UNIVERSITY of NEVADA, RENO Dimensions of Antarctic Microbial
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UNIVERSITY OF NEVADA, RENO Dimensions of Antarctic microbial life revealed through microscopic, cultivation-based, molecular phylogenetic and environmental genomic characterization A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry by Emanuele Kuhn Dr. Alison E. Murray/Dissertation Advisor May, 2014 Copyright by Emanuele Kuhn 2014 All Rights Reserved P age | i Abstract Extreme cold temperatures have shaped Antarctic environments and the life that lives within them. Microorganisms affiliated with the three domains of life - Bacteria, Archaea, and Eukaryote - can be found in Antarctic environments from deep subglacial lakes to dry deserts and from deep oceans to cold and dark winter surface seawaters. This dissertation focused on the investigation of the microbial assemblage in two Antarctic environments: Lake Vida, located in the McMurdo Dry Valleys, and the surface seawater from the Antarctic Peninsula. Lake Vida has a thick (27+ m) ice cover which seals a cryogenic brine reservoir within the lake ice below 16 m. This brine’s environment challenges the conditions for the existence of life. Despite the perceived challenges of aphotic, anoxic and freezing conditions, the brine contained an abundant assemblage (6.13 ± 1.65 × 10 7 cells mL -1) of ultra-small cells 0.192 ± 0.065 μm in diameter and a less abundant assemblage (1.47 ± 0.25 × 10 5 cells mL -1) of microbial cells ranging from > 0.2 to 1.5 μm in length. Scanning electron microscopy provided supporting evidence for cell membranes associated with the ~ 0.2 μm cells and helped discern a second smaller size class of particles (0.084 ± 0.063 μm). 16S rRNA clone library analyses indicated that the ultra-small cell-size assemblage was dominated by the Proteobacteria-affiliated genera Herbaspirillum, Pseudoalteromonas, and Marinobacter . Cultivation efforts of the 0.1 – 0.2 μm size fraction led to the isolation of Actinobacteria-affiliated genera Microbacterium and Kocuria . Based on phylogenetic relatedness and microscopic observations, we P age | ii hypothesize that the ultra-small cells in Lake Vida brine are ultramicrocells that are likely in a reduced size state as a result of environmental stress or life-cycle related conditions. The previously unexplored deeper ice of Lake Vida (from 18 to 27 m) revealed an ice column banded by sediment layers up to 15 cm in length and a diverse and cell-rich microbial assemblage with cell counts ranging from 3.73 × 10 4 to 8.58 × 10 6 cells mL -1. Illumina tag sequencing (iTag) targeting the 16S rRNA gene and RNA (as cDNA) indicated that the microbial assemblage from the lake ice and four sediment layers below 21 m was dominated by organisms capable of the reduction and oxidation of sulfur compounds in addition to high molecular weight complex polymer degradation. Proteobacteria, Bacteroidetes , Actinobacteria , and Firmicutes-affiliated genera were the most abundant bacterial phyla detected. The distribution of the microbial assemblage within the ice and sediment layers was correlated with the presence of sediment particles, 2- + total dissolved solids (TDS), total carbon, SO 4 , and Na concentration. Chemolithoautotrophic and heterotrophic genera such as Sulfurovum, Desulfocapsa, Lutibacter, and Desulforomonas dominated the ice segments and heterotrophic genera such as Cellulomonas and Conexibacter dominated the sediment layers. Sediment layer cDNA iTag sequences indicated that the taxa carrying the potential for metabolic capacity were mostly the Proteobacteria-affiliated genera Pseudoalteromonas and Vibrio . Representatives of these genera are often identified in Antarctic marine environments such as sea-ice, seawater, and marine sediment and adapted to low temperatures and high salinity. Therefore, the detection of abundant and considerably diverse microbial assemblages in Lake Vida brine, ice and sediment layers indicates that life is likely sustained in isolated deep, icy and dark anoxic environments. This also suggests that if similar conditions are found elsewhere beyond Earth, there is the possibility to find life. The second subject in this dissertation research was to conduct an in-depth assessment of the environmental genomics of the archaeal phylum Thaumarchaeota within Antarctic surface seawaters during the wintertime. Thaumarchaeota (earlier identified as Marine Group I.1a) composes 10-30% of the bacterioplankton during the P age | iii winter in Antarctic surface seawaters playing a noteworthy role in carbon fixation coupled to ammonia oxidation. By comparative genomic analyses, we found that the Antarctic Group I.1a genome fragments represent a unique low diversity Group I.1a cluster affiliated with a “ Ca. Nitrosopumilus” that has not yet been cultivated. Antarctic Group I.1a exhibited highly conserved genes in a rearranged genomic structure when compared to the genome of Nitrosopumilus maritimus SCM1indicating a high frequency of recombination (30% of the Antarctic Group I.1a open reading frames). Overall, our results provided insights into the genomic variability of a thaumarchaeal Antarctic assemblage indicating very low diversity that motivates the importance of acquiring the Antarctic strain in pure culture for further analysis of its physiological capacities and evolutionary history. P age | iv Acknowledgments C A P E S Brazil P age | v They are always around you To give you the support that you need So you can do the best of your best . P age | vi Content Chapter I Introduction: Life under subzero conditions and study sites …………………………. 1 1.1. Lake Vida, McMurdo Dry Valleys, Antarctica……………………………….. 4 1.2. Antarctic Peninsula coastal surface seawater and its Thaumarchaeota Group I.1a assemblage………………………………………………….……………. 7 1.3. Objectives……………………………………………………………………… 10 1.4. References……………………………………………………………………... 11 Chapter II The ultra -small microbial assemblage of Lake Vida encapsulated brine ……………. 14 Abstract……………………………………………………………………………........... 14 2.1. Introduction……………………………………………………………………. 15 2.2. Materials and methods………………………………………………………… 19 2.2.1. Brine sampling and cell size fractionation……………………………….. 19 2.2.2. Microscopy (Confocal, SEM, and STEM) and EDS analysis …………… 21 2.2.3. Grazing angle X-ray diffraction………………………………………….. 23 2.2.4. Cultivation of heterotrophic bacteria from brine fractions……………….. 23 2.2.5. Nucleic acid extractions, 16S rRNA gene and phylogenetic analyses …... 24 2.3. Results…………………………………………………………………………. 28 2.3.1. Microscopy and elemental analyses …………………………..…………. 28 2.3.2. Heterotrophic bacteria cultivation ……………………………………….. 36 2.3.3. Molecular analyses……………………………………………………….. 38 2.4. Discussion……………………………………………………………………... 43 2.4.1. Electron microscopy and elemental analyses…………………………….. 43 2.4.2. Heterotrophic bacteria cultivation………………………………………... 48 2.4.3. Molecular analyses……………………………………………………….. 50 2.4.4. Possible nature of the ultra-small cells…………………………………… 51 2.5. Summary and conclusions……………………………………………………... 54 2.6. Acknowledgments……………………………………………………………... 56 2.7. References……………………………………………………………………... 57 P age | vii Chapter III Microbial assemblage within deep ice and deep sediment layers from a frozen lake: Lake Vida, Antarctica …………………………………………………………………… 65 Abstract…………………………………………………………………………….. 65 3.1. Introduction……………………………………………………………………. 67 3.2. Materials and methods………………………………………………………… 70 3.2.1. Ice core sampling and processing………………………………………… 70 3.2.2. Geochemical analyses………………..…………………………………... 72 3.2.3. Cell enumeration and scanning electron microscopy…………………….. 73 3.2.4. DNA preparation ………………………………………………………… 74 3.2.5. RNA and cDNA preparation…………………………………………….. 75 3.2.6. Illumina tag sequencing and pre-processing of reads……………………. 77 3.2.7. Microbial assemblage structure and composition………………………... 79 3.2.8. Statistical analyses………………………………………………………... 81 3.3. Results and discussion……….………………………………………………… 82 3.3.1. Geochemical characterization of the ice and sediment layers..…………... 82 3.3.2. Microscopy observations…………………………………………………. 92 3.3.3. Phylotype-based and OTU-based microbial assemblage composition..…. 99 3.3.4. Microbial assemblage diversity and structure within the core…...………. 111 3.3.5. Relative abundance of rRNA (as cDNA) in the sediment layers…...…..... 117 3.4. Summary and conclusions……………………………………………………... 125 3.5. Acknowledgments …………………………………………………………….. 129 3.6. References……………………………………………………………………... 130 Chapter IV Characterization of Thaumarchaeota marine Group I.1a environmental genome fragments from Antarctic Peninsula coastal surface seawaters ……………………… 139 Abstract…………………………………………………………………………….. 139 4.1. Introduction……………………………………………………………………. 141 4.2. Materials and methods………………………………………………………… 144 4.2.1. Antarctic Group I.1a data set, assemblage, and recruitment …………….. 144 4.2.2. Gene content within Antarctic Group I.1a genome fragments and comparative genomic analyses.……………………………………..…… 145 4.2.3. Protein homology analysis …………………………………….………… 147 4.3. Results and discussion…………………………………………………………. 148 4.3.1. Recruitment of Antarctic Group I.1a genome fragments to SCM1 genome ………………………………………………………..………… 148 4.3.2. Gene content and comparative genomic analyses……………………….. 150 4.3.3. Protein homology analyses ………………………………………………. 160 4.3.4. Categorizing the Antarctic Group I.1a assemblage ……………………… 166 4.4. Acknowledgements……………………………………………………………. 174 4.5. References……………………………………………………………………... 175 4.6. Supplemental material………………………………………………………….