A Genetic Investigation of Archaeal Information-Processing Systems
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A genetic investigation of archaeal information-processing systems. Thesis Presented in partial fulfillment of the requirements for the degree master of science in the Graduate School of The Ohio State University Travis H. Hileman B.S. Graduate Program in Microbiology The Ohio State University 2013 Thesis Committee: Dr. Thomas Santangelo, Advisor Dr. Irina Artsimovitch Dr. Tina Henkin Dr. Michael Ibba Copyright by Travis H. Hileman 2013 Abstract Studies of Archaea and their biology have been hindered by the lack of defined genetic systems. Thermococcus kodakarensis has emerged as a model organism with a near complete suite of genetic tools that can be used to investigate basic biological mechanisms in Archaea. This thesis is centered on two aspects of archaeal information processing systems, namely transcription and DNA replication. Properly regulated gene expression is necessary for cellular homeostasis and response to external signals. Much regulation occurs at the level of transcription initiation, but post-initiation events can also dramatically alter gene expression. Transcription termination represents a regulatory event; however, the mechanisms employed to direct transcription termination in Archaea remain undefined. Intrinsic transcription termination occurs within poly-T tracts encoded on the non-template strand of DNA, but the mechanism by which termination occurs is unknown. Utilizing the genetic system unique to T. kodakarensis, two selective schemes were constructed, and continued efforts should permit isolation of RNA polymerase variants that have aberrant termination phenotypes. DNA replication is similarly subject to many regulatory inputs, and these inputs are received by different components of the replication apparatus. The protein encoded by TK0808, a protein of previously unknown function, was shown to stably interact with replisome components in vivo. To investigate the function of the protein encoded by TK0808, the gene was deleted from the chromosome. Deletion strains are viable, with no obvious growth defect; however, ii biochemical studies demonstrate that the TK0808 encoded protein can exert strong regulatory effects on replisome components in vitro. iii Dedication I dedicate this to my wife, who has supported me through this adventure with words of encouragement and the occasional kick in the pants. iv Acknowledgements I would like to thank Dr. Tom Santangelo for allowing me to be a part of his laboratory and training me to be a better scientist. I would also like to thank the members of the Santangelo lab for the meaningful discussions and at times arguments about life, the universe and everything. Especially Chandni Pawar, for all the help in the lab. I would also like to thank my committee members for their patience and support. v Vita June 2001……………………………………………………………Harrison High School April 2009……………………………………………………..Brigham Young University 2010 to present………………………………………………… The Ohio State University Publications Hileman TH and Santangelo TJ. 2012. Genetic techniques for Thermococcus kodakarensis. Front. Microbiol. 3:195. Erickson DL, Russell CW, Johnson KL, Hileman T, Stewart RM. 2011. PhoP and OxyR transcriptional regulators contribute to Yersinia pestis virulence and survival within Galleria mellonella. Microb Pathog. 51(6):389-95 Fields of Study Major Field: Microbiology vi Table of Contents Abstract…………………………………………………………………………………....ii Dedication...………………………………………………………………………………iv Acknowledgements………………………………………………………………………..v Vita………………………………………………………………………………………..vi Publications……………………………………………………………………….vi Fields of Study……………………………………………………………………vi Table of Contents………………………………………………………………………...vii List of Figures…………………………………………………………………………...viii List of Tables……………………………………………………………………………..ix Introduction………………………………………………………………………………..1 Materials and Methods………………………………………………………...…………20 Results ……………….…………………………………………………………………..26 Perspectives……...……………………………………………………………………….33 References……………………………………………………………………………..…35 vii List of Figures Figure 1. Genomic modification using a single selectable marker………………………..4 Figure 2: Markerless deletion using selectable and counter-selectable markers………….4 Figure 3: RNA polymerases from three Domains and preinitiation complexes…………..7 Figure 4: Schematic of pTS522 containing rpoB (TK1083)…………………………….12 Figure 5: Selection for hyposensitive RNAP variants using TK0149…………………...14 Figure 6: Selection for hypersensitive RNAP variants using TK0664….……………….14 Figure 7: Quantitative assessment of RNAP variant termination propensity……………15 Figure 8: Archaeal replisome…………………………………………………………….16 Figure 9: Interaction network of tagged replisome proteins in T. kodakarensis………...17 Figure 10: Crystal structure of PCNA1 (TK0535)……………….……………………...19 Figure 11: Diagram of PhmtB driven TK0149……………………………………………27 Figure 12: Diagram of PhmtB driven TK0664……………………………………………29 Figure 13: Genomic organization, confirmation, and growth of strains lacking TK0808…………………………………………………………………………………..32 viii List of Tables Table 1: Selectable markers available for use in T. kodakarensis………………………...3 Table 2: Primers used in the construction of plasmids……………………………..……25 Table 3: TK0149 plasmid designations and terminator variant sequences….…………...28 Table 4: TK0664 plasmid designations and terminator variant sequences ……….……..29 Table 5: RNAP subunit containing plasmid designations....…………………………….30 ix Introduction Archaeal Genetics Archaea are typically envisioned as organisms that thrive in extreme environments, and while true for many well-studied clades, Archaea are in fact nearly ubiquitous in mesophilic marine and terrestrial environments (Jarrel 2011). The dominance of the archaea in many more unusual environments argues that these microbes contain biochemical and biophysical adaptations permitting survival in these environments, and the underlying basis for these properties is of significant interest for biotechnology platforms where high temperatures and high pressures are often employed (Bae 2009; Blumer-Schuette 2008; Cho 2007; De Stefano 2008; Fujiwara 1998; Gaidamaviciute 2010; Griffiths 2007; Hashimoto 2001; Hotta 2002; Imanaka 2001, 2002; Izumi 2001; Kelly 2009). Studies using many of the extremophilic Archaea were, and remain, hampered by the complexities of accurately reproducing their natural environments in the laboratory. Once growing, more delays have emerged due to the lack of genetic techniques permitting molecular analyses of archaeal pathways and physiology (Atomi 2012). Recently developed genetic techniques permitting rapid and complex strain construction have allowed the hyperthermophilic, heterotrophic, marine archaeon Thermococcus kodakarensis to come to the forefront in the investigation of 1 archaeal biology and biochemistry (Sato 2003, 2005; Matsumi 2007; Santangelo 2008B, 2010A, 2010A, 2010C; Takemasa 2011). T. kodakarensis provides a unique opportunity to investigate the totality of archaeal physiology, employing genetic techniques similar to those often used with model organisms for Bacteria (e.g.; Escherichia coli and Bacillus subtilis) and Eukarya (e.g.; Saccharomyces cerevisiae, Caenorhabditis elegans, and Drosophila melanogaster). T. kodakarensis can be transformed with both circular and linear DNA molecules (Sato 2003) and readily incorporates donor DNA into a single circular chromosome (2.08 Mbp) (Fukui 2005; Morikawa 1994). Gene expression cassettes, termed genetic markers (Table 1), are available for strain construction, each allowing co-integration of the marker into the genome with another targeted genome modification. A selectable marker(s) is typically flanked by sequences with homology to the desired integration locus, and homologous recombination results in chromosomal modifications (Figure 1). Gene deletions, gene additions, promoter exchanges, introduction of sequences encoding epitope- or affinity-tags and other modifications are possible, as are combinations thereof. Counter-selections have also been developed permitting removal of a marker(s) through a recombination event, thereby allowing unlimited repetitive modifications to a single genome (Sato 2005; Santangelo 2010A) (Figure 2). 2 Selectable Gene(s) Gene Function Strain Advantages Limitations/ Reference(s) Marker (required Disadvantanges genotype) Uracil TK2276 orotidine-5'- KU216 Easily paired with Uracil Sato 2003, phosphate (ΔpyrF) 5-Fluoroorotic contamination 2005 decarboxylase KUW1 acid based yields high (ΔpyrF, counter-selection backgrounds; ΔtrpE) for markerless limited to modifications minimal media; limited host range Tryptophan TK0254 Large subunit of KW128 Rigid selection Limited to Sato 2005 anthranilate (ΔpyrF; requiring no minimal media; synthase ΔtrpE::pyrF) media additions limited host range Arginine/ PF0207 Argininosuccinate Any strain No strain Limited to Santangelo Citrulline synthase restrictions minimal media; 2010C PF0208 Argininosuccinate requires lyase supplementation with citrulline Agmatine TK0149 Pyruvoyl- TS559 Provides selective Limited host Santangelo dependent (ΔpyrF; pressure in rich range 2010C arginine ΔtrpE::pyrF, media decarboxylase ΔTK0664, ΔTK0149) Simvastatin/ PF1848 3-hydroxy-3- Any strain Provides selective Spontaneous Matsumi Mevinolin methylglutaryl pressure in rich Sim/Mev 2007 coenzyme A media; no strain resistance Santangelo reductase restrictions provides a high 2008 background 6-methyl TK0664 Hypoxanthine TS517 Provides counter- Provides no Santangelo purine guanine (ΔpyrF; selective pressure positive selection;