A Genomic Approach to the Role of Type IV Secretion Systems in Bartonella Host Adaptation Inauguraldissertation zur Erlangung der Würde eines Doktors der Philosophie vorgelegt der Philosophisch-Naturwissenschaftlichen Fakultät der Universität Basel von Philipp Engel aus Essen, Deutschland 0000 250 0 000 40 2 26 00 00 25 30 2 0 0 0 24 0 0 2 2 23 0 0 22 0 0 Basel, 2011 0 1 21 2 Originaldokument gespeichert auf dem Dokumentenserver der Universität Basel edoc.unibas.ch 20 0 0 0 0 0 19 0 2 Dieses Werk ist unter dem Vertrag „Creative Commons Namensnennung-Keine kommerzielle Nutzung-Keine Bearbeitung 2.5 Schweiz“ lizenziert. Die vollständige Lizenz18 kann unter creativecommons.org/licences/by-nc-nd/2.5/ch eingesehen werden. 1 9 17 0 0 0 0 16 0 Namensnennung-Keine kommerzielle Nutzung-Keine Bearbeitung 2.5 Schweiz Sie dürfen: das Werk vervielfältigen, verbreiten und öffentlich zugänglich machen Zu den folgenden Bedingungen: Namensnennung. 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Quelle: http://creativecommons.org/licenses/by-nc-nd/2.5/ch/ Datum: 3.4.2009 Genemigt von der Philosophisch-Naturwissenschaftlichen Fakultät auf Antrag von Prof. Christoph Dehio Prof. Urs Jenal Basel, den 08.12.2009 Prof. Dr. Eberhard Parlow (Dekan) for Fabienne Statement to my Thesis Statement to my Thesis This work was carried out in the group of Prof. Christoph Dehio in the Focal area Infection Biology at the Biozentrum of the University of Basel. My PhD thesis committee consisted of: Prof. Christoph Dehio Prof. Urs Jenal Prof. Guy Cornelis Prof. Walter Salzburger My thesis is written in a cumulative format. It consists of a synopsis covering several aspects related to my work. This is followed by result chapters presenting my research consisting of a published research article, a manuscript in preparation, a submitted manuscript, unpublished results, and a published review article. Finally, I recapitulate the major findings of my thesis and discuss some aspects and open questions of this work. i Table of Contents Table of Contents 1. Introduction 1 1.1 Studying bacterial evolution in the genomic era 2 1.2 Genome dynamics: Common strategies for adaptation in bacteria 4 1.2.1 Gene change 4 1.2.2 Gene gain 9 1.2.3 Gene loss 15 1.3 Alphaproteobacterial evolution 18 1.3.1 Genomic plasticity: Driving force of adaptability 18 1.3.2 Persistence: Common theme in infection strategies 21 1.4 Type IV secretion systems: Versatile nanomachines 23 1.4.1 Diversifying evolution of conjugation machineries 23 1.4.2 VirB and Trw T4SSs of Bartonella 25 1.5 References 29 2. Aim of the Thesis 37 3. Results 41 3.1 Research article I (published) 43 3.1.1 Summary 45 3.1.2 Manuscript: “Genomic analysis of Bartonella identifies type IV secretion systems as host adaptability factors” 47 3.2 Research article II (in preparation) 55 3.2.1 Summary 57 3.2.2 Manuscript: “Parallel adaptive radiations in a bacterial pathogen” 59 3.2.3 Supporting Online Material 81 ii Table of Contents 3.3 Research article III (submitted) 127 3.3.1 Summary 129 3.3.2 Manuscript: “The Trw type IV secretion system of Bartonella mediates host-specific infection of erythrocytes” 131 3.4 Unpublished results 181 “The VirB-homologous T4SS of B. schoenbuchensis: An evolutionary link between conjugation machineries and T4SSs adopted for host interaction” 3.5 Review article (published) 193 3.5.1 Summary 195 3.5.2 Manuscript: “Genomics of host-restricted pathogens of the genus Bartonella” 197 4. Summary 209 5. Discussion 213 5.1 Genomic overview of Bartonella and its lifestyle 214 5.2 Adaptive radiations in Bartonella 217 5.2.1 The VirB system: a key innovation driving adaptive radiations 218 5.2.2 The Trw system: a lineage-specific key innovation 222 5.2.3 Ecological opportunities 224 5.2.4 The Vbh system in the ruminant-infecting species 226 5.3 References 228 6. Acknowledgments 233 7. Curriculum Vitae 237 iii Introduction 1. Introduction 1 Introduction – Studying bacterial evolution in the genomic era 1.1 Studying bacterial evolution in the genomic era How bacteria and their traits are shaped by evolution has always been an important question. Particularly, the understanding of the evolutionary mechanisms underlying the occurrence and emergence of bacterial pathogens is of general importance. Since evolution occurs through changes in heritable traits encoded on the DNA, a prerequisite for studying bacterial evolution is the access to DNA sequence information. For long time, DNA sequences were available for only a few genes or taxa, and systematic or representative data which would allow inference of evolutionary aspects was missing (Seifert and DiRita 2006). The appearance of the first complete genome sequence in 1995, the one of Haemophilus influenzae, has dramatically changed the possibilities to examine evolutionary patterns (Fleischmann et al. 1995). In this study, the complete sequence of the attenuated laboratory strain Rd was compared to the sequences of known virulence genes cloned from more pathogenic clinical isolates providing insights into the molecular evolution of this pathogen. In the following, new genome projects were initiated and the continuous improvement of existing as well as the establishment of new sequencing technologies (Schuster 2008) has resulted in more and more genomic sequences deposited in databases (Fig. 1) . At the time of writing, 1001 complete microbial genomes (archea and bacteria) were found to be stored in public databases (http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi). The availability of genomic sequence data did not only provide more than enough information to infer comprehensive phylogenic relationships among bacterial species, but was also the onset for a new way of studying bacterial evolution (Lawrence 2005). Comparative genomics - the comparison of different genome sequences across biological species or strains - is nowadays an important research field mainly aiming at the understanding of molecular mechanisms underlying evolution of biological organisms (Koonin and Wolf 2008). By these means, certain biological questions about evolution could be addressed the first time and unexpected genomic characteristics revealed. Bacterial chromosomes have been viewed for long time as collections of a defined set of genes (Lawrence and Hendrickson 2005). However, the unprecedented view into the genomes of bacteria discovered a high degree of 2 Introduction – Studying bacterial evolution in the genomic era plasticity reflected by the difference in size among sequenced bacterial genomes ranging from 180 kb in the intracellular symbiont Carsonella rudii to 13 Mb in the soil bacterium Sorangium cellulosum (Fig. 1). The high abundance and importance of horizontal gene transfer (HGT), i.e. the lateral acquisition of foreign DNA, might be the biggest conceptual novelty brought about by comparative genomics of prokaryotes (Lawrence 1999; Ochman et al. 2000). In the pre-genomic area, HGT was viewed as a marginal phenomenon responsible for specific evolutionary events such as the spread of resistances (Koonin and Wolf 2008). Nowadays it is clear that horizontal transfer of genes plays a central role in the evolution of bacteria, and together with the phenomenon of gene loss represents the major factors contributing to the observed differences in genome size among bacteria. This high degree of genomic plasticity found among bacteria seems to reflect their distinct lifestyles and their adaptation to a wide range of ecological niches (Pallen and Wren 2007). Archaea Bacteria Next-generation sequencing Figure 1: Exponential growth of microbial genome sequences in public databases since 1995 (green: genomes/ blue: genera). The onset of next-generation sequencing by the landmark publication of the sequencing-by-synthesis technology (Margulies et al. 2005) resulted in a boost of available genome sequences. Nowadays, genomes of most microbial genera are available. (http://www.ncbi.nlm.nih.gov/genomes/MICROBES/microbial_growth.html). The inset shows the distribution of genome sizes among bacteria and archaea. Sequenced genomes of bacteria show a wide distribution with two peaks at 2 and 5 Mb, whereas archeal