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NOVEL ANAEROBIC THERMOPHILIC BACTERIA; INTRASPECIES HETEROGENEITY AND BIOGEOGRAPHY OF THERMOANAEROBACTER ISOLATES FROM THE KAMCHATKA PENINSULA, RUSSIAN FAR EAST by ISAAC DAVID WAGNER (Under the Direction of Juergen Wiegel) ABSTRACT Thermophilic anaerobic prokaryotes are of interest from basic and applied scientific perspectives. Novel anaerobic thermophilic taxa are herein described are Caldanaerovirga acetigignens gen. nov., sp. nov.; and Thermoanaerobacter uzonensis sp. nov. Novel taxa descriptions co-authored were: Thermosediminibacter oceani gen. nov., sp. nov and Thermosediminibacter litoriperuensis sp. nov; Caldicoprobacter oshimai gen. nov., sp. nov. More than 220 anaerobic thermophilic isolates were obtained from samples collected from 11 geothermal springs within the Uzon Caldera, Geyser Valley, and Mutnovsky Volcano regions of the Kamchatka Peninsula, Russian Far East. Most strains were phylogenetically related to Thermoanaerobacter uzonensis JW/IW010T, while some were phylogenetically related to Thermoanaerobacter siderophilus SR4T. Eight protein coding genes, gyrB, lepA, leuS, pyrG, recA, recG, rplB, and rpoB, were amplified and sequenced from these isolates to describe and elucidate the intraspecies heterogeneity, α- and β-diversity patterns, and the spatial and physicochemical correlations to the observed genetic variation. All protein coding genes within the T. uzonensis isolates were found to be polymorphic, although the type (i.e., synonymous/ nonsynonymous substitutions) and quantity of the variation differed between gene sequence sets. The most applicable species concept for T. uzonensis must consider metapopulation/ subpopulation dynamics and acknowledge that physiological characteristics (e.g., sporulation) likely influences the flux of genetic information between subpopulations. Spatial variation in the distribution of T. uzonensis isolates was observed. Evaluation of T. uzonensis α-diversity revealed a range of genetic variation within a single geothermal spring. β-diversity measurements revealed that while most of the molecular variance came from inter-regional comparisons, high diversity measures between populations within a region were also observed. Between geothermal springs from different regions, T. uzonensis genetic divergence was correlated with an increase in spatial separation. However, the trend was not observed when only the isolates between the geothermal springs within a region were considered. When 27 physicochemical properties from four geothermal springs in the Uzon Caldera were matched to a corresponding biological distribution pattern, high rank correlation values, calculated as Spearman's ρ, were observed. Together, these analyses suggest that the spatial variation of T. uzonensis was influenced by both environmental differences and spatial separation. INDEX WORDS: thermophiles, microbial biogeography, microbial diversity, anaerobic thermophiles, biogeochemistry NOVEL ANAEROBIC THERMOPHILIC BACTERIA; INTRASPECIES HETEROGENEITY AND BIOGEOGRAPHY OF THERMOANAEROBACTER ISOLATES FROM THE KAMCHATKA PENINSULA, RUSSIAN FAR EAST by ISAAC DAVID WAGNER B.S., Concordia University, Nebraska, 2004 A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY ATHENS, GEORGIA 2010 © 2010 Isaac David Wagner All Rights Reserved NOVEL ANAEROBIC THERMOPHILIC BACTERIA; INTRASPECIES HETEROGENEITY AND BIOGEOGRAPHY OF THERMOANAEROBACTER ISOLATES FROM THE KAMCHATKA PENINSULA, RUSSIAN FAR EAST by ISAAC DAVID WAGNER Major Professor: Juergen Wiegel Committee: Mary Ann Moran Christopher S. Romanek William B. Whitman Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia May 2010 DEDICATION For their constant support and encouragement, this dissertation is dedicated to my parents, Kim and David Wagner. iv ACKNOWLEDGEMENTS Foremost, I would like to acknowledge my major professor J. Wiegel, and my committee members, M. A. Moran, C. S. Romanek, and W. B. Whitman. Thank you for your instrumental guidance and assistance. I would also like to thank my co-authors, W. Zhao, H. Yokoyama, E. A. Burgess, C. L. Zhang, M. Rohde, Y.-J. Lee, M. E. Brice, V. V. Kevbrin, G. L. Mills, and S. Ahmed. I thank D. E. Crowe for the assistance he provided concerning analyses with the physicochemical properties of the geothermal springs. I am grateful for the help provided by members of the Wiegel laboratory, including N. Mesbah, K. Bowers, M. Salameh, E. A. Burgess, Y.-J. Lee, R. Onyenwoke, B. Zhao, B. Lupa, M. Hodges, J. Fiser, F. Sarmiento, L. Varghese, and B. Pugin; C. L. Hemme for sharing unpublished sequence data; E. Yokoyama and A. Torrens for their preliminary work on the sequencing and analysis of the universally conserved protein coding genes; the participants of the Kamchatka field expeditions, including the fellow graduate students, and P. A. Schroeder, F. T. Robb, and our Russian colleagues, especially E. Bonch-Osmolovskaya and T. Sokolova; and the University of Georgia, Athens, Microbiology Department. I am also grateful to my friends and family, including A. Wagner, L. Wagner, and M. Szarke. I am especially thankful to M. Heider for the support and encouragement she has given over the years. This research was funded the National Science Foundation (NSF-MCB0238607 – Kamchatka Microbial Observatory) as well as a UGA Dissertation Completion Award. v TABLE OF CONTENTS Page ACKNOWLEDGEMENTS .............................................................................................................v LIST OF TABLES ....................................................................................................................... viii LIST OF FIGURES .......................................................................................................................xv CHAPTER 1. DISSERTATION STRUCTURE...................................................................................1 2. DIVERSITY OF THERMOPHILIC ANAEROBES ....................................................4 3. CALDANAEROVIRGA ACETIGIGNENS GEN. NOV., SP. NOV., AN ANAEROBIC, XYLANOLYTIC, ALKALITHERMOPHILIC BACTERIUM ISOLATED FROM TREGO HOT SPRING, NEVADA, USA ...........................123 4. THERMOANAEROBACTER UZONENSIS SP. NOV., AN ANAEROBIC THERMOPHILIC BACTERIUM ISOLATED FROM A HOT SPRING WITHIN THE UZON CALDERA, KAMCHATKA, FAR EAST RUSSIA .......................145 5. VARIATIONS OF EIGHT UNIVERSALLY CONSERVED PROTEIN CODING GENES AMONG 123 THERMOANAEROBACTER UZONENSIS ISOLATES FROM GEOTHERMAL SPRINGS OF KAMCHATKA, RUSSIAN FAR EAST .....................................................................................................................171 6. THE POPULATION STRUCTURE AND SPATIAL DIVERSITY OF THERMOANAEROBACTER ISOLATES FROM GEOTHERMAL SPRINGS OF KAMCHATKA, RUSSIAN FAR EAST, BASED ON ANALYSES WITH EIGHT UNIVERSALLY CONSERVED PROTEIN CODING GENES ..........................222 vi 7. SPATIAL AND PHYSICOCHEMICAL CORRELATIONS TO THE GENETIC DIVERSITY OF THERMOANAEROBACTER UZONENSIS ISOLATES FROM GEOTHERMAL SPRINGS OF THE UZON CALDERA, GEYSER VALLEY, AND MUTNOVSKY REGIONS OF KAMCHATKA, RUSSIAN FAR EAST .....................................................................................................................301 8. DISSERTATION CONCLUSION ............................................................................324 APPENDIX A. THERMOSEDIMINIBACTER OCEANI GEN. NOV., SP. NOV. AND THERMOSEDIMINIBACTER LITORIPERUENSIS SP. NOV., NEW ANAEROBIC THERMOPHILIC BACTERIA ISOLATED FROM PERU MARGIN ..............................................................................................................327 B. CALDICOPROBACTER OSHIMAI GEN. NOV., SP. NOV., AN ANAEROBIC, XYLANOLYTIC, EXTREMELY THERMOPHILIC BACTERIUM ISOLATED FROM SHEEP FAECES, AND PROPOSAL OF CALDICOPROBACTERACEAE FAM. NOV. ...........................................................................................................353 vii LIST OF TABLES Page Table 2.1: Validly described thermophilic anaerobes ........................................................................88 Table 3.1: Phospholipid fatty acid contents (%) of strains JW/SA-NV4T, JW/IW-1228PTand JW/YJL-1230-7/2T. ....................................................................................................139 Table 3.2: Morphological and physiological characteristics of strain JW/SA-NV4T and close relatives .......................................................................................................................140 Table 4.1: Phospholipid fatty acid contents (%) of strains JW/IW010T and 1501/60. ................165 Table 4.2: Characteristics of JW/IW010T, strain 1501/60, and selected members of the genus Thermoanaerobacter. ..................................................................................................166 Table 5.1: Summary of Thermoanaerobacter uzonensis isolates universally conserved protein coding gene sequence heterogeneity ...........................................................................195 Table 5.2: Comparisons of the divergence between the Thermoanaerobacter uzonensis universally conserved protein coding gene sequence sets ...........................................196 Table 5.S1: Oligonucleotide primers for the amplification of universally conserved protein coding genes from Thermoanaerobacter taxa .............................................................202 Table 5.S2: Distribution of rplB gene sequence variants