E.K. Fowler 2015 On the molecular diversity of dimethylsulphoniopropionate catabolism by marine bacteria Emily Kate Fowler A thesis submitted to the University of East Anglia for the Degree of Doctor of Philosophy April 2015 © This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with the author and that use of any information derived there from must be in accordance with current UK Copyright Law. In addition, any quotation or extract must include full attribution i E.K. Fowler 2015 Declaration I declare that the work in this thesis has not been previously submitted for a degree at the University of East Anglia or any other university, and all research has been carried out by myself, unless otherwise stated. Signed Emily Kate Fowler ii E.K. Fowler 2015 Abstract Dimethylsulphoniopropionate (DMSP) is the most abundant organic sulphur molecule in the oceans. Its breakdown by marine organisms is important for the global cycling of sulphur, and as a nutrient source for microbial life. In recent years, the molecular basis of DMSP catabolism by marine bacteria has begun to be unravelled, through the discovery of six different DMSP lyases and a DMSP demethylase, as well as downstream pathways. From these studies, it is becoming evident that there is great diversity in the way bacteria breakdown this important molecule. The work presented here further explores and expands our knowledge of this diversity. I have identified a novel DMSP lyase (DddK), which catalyses the cleavage of DMSP into acrylate and dimethyl sulphide (DMS) in the DMS-producing Candidatus Pelgaibacter ubique HTCC1062 - one of the most prolific bacteria on this planet. I have also shown that the γ-proteobacterium Oceanimonas doudoroffii, which has long been a study species for DMSP catabolism, has no fewer than three functional DMSP lyases - DddD, DddP1 and DddP2 - this being the first example of a species outside of the α-proteobacteria having multiple lyases. Additionally, I have presented a thorough bioinformatics analysis of the occurrence and synteny of genes associated with DMSP catabolism within sequenced members of the abundant Roseobacter clade, revealing some interesting patterns which warrant further experimental investigation. Finally, I have shown that the model marine Roseobacter species Ruegeria pomeroyi DSS-3 is able to use DMSP-derived acrylate as a sole carbon source via a fatty acid biosynthesis route, linked to propionate catabolism. iii E.K. Fowler 2015 List of Contents Title page ....................................................................................................................................... i Declaration ................................................................................................................................... ii Abstract .......................................................................................................................................iii List of contents ............................................................................................................................ iv List of figures .............................................................................................................................. xii List of tables ............................................................................................................................... xv Acknowledgements ................................................................................................................... xvi Chapter 1: Introduction .............................................................................................................. 1 1.1 Dimethylsulphoniopropionate ......................................................................................... 2 1.2 Biosynthesis of DMSP ....................................................................................................... 3 1.2.1 Algae ........................................................................................................................... 3 1.2.2 Angiosperms ............................................................................................................... 3 1.2.3 Corals .......................................................................................................................... 4 1.2.4 Cyanobacteria ............................................................................................................. 4 1.3 Pathways of DMSP biosynthesis ...................................................................................... 4 1.3.1 DMSP biosynthesis in Wollastonia biflora ................................................................ 4 1.3.2 DMSP biosynthesis in Spartina alterniflora .............................................................. 5 1.3.3 DMSP biosynthesis in Ulva intestinalis ..................................................................... 5 1.3.4 DMSP biosynthesis in Fragilariopsis cylindrus......................................................... 5 1.3.5 DMSP biosynthesis in dinoflagellates ........................................................................ 7 1.4 Functions of DMSP ........................................................................................................... 7 1.4.1 DMSP as an osmoprotectant ....................................................................................... 7 1.4.2 DMSP as an antioxidant ............................................................................................. 8 1.4.3 DMSP as a herbivore grazing deterrent ...................................................................... 9 1.4.4 DMSP as a cryoprotectant .......................................................................................... 9 1.5 Environmental fate of DMSP .......................................................................................... 9 1.6 DMSP demethylation ...................................................................................................... 12 1.6.1 DmdA – discovery of the DMSP demethylase and the corresponding gene ............ 12 1.7 Downstream steps in the DMSP demethylation pathway ........................................... 12 1.7.1 The MMPA demethiolation pathway enzymes DmdB, DmdC and DmdD.............. 13 1.8 Distribution of DmdA, DmdB, DmdC and DmdD ....................................................... 15 1.9 Alternative pathways of MMPA degradation .............................................................. 18 1.10 DMSP cleavage.............................................................................................................. 19 1.11 Molecular genetics reveals diversity of bacterial DMSP lyases ................................ 20 1.12 DddY .............................................................................................................................. 21 iv E.K. Fowler 2015 1.12.1 Distribution of DddY .............................................................................................. 22 1.13 DddD .............................................................................................................................. 24 1.13.1 Discovery of the dddD gene ................................................................................... 24 1.13.2 Distribution of DddD in different organisms .......................................................... 28 1.14 DddP .............................................................................................................................. 32 1.14.1 DddP is a member of the metallopeptidase family ................................................. 32 1.14.2 Distribution of DddP .............................................................................................. 33 1.15 The cupin DMSP lyases DddL, DddQ and DddW ..................................................... 35 1.15.1 DddL ....................................................................................................................... 35 1.15.2 Distribution of DddL .............................................................................................. 36 1.15.3 DddW ..................................................................................................................... 38 1.15.4 Distribution of DddW ............................................................................................. 38 1.15.5 DddQ ...................................................................................................................... 38 1.15.6 Distribution of DddQ .............................................................................................. 39 1.15.7 Structure and mechanism of DddQ ........................................................................ 40 1.16 Fate of DMSP cleavage products ................................................................................. 41 1.16.1 DMS is an environmentally important gas ............................................................. 41 1.16.2 Fate of acrylate and 3-hydroxypropionate .............................................................. 43 1.16.3 DMSP catabolism in Halomonas HTNK1 ............................................................. 43 1.16.4 DMSP catabolism in Alcaligenes faecalis .............................................................. 44 1.17 Regulation of DMSP catabolism .................................................................................. 46 1.18 DMSP transport ...........................................................................................................