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Characterization of Swarm-Colony Development Reveals the Release of a Distinct Cell Type Facilitating Dissemination of Vibrio Parahaemolyticus

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Characterization of Swarm-Colony Development Reveals the Release of a Distinct Cell Type Facilitating Dissemination of Vibrio Parahaemolyticus Characterization of swarm-colony development reveals the release of a distinct cell type facilitating dissemination of Vibrio parahaemolyticus Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) dem Fachbereich Biologie der Philipps-Universität Marburg vorgelegt von Carolina Duarte de Freitas aus Lisboa, Portugal Marburg (Lahn), im Juli 2019 Originaldokument gespeichert auf dem Publikationsserver der Philipps-Universität Marburg http://archiv.ub.uni-marburg.de Dieses Werk bzw. Inhalt steht unter einer Creative Commons Namensnennung Keine kommerzielle Nutzung Weitergabe unter gleichen Bedingungen 3.0 Deutschland Lizenz. Die vollständige Lizenz finden Sie unter: http://creativecommons.org/licenses/by-nc-sa/3.0/de/ Die Untersuchungen zur vorliegenden Arbeit wurden von Oktober 2015 bis Juli 2019 am Max-Planck-Institut für Terrestrische Mikrobiologie unter der Leitung von Dr. Simon Ringgaard durchgeführ. Vom Fachbereich Biologie der Philipps-Universität Marburg als Dissertation angenommen am: Erstgutachter: Dr. Simon Ringgaard Zweitgutachter: Prof. Dr. Martin Thanbichler Weitere Mitglieder der Prüfungskommission: Prof. Dr. Knut Drescher Prof. Dr. Michael Bölker Tag der mündlichen Prüfung am: 24.09.2019 Die während der Promotien erzielten Erbebnisse wurden zum Teil in folgenden Originalpublikationen veröffentlicht: Muraleedharan, S., Freitas, C., Mann, P., Glatter, T. and Ringgaard, S. (2018). A cell length-dependent transition in MinD-dynamics promotes a switch in division-site placement and preservation of proliferating elongated Vibrio parahaemolyticus swarmer cells. Molecular Microbiology 109(3): 365-384 Unpublished work: Freitas, C., Glatter, T. and Ringgaard, S. The release of a distinct cell type from swarm colonies facilitates dissemination of Vibrio parahaemolyticus in the environment. Under review, The ISME Journal To my family Table of contents Table of contents Contents Table of contents.............................................................................................................. I List of figures ................................................................................................................. IV List of tables ................................................................................................................... V Abbreviations ................................................................................................................ VI Abstract ......................................................................................................................... IX Zusammennfassung...................................................................................................... XI 1. Chapter I - Introduction ............................................................................................... 1 1.1 Biofilm dispersal ....................................................................................................... 3 1.1.1 Signals and Regulatory Networks ...................................................................... 3 1.1.1.1 Nutritional cues ............................................................................................ 3 1.1.1.2 Oxygen depletion, oxidative stress and nitric oxide ..................................... 4 1.1.1.3 c-di-GMP ...................................................................................................... 5 1.1.1.4 Quorum sensing and RpoS stress response ............................................... 5 1.1.2 Mechanisms of dispersal .................................................................................... 7 1.1.3 Effect of nitroxides on swarming motility and biofilm dispersal of Bacillus subtilis and Pseudomonas aeruginosa .................................................................................... 8 1.1.4 Dispersed bacteria, a specific cell type .............................................................. 9 1.2 Swarming motility .................................................................................................... 12 1.2.1 Gram-positive swarming bacteria ..................................................................... 16 1.2.1.1 Paenibacillus .............................................................................................. 16 1.2.1.2 Bacillus subtilis........................................................................................... 18 1.2.2 Gram-negative swarming bacteria .................................................................... 23 1.2.2.1 Proteus mirabilis ........................................................................................ 23 1.2.2.2 Vibrio species ............................................................................................. 26 1.2.2.2.1 Vibrio alginolyticus .................................................................................. 27 1.3 Vibrio parahaemolyticus as model organism ......................................................... 29 1.3.1 Ecology of Vibrio parahaemolyticus ................................................................. 30 1.3.2 Differentiation of Vibrio parahaemolyticus ....................................................... 35 1.3.3 Regulation of swarming .................................................................................... 40 1.3.3.1 Physiological conditions ............................................................................. 40 1.3.3.2 Quorum sensing and c-di-GMP ................................................................. 41 1.3.3.3 The autoinducer S-signal ........................................................................... 42 1.3.4 Swarming and Virulence ................................................................................... 44 2. Chapter II - Aim and scope ....................................................................................... 47 I Table of contents 3. Chapter III - Release and dissemination of distinct cells from swarm-colonies ........ 51 3.1 Results – Part I ................................................................................................... 53 3.1.1 Cells are released from flooded swarm colonies into their liquid surroundings .............................................................................................................................. 53 3.1.2 Release of cells from swarm colonies facilitates dissemination of V. parahaemolyticus in the environment and its re-attachment to new submerged surfaces ................................................................................................................. 54 3.1.3 Cells released from swarm colonies possess a length distinct from cells from the center and from the periphery of swarm colonies ........................................... 54 3.1.4 Cells released from swarm colonies comprise a distinct cell type ............... 56 3.1.5 Cells released from swarm colonies are highly swimming proficient ........... 59 3.1.6 Cells released from swarm colonies chemotax towards the chitin component N-acetylglucosamine. ............................................................................................ 60 3.1.7 A distinct regional architecture of mature swarm colonies ........................... 61 3.1.8 Temporal architecture development of swarm colonies ............................... 62 3.1.9 V. parahaemolyticus colonies act as recurrent sources of swarmer cells during fluctuations in the external environment ..................................................... 67 3.2 Discussion .......................................................................................................... 70 4. Chapter IV - Potential swarm-regulators and distinct proteomic identities of Vibrio parahaemolyticus ......................................................................................................... 76 4.1 Results – Part II .................................................................................................. 78 4.1.1 Set of periphery-specific proteins ................................................................. 78 4.1.2 Potential swarm-regulators .......................................................................... 80 4.1.3 Set of center-specific proteins ...................................................................... 84 4.1.4 Set of liquid growth specific proteins ............................................................ 86 4.1.5 Housekeeping proteins ................................................................................ 88 4.2 Discussion .......................................................................................................... 90 4.2.1 Set of periphery-specific proteins ................................................................. 90 4.2.2 Set of center-specific proteins ...................................................................... 95 4.2.3 Set of liquid growth specific proteins ............................................................ 96 4.2.4 Housekeeping proteins ................................................................................ 97 5. Chapter V - Conclusions and future prospects ......................................................... 98 6. Chapter VI - Materials and methods ....................................................................... 104 6.1 Chemicals, equipment and software ................................................................. 106 6.2 Media, buffers and solutions ............................................................................. 110 6.3
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