The genus The Bonsai For hundreds of years the image of a tree has been used as a visual representation of (evolutionary) Romboutsia relationships among organisms (‘The Tree of Life’). The leaves of the tree represent individual species, while the splitting branches represent divergence events. Also relationships among bacterial - genomic and functional characterization Jacoline Gerritsen Jacoline and functional characterization - genomic species are often visualized in tree-shaped diagrams. It is not just a tree that decorates the cover of this thesis, it is a bonsai. The Japanese word ‘bonsai’ is used to describe a tree which is planted in a shallow container. The bonsai metaphorically depicts the bacterial tree of life, since a number of techniques used for the care of bonsai are similar to those applied in bacterial classification. Selective removal of parts of a tree, known as pruning, is performed to maintain and refine the shape of the tree. In addition, pruning of leaves (species) is sometimes needed to restrain excessive growth. Furthermore, branches are wired to bend and reposition them into the desired shape. This is similar to the creation of higher- level groups in the bacterial tree of life to support the formation of small branches and new leaves on specific locations in the tree. Grafting, the placement of new growing material into a prepared area on the trunk, resembles the reclassification of bacterial taxa. Deciding which branches or The genus Romboutsia leaves should stay or be removed can be challenging since there Genomic and functional characterization of novel are no rules but only guidelines to support decision making, as is also the case for the classification bacteria dedicated to life in the intestinal tract of bacteria. So in the end, the shapes of the tree is strongly dependent on choices that have been made by the artist/scientist Jacoline Gerritsen in the creative process. The genus Romboutsia Genomic and functional characterization of novel bacteria dedicated to life in the intestinal tract Jacoline Gerritsen THESIS COMMIttEE PROMOTORS Prof. Dr H. Smidt Personal chair at the Laboratory of Microbiology Wageningen University Prof. Dr W.M. de Vos Professor of Microbiology Wageningen University CO-PROMOTOR Prof. Dr G.T. Rijkers Professor of Medical Biology and Life Sciences University College Roosevelt, Utrecht University OTHER MEMBERS Prof. Dr E.J.M. Feskens, Wageningen University Prof. Dr I.C.W. Arts, Maastricht University Dr K. Scott, Rowett Institute of Nutrition and Health, Aberdeen, Scotland Dr E.E. Vaughan, Sensus, Roosendaal This research was conducted under the auspices of the Graduate School VLAG (Advanced studies in Food Technology, Agrobiotechnology, Nutrition and Health Sciences). The genus Romboutsia Genomic and functional characterization of novel bacteria dedicated to life in the intestinal tract Jacoline Gerritsen THESIS submitted in fulfilment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus Prof. Dr M.J. Kropff, in the presence of the Thesis Committee appointed by the Academic Board to be defended in public on Friday 23 January 2015 at 11 a.m. in the Aula. Jacoline Gerritsen The genus Romboutsia - Genomic and functional characterization of novel bacteria dedicated to life in the intestinal tract. 280 pages PhD thesis, Wageningen University, Wageningen, NL (2015) With references, with summaries in English and Dutch ISBN 978-94-6257-242-3 Table of Contents Chapter 1 General introduction and thesis outline 7 Chapter 2 Diversity of the intestinal microbiota in health and disease - 39 impact of probiotics Chapter 3 Probiotics stimulate a commensal gut bacterium which protects 85 the host from severe pancreatitis Chapter 4 Characterization of Romboutsia ilealis gen. nov., sp. nov., 109 isolated from the gastro-intestinal tract of a rat, and proposal for the reclassification of five closely related members of the genus Clostridium into the genera Asaccharospora gen. nov., Intestinibacter gen. nov., Romboutsia gen. nov., and Terrisporobacter gen. nov. Chapter 5 Genomic and functional analysis of Romboutsia ilealis CRIBT 143 reveals adaptation to the small intestine Chapter 6 Comparative genomics and functional analysis of the genus 187 Romboutsia provides insight into adaptation to an intestinal lifestyle. Chapter 7 General discussion 233 Chapter 8 Appendices: Summary 262 Nederlandse samenvatting 264 List of abbreviations 278 Co-author affiliations 271 Acknowledgements / Dankwoord 272 About the author 276 List of publications 277 Overview of completed training activities 278 5 C H A P T E 1R General introduction and thesis outline 7 CHAPTER 1 Intestinal microbes: they outnumber us! A thesis that focusses on specific members of the bacterial communities residing in 1 the mammalian intestinal tract has to start with a general introduction on intestinal microbiota, the microbial populations living in the intestinal tract. So, let’s start with some numbers. The human body contains at least tenfold more microbial cells (1014) than human cells 124. Microbes colonize every surface that is exposed to the external environment. Of these surfaces, the intestinal tract is by far the most densely colonized. Furthermore, the microbes residing in the intestinal tract add ~150 times more genes to our total gene pool than our own human genome 46, 112. Maybe these numbers have been quoted too often already, but they still illustrate very nicely that humans, like other mammals, are not single-species organisms, but in fact they constitute very complex ecosystems. The complex network of host-microbe and microbe-microbe interactions is tremendously important for host health, as will be discussed in Chapter 2. The microbes in our intestinal tract are our first line of defence against other (pathogenic) microbes and in this sense they supplement our immune system. Microbes are in constant interaction and competition with each other for nutrients and attachment sites 4, 65. In addition, they are constantly communicating with each other, for example by the secretion of anti-microbial compounds such as short chain fatty acids (SCFA) and bacteriocins 22, 28. In addition, they are not only communicating with each other, but also with their host. Especially in the intestinal tract microbes have the opportunity to communicate with their host, as the intestinal tract contains most of the body’s immune cells, hormone producing cells, and the body’s nervous system is embedded throughout the lining of the intestinal tract 87, 98, 149. Last but certainly not least, the intestinal microbes contribute significantly to the metabolic capacities of the host by producing a wide array of compounds that are important nutrients for the host, such as SCFA and vitamins 39, 103, 161. Metaphorically speaking, we are maybe nothing more than walking bioreactors; microbes outnumber us by at least one order of magnitude, and maybe the only way to control them is by how and what we feed them. It is important to mention that (micro)organisms from all three domains of life are found in the intestinal tract: bacteria, archaea, and eukaryotes. The eukaryotes found in the intestinal tract consist of fungi, protozoa, helminths and others 125. In addition, viruses are found abundantly in the intestinal tract, including bacteriophages, which are viruses that specifically infect bacteria 1, 10. Future studies will provide much more insight in the role of each of these (micro)organisms in the intestinal ecosystem 153. However, this thesis will focus only on bacterial members of the intestinal microbiota. The intestinal tract contains distinct microenvironments Factors such as nutrient availability, pH, redox potential, host secretions, and peristalsis strongly influence the composition of bacterial communities in the intestinal tract as 8 GENERAL INTRODUCTION will be discussed next. However, the intestinal tract is not simply one big hollow tube that starts after the stomach and ends with the anal canal, but it is in fact divided into several distinct segments. Each segment has its own functionality, which is reflected in 1 the anatomy of that segment. Although the anatomy of the intestinal tract varies greatly between different animals, for the purposes of this thesis, only the mammalian intestinal tract will be discussed in the next paragraphs, with a specific focus on the human intestinal tract. Along the longitudinal axis, the intestinal tract can roughly be divided into two segments, the small intestine and the large intestine (Figure 1). The small intestine can be further subdivided into the duodenum, jejunum, and ileum. These are regions where most of the enzymatic digestion takes place, and host secretions provide a strong selective pressure on the microbes in these niches 6, 124, 154. Increasing numbers of microbes have been found along the length of the small intestine 6, 27. Gastric acid from the stomach ensures a low pH in the small intestine that gradually increases along the intestinal tract, reaching neutral pH at the terminal ileum 35. Bile and pancreatic secretions containing digestive enzymes enter the intestinal tract in the duodenum. The small intestinal epithelium is extensively folded into crypts, and the luminal surface is covered with finger- like projections (villi) that increase the surface area available for nutrient absorption. The jejunum and ileum are regions where active and passive nutrient absorption takes place by the host. In these regions, the microbes are in strong competition with the host for these nutrients. It is observed that the microbes in the small intestine are adapted to this dynamic environment by their ability to rapidly take in and convert simple carbohydrates 166. Most of the bile is reabsorbed in the ileum together with vitamin B12, an important vitamin for the host that can only be produced by microbial activities. One of the main characteristics of the ileum is the presence of Peyer’s patches, which are organized lymphoid nodules that contain large numbers of lymphocytes and other immune cells 9. This indicates that the ileum is an important region for immune-system related host-microbe interactions.