The lichen-associated microbiome: taxonomy and functional roles of lichen-associated bacteria Margrét Auður Sigurbjörnsdóttir Faculty of Life and Environmental Sciences University of Iceland 2016 The lichen-associated microbiome: taxonomy and functional roles of lichen-associated bacteria Margrét Auður Sigurbjörnsdóttir Dissertation submitted in partial fulfillment of a Philosophiae Doctor degree in Biology Supervisors Professor Oddur Vilhelmsson Professor Ólafur S. Andrésson PhD Committee Professor Oddur Vilhelmsson Professor Ólafur S. Andrésson Professor Kristinn P. Magnússon Starri Heiðmarsson Opponents Gabrielle Berg Viggó Marteinsson Faculty of Life and Environmental Sciences School of Engineering and Natural Sciences University of Iceland Reykjavik, January 2016 The lichen-associated microbiome: taxonomy and functional roles of lichen-associated bacteria The biodiversity of bacterial lichen-associates (Short title) Dissertation submitted in partial fulfillment of a Philosophiae Doctor degree in Biology Copyright © 2016 Margrét Auður Sigurbjörnsdóttir All rights reserved Faculty of Life and Environmental Sciences School of Engineering and Natural Sciences University of Iceland Sturlugötu 7 101, Reykjavik Iceland Telephone: 525 4000 Bibliographic information: Margrét Auður Sigurbjörnsdóttir, 2016, The lichen-associated microbiome: taxonomy and functional roles of lichen-associated bacteria, PhD dissertation, Faculty of Life and Environmental Sciences, University of Iceland, 199 pp. ISBN 978-9935-9283-7-5 Printing: Háskólaprent Reykjavik, Iceland, January 2016 ii Abstract Although lichens are generally characterized as the symbiotic association of a fungus (mycobiont) and a photobiont (green algae and/or cyanobacteria), species-specific communitites of other endolichenic bacteria, typically dominated by Proteobacteria, can also be found within the symbiosis. The roles that the non-phototrophic microbiota of lichens play in the symbiotic association is, however, not fully understood. This thesis describes studies that aim to characterize the non-phototrophic microbiota of lichens and elucidate some of their roles in the symbiotic association. Firstly, bacteria were cultured from four saxicolous seashore lichens; Hydropunctaria maura, Verrucaria ceuthocarpa, Caloplaca verruculifera and Lecanora helicopis, which are all commonly found along the Icelandic seashore. A total of 168 strains were isolated and selected isolates were further analysed. Secondly, lichen thalli from the membranous dog lichen, Peltigera membranacea, were investigated using culture- independent techniques, as well as characterizing cultured isolates and some of the roles the constituent bacteria play in the symbiotic association elucidated. Total DNA was extracted and a metagenomic library of 150 thousand contigs constructed and binned against 15 reference genomes. The BlastX algorithm was used for analyses of the selected contigs against the protein database. Taxonomically, the majority of the bacterial associates belong to Alphaproteobacteria, although members of Acidobacteria, Actinobacteria, Bacteroidetes and Verrucomicrobia are also present. Analysis of 28,000 bacterial contigs from the Peltigera membranacea metagenome yielded multiple hits on several genes involved in lichen secondary metabolite resistance, inorganic phosphate mobilization, biopolymer degradation and several other potentially important functions in thallus colonization and symbiosis. Samples of P. membranacea thalli were plated on various media types and 113 bacterial strains isolated. Phenotypic analyses of selected strains indicates that hydrolytic activity is common among these bacteria, as is inorganic phosphate mobilization and nitrogen fixation. Scavenging of organic nutrients may also constitute an important role for Peltigera-associated bacteria, with many of the strains producing strong biosurfactants and being able to degrade naphthalene. iii iv Útdráttur Fléttur eru sambýli sveppa og ljóstillífandi lífvera, ýmist grænþörunga eða blábaktería. Í þeim er þó einnig að finna stórt og fjölbreytt samfélag annarra baktería, en hin ljósóháða bakteríubíóta fléttna hefur afar lítið verið rannsökuð þar til á allra síðustu árum. Í fyrri hluta verkefnisins var sýnum af fjórum íslenskum strandfléttum (Hydropunctaria maura, Verrucaria ceuthocarpa, Caloplaca verruculifera and Lecanora helicopis) safnað og ræktanleg bakteríubíóta þeirra greind. Einangraðir voru 168 bakteríustofnar og valdir stofnar teknir til greiningar. Í síðari hluta verkefnisins var sýnum af himnuskóf, Peltigera membranacea, safnað. Erfðaefni fléttunnar var einangrað og raðgreint. Gerður var gagnagrunnur samfellna (contigs) sem flokkaðar voru úr rúmlega 150 þ. samfella samkvæmt einsleitni við 15 valin bakteríuerfðamengi. BLASTx algóriþmi var notaður til að leita gegn þekktum röðum í prótein gagnabanka. Niðurstöður sýna að meirihluti bakteríubíótunnar tilheyrir Alphaproteobacteria en jafnframt er þar að finna Acidobacteria, Actinobacteria, Bacteroidetes og Verrucomicrobia. Einnig sést að þar er að finna gen sem taka þátt í grunnefnaskiptum, áreitissvörun (stress response), boðferlum (signal transduction), niðurbroti fjölliða, nýmyndun próteina og DNA og fleira. Að auki var sýni úr P. membranacea sáð á mismunandi ætistegundir. Einangraðir stofnar úr himnuskóf voru samtals 113 og voru framkvæmd ýmis greiningapróf á þeim, t.a.m. niðurbrot fjölliða og olíuefna, og fleiri lífefnafræðilegir þættir. v vi To my family and my friends… …for their incredible support over the years vii viii List of papers The thesis is based on four papers of which two are published and two are submitted to scientific journals. The papers will be referred in the text by their respective numbers as following: Paper I: Sigurbjörnsdóttir, M. A., Heiðmarsson, S., Jónsdóttir, A. R. and Vilhelmsson, O. 2014. Novel bacteria associated with Arctic seashore lichens have potential roles in nutrient scavenging. Canadian Journal of Microbiology, 60: 307-317. Paper II: Sigurbjörnsdóttir, M. A., Andrésson, Ó. S. and Vilhelmsson, O. 2015. Analysis of the Peltigera membranacea metagenome indicates that lichen-associated bacteria are involved in phosphate solubilization. Microbiology, 161: 989-996. doi: 10.1099/mic.0.000069 Paper III: Sigurbjörnsdóttir, M. A. & Vilhelmsson, O. (SUBMITTED). Culturable phosphate-mobilizing, biosurfactant-producing and biodegradative bacteria associated with a sub-Arctic, terricolous lichen, Peltigera membranacea. Submitted to FEMS Microbial Ecology November 24th 2015. Paper IV (Invited mini-review): Sigurbjörnsdóttir, M. A., Andrésson, Ó. S., Grube, M. & Vilhelmsson, O. (IN PRESS). Nutrient scavenging activity and antagonistic factors of non-photobiont lichen-associated bacteria: a review. Accepted for publication in World Journal of Microbiology Biotechnology. All papers are reprinted by kind permission of the publishers. Peer reviewed paper not included in the thesis: Paper V: Vilhelmsson, O, Sigurbjörnsdóttir, M. A., Grube, M. & Höfte, M. (IN PRESS). Are lichens natural reservoir for plant pathogens? Accepted for publication in Molecular Plant Pathology. ix x Declaration of contribution Paper I Jónsdóttir and Vilhelmsson designed the study and were responsible for collecting samples with Heiðmarsson. Jónsdóttir was responsible for the isolation procedure and analysing the data. Sigurbjörnsdóttir was responsible for sample collection when repeating the study and wrote the first draft of the paper, with supervision from Vilhelmsson and Heiðmarsson. Vilhelmsson and Heiðmarsson critically revised the manuscript, edited it and approved the final version of manuscript. Paper II Andrésson designed the study, collected samples, and was responsible for DNA isolation. Snæbjarnarson constructed the metagenomic database with assistance from Andrésson and Vilhelmsson. Sigurbjörnsdóttir analysed the bacterial metagenome obtained from the larger metagenomic dataset, with supervison from Vilhelmsson. Sigurbjörnsdóttir wrote the first draft of the paper, with supervision from Vilhelmsson and Andrésson, which critically revised the manuscript. Sigurbjörnsdóttir worked on the final version of the manuscript. All authors gave final approval of the manuscript to be published. Paper III Sigurbjörnsdóttir designed the study and was responsible for collecting samples, with supervision from Vilhelmsson. Ásgeirsdóttir, Jónsdóttir, Þorsteinsdóttir, and Kjartansdóttir assisted with strain isolation and phenotypic analysis, with supervision from Sigurbjörnsdóttir. Sigurbjörnsdóttir was responsible for analysing the data and writing the first draft of paper, with supervision from Vilhelmsson. Vilhelmsson critically revised the manuscript and edited it. Sigurbjörnsdóttir worked on the final version and Vilhelmsson approved the final version of manuscript to be published. xi Paper IV Sigurbjörnsdóttir was responsible for writing the paper. Vilhelmsson and Andrésson critically revised the manuscript and edited it. Sigurbjörnsdóttir worked on the final version and all authors approved the final version submitted. xii Table of Contents ABSTRACT III ÚTDRÁTTUR V LIST OF PAPERS IX DECLARATION OF CONTRIBUTION XI LIST OF FIGURES XV LIST OF TABLES XVI ACKNOWLEDGEMENTS XVII 1 INTRODUCTION 1 1.1 Symbiosis 1 1.2 Lichens 1 1.3 General description of the lichen symbiosis 2 1.4 Lichen morphology 4 1.4.1 Crustose lichens 5 1.4.2 Foliose lichens 6 1.4.3 Fruticose lichens 7 1.5 Lichen growth and resources 8 1.5.1 Water 9 1.5.2 Carbon 9 1.5.3 Elements 10 1.6 Lichen metabolites