Supervising Professors Dr. Tao Wei, Chair Dr. Neal Guentzel Dr. Jilani Chaudry Nagarjun Kasaraneni, B.Tech
  • 2.
  • 3.
  • 4.
  • 5. Biofilm Matrix-enclosed bacterial populations adherent to each other and/or to surfaces Singh et al . (2002) Nature 417: 552
  • 6. Developmental cycle of a biofilm J.W.Costerton et al., Annu. Rev. Microbiol, 1995
  • 7. Strategies to prevent biofilm formation Rickard et al. (2003) TIM 11: 94 .
  • 8.
  • 9. Riboncleiotide reductase & Hydroxyurea Riboncleiotide reductase (RNR) Cysteine thiyl radical attacks OH-C3’ leading to removal of hydroxide ion of C-2 ’ NTPs dNTPs
  • 10.
  • 11. Mechanism of SOS response Bianco and Kowalczykowski, 1998
  • 12.
  • 13. Cyclic-di-GMP signaling proteins in different bacteria Simm etal., Mol.Microbiol, 2004
  • 14. Aminoglycoside response regulator (Arr) Hoffman et al., Nature 436, 1171, 2005
  • 15.
  • 16. Biofilm stimulation is RecA dependent Hydroxyurea –inducible biofilm formation appears RecA-dependent .
  • 17. SOS involvement in biofilm stimulation
  • 18.
  • 19. DNA release in response to hydroxyurea Whether the release of DNA is under SOS control? [DNA μ g/ml]/cell
  • 21.
  • 22. Protein release in response to hydroxyurea 60 kDa : Chaperonin GroEL 30 kDa : Elastase elsB 47 kDa : Elongation factor-TU 1: Cell lysate proteins HU- 2: Cell lysate proteins HU+ 3: Wt -HU Supernatent proteins 4: Wt+HU Supernatent proteins 5: RecA –HU supernatent proteins 6: RecA +HU Supernatent proteins 7: RecA Comp –HU Supernatent proteins 8: RecA Comp +HU Supernatent proteins.
  • 23.
  • 24.
  • 25. Correlation of protein release with biofilm stimulation
  • 26.
  • 27. Complementation of the recA mutant biofilm with wild type proteins
  • 28.
  • 29. Protein release is regulated by Arr Summary From the Protein concentrations and SDS gel profiles suggests the Protein release in response to hydroxyurea appears to be regulated by Arr Wt arr 1 : Wt –HU Supernatent proteins 2 : Wt+HU Supernatent proteins 3 : arr –HU Supernatent proteins 4 : arr +HU Supernatent proteins Extracted at 8 hr time period.
  • 30.
  • 31.
  • 32.
  • 34. Cynthia j.Kenyon. , Biomedical press, 1983 Cartoon of the SOS response

Notas do Editor

  1. Ribonucleotide reductase (RNR) is an enzyme that controls the cellular concentration of deoxyribonucleotides . Biosynthesis begins with the building up of essential molecules that RNR processes in a catalyzed reaction to make deoxyribonucleotides. RNR assembles deoxyribonucleotides for the synthesis of DNA . The processes are identical in all living organisms. What makes RNR unique from other enzymes is the need for a free radical . Hydroxyurea is a scavenger of free radicals.