Structural and Functional relationship of OprP and OprO mutants of Pseudomonas aeruginosa and genome annotation of Dietzia maris By Sonalli Ganguly a Thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemical Engineering Approved Dissertation Committee Prof. Dr. Roland Benz, Jacobs University, Bremen ________________________________ Name, title and affiliation of Chair Prof. Dr. Mathias Winterhalter Jacobs University, Bremen Name title and affiliation of Committee Member Prof. Dr. Tilman Achstetter Hoschule Bremen, Bremen Name title and affiliation of Committee Member Prof. Dr. Ulrich Kleinekathöfer Jacobs University, Bremen Name title and affiliation of Committee Member Date of Defense: 29.06.2016 Department of Life Sciences and Chemistry 2 Statutory Declaration (Declaration on Authorship of a Dissertation) I, Sonalli Ganguly hereby declare, under penalty of perjury, that I am aware of the consequences of a deliberately or negligently wrongly submitted affidavit, in particular the punitive provisions of § 156 and § 161 of the Criminal Code (up to 1 year imprisonment or a fine at delivering a negligent or 3 years or a fine at a knowingly false affidavit). Furthermore, I declare that I have written this PhD thesis independently, unless where clearly stated otherwise. I have used only the sources, the data and the support that I have clearly mentioned. This PhD thesis has not been submitted for the conferral of a degree elsewhere. Bremen December 6, 2016 __________________ ____________________ Place Date Signature 3 Dedicated to my lovely aunt Meenaa Gangully (Pisi) 4 Table of Contents Statutory Declaration .................................................................................................. 3 ABSTRACT .............................................................................................................. 10 ACKNOWLEDGEMENTS ........................................................................................ 11 AIM AND OBJECTIVES ........................................................................................... 14 CHAPTER 1 ............................................................................................................. 15 INTRODUCTION ...................................................................................................... 15 1.1 The bacterial cell envelope ............................................................................. 15 1.2 The Gram-negative bacteria cell envelope...................................................... 15 1.2.1 The outer membrane ................................................................................ 16 1.2.2 The peptidoglycan layer ............................................................................ 17 1.2.3 The periplasm ........................................................................................... 18 1.2.4 The inner membrane ................................................................................ 18 1.3 The phylum proteobacteria ............................................................................. 19 1.3.1 Pseudomonas aeruginosa: A Bad bug ..................................................... 20 1.3.2 Pathogenicity and drug resistance of Pseudomonas aeruginosa ............. 20 1.3.3 Porins of Pseudomonas aeruginosa ......................................................... 20 1.4 The cell wall of Gram-positive bacteria ........................................................... 22 1.4.1 Mycolata as members of Gram-positive bacteria ...................................... 23 1.5 The genus Dietzia ........................................................................................... 24 1.6 The genus Rhodococcus ................................................................................ 25 1.6.1 MspA like porins in Rhodococcus species ................................................ 26 1.7 Prokaryotic gene sequencing and prediction .................................................. 26 1.8 REFERENCES: - ............................................................................................ 29 CHAPTER 2 ............................................................................................................. 32 STUDY OF ORTHOPHOSPHATE SPECIFIC PORIN OprP MUTANTS OF PSEUDOMONAS AERUGINOSA ............................................................................ 32 2.1 Summary ......................................................................................................... 32 2.2 Introduction ..................................................................................................... 33 2.3 Materials and methods .................................................................................... 36 2.3.1 Bacterial strains and plasmid .................................................................... 36 5 2.3.2 pAS27 OprP single and double mutagenesis using the oprP gene in the E. coli plasmid ........................................................................................................ 37 2.3.3 Growth media, enzymes and reagents ..................................................... 38 2.3.4 Extraction of cell wall proteins of OprP wild type and mutants from E. coli38 2.3.5 Purification of cell wall proteins of wild type and mutants from E. coli ...... 39 2.3.5.1 Mono Q and Fast-Protein Liquid chromatography (FPLC) ..................... 39 2.3.5.2 SDS-PAGE and Western immunoblotting (WB) ..................................... 41 2.3.6 Bilayer assays ........................................................................................... 41 2.3.6.1 Single channel measurements ............................................................... 42 2.3.6.2 Titration Experiments ............................................................................. 44 2.4 Results ............................................................................................................ 45 2.4.1 Colony PCR confirmation of mutants and sequencing .............................. 45 2.4.2 Extraction and purification of cell wall proteins of wild type and mutant proteins .............................................................................................................. 46 2.4.3 Channel forming activity of single and double mutants ............................. 50 2.4.4 Comparison studies of substrate specificity of OprP wild type with its single and double mutants ........................................................................................... 51 2.5 Discussion ....................................................................................................... 53 2.5.1 Swapping of the specificity with the mutants from wild type OprP and OprO ........................................................................................................................... 53 2.6 REFERENCES ............................................................................................... 54 CHAPTER 3 ............................................................................................................. 67 MUTANTS OF DIPHOSPHATE SPECIFIC PORIN OprO OF PSEUDOMONAS AERUGINOSA ......................................................................................................... 67 3.1 Summary ......................................................................................................... 67 3.2 Introduction ..................................................................................................... 68 3.3 Materials and methods .................................................................................... 69 3.3.1 Bacterial strains and plasmids .................................................................. 69 3.3.2 Development of OprO wild type gene in E.coli .......................................... 71 3.3.3 Transformation of pTZ19R plasmid with oprO gene into E.coli cells ......... 72 3.3.4 Site- directed mutagenesis in oprO gene using mismatch nucleotides ..... 72 3.3.5 Expression and purification of OprO and OprO mutant recombinant proteins .............................................................................................................. 74 3.3.6 Lipid bilayer experiments .......................................................................... 75 3.4 Results ............................................................................................................ 77 6 3.4.1 Confirmation of presence of OprO wild type and tyrosine mutant genes in pTZ19R vector plasmid ...................................................................................... 77 3.4.2 Cell wall extraction and purification of OprO and its mutants .................... 77 3.4.3 Single channel conductance of OprO double mutant does not show similar conductance as of OprP wild type. .................................................................... 79 3.4.4 Substrate specificity studies of the proteins .............................................. 82 3.5 Discussion ....................................................................................................... 84 3.5.1 OprO mutants shows similar specificity as of OprP wild type and future aspects .............................................................................................................. 84 3.6 REFERENCES ............................................................................................... 85 CHAPTER 4 ............................................................................................................