Metagenomic/Metatranscriptomic Study of Organisms Entrapped
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METAGENOMIC/METATRANSCRIPTOMIC STUDY OF ORGANISMS ENTRAPPED IN ICE AT FOUR LOCATIONS IN ANTARCTICA Sammy O. Juma A Thesis Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of Master of Science August, 2013 Committee: Dr. Scott O. Rogers, Advisor Dr. Paul Morris Dr. Vipaporn Phuntumart © 2013 Sammy Juma All Rights Reserved iii ABSTRACT Dr. Scott O. Rogers, Advisor Antarctica has one of the most extreme environments on Earth. The biodiversity and the species richness on the continent are low and decrease with increases in elevation and distance from the coastal regions. Previous scientific research in Antarctica has been used to understand the past climatic conditions, survival mechanisms used by the microbial communities and various environmental factors that contribute the dispersal of microorganisms. The research presented here is a comparison of microbial inclusions in ice at four locations in Antarctica (Byrd, Taylor Dome, Vostok and J-9) to identify the factors that influence the microbial distribution patterns and to investigate survival of the micobes under harsh conditions. Culture- dependent and culture independent techniques (e.g., metagenomics and metatranscriptomics) were used to analyze sequences present in ice cores from Antarctica. The sequences analyzed matched those from Spirochaetes, Verrucomicrobia, Bacteroideters, Cyanobacteria, Deinococcus-Thermus, Proteobacteria, Firmicutes, Actinobacteria, Euryarchaeota and Ascomycota. Analysis of the metagenomic/metatranscriptomic sequences was also carried out to characterize the various pathways represented in the diverse communities. Analysis of the data revealed that the numbers of unique sequences obtained from the samples were few (Taylor Dome (51), Byrd (43), Vostok (33) and J-9 (40). The number of unique sequences was lowest from the sample obtained at the most elevated location in the interior of Antarctica (Vostok (33)) and highest from the sample that was closest to the Antarctic coast (Taylor Dome (51)). The samples from all the four locations appeared to harbor very few species of microorganisms (Taylor Dome (12), Byrd (13), Vostok (6), and J-9 (7)). Analysis of the microbial pathways revealed that the microorganisms are able to utilize various sources of carbon, recycle nitrogen iv and had unique enzymes and cell structures that have previously been reported to be important for microbial survival at very low temperatures. v TABLE OF CONTENTS CHAPTER 1: INTRODUCTORY REVIEW………...………………………………...…………1 1.1 ANTARCTICA: A COLD DESERT…………………………………………………........1 1.2 DISTRIBUTION OF MICROBES IN COLD ECOSYSTEMS………...…….…………..2 1.3 INVASION OF MICROBES IN TO ANTARCTICA...……………..…………………...3 1.3.1 Storm system and circumpolar currents……………………………………..................3 1.3.2 Wind transport………….………………………………………………………………4 1.3.3 Migratory birds……….………………………………………………………………...5 1.4 METHODS OF CHARACTERIZING MICROBIAL DISTRIBUTION……….………...5 1.4.1 Culture-dependent methods…………………………………………………………….5 1.4.2 Culture-independent method…………………………………………………………...6 1.4.2.1 454 pyrosequencing…………………………………………………………….......7 1.4.2.2 rRNA (ribosomal RNA)……………………………………………………………8 1.5 ANALYSIS OF METABOLIC FUNCTIONS……..………………...…………………...9 1.6 HYPOTHESIS AND GOALS OF THIS PROJECT……….……...…..………………...10 1.7 REFERENCES…………………………………………………………...……………...12 CHAPTER 2: COMPARISON STUDY OF THE SEQUENCE DISTRIBUTION AND METABOLIC PATHWAYS PRESENT IN FOUR LOCATIONS OF ANTARCTICA……….17 2.1 ABSTRACT………………….……………………………………………...…………...17 2.2 INTRODUCTION…………….…………………………………………...…………….19 vi 2.3 METHODOLOGY………………...……………………………………………………..23 2.3.1 Ice core section handling and culturing…...…………………………………………..23 2.3.2 DNA extraction of the pure cultures…………………………………………………..24 2.3.2.1 PCR (polymerase chain reaction)…………………………………………………25 2.3.2.2 PCR product purification……………………………………………….…………26 2.3.3 Ultracentrifugation…………………………………………………………………….26 2.3.4 DNA extraction………………………………………………………………………..27 2.3.5 RNA extraction.……………………………………………………………………….27 2.3.6 cDNA synthesis….……………………………………………………………………28 2.3.6.1 First strand synthesis…………………………………………………………...…28 2.3.6.2 Second strand synthesis………………………………………………….………..29 2.3.7 EcoRI (NotI) adapter addition………………………………………………………...30 2.3.8 Column chromatography……….……………………………………………………..30 2.3.9 Amplification and quantitation………………………………………………………..31 2.3.10 Addition of 454 adapters…………………………………………………………….32 2.3.11 Sequencing…………………………………………………………………………...33 2.3.12 Analysis of the sequences….………………………………………………………...35 2.4 RESULTS………………………………………………………………………...…….37 2.4.1 Sequences obtained……….…………………………………………………………...37 vii 2.4.2 Community structure………………………………………………………………….40 2.4.2.1 Taxonomic groups represented…………………………………………………...40 2.4.2.2 Phylogenetic analysis……………………………………………………………..42 2.4.3 Statistical analysis……………………………………………………………………..46 2.4.4 Metabolic analysis…………………………………………………………………….46 2.5 DISCUSSION…………………………………………………………………...……...51 2.5.1 Factors responsible for sequence distribution in Antarctica…………………………..52 2.5.2 Metabolic analysis…………………………………………………………………….59 2.5.2.1 Metabolism in cold habitats…………………………….…………………….......59 2.5.3 Adaptation to cold habitats……………………………………………………………62 2.5.4 Important Enzymes in cold habitats…………………………………………………..63 2.6 CONCLUSION……………………………………………………………………..…...64 2.7 REFERENCES…………………………………………………………………...……...66 viii APPENDICES………..………………………………………………………………………….73 A. Numbers of sequences matching those for genes in specific pathways that were recovered from the Taylor Dome, Bryd, Vostok and J-9 samples………………………………….73 B. Description of the sequences found in the Taylor Dome, Bryd, Vostok and J-9 ice core section samples…………………………………………………………………………..76 C. Enzyme gene sequences identified from the Byrd, Vostok, J-9 and Taylor Dome samples…………………………………………………………………………………...86 D. Statistical analysis of factors influencing sequence distribution in Antarctica…………..96 ix LIST OF TABLES Table 1: Ice core information for the core sections used in the research…………..……............11 Table 2: MID primer sequences used for each sample. The primers have the form A-key-MID- EcoRI and B-key-MID-EcoRI…………………………………………….…………….……….34 Table 3: Taxonomic affiliations of bacteria at Taylor Dome, Byrd, Vostok and J-9 determined by phylogenetic analysis of LSU and SSU rRNA……………….………………………………… 38 Table 4: Taxonomic affiliations of bacteria sequences amplified in the metagenomic/metatranscriptomic analysis at Taylor Dome, Byrd, Vostok and J-9 determined by phylogenetic analysis of LSU and SSU rRNA…………………………………………………..39 Table 5: Percentage representation of the major metabolic pathways from the four locations based on the number of enzyme sequences found from the metagenomic/metatranscriptomic analysis.......................................................................................................................................... 50 Table 6: Comparison of the sampled locations in terms of site elevation, distance from the coast, number of unique sequences recovered and number of species identified using the ribosomal subunits………………………………………………………………………….……………… 54 x LIST OF FIGURES Figure 1: Locations in Antarctica from which the ice cores were obtained. Vostok and Taylor Dome are in East Antarctica, while Byrd and J-9 are in West Antarctica. In general, East Antarctica is higher in elevation than West Antarctica…………………………….……………22 Figure 2: Bar chart of the microbial composition at the phylum level from the four sampled locations in Antarctica. Percentage distribution was calculated on the basis of the total number of sequences obtained from each sampled location…………………………………………...……41 Figure 3: Phylogenetic analysis of LSU rRNA gene sequences isolated from Antarctica by metagenomic/metatranscriptomic analysis……………………………….………………….......44 Figure 4: Neighbor joining tree of SSU rRNA gene sequences isolated from Antarctica by the culture and metagenomic/metatranscriptomic analysis methods………………………...………45 Figure 5: Bar chart of the major metabolic pathways representation at the four sampled locations in Antarctica………………………………………………………………………………….......49 Figure 6: Prevailing winds in Antarctica during winter…………………………………………56 Figure 7: Distribution of the number of sequences obtained from the sampled locations versus the distance from Antarctic coast and elevation of the sampled locations …………….......……58 1 CHAPTER 1: INTRODUCTORY REVIEW 1.1 ANTARCTICA: A COLD DESERT Antarctica is the fifth largest continent, with a land-mass of more than 14,000,000 km2 (5,400,000 sq mi). It is the southernmost continent, and is surrounded by the Southern Ocean, which causes only cold ocean currents to reach the continent. Unlike other continents, 98% of Antarctica is covered by ice that averages 1.6 kilometers in thickness. Antarctica is considered the coldest, driest, windiest desert on Earth, with annual precipitation of only 200 mm (8 inches) along the coast. The inland temperatures that can reach to as low as -89°C (11°C colder than subliming ice) in winter and as high as 15°C near to the coast in the summer (Bratchkova and Ivanova 2011). It is much colder than the Arctic because parts of East Antarctica are up to 3 km above sea level (Bratchkova and Ivanova 2011). Because of its elevation, East Antarctica is much colder than West Antarctica. The frozen environment in Antarctica had once been considered to be devoid of life (Cowan and Ah Tow 2004), but in 1908 Tsiklinsky published