Molecular and Cultivation-based Characterization of Ancient Algal Mats from the McMurdo Dry Valleys, Antarctica A thesis submitted to Kent State University in partial fulfillment of the requirements for the degree of Master of Science by Doug Antibus December, 2009 Thesis written by Doug Antibus B.S., Kent State University, 2007 M.S., Kent State University, 2009 Approved by Dr. Christopher B. Blackwood Advisor Dr. James L. Blank Chair, Department of Biological Sciences Dr. Timothy Moerland Dean, College of Arts and Sciences iii TABLE OF CONTENTS LIST OF TABLES………………………………………………………………………..iv LIST OF FIGURES ……………………………………………………………………...vi ACKNOWLEDGEMENTS…………………………………………………………......viii CHAPTER I: General Introduction……………………………………………………….1 CHAPTER II: Molecular Characterization of Ancient Algal Mats from the McMurdo Dry Valleys, Antarctica: A Legacy of Genetic Diversity Introduction……………………………………………………………....22 Results and Discussion……………………………………………..……27 Methods…………………………………………………………………..51 Literature Cited…………………………………………………………..59 CHAPTER III: Recovery of Viable Bacteria from Ancient Algal Mats from the McMurdo Dry Valleys, Antarctica Introduction………………………………………………..……………..78 Methods…………………………………………………………………..80 Results……………………………………………………………...…….88 Discussion…………………………………………………………...….106 Literature Cited………………………………………………………....109 CHAPTER IV: General Discussion…………………………………………………….120 iii LIST OF TABLES Chapter II: Molecular Characterization of Ancient Algal Mats from the McMurdo Dry Valleys, Antarctica: A Legacy of Genetic Diversity Table 1. DNA yield of samples during successive rounds of extraction…..…….28 Table 2. Diversity and richness estimates of bacterial 16S rRNA gene clone libraries …………………………………………………………….……33 Table 3. Results of permutation tests of RDA (Redundancy Analysis) significance………………………………………………………………36 Table 4. Phylogenetic affiliation of groups unevenly distributed over sample age classes…………………………………………………………………....42 Table 5. Distribution of families within the Firmicutes in 16S rRNA gene clone libraries…………………………………………………………………..45 Table 6. Occurrence of selected phyla in 16S rRNA gene clone libraries from Antarctic habitats………………………………………………………...46 Table 7. Sample 14 C ages…………………………………………………...……52 iv Chapter III: Recovery of Viable Bacteria from Ancient Algal Mats from the McMurdo Dry Valleys, Antarctica Table 8. Summary of bacterial recovery from algal mat samples……………….89 Table 9. Phylogenetic affiliation of recovered ARDRA types as determined by BLAST searches……………………………………………………...….93 Table 10. Sample genotype richness and G tests of genotype distribution by temperature and medium…………………………………………..…..97 Table 11. Distribution of genotypes from sample 8643 among temperature treatments……………………………………………………………..97 Table 12. Abundance of genotype temperature response classes across age classes of samples………………………………………………………………105 Table 13. Unifrac distances (based on Euclidean distance of BOX-PCR profiles) among genotype temperature response classes…………………..……..105 v LIST OF FIGURES Chapter I: General Introduction Figure 1. Location of the McMurdo Dry Valleys in Antarctica…………………26 Chapter II: Molecular Characterization of Ancient Algal Mats from the McMurdo Dry Valleys, Antarctica: A Legacy of Genetic Diversity Figure 2. DNA yield of algal mat samples………………………………………29 Figure 3. Bacterial 16S rRNA gene copy number of DNA templates…………...30 Figure 4. Rarefaction analysis of bacterial 16S rRNA gene clone libraries……..32 Figure 5. Composition of bacterial 16S rRNA gene clone libraries by phylum…35 Figure 6. Distribution of samples on the age class canonical axis from redundancy analysis……………………………………………………...37 Figure 7 a-c. Abundance of 16S rRNA gene sequence groups distributed unevenly among sample age classes………………………………………………..41 Chapter III: Recovery of Viable Bacteria from Ancient Algal Mats from the McMurdo Dry Valleys, Antarctica Figure 8. Growth curves of selected isolates in TSB at 15 °C……………..……..87 Figure 9 a-b. CFU abundance from heterotrophic plate counts on a) R2A and b) 1/10 strength R2A medium………………………………90 vi Figure 10. Rarefaction analysis of BOX-PCR genotype occurrence by sample…………………………………………………………………96 Figure 11. Estimated phylogeny of 16S rRNA sequences from ARDRA type 1 isolates……………………………..…………………………………..99 Figure 12. Correlation of pairwise BOX-PCR profile Euclidean distance with 16S rRNA gene sequence p distance……………………………….….……100 Figure 13. Time to colony formation during growth temperature screening.….102 Figure 14. UPGMA tree of genotype BOX-PCR profiles.………….……...….109 vii ACKNOWLEDGEMENTS This work would not have been possible without the assistance of many people at Kent State and other institutions; too many to name, actually. I first of all would like to thank my guidance committee: Dr. Chris Blackwood, Dr. Laura Leff, and Dr. Christopher Woolverton for their encouragement and assistance. I also need to thank colleagues at other institutions: Dr. Brenda Hall at the Univeristy of Maine, Orono and Dr. Jennifer Baeseman at the University of Tromsø, Norway. I received a great deal of assistance, advice, and comaraderie from colleagues in the KSU Biology Department; I will certainly miss all of my friends in the department when I leave. I would particularly like to thank Larry Feinstein and Oscar Valverde who were always working alongside me and kept me company during long hours in the lab. Funding for this research came from a Kent State startup grant to Dr. Chris Blackwood and partially from a NSF grant to Dr. Jennifer Baeseman. I am also grateful for travel support received from Kent State University, the American Society for Microbiology, and the NSF Office of Polar Programs. viii Chapter I General Introduction Terrestrial Antarctic habitats present a challenge to microbial growth and survival because of low temperatures, freeze-thaw cycles, and the scarcity of liquid water. In spite of these conditions, molecular and cultivation-based studies have revealed a diverse microbial flora on the Antarctic continent, including the McMurdo Dry Valleys (reviewed in Vincent, 2000; Tindall, 2004). The McMurdo Dry Valleys represent a particularly harsh environment within the Antarctic in which resources needed for microbial metabolic activity are often unavailable (e.g. liquid water), forcing organisms to undergo periods of dormancy (Horowitz et al. , 1972; Kennedy, 1993; Treonis et al. , 2002). For example, metabolic activity in Dry Valley cryptoendolithic communities may only be possible for 400-1050 hours per year (Friedman et al. , 1993). Sun and Friedmann (1999) noted that in Dry Valley cryptoendolithic communities, ‘biological and geological time scales overlap’ due to the extremely short growing seasons. Adaptation to withstand environmental stresses is thought to be a significant factor in the evolution of Antarctic microbes and an underlying cause for the existence of endemic Antarctic microbial taxa. The psychrophilic or psychrotolerant characteristics of Antarctic microbes (and metazoans as well) have received a substantial amount of scientific interest (reviewed by Deming, 2002). Many Antarctic bacteria (Franzmann and Dobson, 1993; Nadeau et al. , 2001; Spring et al. , 2003) and algae (Seaburg et al. , 1981) 1 2 possess lower optimal growth temperatures than related strains from temperate climates. Other Antarctic microorganisms are closely-related to microbes isolated from non- Antarctic cold environments including Arctic sea ice (Staley and Gosink, 1999), refrigerated foods (Franzmann et al. , 1991; Spring et al. , 2003), and permafrost (Fruhling et al. , 2002). Antarctic microbes also appear to be adapted to withstand stresses associated with dormancy and dormancy-inducing conditions, namely desiccation/rehydration and freeze/thaw cycles (Davey, 1989; Šabacká and Elster, 2006). Antarctic algal mats can rapidly resume metabolic activity after years of dormancy (Hawes et al. , 1992) and can survive nearly-intact after more than two decades of dormancy (McKnight et al. , 2007). In addition, long-term microbial dormancy has been examined in Arctic and Antarctic glacial ice and permafrost (Johnson et al. , 2007; Willerslev et al. , 2004; reviewed in Price, 2007). Glacial ice and permafrost contain unusual environmental conditions which are likely to influence microbial preservation; hence, studies of dormancy in a desiccating environment provide a point of comparison to glacial ice and permafrost. In spite of this potential importance, the response of Antarctic microbes to long-term (millennial-scale) dormancy imposed by desiccating conditions has not been studied. The research presented in this thesis was carried out to evaluate the possibility for Antarctic microbes and microbial DNA to be preserved under long-term desiccation-imposed dormancy. Emphasis was placed on evaluating factors affecting community composition within samples, including differences in the robustness of taxa to dormancy. Additionally, I sought to evaluate the relatedness of isolates from 3 ancient samples to those from the modern sample using both a genotyping technique (BOX-PCR) and temperature-growth relationships. Cellular Dormancy and Preservation Dormant cells are commonly defined as cells that have ceased metabolic activity but retain viability (Roszak and Colwell, 1987). Endospores are the most well-known bacterial dormant state of bacteria (Gould, 2006), but other dormant cell types have been described for diverse bacterial groups, including cyanobacterial akinetes