Intercellular Bacterial Signalling in Activated Sludge by Grace Chong A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy School of Biotechnology and Biomolecular Sciences Faculty of Science The University of New South Wales Sydney, Australia March 2010 Table of Contents Table of Contents ........................................................................................................ 2 Acknowledgements ...................................................................................................... 7 Abstract ........................................................................................................................ 9 Originality Statement................................................................................................ 11 List of Figures ............................................................................................................ 12 List of Tables ............................................................................................................. 14 List of Abbreviations ................................................................................................ 15 1 General introduction and literature review ...................................................... 18 1.1 Introduction ........................................................................................... 18 1.2 Introduction to cell-cell communication ............................................... 18 1.2.1 Quorum sensing paradigm ................................................................ 20 1.2.2 Vibrio fischeri ................................................................................... 21 1.2.2.1 LuxR, the acyl-HSL response regulator .................................... 23 1.2.2.2 LuxI, the acyl-HSL synthase ..................................................... 25 1.2.2.3 luxICDABEG, the bioluminescence operon .............................. 28 1.2.3 Quorum sensing in Gram-negative bacteria ...................................... 29 1.3 Ecological aspects of bacterial intercellular signalling ......................... 30 1.3.1 Intraspecies signalling ....................................................................... 31 1.3.2 Interspecies signalling ....................................................................... 32 1.3.2.1 LuxR interacting with more than one species of luxI homologue gene product .............................................................................................. 32 1.3.2.2 Bacterial strains producing more than one luxI gene product ... 33 1.3.2.3 The lack of luxI homologues in some bacterial strains that possess luxR homologues .......................................................................... 34 1.3.4 Quorum sensing interference ............................................................ 36 1.4 Wastewater treatment process ............................................................... 38 1.4.1 Activated sludge ................................................................................ 41 1.4.2 Organic carbon removal .................................................................... 42 1.4.3 Nitrogen removal .............................................................................. 45 1.4.4 Acyl-HSLs and activated sludge ....................................................... 46 1.5 Bacterial acyl-HSL biosensors .............................................................. 48 2 1.5.1 Thin-layer chromatography ............................................................... 50 1.5.2 In situ applications of acyl-HSL biosensors...................................... 51 1.5.3 Advantages of biosensors .................................................................. 51 1.6 Evolutionary biology of microbial social behaviour ............................. 52 1.6.1 Costs of cell-cell communication ...................................................... 53 1.6.2 Explaining cooperation ..................................................................... 55 1.6.2.1 Mutual benefits.......................................................................... 55 1.6.2.2 Kin selection.............................................................................. 56 1.6.3 Other forms of cooperation ............................................................... 58 1.6.4 Multi-species cooperation in activated sludge .................................. 58 1.7 Aims and objectives .............................................................................. 60 1.7.1 Chapter synopses ............................................................................... 61 2 Detection of acyl-HSL-like activity in activated sludge ................................... 63 2.1 Introduction ........................................................................................... 63 2.2 Material and Methods ........................................................................... 66 2.2.1 Bacterial strains and culture conditions ............................................... 66 2.2.2 Activated sludge sampling ................................................................ 68 2.2.3 Isolation of bacterial strains from activated sludge........................... 68 2.2.4 Assays for detection of acyl-HSL molecules .................................... 68 2.2.4.1 Screening for acyl-HSL in agar systems ................................... 68 2.2.4.2 Screening for acyl-HSLs in liquid systems ............................... 69 2.2.5 Construction of acyl-HSL monitor strain .......................................... 69 2.2.6 Characterization of acyl-HSL monitor strain .................................... 70 2.2.7 Masking effects ................................................................................. 70 2.2.8 Acyl-HSL detection in sludge samples ............................................. 71 2.2.8.1 Epifluorescence microscopy ..................................................... 71 2.2.9 Extraction of activated sludge supernatant ....................................... 71 2.2.9.1 Acyl-HSL screening of sludge extract using agar systems ....... 72 2.2.9.2 Acyl-HSL screening of sludge extract using thin-layer chromatography (TLC) ............................................................................. 72 2.3 Results ................................................................................................... 73 2.3.1 Construction of acyl-HSL monitor strain .......................................... 73 2.3.2 Characterization of the acyl-HSL monitor strain .............................. 73 2.3.3 Masking effects ................................................................................. 78 3 2.3.4 Detection of acyl-HSLs in sludge ..................................................... 80 2.3.5 Identification of acyl-HSL profiles in activated sludge .................... 83 2.4 Discussion ............................................................................................. 85 3 Testing for acyl-HSL activity and acyl-HSL regulated phenotypes in activated sludge isolates ............................................................................................ 88 3.1 Introduction ........................................................................................... 88 3.2 Materials and methods .......................................................................... 91 3.2.1 Bacterial strains and culture conditions ............................................ 91 3.2.2 Activated sludge collection ............................................................... 93 3.2.3 Isolation and identification of bacterial strains from activated sludge 93 3.2.4 Assays for detection of acyl-HSL molecules .................................... 94 3.2.4.1 Screening of acyl-HSL in agar systems .................................... 94 3.2.4.2 Screening of acyl-HSL in liquid systems .................................. 95 3.2.5 Phenotypic characterization of sludge isolates ................................. 95 3.2.5.1 Cellulase assay .......................................................................... 95 3.2.5.2 Lipase assay .............................................................................. 96 3.2.5.3 Chitinase assay .......................................................................... 96 3.2.5.4 Elastase assay ............................................................................ 96 3.2.5.5 Surfactant production ................................................................ 97 3.2.5.6 Antimicrobial assay ................................................................... 97 3.3 Results ................................................................................................... 98 3.3.1 Characterisation of sludge floc community ...................................... 98 3.3.2 Identification of acyl-HSL-producers in the collection of isolates ... 98 3.3.3 Phenotypic characterization of sludge isolates ............................... 103 3.4 Discussion ........................................................................................... 108 4 Acyl-HSL responses in activated sludge ......................................................... 112 4.1 Introduction ......................................................................................... 112 4.2 Materials and Methods ........................................................................ 115 4.2.1 Bacterial strains ............................................................................... 115 4.2.2 Activated