Identification of Bacterial Genes Required for Identification Of
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Identification of bacterial genes required for diatomdiatom----bacteriabacteria interactions by Ingrid TorresTorres----MonroyMonroy A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy iiininnn Marine Microbiology Approved Thesis Committee Prof. Dr. Matthias Ullrich Jacobs University Bremen Prof. Dr. Frank-Oliver Glöckner Jacobs University Bremen Prof. Dr. Meinhard Simon Carl-von-Ossietzky-University Oldenburg Date of defense: May 28, 2013 Jacobs University Bremen - School of Engineering and Science International Max Planck Research School of Marine Microbiology (marmic) SUMMARY Aggregate formation in form of marine snow is an essential mechanism in the oceans that mediates the sinking of organic carbon to depth. Interactions between bacteria and diatom play an important role during this process by inducing secretion of different polymers, which increase the size of marine aggregates. Not much is known about the molecular mechanisms responsible for the diatom-bacteria interaction. To address this issue, a bilateral model system consisting of Marinobacter adhaerens HP15 and the diatom Thalassiosira weissflogii has been established. This bacterium specifically attaches to T. weissflogii cells thereby increasing its aggregation and inducing an increased formation of transparent exopolymeric particles. A genetic tool system was developed for M. adhaerens HP15, in which successful expression of reporter genes revealed useful tools for gene expression analyses. In addition, several bacterial genes potentially important during the interaction have been investigated. However, genes specifically expressed in vivo are still unknown. In this work we identified bacterial genes that are induced during the interaction with T. weissflogii by two different approaches. First, an In vivo expression technology (IVET) screening was conducted, constructing a promoter-trap vector containing a fusion between a promoterless selection marker gene and a reporter gene. Generation of a library of plasmids carrying genomic fragments upstream of the fusion and its subsequent transformation into a selection marker mutant allowed the selection of bacterial promoters specifically expressed during the interactions with T. weissflogii . Second, bacterial proteins expressed in response to the presence of T. weissflogii were identified by comparison of protein profiles of bacterial cultures incubated with or without diatom cells and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). By these approaches several genes expressed during the co-cultivation with diatoms were identified. Sequence analyses of these genes showed that they are required for central intracellular metabolism, cell envelope structure, nutrient scavenging, regulation, chemotaxis, secretion, stress response and DNA transfer. These genes may play an important role during the interaction between M. adhaerens HP15 and T. weissflogii . The results obtained in this study contribute to a better understanding of the molecular and biochemical mechanisms responsible for diatom-bacteria interactions. Additionally, the tight adherence (tad ) gene locus present on the 187-kb HP15 plasmid and consisting of 9 genes ( flp , rcpCA , tadZABCDG ), was analyzed. This locus is found in several Gram-negative bacteria encoding for a type IVb fimbrial low-molecular- weight (Flp) pilus, which plays a role in adherence and biofilm formation. The identification of a constitutively active promoter upstream the flp gene of M. adhaerens HP15 suggested the tad locus is transcribed. A flp -rcpCA deletion mutant was generated and analyzed in terms of its motility and attachment to abiotic and biotic surfaces. Under the experimental conditions tested, the mutant did neither show a phenotype in terms of surface attachment nor motility. However, the preliminary results of the current study encourage an in-depth analysis of the role of the tad locus in M. adhaerens HP15. II ACKNOWLEDGEMENTS This PhD thesis was support by Jacobs University Bremen, International Max Planck Research School of Marine Microbiology (marmic) and the Deutsche Forschungsgemeinschaft (DFG). I would like to express my gratitude to Prof Dr. Matthias Ullrich for giving me the opportunity to work in such an interesting project and for supervise my work. I would also like to thank my committee members Prof Dr. Frank-Oliver Glöckner and Prof Dr. Meinhard Simon for evaluating my thesis and for all the discussions and advises during this time. My special thanks to Dr. Christiane Glöckner for the constant help, in particular during my first months in Germany, and also for the great marmic program. I am also grateful to all present and former members of the Microbiology Laboratory at Jacobs University, especially Amna Mahmood, Daniel Pletzer, Shaunak Khandekar, Antje Stahl, Gabriela Alfaro, Maria Johansson, Delasegne Abebew Syit, Khaled Abdallah, Ahmen Sayed Deyab, San San Yu, Dr. Shalin Seebah, Dr. Eva Sonnenschein for a great working atmosphere, for all the help and discussion during these three years. I want to give special thanks to Dr. Shalin Seebah for her great support and friendship. I want to thank the marmic class 2012, especially Zainab Beiruti, Sandra Havemeyer and Andreas Krupke for their friendship. My lab rotation students, Antje Stahl, Joe Marshall and Nikki Atanasova, are also thanked for giving me the opportunity to supervise their work and for their help in my project. I would like to express my deepest gratitude to my family, for supporting me during all my studies, in particular these 5 years away from home. To my parents because without their constant effort, love and sacrifices I would not have been able to reach this stage on my career. I am also grateful to Martin for being a great company and moral support during this time. Finally, I want to thank God to give me all the best opportunities in live. III LIST OF ABBREVIATIONS 2-DE Two-dimensional protein electrophoresis ABC ATP-binding cassette ACN Acetonitrile ALA 5-aminolevulinic acid Amp Ampicillin ATP Adenosine-5'-triphosphate CE4 Carbohydrate Esterase family 4 CFU Colony Forming Unit Cm Chloramphenicol Cm R Chloramphenicol resistance CO 2 Carbon dioxide DHDPS Dihydropicolinate synthase DIC Dissolved Inorganic Carbon DNA Deoxyribonucleic acid DOC Particulate Organic Matter DOM Dissolved Organic Matter EDTA Ethylenediamine Tetra-acetic acid EPA Eicosapentaenoic acid EPS Extracellular Polymeric Substances ETF Electron Transfer Flavoprotein f/2 GLUT f/2 medium supplemented with 5 g l -1 glutamate Flp Fimbrial low-molecular-weight HCCA α-cyano-4-hydroxycinnamic acid HTA Hexadecatrienoic acid IVET in vivo Expression Technology Km Kanamycin LB Luria-Bertani IV MALDI-TOF Matrix-assisted laser desorption/ionization time-of-flight MB Marine broth MIC Minimal inhibitory concentrations MMSDH Methylmalonate-semialdehyde dehydrogenase MS Mass Spectrometry MSHA Mannose-sensitive haemagglutinin NCBI National Center of Biotechnology Information NGS Next-Generation Sequencing OD Optical density ON Over night PAGE Polyacrylamide gel electrophoresis PCR Polymerase Chain Reaction POM Particulate Organic Matter PPP Prokaryotic Promoter Prediction PTS Phosphoenolpyruvate:carbohydrate phosphotransferase system PUA Polyunsaturated aldehydes RDOM Recalcitrant Dissolved Organic Matter RNA Ribonucleic acid RNA-seq RNA sequencing RT Room temperature RT-qPCR Reverse-transcription quantitative real-time PCR SAGE Serial analysis of gene expression SDS Sodium dodecyl sulfate T2SS Type II secretion system TEP Transparent Exopolymeric Particles Tet Tetracycline TFA Trifluoracetic acid TRAP Tripartite ATP-independent periplasmic X-Gal 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside V CONTENTS SUMMARY ....................................................................................I ACKNOWLEDGEMENTS.................................................................. III LIST OF ABBREVIATIONS ............................................................... IV CONTENTS ................................................................................. VI 1. INTRODUCTION ........................................................................ 1 1.1. The biological Pump ................................................................... 1 1.2. Marine snow ............................................................................. 5 1.3. Diatom-bacteria interactions......................................................... 9 1.4. The in vitro model system............................................................13 1.4.1. Thalassiosira weissflogii ............................................................13 1.4.2. Marinobacter adhaerens HP15 ...................................................14 1.4.3. Interaction between T. weissflogii and M. adhaerens HP15.................16 1.5. Methods for the analysis of differential gene expression during cell-to-cell interactions ......................................................................................20 1.5.1. Promoter trap techniques..........................................................20 1.5.1.1 In vivo expression technology (IVET) ................................................. 21 1.5.2. Transcriptomics .....................................................................24 1.5.2.1 Reverse-Transcription quantitative Polymerase Chain Reaction (RT-qPCR) 214 1.5.2.2 Microarrays ................................................................................ 21 1.5.2.3 Tad-based