Bacterial adaptation strategies and interactions in different soil habitats Von der Fakultät für Lebenswissenschaften der Technischen Universität Carolo-Wilhelmina zu Braunschweig zur Erlangung des Grades einer Doktorin der Naturwissenschaften (Dr. rer. nat.) genehmigte D i s s e r t a t i o n von Selma Gomes Vieira aus Leiria / Portugal 1. Referent: Professor Dr. Jörg Overmann 2. Referent: Professor Dr. Dieter Jahn eingereicht am: 25.06.2018 mündliche Prüfung (Disputation) am: 22.10.2018 Druckjahr 2019 Vorveröffentlichungen der Dissertation Teilergebnisse aus dieser Arbeit wurden mit Genehmigung der Fakultät für Lebenswissenschaften, vertreten durch den Mentor der Arbeit, in folgenden Beiträgen vorab veröffentlicht: Publikationen Vieira, S., Luckner, M., Wanner, G., & Overmann, J. Luteitalea pratensis gen. nov., sp. nov. a new member of subdivision 6 Acidobacteria isolated from temperate grassland soil. Int J Syst Evol Microbiol 67: 1408-1414 (2017). Huang, S., Vieira, S., Bunk, B., Riedel, T., Spröer, C. & Overmann, J. First Complete Genome Sequence of a Subdivision 6 Acidobacterium Strain. Genome Announc. 4: e00469-16 (2016). Posterbeiträge Vieira, S., Sikorski, J. & Overmann, J., Cultivation of environmental and taxonomically important soil bacteria from the Biodiversity Exploratories (Poster) 12th Biodiversity Exploratories Assembly, Wernigerode (2015). Vieira, S., Sikorski, J. & Overmann, J., Drivers of bacterial communities in grassland plant rhizospheres (Poster) 13th Biodiversity Exploratories Assembly, Wernigerode (2016). Vieira, S., Sikorski, J. & Overmann, J., Drivers of bacterial communities in grassland plant rhizospheres (Poster) 14th Biodiversity Exploratories Assembly, Wernigerode (2017). Vieira, S., Sikorski, J. & Overmann, J., Dynamics of bacterial colonisation of novel mineral surfaces in soils (Poster) 15th Biodiversity Exploratories Assembly, Wernigerode (2018). Vieira, S., Sikorski, J., Dietz, S., Herz, K., Bruelheide, H., Scheel, D., & Overmann, J., Governing factors of bacterial communities in temperate grassland plant rhizospheres (Poster) Vereinigung für Allgemeine und Angewandte Mikrobiologie Jahrestagung 2018, Wolfsburg (2018). Dedicated to my late brother, Duarte Gomes Vieira Table of Contents Chapter 1 – Summary ............................................................................................................................. 1 Chapter 2 – Introduction ......................................................................................................................... 3 2.1 – Bacteria in the soil environment ................................................................................................ 3 2.1.1 – Roles and significance of bacteria in soils ........................................................................... 3 2.1.2 – Soil heterogeneity and bacterial hotspots .......................................................................... 5 2.1.3 – Determinants of bacterial community structure in soil ...................................................... 6 2.2 – Bacterial association with plant roots ........................................................................................ 8 2.2.1 – Rhizosphere effect .............................................................................................................. 9 2.2.2 – Root exudation .................................................................................................................. 11 2.2.3 – Bacteria – exudate interactions in the rhizosphere .......................................................... 12 2.2.4 – Implications of rhizosphere engineering for agriculture .................................................. 15 2.3 – Microbial community assembly ............................................................................................... 16 2.3.1 – Ecological processes .......................................................................................................... 17 2.3.1.1 – Selection ..................................................................................................................... 17 2.3.1.2 – Dispersal ..................................................................................................................... 18 2.3.1.3 – Diversification ............................................................................................................ 19 2.3.1.4 – Drift ............................................................................................................................ 19 2.3.2 – Ecological succession ........................................................................................................ 19 2.3.2.1 – Bacterial primary succession in soil ........................................................................... 20 2.3.2.2 – Artificial soils .............................................................................................................. 21 2.4 – Culture-independent approaches to study soil bacterial communities .................................. 21 2.4.1 – Assessing biomass ............................................................................................................. 22 2.4.2 – Assessing diversity............................................................................................................. 23 2.4.3 – Assessing activity ............................................................................................................... 24 2.5 – Culture-dependent approaches to study soil bacterial communities ..................................... 25 2.5.1 – Reasons for low cultivation success .................................................................................. 26 2.5.1.1 – Physiological state and adaptation of bacteria to the heterogeneous environment 27 2.5.1.2 – Adaptation to oligotrophy.......................................................................................... 28 2.5.1.3 – Bacterial interactions ................................................................................................. 28 2.6 – Aims of the study ..................................................................................................................... 30 2.7 – References ................................................................................................................................ 32 Chapter 3 – Experimental procedures .................................................................................................. 46 3.1 – Rhizosphere .............................................................................................................................. 46 3.1.1 – Sampling ............................................................................................................................ 46 3.1.2 – Plant root exudates ........................................................................................................... 47 3.1.2.1 – Collection ................................................................................................................... 47 3.1.2.2 – Gas Chromatography / Mass Spectrometry (GC/MS) ................................................ 47 3.1.3 – Plant traits and soil parameters ........................................................................................ 48 3.1.4 – RNA extraction .................................................................................................................. 48 3.1.4.1 – RNA / DNA co-extraction from rhizosphere samples ................................................ 48 3.1.4.2 – DNA digestion ............................................................................................................ 49 3.1.4.3 – Reverse transcription ................................................................................................. 49 3.1.5 – Preparation of 16S rRNA V3 amplicon libraries and sequencing ...................................... 49 3.1.5.1 – Indexing PCR (Bartram method) ................................................................................ 49 3.1.5.2 – Metaphor agarose purification of amplicons............................................................. 50 3.1.5.3 – Illumina HiSeq sequencing ......................................................................................... 50 3.1.6 – Downstream processing of amplicons sequencing data ................................................... 50 3.1.6.1 – Pre-processing of sequencing reads .......................................................................... 50 3.1.6.2 – OTU clustering and taxonomy assignment ................................................................ 50 3.1.7 – Statistical analysis ............................................................................................................. 50 3.2 – Mineral containers ................................................................................................................... 52 3.2.1 – Experimental set up .......................................................................................................... 52 3.2.2 – Sampling of mineral containers ........................................................................................ 53 3.2.3 – DNA extraction .................................................................................................................. 53 3.2.4 – qPCR .................................................................................................................................. 53 3.2.5 – Preparation of 16S rRNA V3 amplicon libraries