Acyl homoserine lactone signaling in members of the Vibrionaceae family Amit Anand Purohit “In the fields of observation chance favors only the prepared mind.” - Louis Pasteur, French chemist and microbiologist "What the mind of man can conceive and believe, it can achieve" - Napoleon Hill, author of many books on formulas to achieve success for average person Content Acknowledgement ...................................................................................................................................... 4 Abbreviations .............................................................................................................................................. 5 List of Papers .............................................................................................................................................. 6 Summary ..................................................................................................................................................... 7 Sammendrag ............................................................................................................................................... 9 I. Introduction ...................................................................................................................................... 11 Part 1. Vibrionaceae family and Quorum Sensing ................................................................................ 11 1.1. Vibrionaceae family .................................................................................................................. 11 1.1.1. General description of Vibrionaceae family ......................................................................... 12 1.1.2. Members of Vibrionaceae family ........................................................................................... 12 1.1.3. Bacteria involved in this study from Vibrionaceae family .................................................. 14 1.2. Quorum sensing ............................................................................................................................. 14 1.2.1. QS in bacteria ......................................................................................................................... 15 1.2.2. Definition of a QS signal ........................................................................................................ 16 1.2.3. Different QS systems in Vibrionaceae family ....................................................................... 16 1.2.4. LuxS/LuxPQ system in prokaryotes ..................................................................................... 17 1.2.5. AHL and different AHL producing systems ........................................................................ 19 (a). LuxI/LuxR system ................................................................................................................. 20 (b). LuxM/LuxN system ............................................................................................................... 20 (c). HdtS system ............................................................................................................................ 21 1.2.6. CqsA/S system ......................................................................................................................... 21 1.2.7. QS in Vibrionaceae family members ..................................................................................... 22 (i). QS in A. fischeri ...................................................................................................................... 22 (ii). QS in V. harveyi ..................................................................................................................... 24 (iii). QS in V. anguillarum ........................................................................................................... 26 (iv). QS in A. salmonicida ............................................................................................................ 27 Part 2. AHL biochemistry ........................................................................................................................ 29 2.1. AHL synthases ............................................................................................................................... 29 2.1.1. LuxI family of synthases ........................................................................................................ 29 2.1.2. LuxM family of synthases ...................................................................................................... 29 2.1.3. Comparisions of LuxI and LuxM families ........................................................................... 30 2.2. Substrates ....................................................................................................................................... 30 1 2.2.1. SAM (S-adenosylmethionine) ................................................................................................ 31 2.2.2. Acyl carriers ............................................................................................................................ 32 2.3. Mechanism of AHL synthesis ....................................................................................................... 33 2.4. Enzyme kinetics and cold adapted regulation of QS .................................................................. 34 Part 3. Detection and verification of AHLs ............................................................................................ 35 3.1. Introduction ................................................................................................................................... 35 3.2. Extraction ....................................................................................................................................... 36 3.3. Methods for identification ............................................................................................................ 36 3.3.1. Biological methods .................................................................................................................. 36 A. Bioassay (Biosensing on solid medium) ................................................................................. 36 B. Bioluminescence (Biosensing in liquid medium) ................................................................... 37 3.3.2. Non-biological methods .......................................................................................................... 37 A. Separation techniques ............................................................................................................. 37 (i). Thin layer chromatography (TLC) ................................................................................... 37 (ii). Liquid chromatography (LC) ........................................................................................... 38 B. Analytical methods .................................................................................................................. 40 Mass spectrometry ................................................................................................................... 40 3.4. Methods to study QS ..................................................................................................................... 40 II. Aims of the study ................................................................................................................................. 45 III. Summary of the main results ............................................................................................................ 46 Paper1: Presence of acyl-homoserine lactones in 57 members of the Vibrionaceae family ........... 46 Paper 2: Temperature dependent regulation of quorum sensing in Aliivibrio salmonicida LFI1238 ................................................................................................................................................. 47 Paper 3: Preliminary analysis of the substrate specificity of acyl homoserine lactone (AHL) synthase VanM from Vibrio anguillarum ........................................................................................... 48 IV. Discussion ........................................................................................................................................... 49 4.1. General Discussion ........................................................................................................................ 49 4.2. Detection of AHLs ......................................................................................................................... 49 4.3. AHLs extraction ............................................................................................................................ 51 4.4. AHL stability ................................................................................................................................. 51 4.4.1. pH ............................................................................................................................................. 51 4.4.2. Solvent ..................................................................................................................................... 51 4.5. Has A. salmonicida a novel LuxR type of regulator? ................................................................. 51 4.6. Why do bacteria produce different AHLs and at different concentrations? ........................... 53 4.7. LitR is temperature regulated and effects AHL