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Acyl Homoserine Lactone Signaling in Members of the Vibrionaceae Family

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Acyl Homoserine Lactone Signaling in Members of the Vibrionaceae Family 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
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