Genomic Analyses of Polysaccharide Utilization in Marine Flavobacteriia Dissertation zur Erlangung des Grades eines Doktors der Naturwissenschaften - Dr. rer. nat. - dem Fachbereich 2 Biologie/Chemie der Universität Bremen vorgelegt von Lennart Kappelmann Bremen, April 2018 Die vorliegende Doktorarbeit wurde im Rahmen des Programms International Max Planck Research School of Marine Microbiology“ (MarMic) in der Zeit von August 2013 bis April 2018 am Max-Planck-Institut für Marine Mikrobiologie angerfertigt. This thesis was prepared under the framework of the International Max Planck Research School of Marine Microbiology (MarMic) at the Max Planck Institute for Marine Microbiology from August 2013 to April 2018. Gutachter: Prof. Dr. Rudolf Amann Gutachter: Prof. Dr. Jens Harder Prüfer: Dr. Jan-Hendrik Hehemann Prüfer: Prof. Dr. Rita Groß-Hardt Tag des Promotionskolloquiums: 16.05.2018 Inhaltsverzeichnis Summary ............................................................................................................................................. 1 Zusammenfassung .............................................................................................................................. 3 Abbreviations ..................................................................................................................................... 5 Chapter I: General introduction ...................................................................................................... 6 1.1 The marine carbon cycle ......................................................................................................... 6 1.2 Marine microbial life and the marine food web .................................................................... 8 1.3 The North Sea as a coastal shelf sea ....................................................................................... 9 1.4 Algal primary production ..................................................................................................... 10 1.5 Carbohydrates ........................................................................................................................ 14 1.6 Carbohydrate-active enzymes ............................................................................................... 17 1.7 Polysaccharide utilization loci............................................................................................... 20 1.8 PUL analyses provide insights into the structure of polysaccharides and their decomposition by adapted microbes .......................................................................................... 22 1.9 Aims of this thesis ................................................................................................................... 25 Chapter II: Polysaccharide Utilization Loci of Bacteroidetes from two contrasting open ocean sites in the North Atlantic ................................................................................................................ 27 2.1 Summary ................................................................................................................................. 29 2.2 Introduction ............................................................................................................................ 30 2.3 Experimental Procedures ...................................................................................................... 33 2.3.1 Study sites and fosmid library preparation .................................................................. 33 2.3.2 Selection and sequencing of fosmids.............................................................................. 34 2.3.3 Fosmid re-assembly......................................................................................................... 35 2.3.4 Taxonomic classification................................................................................................. 35 2.3.5 Automated gene prediction and annotation ................................................................. 35 2.3.6 Manual CAZyme annotation ......................................................................................... 36 2.4 Results and Discussion ........................................................................................................... 37 2.4.1 Characterization of the dataset ...................................................................................... 37 2.4.2 Bacteroidetes' peptidases and CAZymes ...................................................................... 40 2.4.3 Commonalities between PULs from both stations ....................................................... 44 2.4.4 Additional PULs from BPLR station 3 ......................................................................... 48 2.4.5 Additional PULs from NAST station 18 ....................................................................... 50 2.4.6 Comparative analysis of PULs ....................................................................................... 52 2.5 Conclusions ............................................................................................................................. 53 2.6 Acknowledgements................................................................................................................. 56 2.7 References ............................................................................................................................... 56 Chapter III: Recurring patterns in bacterioplankton dynamics during coastal spring algae blooms ............................................................................................................................................... 66 3.1 Abstract ................................................................................................................................... 68 3.2 Introduction ............................................................................................................................ 68 3.3 Materials and methods .......................................................................................................... 72 3.3.1 Phytoplankton and physicochemical data .................................................................... 72 3.3.2 Bacterioplankton ............................................................................................................. 73 3.3.3 Microscopy: total cell counts, CARD-FISH ................................................................. 73 3.3.4 16S rRNA V4 gene tag sequencing ................................................................................ 74 3.3.5 16S rRNA gene tag analysis ........................................................................................... 75 3.3.6 Metagenome sequencing ................................................................................................. 76 3.3.7 Metagenome analysis ...................................................................................................... 78 3.3.8 Statistical analyses........................................................................................................... 80 3.4 Results ..................................................................................................................................... 81 3.4.1 Sampling site characteristics .......................................................................................... 81 3.4.2 Phytoplankton - diversity and bloom characteristics .................................................. 82 3.4.3 Bacterioplankton - diversity and bloom characteristics .............................................. 85 3.4.4 Bacterioplankton - genetic repertoires .......................................................................... 93 3.5 Discussion .............................................................................................................................. 104 3.6 Concluding remarks ............................................................................................................ 109 3.7 Acknowledgements............................................................................................................... 110 3.8 References ............................................................................................................................. 110 Chapter IV: Polysaccharide Utilization Loci of 53 North Sea Flavobacteriia as Basis for Using SusC/D-Protein Expression for Predicting Major Phytoplankton Glycans ............................. 118 4.1 Abstract ................................................................................................................................. 120 4.2 Introduction .......................................................................................................................... 121 4.3 Materials and methods ........................................................................................................ 123 4.3.1 Isolation and sequencing of North Sea Flavobacteriia .............................................. 123 4.3.2 Gene and PUL annotation ............................................................................................ 124 4.3.3 Gene expression analyses of Flavobacteriia-rich North Sea bacterioplankton using metaproteomics ...................................................................................................................... 125 4.3.4 SusC/D homolog tree reconstruction ........................................................................... 126 4.4 Results ..................................................................................................................................