A Genetic Investigation of Archaeal Information-Processing Systems

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

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.

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.

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

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

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).

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; 2010A phosphoribosyl- ΔtrpE::pyrF, counter-selection transferase ΔTK0664) requires minimal media Table 1: Selectable markers available for use in T. kodakarensis; adapted from (Hileman 2012).

Figure 1. Genomic modification using a single selectable marker. The expression of the selectable marker (grey arrow) is driven by a strong or constitutive promoter (bent arrow) to provide positive selection for recombination into the genome. This scheme illustrates a gene deletion with recombination events (dashed lines) occurring between homologous DNA flanking the selectable marker and target gene. This methodology can be used to introduce allelic variants, promoter alterations, and epitope tags.

Figure 2: Markerless deletion using selectable and counter-selectable markers. Hypothetical recipient, intermediate, and final genomes are shown at the target locus; donor DNA is shown above. Intermediate strain construction results from homologous recombination (dashed lines) between regions of homology on the donor and the recipient DNA (green and blue arrows). Final strains are generated by removing the selectable and counter-selectable markers through homologous recombination between direct repeats flanking the markers.

T. kodakarensis can also maintain replicative plasmids, although it does not harbor any plasmids naturally (Santangelo 2008B). A related species, Thermococcus nautilus, contains three distinct plasmids (Soler 2007), and the smallest of these, pTN1, was converted into an E. coli-T. kodakarensis shuttle vector (Santangelo 2008B).

Variants of this shuttle vector have been generated carrying combinations of selective markers (Santangelo 2010A). These shuttle vectors can be used to ectopically express a gene(s), and the chromosomal copy of the same gene(s) can then be modified or deleted

(Raina 2012; Santangelo 2008B, 2010B; Takemasa 2011). Viability of the transformed strain may then be dependent on retention of the plasmid. Replicative expression vectors also permit expression of archaeal genes in an archaeal host, eliminating a range of concerns associated with the more common technique of recombinant expression of archaeal proteins in bacterial hosts (Santangelo 2008B, 2010; Takemasa 2011). The

ColE1 origin permits replication in E. coli, simplifying plasmid construction and variant construction, as conventional molecular cloning procedures are available (Santangelo

2008). Finally, collections of plasmids, for example a plasmid library containing randomly mutagenized variants of a gene, can be quickly produced in E. coli, and the efficiency of T. kodakarensis transformation (Sato 2003) supports introduction of this library for use in screens and selections investigating archaeal physiology.

These advances in T. kodakarensis genetics allow the direct characterization of archaeal enzymes, provide a means to study and engineer biofuel production, give access to natural products encoded by Archaea, and permit opportunities to study information processing systems specific to the third Domain of life (Atomi 2001, 2004, 2005, 2011;

Bae 2012; Borges 2010; Chou 2008; Danno 2008; Davidova 2012; Dev 2009; Fujikane

2010; Fukuda 2004, 2008; Hirata 2008; Imanaka 2006; Ishino 2011; Kanai 2005, 2007,

2010, 2011; Kim 2006; Kim 2010; Kobori 2010; Li 2011; Littlechild 2011; Louvel 2009;

Matsubara 2011; Morimoto 2010; Nunoura 2011; Orita 2006; Pan 2011; Rashid 2004;

Santangelo 2006, 2007, 2008A, 2009, 2010B, 2011A, 2011B; Sato 2004, 2007, 2011;

Shiraki 2003; Yamaji 2009; Yokooji 2009). This last opportunity, genetic information processing, is the focus of this thesis. Two different lines of study are described, one generating a scheme to investigate aspects of transcription regulation and transcription termination, and the second one constituting a genetic investigation of a novel component and regulatory factor of the Thermococcales replisome. Each study is prefaced with a more detailed introduction in the following sections.

Transcription and Transcription Termination

Transcription in all domains of life is catalyzed by a multi-subunit RNA polymerase (RNAP) (Werner 2011). While the exact subunit composition varies between the domains, significant subunit sequence and structural conservation, as well as a conserved core structure for the complete enzyme, supports a common ancestry for all extant multi-subunit RNAPs. RNA synthesis and chain elongation are catalyzed by the same reaction in all domains (Steitz 1993), but the mechanisms of initiation and termination differ between RNAPs from each domain (Hirata 2009; Santangelo 2009); each eukaryotic RNAP also relies on a different set of factors for initiation and termination (Kuehner 2011; Németh 2013; Orioli 2012; Vannini 2012). Archaea and

Bacteria share many gross morphological traits, both rely on a single RNAP for all 6 transcription, and both have evidence for abundant coupling of transcription and translation (French 2007; Miller 1970). However, whereas bacterial transcription initiation is dependent on the initiation factor sigma (Mooney 2005), archaeal RNAPs are directed to promoter sequences by transcription factors that are conserved in Eukarya and notably absent in Bacteria. Transcription initiation in Archaea is perhaps best described as a simplified eukaryotic-like system requiring only two basal factors, the TATA box binding protein (TBP) and Transcription Factor B (TFB), a TFIIB homolog (Grohmann

2011). Together with RNAP and a TATA-containing DNA template, these factors are sufficient for accurate promoter-directed transcription initiation in vitro (Figure 3).

Figure 3: RNA polymerases from three Domains and preinitiation complexes. A. Crystal structures of RNAPs from (left to right) Thermus aquaticus, Sulfolobus solfataricus, and Saccharomyces cerevisiae. Each RNAP subunit is labeled and colored, with orthologous subunits between Domains colored identically. B. Preinitiation complexes from (left to right) Bacteria, Archaea, and Eukarya. Orange and blue boxes designate -35 and -10 sites (Bacteria) and B-recognition element and TATA-box sequences (Archaea and Eukarya) respectively. Grey line designates DNA. Dashed line labeled CTD denotes the C- terminal domain of eukaryotic RNAP II. Adapted from (Hirata 2009). 7

Following transcription initiation and promoter clearance, the archaeal transcription initiation factors TBP and TFB are released from the template (Xie 2004) and transcription elongation can continue uninterrupted for thousands of base pairs.

Processive RNA synthesis is necessary to complete transcription of long genes and operons, and thus the transcription elongation complex represents a remarkably long- lived, stable complex (Levin 1987) that receives many regulatory inputs affecting its movement and activity, including signals to release the nascent transcript and template

DNA when necessary (Roberts 2008; Santangelo 2011; Schneider 2012; Uptain 1997;

Werner 2012). The regulation imposed via transcription elongation-termination decisions in Archaea is poorly understood, as are the mechanisms employed to disrupt the normally very stable transcription elongation complex.

Initial assumptions that archaeal transcription termination would largely mimic bacterial intrinsic termination mechanisms perhaps limited progress in the field (Muller

1985). In addition, no known Rho homologs have been identified in Archaea

(Santangelo 2006) and thus far only an intrinsic termination mechanism has been identified, although factor-dependent termination is likely (Santangelo 2009). An intrinsic termination mechanism was first proposed in the organism Methanococcus voltae to terminate transcription downstream of the Methyl CoM reductase alpha subunit encoding sequence (Muller et al. 1985). The proposed termination sequence contains an inverted repeat followed by a poly-T tract that conforms to the conventions of Rho- independent intrinsic terminators in Bacteria (d'Aubenton Carafa 1990). The poly-T tract

(TTTTAATTTT) of the tRNAVal gene from Methanococcus vannielii was shown to 8 promote transcription termination in vitro (Thomm 1994). Transcription termination was further investigated in vitro using components purified from Methanothermobacter thermoautotrophicus (Santangelo 2006). These results showed that while an inverted repeat may exist in the sequence, it is not required to terminate transcription and that the composition and length of the poly-T tract affected the efficiency of termination. Until the genetic tools for T. kodakarensis were developed, all assays used to specifically investigate mechanistic properties of transcription termination in Archaea were performed in vitro. The in vivo studies conducted in T. kodakarensis involved replacing a native promoter with a constitutive non-endogenous promoter, introducing a terminator variant upstream of the reporter gene, and comparing levels of β-glycosidase activity of a strain with no terminator to those with potential intrinsic termination sequences

(Santangelo 2009). This assay found that reporter activity was reduced up to 97% when select sequences were incorporated between the promoter and reporter construct.

The compact, gene-dense nature of archaeal chromosomes suggests that in order to avoid unregulated transcription of neighboring genes and operons, transcription complexes should be efficiently disrupted quite near the end of genes or operons. Recent in vitro and in vivo studies revealed at least two mechanisms employed in T. kodakarensis to terminate transcription, although neither mechanism has been described in molecular detail (Santangelo, 2008A, 2009).

The first described mechanism of transcription termination in the archaeal system is termed intrinsic termination, as no auxiliary factors are necessary for RNAP to release the nascent transcript. Intrinsic termination is a mechanism employed by Bacteria to 9 regulate transcription (Santangelo 2011). Despite the common name, the mechanisms underlying intrinsic termination in Archaea and Bacteria are quite distinct, with the most notable differences being: I) the necessity for an RNA structural element, an RNA hairpin, for efficient termination in Bacteria (Yarnell 1999), whereas no such structural element is required to disrupt the archaeal elongation complex (Santangelo 2006); and II) the very limited range of termination positions demonstrated for bacterial transcription at set distances from RNA structural elements (Yarnell 1999), which are in contrast to the range of adjacent termination positions observed with archaeal components (Santangelo

2009). Intrinsic termination mechanisms are also found in Eukarya; however, this mechanism of termination is only well defined for RNA polymerase III (Pol III)

(Arimbasseri 2013; Cozzarelli 1983; Hamada 2000; Rijal 2013). For both eukaryotic Pol

III and archaeal RNAPs, a poly-T tract in the non-template strand of DNA is sufficient to terminate transcription in vitro (Huang 2005; Santangelo 2009), and when such a sequence was placed between the promoter and coding region of a reporter gene in vivo, reporter gene expression was essentially eliminated (Santangelo 2009). These results clearly demonstrate a requirement for a poly-T tract for intrinsic termination, but the energetics and mechanism of transcription termination remain under study.

Archaea employ a second mechanism of regulating transcription elongation- termination decisions. Evidence for polarity in vivo, and recent biochemical evidence supporting factor-dependent transcription termination in vitro, both support a governance mechanism regulating transcription in archaeal cells (Santangelo 2008A, 2009). The interdependence, or lack thereof, of the two transcription termination systems is

10 unknown, and the details surrounding the factor(s) and requirements for factor-dependent termination are not the focus of this document. However, better understanding of this mechanism of termination is likely to elucidate shared activities that may regulate eukaryotic Pol II transcription.

In the materials and methods section, I describe construction of plasmids and T. kodakarensis strains that can be employed to isolate RNAP variants with altered termination properties. The identification and characterization of such mutants should shed light on the mechanisms of intrinsic termination in Archaea, and given the structural and biochemical similarities of all multi-subunit RNAPs, this information will also likely translate into universal mechanisms underlying the stability of the transcription elongation complex.

Expression vectors containing RNAP subunits

Genetic strategies have allowed isolation of variant RNAPs with distinctive phenotypes from generated libraries of plasmids encoding RNAP subunits in Bacteria

(Jin 1988; Santangelo 2003; Tavormina 1996; Weilbaecher 1994). Similar strategies have yet to be applied to Archaea, but by using the genetic tools that are now available for T. kodakarensis, this is now a possibility, and is the central aim of this section. Every

T. kodakarensis RNAP subunit has been cloned into a replicative expression vector, permitting construction of libraries of variants that can be screened and selected under different conditions in vivo (Figure 4). The encoded RNAP subunits include an additional sequence encoding a hexa-histidine epitope tag to facilitate purification and

11 follow-up comprehensive in vitro analyses. These plasmids can be used to express wild- type RNAP subunits, variants with specific modifications, or randomly mutagenized

RNAP subunits in vivo (Santangelo 2008B, 2010B). Compatible selections (below) should permit identification of variants of interest.

Figure 4: Schematic of pTS522 containing rpoB (TK1083). Expression of rpoB is driven by the non- endogenous promoter from M. thermoautotrophicus histone B (PhmtB). trpE and HMG-CoA reductase (MevR) are used to maintain the plasmid in the cell. Adapted from (Santangelo 2010B).

Selections for hyper- and hypo-termination responsive RNAP variants.

Transcription studies in Archaea have been predominately conducted in vitro, largely due to a dearth of genetic accessibility for in vivo studies. In vitro analysis of archaeal transcription termination has yielded important information regarding the DNA sequence requirements for intrinsic termination, but it remains unclear how these sequences signal termination and further, how RNAP normally maintains a stable elongation complex for extended time periods (Muller 1985; Santangelo 2006; Spitalny

2008; Thomm 1994). The role(s) of different archaeal RNAP structural components in stabilizing the transcription elongation complex, and which of these elements may be

12 disrupted to drive a termination event, are unclear from current structural models of the elongation complex from any domain.

Recent work in T. kodakarensis has shown that insertion of a poly-T tract terminator sequence can drastically lower reporter gene expression in vivo (Santangelo

2009). The same scheme can be used to create a selection system that will select for

RNAP mutants that result in termination phenotypes that are hypo- or hypersensitive to termination sequences in vivo. Plasmid-encoded RNAP subunit variants will be transformed into T. kodakarensis and growth of cells on selective media will rely on the mutations within RNAP to support survival by terminating at a weak terminator sequence or resisting termination at a strong terminator. These systems will allow for in vivo selections to identify select variant RNAPs with mutations that alter response to intrinsic terminators from a very large pool of variants.

Hyposensitive RNAP termination mutations will be selected using a strong terminator that prevents wild-type RNAP from transcribing the selection marker, TK0149

(Table 1). TK0149 will be put under control of PhmtB with a terminator sequence located

+18 from the transcription start site. The terminator used in this selection will cause the vast majority of wild-type RNAPs to terminate without transcribing TK0149. Survival will be conferred through a plasmid-encoded termination-resistant RNAP subunit (Figure

5). Plasmids will be isolated from resultant colonies and the desired phenotype confirmed to correspond with expression of the plasmid encoded subunit.

Figure 5: Selection using TK0149. Strains will be constructed with PhmtB controlling TK0149 expression. The terminator (red octagon) introduced upstream of TK0149 will inhibit wild-type RNAP transcription into TK0149, reducing protein synthesis, limiting agmatine production, in turn preventing growth. Mutant RNAPs that confer a hyposensitive termination phenotype will read through the terminator allowing transcription of TK0149 and synthesis of agmatine allowing cellular growth.

Hypersensitive RNAP termination mutations will be selected using a terminator variant that allows wild-type RNAP to read through and express TK0664. Expression of

TK0664 confers 6-methypurine sensitivity (6-MP) (Table 1). Like the hyposensitive selection, TK0664 expression will be driven by PhmtB with a terminator at +18 from the transcription start site. Selection on media containing 6-MP will require the plasmid encoded RNAP subunit to confer a hypersensitive termination phenotype to the RNAP that causes transcription to terminate at weaker termination signals (Figure 6).

Figure 6: Selection using TK0664. Strains will be constructed with PhmtB controlling TK0664 expression. The introduced terminator (red octagon) will not prevent wild-type RNAP from transcribing TK0664, and thus production of protein conferring sensitivity to 6-MP preventing cellular growth on media containing 6- MP. Mutant RNAPs that confer a hypersensitive termination phenotype will terminate transcription at the terminator, preventing protein synthesis and thus promote survival in the presence 6-MP. 14

Phenotypes conferred by plasmid encoded RNAP subunits will be confirmed with the same promoter and terminator constructs used to select these mutants however, the promoter-proximal region will instead be coupled with the reporter gene TK1761.

TK1761 encodes a β-glycosidase that was used in the original in vivo termination assay

(Santangelo 2009). Reporter activity will be measured to compare plasmids with putative termination mutations to those of a plasmid expressing the wild-type subunit (Figure 7).

Confirmation of RNAP subunit encoded phenotypes in vivo will progress to sequencing the plasmid-encoded genes and further in vitro characterization of purified RNAPs containing the mutant subunits.

Figure 7: RNAP phenotypic screens. A. The screen for hyposensitive RNAP mutations will utilize the same terminator used in the hyposensitive selection. The strong terminator (red octagon) will normally prevent wild-type RNAP from transcribing the reporter gene TK1761. Hyposensitive termination variant RNAPs will increase reporter activity compared to wild-type RNAP. B. The screen for hypersensitive RNAP mutations will utilize the same terminator used in the hypersensitive selection. The weak terminator will normally allow wild-type RNAP to express the reporter gene TK1761. Hypersensitive RNAP mutants will terminate transcription before the reporter gene, decreasing reporter activity.

DNA Replication and TK0808 DNA replication is carried out by a complex of proteins termed the replisome

(Figure 8) (Li 2013). As is true for many aspects of the archaeal transcription apparatus, the archaeal replisome exhibits characteristics more in line with the eukaryotic replisome than the bacterial replisome (Li 2013). Homology searches and in-silico approaches have identified and assigned putative functions to many proposed archaeal replisome components, but much of this work lacks biochemical evidence (Li 2010). Importantly, homology searches cannot identify archaeal-specific replisome components, and it is important to identify all components - structural, enzymatic, and regulatory - to better understand, and ultimately manipulate, the archaeal DNA replication machinery. Much interest stems from the biotechnological use of thermostable enzymes for DNA amplification reactions, and future technologies dependent on amplification of very large fragments of DNA are predicted to employ entire replisomes rather than individual thermostable DNAPs that lack processivity and show lower fidelity when used in isolation.

Figure 8: Archaeal replisome. Cartoon model of the archaeal replisome. Components are indicated with names and gene designations. All proteins (except TK1902) were tagged and interacting partners identified. Modified from (Li 2010). 16

Using the genetic system that has been developed for T. kodakarensis, nineteen genes encoding known or predicted members of the replisome were modified (one gene was modified in each of nineteen near isogenic strains) with a sequence encoding a hexa- histidine tag. Growth of the resultant 19 strains, and gentle purification of the tagged protein in complex with natively assembled proteins, was used to isolate complexes that were de-convoluted via mass spectrometry to identify a protein interaction network of T. kodakarensis replisome components (Li 2010) (Figure 9).

Figure 9: Interaction network of tagged replisome proteins in T. kodakarensis. Colored ovals designate genes whose sequences were modified to encode proteins that contain affinity tags, while white ovals indicate proteins, identified by the gene number, that were co-isolated. The red box highlights the TK0808-TK0535 interaction. Modified from (Li 2010).

Proliferating cell nuclear antigen (PCNA) is a homotrimer that forms a ring- shaped structure (Figure 10) that encircles double-stranded DNA and is used as an anchor

17 for other members of the replisome (Ladner 2011). These interactions generally stabilize replisome components and increase processivity, but PCNA proteins also can modulate activities of proteins in the replisome via indirect mechanisms (Li 2010). PCNA proteins cannot self-assemble around DNA, and PCNA is loaded onto duplex DNA by replication factor C (RFC), a heteropentamer consisting of four RFC –S proteins and a single RFC-L subunit (Pan 2013). When TK0535, encoding one of two PCNA proteins in T. koakarensis, was modified to encode a protein containing a hexa-histidine tag, several predicted replisome components, i.e., RFC-S and RFC-L, were identified in co-purifying complexes, as expected. However, a small protein of unknown function, encoded by

TK0808, was also found to be associated with PCNA1 (Figure 9, red box). The abundance of the protein encoded by TK0808 in the PCNA1 complexes, combined with the small and unique nature of the predicted protein, stimulated follow-up studies on this apparently archaeal-specific replisome component.

Figure 10: Crystal structure of PCNA1 (TK0535). PCNA1 forms a ring as a homotrimer. The three proteins that make up the homotrimer are labeled A, B, and C. Modified from (Ladner 2011).

In the results and discussion section, I present a genetic profile of the role of

TK0808 in T. kodakarensis that complements biochemical studies from our collaborators that detail the biochemical and biophysical parameters of TK0808 in vitro. Proteins shown to interact with PCNA typically interact via a PCNA interaction peptide (PIP); however, TK0808 does not contain the PIP motif and instead interacts with a novel surface of PCNA. TK0808 is non-essential, and strains wherein TK0808 is deleted show no obvious phenotype; however, TK0808 can significantly impact the activity of several replisome components in vitro.

Materials and Methods

This section details the methodology used to generate selections for RNAP variants with altered transcription termination phenotypes as well as the genetic analyses done for the investigation of TK0808. The selections utilize the selective markers

TK0149 and TK0664. These markers were cloned into plasmids and surrounding sequences were modified to introduce a constitutive promoter, a terminator sequence, and regions of homology for integration into the T. kodakarensis chromosome. Construction of TK0808 strains, confirmation of strains, construction of the complementation plasmid, and growth curve methodology are described. All primers used in this study are listed in

Table 2.

Plasmid construction. Standard molecular biology techniques were used to generate plasmids in E. coli. Details are included below for several plasmids where the constructions were unique or non-conventional.

Plasmids containing terminator variants between PhmtB and TK0149.

TK0149 was amplified from the T. kodakarensis chromosome using primers

0149BamHIpos and 0149EcoRIneg (Table 2). This 1.7 Kbp amplicon was cloned into pCR 2.1 using Invitrogen TA Cloning® Kit following manufacturer’s instructions, generating plasmid pD1. pD1 was used as a template for site-directed mutagenesis

(Agilent Quikchange Mutagenesis kit) to introduce the hmtB promoter according to 20 manufacture’s protocol with primers 0149PhmtBpos and 0149PhmtBneg (Table 2). The resultant plasmid, pDQ1, was again subjected to site-directed mutagenesis, as before, using primers 0149QtoXpos and 0149QtoXneg. These primers contain a mixture of bases (20% A and 80% T) at positions 5-10 within the potential terminator (see Table 2).

The terminator was introduced at +18 from the transcription start site and immediately before the ribosome binding site. The partially randomized primers generated a collection of plasmids, each with a different terminator variant, from which nine terminator variants, designated pDX1-9 respectively, were chosen for additional studies.

Plasmids pDX1-9 were then individually used as templates for PCR using primers

0010149 and 0020149 for introduction into pTS700 using ligation-independent cloning techniques (Hileman 2012). These final plasmids, designated pOSU0149X1-9 (see Table

3), are available for incorporation into T. kodakarensis using the selection and counter- selection methods described previously (Hileman 2012).

Plasmids containing terminator variants between PhmtB and TK0664.

TK0664, together with ~ 500 base pairs of downstream flanking DNA was amplified from T. kodakarensis chromosomal DNA using primers Pos664-665BamHI and Neg664-

665Acc65I (Table 2). The resulting amplicon was digested with BamHI and Acc65I, purified, and directionally cloned into BamHI-Acc65I digested pTS511 to generate pTHH1. pTS511 was derived from pUMT2 (Sato 2003) by replacing trpE with PF0207 and PF0208 (Table 1). A second amplicon was added to pTHH1 by amplification of ~500 bp of DNA upstream of TK0664 (primers Pos0663PstI and Neg0663PstI), digestion with

PstI, and ligation to PstI-digested pTHH1. A clone containing the correctly oriented

21 amplicon was identified, hereafter termed pTHH2, and this DNA was used as template for site-directed mutagenesis to introduce PhmtB (Agilent Quikchange Mutagenesis kit) according to the manufacturer’s protocols (primers 0664hmtbprompos and

0664hmtbpromneg). A clone with the desired promoter modification was identified and was termed pTHHQ. pTHHQ was subjected to site directed mutagenesis using primers

QtoX0664pos and QtoX0664neg; these primers contain a mixture of bases (20% A and

80% T) at positions 5-10 within the potential terminator. The terminator was introduced at +18 from the transcription start site immediately before the ribosome binding site.

Resultant colonies were screened via DNA sequencing and 30 plasmids, each containing a unique terminator variant, were selected for additional studies and designated pTHHX1-30.

RNAP subunit expression vectors. Sequences encoding RNAP subunits A’’

(TK1081), B (TK1083), D (TK1503), E (TK1699), H (TK1084), K (TK1498), L

(TK1167), and N (TK1499) were amplified via PCR with a 5’ primer that contained the

NotI restriction enzyme recognition sequence and the promoter for the hmtB gene from

Methanothermobacter thermautotrophicus (PhmtB) together with a downstream primer that introduced a unique SalI site to the amplicon. The resultant amplicons were individually cloned into pLC70 using NotI and SalI restriction sites, resulting in plasmids pTS414, pCPK1-7 and pCKP11 (Table 5). Sequences encoding RNAP subunits A’

(TK1082), F (TK0901), and P (TK0616) were amplified via PCR with a 5’ primer that contained the recognition site for the NotI restriction enzyme as well as PhmtB together with a downstream primer that also added a NotI recognition sequence. These amplicons

22 were individually used as templates for amplification with primers Rnapinfusionnew1 and Rnapinfusionnew2. The resultant amplicons were cloned into pLC70 at a NotI restriction site using Clontech In-Fusion® HD cloning, resulting in plasmids pCPK8, pCPK9, and pCPK10 (Table 5).

Construction of TK0808 plasmids (deletion, tag, autonomously replicating)

Plasmids pOSU0808A, pOSU0808B, and pOSU0808D were created using standard molecular biology techniques (Hileman 2012) and were maintained in E. coli.

pTHH6 construction and confirmation. Sequences encoding TK0808 were amplified via PCR with a 5’ primer, 1DY0808, that contained PhmtB and a downstream primer, 2DY0808, this amplicon was cloned into pCR 2.1 using Invitrogen TA Cloning®

Kit following manufacturer’s instructions. The resultant plasmid was used as template for PCR with primers 0808infusionnew1 and 0808infusionnew2. This amplicon was cloned into pTS543 at a unique NotI restriction site using Clonetech In-Fusion® HD cloning.

T. kodakarensis strain construction and confirmation of genome sequences.

T. kodakarensis strains were grown in artificial sea water (ASW) with 5 g/l each of yeast extract (Y) and tryptone (T), and 2 g/l sulfur (S˚) at 85˚C with the growth of cultures monitored by an increase in optical density at 600 nm (OD600) as previously described

(Hileman 2012). Transformation of T. kodakarensis (Sato 2005) and strain construction for markerless deletion and introduction of 6-His tags was done essentially as described previously (Hileman 2012). Strains were constructed by transformation of T.

23 kodakarensis TS559 (Hileman 2012) with transformants selected on media lacking agmatine, selecting for plasmid incorporation into the host genome. PCRs using DNA isolated from intermediate strains was used to identify strains where the integration had occurred as predicted, and these confirmed intermediate stains were then subjected to counter-selection on ASW-Amino Acid-S˚ media containing 6-methylpurine to facilitate isolation of a final strain containing the desired genome (Hileman 2012). The addition of sequences encoding an N-terminal His6-HA tag in strain THH1 was confirmed by differential restriction digests based on the introduction of additional BccI sites in the tag- encoding sequence (data not shown) and sequencing of amplicons from the final strain.

Strain THH2 (TK0808) was generated by deleting the first 189 bp of the TK0808 ORF; the final 6 bp overlap with TK0807 and were thus retained.

Southern blots. The genomic organization of strain THH2 was confirmed via

Southern blots of Acc65I and BglII digested genomic DNA. The Acc65I and BglII restriction fragments that hybridized to a digoxigenin (DIG)-labeled amplicon probe,

PCR-generated from within TK0808 (probe B/C) and a flanking region (probe E/F)

(Table 2; Figure 13), were identified by using anti-DIG antibodies coupled to alkaline phosphatase as previously described (Cubonova 2012).

T. kodakarensis growth curves. T. kodakarensis strains were grown overnight in

20 ml ASW-YT-S˚ medium, and strains lacking pTHH6 were supplemented with 1 mM agmatine sulfate. Growth at 85˚C of triplicate cultures was monitored at OD600 nm of

1:100 inoculated cultures.

Table 2: Primers used in the construction of plasmids. W represents a mixed base position of 20% A and 80% T.

Results

Transcription Termination Selections

Previous studies of archaeal transcription termination have identified the minimal template requirements (a poly-T tract) that induce intrinsic transcription termination in vivo and in vitro (Muller 1985; Santangelo 2006, 2009; Spitalny 2008; Thomm 1994). I aimed to develop an in vivo selective scheme to identify RNAP variants with altered termination phenotypes, both to better understand the mechanics of transcription termination and to determine the protein-nucleic acid interactions that normally provide such dramatic stability to the transcription elongation complex.

I adapted a previously developed in vivo reporter system (Santangelo 2009) to generate selective conditions meant to identify RNAP variants that do not respond properly to termination signals. Two variations on the reporter system were used: one to identify RNAP variants that result in a termination-resistant phenotype, and a second selection to identify RNAP variants with termination-sensitive phenotypes. The selections were built from a single expression cassette, designed to be inserted into the T. kodakarensis chromosome to provide the most natural setting to explore transcription termination.

Hypo-termination RNAP variant selection

The selection scheme designed for isolation of termination resistant RNAP variants is based on the selection marker TK0149 (Figures 5 and 11). The plasmids generated to facilitate strain construction were designed to place TK0149 under control of

PhmtB with the terminator variants located at +18 from the transcription start site (Figure

11). These plasmids (pOSU0149Q and pOSU0149X1-9; Table 3) were transformed into

T. kodakarensis and have undergone the selection and counter-selection methods based on protocols previously described to generate strains wherein TK0149 is reinserted to the

T. kodakarensis TS559 genome, at its original locus, but now under the control of PhmtB, and subject to promoter-proximal transcription termination limiting expression of

TK0149. These strains are pending confirmation.

Figure 11: Diagram of PhmtB driven TK0149. Sequence of PhmtB and 5’ end of TK0149. TATA-Box, transcription start site ( ), ribosome binding site (RBS), and 5’ end of TK0149 are indicated. Upward arrow ( ) indicates introduction of terminator variants at +18. These sequences are listed in Table 3.

Table 3: TK0149 plasmid designations and terminator variant sequences

Hyper-termination RNAP variant selective scheme

The markerless selection/counter-selection most commonly employed for T. kodakarensis genetic selections at Ohio State University was impractical for construction of the termination-sensitive selective strains, as both the commonly employed selective- counter selective scheme and the termination phenotype scheme employ TK0664. The selective marker was thus designed to be inserted into the chromosome using an alternative marker that is useful in any strain background, the arginine/citrulline marker

(Table 1). The plasmids generated for this selection include the upstream flanking region of TK0664 preceding the arginine/citrulline marker and TK0664 with the downstream flanking region following the marker. The TK0664 promoter was replaced with PhmtB and further modified by introduction of terminator variants at +18 from the transcription start site.

Figure 12: Diagram of PhmtB driven TK0664. Sequence of PhmtB and 5’ end of TK0664. TATA-Box, transcription start site ( ), ribosome binding site (RBS), and 5’ end of TK0664 are indicated. Upward arrow ( ) indicates introduction of terminator variants at +18. These sequences are listed in Table 4.

Plasmid Terminator Plasmid Terminator pTHHQ None pTHHX16 (T)5ATTAT pTHHX1 (T)10 pTHHX17 (T)5AA(T)3 pTHHX2 (T)9A pTHHX18 (T)5AATTA

pTHHX3 (T)8AT pTHHX19 (T)4ATA(T)3

pTHHX4 (T)8AA pTHHX20 (T)4ATAATT pTHHX5 (T)7ATT pTHHX21 (T)4A(T)5 pTHHX6 (T)7AAT pTHHX22 (T)4A(T)3AT

pTHHX7 (T)6ATAT pTHHX23 (T)4ATTATT

pTHHX8 (T)6A(T)3 pTHHX24 (T)4ATT(A)3

pTHHX9 (T)6ATTA pTHHX25 (T)4ATTAAT pTHHX10 (T)6(A)3T pTHHX26 (T)4(A)3(T)3 pTHHX11 (T)6AATA pTHHX27 (T)4AATATA

pTHHX12 (T)6AATT pTHHX28 (T)4AA(T)4

pTHHX13 (T)5ATATT pTHHX29 (T)4AATTAT pTHHX14 (T)5A(T)4 pTHHX30 (T)4AATTAA pTHHX15 (T)5A(T)3

Table 4: TK0664 plasmid designations and terminator variant sequences.

RNAP expression vector construction

T. kodakarensis shuttle vectors containing each of the RNAP subunit genes have been constructed (Figure 4 and Table 5). Sequences encoding each gene were cloned into pLC70 under the control of PhmtB, a non-endogenous promoter that has been used to drive expression of plasmid-based genes (Santangelo 2008B). These plasmid-encoded epitope-tagged RNAP subunits can now be modified to generate library collections of plasmid variants that can be employed in the selections described above to isolate RNAP variants that are likely to provide insights into mechanisms of archaeal transcription.

Plasmid Encoded Gene subunit pTS414 RpoL TK1167 pCKP1 RpoA’’ TK1081 pCKP2 RpoB TK1083 pCKP3 RpoH TK1084 pCKP4 RpoK TK1498 pCKP5 RpoN TK1499 pCKP6 RpoE’ TK1699 pCKP8 RpoP TK0616 pCKP9 RpoF TK0901 pCKP10 RpoA’ TK1082 pCKP11 RpoD TK1503

Table 5: RNAP subunit containing plasmid and gene designations.

Strain construction, expression vector and growth curves to investigate TK0808

PCNA is required to encircle duplex DNA to interact with and modulate the activities of replisome proteins. Collaborative biochemical data indicated that adding the protein encoded by TK0808 (TK0808p) to in vitro assays abolished PCNA-dependent stimulation of the activities of DNA polymerase B (DNA pol B) and flap endonuclease 1

(Fen1). TK0808p prevented PCNA-stimulated activity in both assays but had no detrimental effect on reaction kinetics when added alone. Size exclusion chromatography analyses of PCNA with DNA pol B showed complex formation between the proteins, but when TK0808p was incubated with the PCNA and DNA pol B, the proteins eluted separately. A proposed mechanisms for TK0808p include (I) the dissociation of the

PNCA ring, and thus a release of the duplex DNA or (II) TK0808p binding prevents binding of other PCNA-interacting proteins. The data described above are pending publication.

TK0808 was deleted from the T. kodakarensis genome using a selection/counter- selection scheme (Hileman 2012), generating strain THH1. The structure of the chromosome in the deletion strain was confirmed via diagnostic PCR as well as with a

Southern blot (Figure 8, B and C). THH1 was still viable, indicating a non-essential role for the protein and encoding gene. A tagged copy of the gene was also introduced into the chromosome replacing the original locus; this strain was designated THH2. Strain construction was confirmed via DNA sequencing and a diagnostic restriction digests with

BccI (data not shown). A growth curve of the strain deleted for TK0808 was conducted to determine any impact on growth. The growth curve was conducted in triplicate, along 31 side growth monitoring of the parental strain, TS559 (Hileman 2012). Growth of THH1 and THH2 were included, and complemented versions of these strains with pTHH6 were also followed. The results obtained showed no significant differences between any the strains, whether or not they were complemented.

Figure 13. Genomic organization, confirmation and growth of strains lacking TK0808. (A) Annotated sections of the T. kodakarensis strains TS559 (top) and ΔTK0808 (bottom) genomes, highlighting the locations of genes (open arrows) and oligonucleotide primers (black arrows; labeled A-F; Table 2). The approximate locations of relevant Acc65I and BglII restriction sites are shown. (B) Ethidium-stained, agarose gel electrophorectic separation of amplicons generated using primers A and D from chromosomal DNA templates from strains TS559 and ΔTK0808 results in a ~190 bp smaller amplicon from T. kodakarensis strains lacking TK0808 than the parental strain, TS559. (C) Southern blots confirm the genome structures of T. kodakarensis strains TS559 and ΔTK0808. Probes were generated with primers B/C (right) and E/F (left). M = DNA markers, in bp. (D) Growth curves of T. kodakarensis strains TS559 and DTK0808, alone and when containing plasmid pTHH6. The moving average (period = 2) is shown as a trend line for each curve.

Perspectives

The advances made in establishing a genetic system for T. kodakarensis have bolstered the ability to investigate many aspects of Archaea that have been previously intractable. In vivo selections were applied to investigate transcription termination twenty years ago in Bacteria, and have now become possible in Archaea. The preliminary groundwork for these selections has been laid. This work has the potential to elucidate key components of RNAP that are responsible for recognizing terminator sequences in Archaea. Plasmids to construct strains for both selection schemes have been generated. Strain creation can begin immediately, followed by selection of the terminators to use for further studies.

The selection scheme requires mutant RNAPs to confer a termination-altering phenotype. Shuttle vector expression of RNAP subunits facilitates introduction of mutations within specific subunits of RNAP to be used in the selection schemes. Each of the eleven RNAP subunits has been cloned into the T. kodakarensis expression vector under control of a constitutive promoter with an epitope tag. These vectors can be used to probe function of the archaeal RNAP in the selection schemes described in this thesis.

TK0808 is a small gene found in the Thermococcales whose protein product co- isolated with PCNA1, an essential component for DNA replication (Li 2010; Pan 2013).

This gene was found to be non-essential and the deletion of TK0808 did not affect growth. Biochemical data of the protein encoded by TK0808 did show inhibitory effects of PCNA in vitro, suggesting a novel mechanism for PCNA inhibition. This protein does not interact with PCNA using a conserved peptide sequence the PCNA-interacting peptide (PIP) nor does it interact with the conserved region of PCNA the inter domain connecting loop (IDCL). This interaction may indicate a new mechanism for PCNA dissociation or inhibition and may be applicable to other systems as a therapeutic target.

Further analysis of these interactions will be required to determine if this interaction is specific to the Thermococcales or can be applied broadly between Domains.

The studies outlined here are possible using the genetic system available in T. kodakarensis. The ability to study Archaea using these powerful techniques is needed if we are to understand this third and unique domain of life.

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