Siderophores from Neighboring Organisms Promote the Growth Of
Total Page：16
File Type：pdf, Size：1020Kb
Siderophores From Neighboring Organisms Promote the Growth of Uncultured Bacteria A dissertation presented by Anthony D’Onofrio to The Department of Biology In partial fulfillment of the requirements for the degree of Doctor of Philosophy in the field of Biology Northeastern University Boston, Massachusetts November, 2008 1 Siderophores From Neighboring Organisms Promote the Growth of Uncultured Bacteria by Anthony D’Onofrio ABSTRACT OF DISSERTATION Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biology in the Graduate School of Arts and Sciences of Northeastern University, November, 2008 2 ABSTRACT The majority of bacterial cells in an environmental sample will not grow on synthetic media and are known as “unculturable”. As high as 99.9% of total cells counted by microscopy will not form colonies on standard Petri dishes. This phenomenon is known as the Great Plate Count Anomaly and is a significant unsolved problem in microbiology. The data presented in this study tests the hypothesis that some unculturable bacteria fail to grow on synthetic media because they are lacking specific growth factors from neighboring species, which signal a suitable environment to grow. In order to test this hypothesis, environmental bacteria were isolated from intertidal sediment and examined for helper-dependent relationships where an unculturable bacteria would only grow in the presence of a culturable helper species. Several such pairs were identified and a model unculturable, M. polysiphoniae KLE1104, was chosen for further study with model helper strain, M. luteus KLE1011, isolated from the same environment. Filtered spent supernatant of M. luteus KLE1011 as well as E. coli was capable of inducing growth of M. polysiphoniae KLE1104. E. coli knockout strains deficient in production of the iron chelating siderophore, enterobactin, were unable to induce growth. Testing purified enterobactin confirmed the compound was necessary and sufficient to induce growth of macrocolonies of M. polysiphoniae KLE1104. Five siderophores were purified from M. luteus KLE1011, and each was capable of inducing growth of the unculturable. Structure elucidation of the siderophores revealed that they were novel acyl-desferrioxamines with variable terminal modifications to increase hydrophobicity. Several species were then isolated from the same environment, which were dependent on M. luteus KLE1011. Six of these isolates along with two others 3 previously isolated from the same environment were tested for growth induction by a panel of 16 commercial siderophores and the five M. luteus KLE1011 siderophores. Each unculturable isolate was helped by a different set of siderophores, ranging from 6 to all 21 siderophores tested. This growth dependence on a varying set of siderophores suggests a strategy of only growing in the presence of a suitable environment, which is signalled by the presence of siderophores from appropriate neighbors. 4 ACKNOWLEDGEMENTS I would like to thank my advisor Kim Lewis for being an insightful, fair and patient mentor. His dedication to investigate challenging scientific questions was inspiring and provided a wonderful atmosphere to conduct research. I would also like to thank my thesis committee, Slava Epstein, Veronica Godoy-Carter, Jon Clardy and Eric Stewart for their support and helpful advice. Thanks to Eric Stewart for his excellent guidance. The success of the project would not have been possible without him. Thanks also to Kathrin Witt for her many contributions to the project, Jason Crawford for his excellent chemical isolation and structure elucidation work and Ekaterina Gavrish for her 16S sequencing and helpful suggestions. Thanks of course to the rest of the Lewis Lab past and present who provided stimulating conversation, advice and humor. I would like to thank my wife for her love and support. I would also like to thank my parents for always encouraging me to get a higher education and providing me with the resources to do so. 5 DEDICATION I would like to dedicate this thesis to my wife, Melanie D’Onofrio and my parents, Mario and Alison D’Onofrio for their love and support. 6 TABLE OF CONTENTS Abstract 3 Acknowledgements 5 Dedication 6 Table of Contents 7 List of Figures 8 List of Tables 9 Chapter 1. Introduction 10 Chapter 2. Methods 17 Chapter 3. Results 21 Chapter 4. Discussion 31 Figures 40 Tables 55 Appendix I 57 Appendix II 58 Appendix III 59 References 60 7 LIST OF FIGURES Figure 1. Sand Biofilm Community on Canoe Beach Intertidal Sediment Samples. Figure 2. Growth of M. polysiphoniae KLE1104 is Induced by M. luteus KLE1011. Figure 3. Growth Induction of M. polysiphoniae KLE1104 by Enterobactin from E. coli. Figure 4. Siderophores Produced by Helper Strain M. luteus KLE1011. Figure 5. Isolation of Bacteria Dependent on M. luteus KLE1011. Figure 6. Verification of M. luteus KLE1011 Dependent Isolates. Figure 7. Growth Induction of Unculturable Isolates By the Five M. luteus KLE1011 Siderophores. Figure 8. Commercial Siderophores Tested For Growth Induction of Unculturables. Figure 9. Growth Induction of Unculturables By Different Siderophores. Figure 10. Complementation of Siderophore Dependence With Soluble Iron (II). Figure 11. Increased Recovery Adding Soluble Fe(II) to R2Asea Plates. Figure 12. Fe(II) Induction of Rubritalea sp. KLE1210 and Parvularcula sp. KLE1250. Figure 13. Viable Cells Recovered From a Single Intertidal Pebble. Figure 14. Iron Dependence of Isolates Re-Suspended From Pebble Biofilm. Figure 15. Recovery of Intertidal Sediment Biofilm Bacteria From a Single Pebble. 8 LIST OF TABLES Table 1. Closest Relatives of 15 M. luteus KLE1011 Dependent Isolates. Table 2. Closest Relatives of Iron Dependent Isolates From Canoe Beach. 9 CHAPTER 1: INTRODUCTION The “Great Plate Count Anomaly” is an important unsolved problem in microbiology referring to the large discrepancy between cells counted in an environmental sample and the number of colonies formed on solid media (Butkevich, 1932; Staley and Konopka, 1985). It is generally accepted that less than 1% of cells in any given environmental sample will form colonies on synthetic media (Barer and Harwood, 1999; Giovannoni, 2000). Those that fail to grow in the lab are commonly referred to as “unculturable” or “uncultivable” bacteria. Advances in culture independent sequencing and phylogenetic comparison of 16s rDNA have revealed that several deep branching clades of bacteria can be detected but have never been cultured (Colwell and Grimes, 2000; Handelsman, 2004). In 1987 Carl Woese proposed a phylogenetic tree of life based on the comparison of 16s and 18s rRNA sequences (Woese, 1987). In his original tree, three domains were proposed, the Eubacteria (Bacteria), the Archaeabacteria (Archaea) and the Eukaryotes. The Bacteria were divided into 11 phyla based on distinct groups that clustered together when comparing the sequence similarity of 16s rDNA from cultured species (Appendix I, pg. 57). Based on the amplification and sequencing of 16s rDNA directly from the environment, numerous candidate phyla have been added which have no cultured representatives. By 1998, 36 bacterial phyla had been proposed, of which 13 were candidate phyla with no cultured members (Appendix II, pg. 58) (Hugenholtz et al., 1998). By 2003, this number had risen to 52 phlya; 26 being candidate phyla (Appendix III, pg. 59) (Rappe and Giovannoni, 2003). It therefore appears that the large unculturable fraction of cells, which fail to grow on synthetic media are in part comprised of members of species and 10 entire phlya which have never been cultured. Large scale DNA sequencing studies have provided valuable data about the abundance and diversity of biosynthetic operons in various marine environments (Rusch et al., 2007). Bacteria grown in pure culture however, can provide physiological insights that cannot be obtained by sequence data alone and the search for novel secondary metabolites will be greatly aided by the ability to culture new species. Several studies have shown that increased recovery or culturing of rare isolates can be achieved by growing cells in conditions that simulate the natural environment. Pelagibacter ubique, a member of the previously uncultured SAR11 phylum, was grown in pure culture by “extinction culturing” which involves the inoculation of single cells from seawater into small volumes (2 mL) of autoclaved seawater amended with very low carbon sources (Rappe et al., 2002). While growth in liquid media yielded concentrations up to 106 cells/mL, the strain did not grow on solid synthetic media. The authors state that this limitation may suggest the presence of an unusual growth factor in the ecology of the organism. The complete genome of P. ubique was subsequently sequenced, revealing interesting details that would not have been possible without a successful culturing method (Giovannoni et al., 2005). P. ubique has the smallest genome, and the fewest number of predicted open reading frames for any free living organism. It also has the smallest intergenic regions of any organism sequenced to date. The interesting “streamlining” of this organism would not have been discovered without the ability to sequence genomic DNA from a cultured strain. A method for genome sequencing from a single cell has been reported, but complete assembly of the genome was not achieved (Marcy et al., 2007). 11 Another method has been developed to incubate samples in a diffusion chamber, which simulates the natural environment. The