Polysaccharide utilization mechanisms under permanent low-temperature conditions in the Southern Ocean I n a u g u r a l d i s s e r t a t i o n zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) der Mathematisch-Naturwissenschaftlichen Fakultät der Universität Greifswald vorgelegt von Alexandra Dürwald Greifswald, Januar 2021 Dekan: Prof. Dr. Gerald Kerth 1. Gutachter: Prof. Dr. Thomas Schweder 2. Gutachter: Prof. Dr. Matthias Labrenz Tag der Promotion: 07.05.2021 Nicht alle Wege führen zum Ziel. Und wenn man erst einmal auf der anderen Seite des Globus angekommen ist, kann man wenigstens sicher sein, dass man auf dem richtigen Weg ist. Wer diese Gewissheit auf dem Herzen trägt, der bleibt kühl, wenn auch die Magnetnadel des Lebens ab und zu tanzt. Roald Amundsen (1872-1928) Content Content Abbreviations ....................................................................................................................... IV Zusammenfassung .............................................................................................................. VII 1. Introduction .................................................................................................................... 1 1.1 Marine Carbon Cycle and the Southern Ocean ....................................................... 1 1.2 Pectin ...................................................................................................................... 2 1.2.1 Structure of Pectin ............................................................................................ 2 1.2.2 Homogalacturonan ........................................................................................... 2 1.2.3 Rhamnogalacturonans ..................................................................................... 3 1.2.4 Marine pectin sources ...................................................................................... 3 1.2.5 Pectin catabolism ............................................................................................. 4 1.2.6 Biotechnological application of pectin and pectate lyases ................................ 4 1.3 Polysaccharide degradation .................................................................................... 4 1.3.1 Carbohydrate-active enzymes .......................................................................... 4 1.3.2 Polysaccharide lyases ...................................................................................... 5 1.3.3 TonB dependent transporters ........................................................................... 6 1.3.4 Polysaccharide utilization loci ........................................................................... 6 1.4 Pseudoalteromonas distincta ANT/505 ................................................................... 6 2. Aim of the study ............................................................................................................. 8 3. Results ........................................................................................................................... 9 3.1 Functional classification of outer membrane extensions of Pseudoalteromonas distincta and their putative role in polysaccharide utilization....................................... 9 3.1.1 PUL repertoire of P. distincta ............................................................................ 9 3.1.2 Morphological diversity of P. distincta ANT/505 ...............................................10 3.1.3 Subproteomic analyses of P. distincta ANT/505 with three different carbon sources ...........................................................................................................16 3.2 Metaproteogeomics of the Southern Ocean ...........................................................23 3.2.1 Phylogenetic classification of bacteria in the Southern Ocean .........................24 3.2.2 Sequencing results ..........................................................................................25 3.2.3 CAZyme identification and quantification .........................................................26 3.2.4 Identification and quantification of TonB-dependent transporters in the Southern Ocean ..............................................................................................27 3.2.5 PUL identification ............................................................................................27 3.2.6 Identification of bacterial key players in the Southern Ocean ...........................33 3.2.7 Distribution of abundant proteins at the two sampling sites in the Southern Ocean .............................................................................................................36 3.2.8 Functional classification of bacterial proteins in the Southern Ocean ..............36 I Content 3.3 Pectin-dependent enrichment cultures ...................................................................39 3.3.1 16S-rDNA-sequencing ....................................................................................39 3.3.2 Functional potential of enriched cultures .........................................................40 3.3.3 Isolation of new pectin degrading bacteria .......................................................41 3.4 Biphasic cellular adaptations and ecologial implications of Alteromonas macleodii degrading a mixture of algal polysaccharides ...........................................................43 4. Discussion .....................................................................................................................45 4.1 Extracellular extensions and their protentional role in Pseudoalteromonas distincta . ...............................................................................................................................45 4.2 Metaproteogenomics of the Southern Ocean .........................................................48 4.2.1 Phylogenetical classification and distribution of bacteria in the Southern Ocean ........................................................................................................................48 4.2.2 Polysaccharide utilization potential of bacteria in the Southern Ocean ............49 4.2.3 Bacterial key players in the Southern Ocean ...................................................50 4.2.4 Pectin-dependent enrichment cultures ............................................................52 4.3 Proteomic results of biphasic growth of Alteromonas macleodii .............................53 5. Outlook ..........................................................................................................................54 6. Conclusion ....................................................................................................................56 7. Material and Methods ....................................................................................................58 7.1 Material ..................................................................................................................58 7.1.1 Chemicals .......................................................................................................58 7.1.2 Buffers, Media and solutions ...........................................................................58 7.1.3 Kits, Consumables, Devices and Software ......................................................62 7.2 Methods .................................................................................................................66 7.2.1 Strain maintenance / Cryopreservation ...........................................................66 7.2.2 Cultivation of marine strains ............................................................................66 7.2.3 Isolation of subcellular compartments of P. distincta ANT/505 ........................66 7.2.4 Subcellular proteome analysis of P. distincta ANT/505 ....................................67 7.2.5 Scanning electron microscopy .........................................................................67 7.2.6 Transmission electron microscopy (TEM) ........................................................67 7.2.7 Negative staining .............................................................................................68 7.2.8 Sampling of Meta-samples ..............................................................................68 7.2.9 Isolation of genomic DNA ................................................................................68 7.2.10 Agarose electrophoresis ..................................................................................69 7.2.11 Metagenomic sequencing and bioinformatical pipeline ....................................69 7.2.12 PUL identification and analysis ........................................................................70 7.2.13 Taxonomical classification ...............................................................................70 7.2.14 Metaproteomic approach .................................................................................71 8. Literature .......................................................................................................................73 II Content 9. Appendix .......................................................................................................................84 Affirmation ............................................................................................................................98 List of publications ................................................................................................................99