ABSTRACT Title of Document: ARCHAEAL TRANSCRIPTIONAL REGULATION OF CATABOLIC CARBON MONOXIDE DEHYDROGENASE IN METHANOSARCINA SPECIES Kimberly Lynn Anderson, Doctor of Philosophy, 2009 Directed By: Professor Kevin R. Sowers, Marine Estuarine Environmental Sciences In Archaea, the basal transcription machinery is eukaryotic-like, but some components, such as activator and repressor proteins, are bacteria-like. To further gain knowledge into cellular processes of Archaea, the genome of Methanosarcina thermophila was searched for helicase genes. A homolog of yeast RAD25, a gene with helicase and nucleotide excision repair (NER) abilities, was isolated. M. thermophila rad25 has the domains for helicase activity, but the C-terminal end is truncated, indicating that this protein mostly likely does not function in NER. After overexpression, helicase activity assays of Rad25 indicated that it might have helicase activity; however, there appeared to be contaminating proteins in the purification, so it was not possible to assign the activity only to Rad25. Additional work is necessary to characterize this protein. To investigate transcription, catabolic gene regulation was studied, specifically regulation of carbon monoxide dehydrogenase/acetyl CoA synthase (CODH/ACS) from Methanosarcina species. The regions upstream of the transcriptional start site, as well as the 5? leader region of cdhA, were investigated for trans factors and cis elements that might be involved in regulation. Experiments revealed that regulation of cdhABCDE does not appear to involve trans factors upstream of the transcriptional start site. However, deletion analysis indicated that the 5? leader region does have a role in regulation. Comparing the protein levels to the mRNA levels revealed there was no significant difference between the two, indicating that translational regulation was not a factor. Other experiments ruled out differential mRNA stability as a factor in regulation. A region located between +358 and +405 was important in transcriptional regulation, indicating that regulation occurred at the level of transcription elongation. A model for regulation of catabolic CODH/ACS by differential elongation is proposed. Although 5? leader regions identified for other archaeal genes have been postulated to be involved in regulation, this was the first study to demonstrate a regulatory role by an archaeal leader sequence for differential elongation. Identifying regulatory mechanism(s) of catabolic genes such as CODH/ACS is critical for understanding the regulatory strategies employed by the methanoarchaea to efficiently direct carbon and electron flow during biomass conversion to methane. ARCHAEAL TRANSCRIPTIONAL REGULATION OF CATABOLIC CARBON MONOXIDE DEHYDROGENASE IN METHANOSARCINA SPECIES By Kimberly Lynn Anderson Dissertation submitted to the Faculty of the Graduate School of the University of Maryland, College Park, in partial fulfillment of the requirements for the degree of Doctor of Philosophy 2009 Advisory Committee: Dr. Kevin R. Sowers, Chair Dr. Robert Gunsalus Dr. Zvi Kelman Dr. Frank Robb Dr. Harold Schreier Dr. Daniel Terlizzi ? Copyright by Kimberly Lynn Anderson 2009 ii Dedication This work is dedicated to my husband, Matthew. His unwavering support and faith in me made this dissertation possible. iii Acknowledgements This work was funded in part by the U.S. Department of Energy grants #DE- FG02-93ER20106 and #DE-FG02-07ER64502, the National Science Foundation grant #MCB0110762, and Advanced Research International subcontract for DOE grant #DE-FG02-03ER83596. I would like to extend my sincerest appreciation to my advisor, Kevin Sowers, for giving me the opportunity to work in his laboratory. His knowledge and support have taught me so much about critical thinking and experimental design. I am a better scientist because of his patience and guidance, and I could not have asked for a better mentor. I would also like to thank the members of my advisory committee: Rob Gunsalus, Zvi Kelman, Frank Robb, and Harold Schreier. Their comments and suggestions have provided me with more insight into my projects, and helped me to see all of the aspects of my research. There have been many people who helped to move this research forward. I thank Zvi Kelman and Jae-Ho Shin for allowing me to work in their lab and training me in biochemical techniques like EMSA. Thanks to Mark Shirtliff at the University of Maryland Baltimore for training me in two-dimensional gel electrophoresis techniques. David Grahame at Uniformed Services University was very helpful in providing the purified Cdh extract for binding studies. Many thanks go out to all the members of the Sowers lab, both past and present. Ethel Apolinario has been a source of invaluable advice and assistance. She has also been one of my strongest supporters. Thanks go to Birthe Kjellerup for her valuable discussions and for reviewing my dissertation. Sonja Fagervold and iv Sheridan MacAuley, two previous graduate students in the lab, provided support and encouragement, as well as participating in many fruitful discussions. Finally, I would like to thank my family and friends for their constant support and encouragement. Your faith in me gave me the strength and the courage to follow my dreams and achieve my goals. I thank you all from the bottom of my heart. v Statement of Contributions Karin Jackson performed the isolation of rad25 from Methanosarcina thermophila, primer extension analysis, and the northern analysis. Ethel Apolinario was responsible for the construction of the deletion constructs and the beta-galactosidase assays for the leader region deletions. Sheridan MacAuley performed the beta-galactosidase assays on the gross deletion constructs. Jae-Ho Shin (Center of Advanced Research in Biotechnology) performed the helicase assays on M. thermophila Rad25. Tom Santangelo (Ohio State University) performed the in vitro transcription assays. vi Table of Contents Dedication ..................................................................................................................... ii Acknowledgements ...................................................................................................... iii Statement of Contributions ............................................................................................v Table of Contents ......................................................................................................... vi List of Tables ............................................................................................................... ix List of Figures ................................................................................................................x List of Abbreviations .................................................................................................. xii Chapter 1: Introduction ..............................................................................................1 1.1. General introduction ...........................................................................................1 1.2. Methanogenic Archaea and their role in the environment ..................................3 1.3. Substrate utilization by methanogens .................................................................5 1.3.1. Acetate utilization by methanogenic Archaea ...........................................10 1.3.2. Carbon monoxide dehydrogenase/acetyl Co-A synthase ..........................10 1.4. Transcription in Archaea ...................................................................................12 1.4.1. Archaeal transcriptional machinery and mechanisms................................12 1.4.2. Regulation of transcription in Archaea ......................................................13 1.4.3. Role of 5? untranslated leader regions in transcriptional regulation ..........15 1.5. Objectives of Research .....................................................................................18 1.5.1. Objective 1: Identification of putative Rad25 transcription factor from M. thermophila TM1 .................................................................................................18 1.5.2. Objective 2: Identification of cis regions and trans factors involved in the regulation of carbon monoxide dehydrogenase/acetyl Co-A synthase from Methanosarcina species .......................................................................................19 1.5.3. Objective 3: Determination of the role of the 5? leader region of carbon monoxide dehydrogenase/acetyl Co-A synthase .................................................20 Chapter 2: Characterization of a rad25 helicase homolog in Methanosarcina thermophila TM1 ........................................................................................................21 2.1. Abstract .............................................................................................................21 2.2. Introduction .......................................................................................................21 2.3. Materials and Methods ......................................................................................23 2.3.1. Nucleotide sequence accession numbers ...................................................23 2.3.2. Strains and plasmids ..................................................................................24 2.3.3. Media and Growth .....................................................................................24 2.3.4. Cloning and sequence analysis of DNA ....................................................25 2.3.5. Southern analysis .......................................................................................25 2.3.6. RNA isolation, Northern hybridization, and primer extension analysis ....26 2.3.7. Protein purification ....................................................................................27 2.3.8. Site-directed mutagenesis ..........................................................................28 2.3.9. Helicase activity assay ...............................................................................28 2.3.10. Disruption of rad25 from M. thermophila ...............................................29 vii 2.4. Results ...............................................................................................................30 2.4.1. Isolation of archaeal M. thermophila rad25 ..............................................30 2.4.2. Phylogenetic analysis .................................................................................34 2.4.3. Expression analysis of M. thermophila rad25 ...........................................35 2.4.4. Identification of transcription initiation site ..............................................36 2.4.5. Detection of helicase activity .....................................................................38 2.4.6. Knockout of M. thermophila rad25 ...........................................................39 2.5. Discussion .........................................................................................................41 Chapter 3: Identification of trans factors involved in the regulation of carbon monoxide dehydrogenase in Methanosarcina species .............................................45 3.1. Abstract .............................................................................................................45 3.2. Introduction .......................................................................................................46 3.3. Materials and Methods ......................................................................................48 3.3.1. Methanogen strains and media...................................................................48 3.3.2. Electrophoretic mobility shift assay...........................................................48 3.3.3. Affinity chromatography of DNA binding proteins ..................................50 3.3.4. Two-dimensional analysis of DNA binding proteins ................................51 3.3.5. Competition assays ....................................................................................51 3.3.6. Construction of cdh?::lacZ deletion plasmids ............................................52 3.3.7. Reporter gene expression analysis .............................................................53 3.4. Results ...............................................................................................................55 3.4.1. Identification of putative protein binding sites using electrophoretic mobility shift assays .............................................................................................55 3.4.2. Isolation and sequence analysis of DNA binding proteins using affinity chromatography ...................................................................................................58 3.4.3. Specificity of Cdh binding .........................................................................62 3.4.4. Effect of sequence deletions on the regulation of cdh by different substrates ..............................................................................................................65 3.5. Discussion .........................................................................................................67 Chapter 4: A 5? leader region regulates expression of Methanosarcina CO dehydrogenase/acetyl-coenzyme A synthase ...........................................................70 4.1. Abstract .............................................................................................................70 4.2. Introduction .......................................................................................................71 4.3. Materials and Methods ......................................................................................73 4.3.1. Archaeal and bacterial strains ....................................................................73 4.3.2. Media and cell growth ...............................................................................74 4.3.3. Sequence analysis ......................................................................................74 4.3.4. Construction of cdh?::lacZ deletion plasmids ............................................75 4.3.5. Identification and deletion of putative mRNA secondary structures .........77 4.3.6. Reporter gene expression analysis .............................................................78 4.3.7. In vitro transcription assays .......................................................................78 4.3.8. RNA extraction and quantitative reverse transcriptase PCR .....................79 4.3.9. Transcript stability studies .........................................................................80 4.3.10. Construction of TBP::lacZ reporter plasmids ..........................................80 viii 4.4. Results ...............................................................................................................82 4.4.1. Identification of conserved sequence in carbon monoxide dehydrogenase/acetyl Co-A synthase (CODH/ACS) orthologs .........................82 4.4.2. Effect of sequence deletions on the regulation of cdh by different substrates ..............................................................................................................84 4.4.3. Effect of deletions in the sequence within the 5? leader region of cdhA ...86 4.4.4. Comparison of Cdh::LacZ fusion protein and cdh transcript levels with different substrates ...............................................................................................89 4.4.5.In vitro transcription of 5? leader region .....................................................89 4.4.6. Transcript stability of cdh during growth with different substrates ...........92 4.4.7. Effect of substrates on elongation of cdh transcript ...................................94 4.4.8. Regulation of different TATA-binding proteins by different substrates ...95 4.5. Discussion .........................................................................................................97 Chapter 5: Discussion and future perspectives .....................................................102 Appendix: Supplemental Information........................................................................111 Bibliography ..............................................................................................................113 ix List of Tables Table 3.1. E. coli and M. acetivorans plasmid constructs???????..53 Table 3.2. Identification of protein spots from two-dimensional gel electrophoresis????????????????????62 Table 4.1. E. coli and M. acetivorans plasmid constructs??????......75 Table S2.1. Primers used in Chapter 2 for characterization of rad25 from M. thermophila?.???????????????????...111 Table S3.1 Primers used in Chapter 3???????????.???....111 Table S4.1. Primers used to construct deletion mutants, TBP constructs, and qRTPCR products in Chapter 4?????????????112 x List of Figures Figure 1.1. Coenzymes in the methanogenic pathway??????????.. 7 Figure 1.2. Four methanogenic pathways found in Methanosarcina????? 9 Figure 2.1. Alignment of Rad25 protein domains???????????... 32 Figure 2.2. Alignment of helicase protein sequences??????????.. 33 Figure 2.3. Northern analysis of M. thermophila rad25 expression????.... 35 Figure 2.4. Primer extension analysis of the 5? terminus of rad25 mRNA from M. thermophila?????????????????????... 37 Figure 2.5. DNA helicase activity of Rad25 from M. thermophila?????. 38 Figure 2.6. Polyacrylamide gel electrophoresis of purified Rad25?????. 40 Figure 3.1. Alignment of cdhA promoter region from four Methanosarcina species???????????????????????... 56 Figure 3.2. Electrophoretic mobility shift assay of DNA fragments from the region upstream of the cdhA promoter??????????????..57 Figure 3.3. Polyacrylamide gel electrophoresis of proteins isolated from affinity chromatography with the 5? 371 bp leader sequence DNA of the cdh operon bound to biotin beads???????????????.59 Figure 3.4. Two-dimensional gel electrophoresis (2DGE) of DNA-binding proteins from acetate- and TMA-grown cell free extracts????..61 Figure 3.5. Electrophoretic mobility shift assay with 16 ?g partially purified Cdh complex binding to region upstream of cdhA transcriptional start site??????????????????????.....64 xi Figure 3.6. Map of deletions upstream of the Methanosarcina thermophila TM1 cdhA transcriptional start site and the effects of those deletions on regulation by different substrates????????..66 Figure 4.1. Alignment of cdhA promoter region from four different Methanosarcina species?????????.???????.. 83 Figure 4.2. Map of deletions within the 5? leader region of M. thermophila TM1 cdhA ????????????????????????. 84 Figure 4.3. Effects of deletions downstream of the transcriptional start site on regulation by different substrates?????.???????.... 86 Figure 4.4. Effects of deletions in putative mRNA hairpin loops in the UTR of M. thermophila TM1 cdhA????????????????.?88 Figure 4.5. In vitro transcription assay of cdhA promoter and 5? leader region??????????????????????..?.. 91 Figure 4.6. Differences in transcript levels after addition of actinomycin D?...93 Figure 4.7. Differences in levels of transcripts of varying lengths demonstrated by qRTPCR????????????????????...... 91 Figure 5.1. Proposed model for regulation of cdhABCDE????????.105 xii List of Abbreviations 2DGE two-dimensional gel electrophoresis aa amino acid bp base pair cDNA complementary DNA CODH/ACS carbon monoxide dehydrogenase/acetyl Co-A synthase CSPD disodium 3-(4-methoxyspiro(1,2-dioxethane-3,2?-(5?- chloro)tricyclo[3.3.1.1.3,7]decan)-4-yl)phenyl phosphate dNTP dinucleoside triphosphate DTT dithiothreitol EMSA electrophoretic mobility shift assay kb kilobase LacZ ?-galactosidase NER nucleotide excision repair PAGE polyacrylamide gel electrophoresis PCR polymerase chain reaction qRT-PCR quantitative reverse transcriptase polymerase chain reaction SDS sodium dodecyl sulfate TAE tris acetate EDTA TBE tris borate EDTA TBP TATA-binding protein TMA trimethylamine UTR untranslated leader region 1 Chapter 1: Introduction 1.1. General Introduction ? Archaea The Archaea are the third lineage of the modern tree of life. This group was first proposed by Carl Woese and colleagues based on phylogenetic analysis of 16S rRNA gene sequences (36, 130). Although the Archaea are phylogenetically distinct from Bacteria and Eukaryotes, they have many characteristics in common with both. Features of Archaea that are similar to Eukaryotes include a multi-subunit RNA polymerase, eukaryotic-like basal transcription components for initiation, and employment of an unmodified methionine for translation. However, the morphology of Archaea is similar to Bacteria, as they both are unicellular organisms that lack intracellular compartmentalization and nuclear membranes. In addition, Archaea are also capable of many metabolic processes that are found in Bacteria, such as nitrogen fixation. Archaea have their own unique characteristics as well. These characteristics include unique cofactors and enzymes and the occurrence of phytanyl ether lipids instead of fatty ester lipids in membranes. A majority of these organisms grow in conditions that were once considered the extremes for life, such as extreme temperatures, high salinity, and highly reduced and anoxic environments. Members of the Archaea include non-methanogenic hyperthermophiles and thermophiles, halophiles, and methanogens. The original division of Archaea was based on physiology and included three groups: methanogens, haloarchaea, and sulfur-dependent thermophiles (36). In the 2 current classification of the Archaea, which is based on phylogeny, the domain is now divided into four different kingdoms: Euryarchaeota, Crenarchaeota, Korarchaeota, and Nanoarchaeota (24, 105). The two kingdoms, Euryarchaeota and Crenarchaeota, contain the majority of known Archaeal species. These two kingdoms, while defined by certain Archaeal species, are also defined by different biochemical properties, such as differences in DNA replication machinery (35). Euryarchaeota have ssDNA-binding protein RPA, a heterotrimer PCNA sliding clamp, and no identified cell division protein. Crenarchaeota, on the other hand, have a ssDNA-binding protein SSB, a homotrimeric PCNA, and the FtsZ cell division protein (57). The kingdom Euryarchaeota includes all known species of extreme halophiles and methanogens, and some species of non-methanogenic extreme thermophiles. Examples of organisms belonging to this kingdom include members of the genera Halobacteria, Methanosarcina, and Pyrococcus. The Crenarchaeota are made up of thermophiles, including members of the genera Sulfolobus and Pyrobaculum. Some potential psychrophilic Crenarchaeotes have been identified through phylogenetic analysis, such as Cenarchaeum symbiosium, which was identified in a deep cold water marine sponge, but these organisms have not yet been cultured in the laboratory (91). The remaining two kingdoms contain fewer members and have been described more recently. Nanoarchaeum equitans is the sole identified member of the kingdom Nanoarchaeota (53). This organism is a symbiote of the Crenarchaeote, Ignicoccus spp. It is a hyperthermophilic organism with a rapid rate of evolution and is one of the smallest known living organisms. The evidence for whether Nanoarchaeum 3 belongs in a separate kingdom depends on which phylogenetic marker is used. 16S rDNA phylogenies indicate that this organism belongs in a separate kingdom (25). However, using different ribosomal proteins to determine the relationship of Nanoarchaeum to other Archaea indicates that this organism may be more closely related to Euryarchaeotes, namely Thermococcales. Members of the fourth kingdom Korarchaeota were identified in hot environments (11). This phylogeny is based on comparative sequence analyses of 16S rRNA genes, as these organisms have not been cultured in the laboratory. 1.2. Methanogenic Archaea and their role in the environment Methanogenic Archaea, or methanogens, were the first members of the Archaea to be described. Biological methane production is an anaerobic microbial process that occurs ubiquitously on Earth. This process was first demonstrated by Alessandro Volta, who collected the gas from disturbed lake sediments and showed that it was flammable. The gas was originally called ?combustible air? and later shown to be methane gas. Approximately 80% or more of the methane produced yearly is produced through biological pathways, making methanogens key producers of this greenhouse gas (51). Methanogens are found in anoxic environments that contain organic material, such as swamps, aquatic sediments, digestive tracts of animals, and near hydrothermal vents (127). These microorganisms also have the ability to grow in 4 different environmental conditions, such as varying salt concentrations, from fresh to marine water, and at different temperatures, ranging from -2.5 to 110 ?C. In the environment these organisms are often part of a consortium of anaerobic microorganisms that break down organic matter into carbon dioxide and methane. The first group of organisms in this process, the fermentative bacteria, convert complex organic matter into hydrogen and carbon dioxide and partially oxidized intermediates such as fatty acids and primary alcohols. The second physiological group, the acetogenic bacteria, further oxidizes the intermediates to hydrogen, acetate, and carbon dioxide. The third physiological group, the methanogenic Archaea, converts acetate, hydrogen, formate, and carbon dioxide formed by the first two groups into methane. These organisms exist as syntrophs, meaning methanogens require the other groups to produce the substrates for their growth and methanogenesis. The other two groups benefit from the utilization of hydrogen by the methanogens, as the production and accumulation of hydrogen creates a thermodynamically unfavorable environment. Methanogens maintain a low hydrogen partial pressure environment that allows reactions to take place that would normally not be thermodynamically feasible. For fermentors, the ability to reduce protons under low hydrogen partial pressure generated by the methanogens enable them to generate more energy by producing more oxidized fermentation products such as acetate. For the hydrogen producing acetogens that are growth-limited by the accumulation of hydrogen, methanogens use the hydrogen, removing the growth inhibition. This process is called interspecies hydrogen transfer. 5 1.3. Substrate utilization by methanogens In the environment, the two most utilized substrates for methanogenesis are acetate and hydrogen. Of these two substrates, the majority of methane produced by these organisms in the environment is derived from acetate, with approximately 75% of methane coming from this source (71). Methanogens are also capable of using a few other substrates for methanogenesis, including formate, methylamines, methylsulfides, and methanol. Recent studies have also reported the ability of some methanogenic Archaea species to use carbon monoxide as a substrate, although this process does not produce methane, but rather acetate and formate as end products. However, not all methanogenic Archaea can use all substrates. Even though the majority of methane produced in the environment is derived from acetate, there are only two genera currently known that are capable of using acetate as a substrate: Methanosarcina and Methanosaeta. For Methanosarcina, which are able to utilize multiple substrates, acetate is not the most energetically favorable, and when two substrates are available, these organisms will exhibit diauxic growth, consuming the other substrate before utilizing acetate (17, 34, 60, 74, 107). These organisms will use the substrate with the highest free energy, such as methanol or trimethylamine, before using acetate as a substrate for methanogenesis. There are four main catabolic pathways for methanogenesis: carbon dioxide (CO2) reduction with hydrogen (H2), methyl reduction using H2, dismutation of methanol or methylated amines, and fermentation of acetate. These pathways all involve multiple coenzymes and cofactors (Figure 1.1) (94). Methanofuran (MFR) is a one-carbon carrier similar to molybdopterins. Tetrahydromethanopterin (H4MPT) 6 is a single carbon carrier which is analogous to tetrahydrofolate in bacterial and eukaryotic systems. Originally, this cofactor was thought to be unique to methanogenic Archaea, but it has since been found in other Archaea. Coenzyme M (HS-CoM) was also thought to be unique to Archaea, but it has been found in methylotrophs and in bacteria capable of epoxy degradation. There are two factors used in methanogenesis, 7-mercaptoheptanoylthreonine phosphate (HS-HTP) and cofactor F430, that are unique to methanogenic Archaea. 7 Figure 1.1. Coenzymes in the methanogenic pathway [In (94)] 8 Each of the four pathways of methanogenesis involves multiple steps (Figure 1.1). Some steps are unique to each pathway, while other steps are shared amongst all the pathways. The carbon dioxide reduction pathway involves the sequential reduction of CO2 via electrons from H2 to produce methane (Figure 1.2A). CO2 is first reduced, along with coenzyme MFR, to generate formyl-MFR. The formyl group is transferred to H4MPT to yield formyl-H4MPT, which is cyclized to methenyl-H4MPT. This substrate is reduced twice to methyl-H4MPT. The methyl group from this compound is transferred to coenzyme CoM-SH, which is further reduced to CH4. The other product of this final step, CoM-SS-HTP, is reduced to regenerate CoM-SH and HPT-SH. There are two pathways through which methylotrophic substrates, such as methanol and trimethylamine, can be converted to methane. The first pathway involves the transfer of the methyl group from these substrates to a corrinoid protein (Figure 1.2.B). The corrinoid proteins are substrate specific. The methyl group from the corrinoid protein is transferred to coenzyme CoM-HS. The methyl CoM-SH is reduced to CH4 as described in the final steps of the process above. Some of the methyl groups that are generated from this process are oxidized in reverse through a pathway identical to the reduction of CO2, which generates electrons to reduce the CoM-SS-HTP. The second pathway involves the reduction of the methyl groups from the methylotrophic substrates through the oxidation of hydrogen (Figure 1.2.C). The fourth pathway of methanogenesis, the fermentation of acetate, is shown in Figure 1.2.D and is discussed at length in the next section. Figure 1.2. Four methanogenic pathways found in Methanosarcina. A, the reduction of CO2 to methane using via oxidation of H2. B, the methylotrophic pathway. C, the methyl reduction pathway. D the aceticlastic pathway. Steps in black are those specifically involved in each pathway. Ech, ferredoxin-dependent hydrogenase; Frh, F420 -dependent hydrogenase; Vho, methanophenazine-dependent hydrogenase; Fpo, F420 dehydrogenase; CHO-MF, formyl-methanofuran; CHO-H4 MPT, formyl- tetrahydromethanopterin; CH ? H4MPT, methenyl tetrahydromethanopterin; CH2 = H4MPT, methylene-tetrahydromethanopterin; CH3- H4MPT, methyl-tetrahydromethanopterin; CH3-CoM, methyl-coenzyme M; CoM, coenzyme M; CoB, coenzyme B; CoM-CoB, mixed disulphide of CoM and CoB; Mph/MphH2, oxidized and reduced methanophenazine; F420/F420H2, oxidized and reduced Factor 420; Fd(ox)/Fd(red), oxidized and reduced ferredoxin; Ac, acetate; Ac-Pi, acetyl-phosphate; Ac-CoA, acetyl-coenzyme A. [Modified from (44) in (109)]. 10 1.3.1. Acetate utilization by methanogenic Archaea The acetate utilization pathway in methanogens is a very important pathway, as the majority of the biologically produced methane is derived from acetate. Of the two genera that can utilize acetate as a substrate for methanogenesis, Methanosaeta is an obligate acetotroph (58). Members of this genus can only grow using acetate as a substrate. In contrast, members of the genus Methanosarcina are able to use most substrates, including methylamines, methanol, carbon dioxide/hydrogen, carbon monoxide, and acetate. Because of this, Methanosarcina species are the most metabolically diverse of all the methanogens. Because of its important role in global methanogenesis, the catabolic acetate utilization has been extensively studied (Figure 1.2D). The first step involves the activation of acetate to form acetyl-CoA. In Methanosarcina species, this is accomplished by the enzymes phosphotransacetylase and acetate kinase. The enzyme acetyl-CoA synthase is responsible for this step in Methanosaeta species. The next enzyme, carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS), catalyzes the release of CO2 and transfers a methyl group onto H4MPT, where the methyl group subsequently proceeds through the reductive pathway discussed above. 1.3.2. Carbon monoxide dehydrogenase/acetyl co-A synthase In acetate catabolism, one of the limiting steps involves the enzyme carbon monoxide dehydrogenase/acetyl CoA synthase (CODH/ACS). This enzyme contains five subunits, including a nickel/iron-sulfur component, a corrinoid/iron-sulfur 11 component, and a component of unknown function. In 1986, CODH/ACS was first detected in Methanosarcina thermophila (121). It was also demonstrated that this protein was much more abundant when cells were grown under acetate as opposed to methanol. Later, genetic analysis of the gene encoding this protein, cdhABCDE, indicated that cdhABCDE was differentially expressed based on substrate, and that the regulation of this operon appeared to be at the level of transcription (114). A previous study using a plasmid-mediated lacZ fusion system revealed that expression of LacZ while under the control of the M. thermophila cdhABCDE promoter was 54- fold lower when cells were grown on methanol versus acetate (5). These same results have been seen in proteomic and microarray studies, confirming that this operon is more highly expressed during growth under aceticlastic conditions than methylotrophic conditions (65). The genomes of Methanosarcina mazei, Methanosarcina acetivorans, and Methanosarcina barkeri have all been sequenced (30, 37, 73). The genomes of all three of these organisms have two full copies of cdhABCDE. These two copies have greater than 95% identity, making it hard to distinguish between the two copies. Proteomic studies of M. acetivorans have indicated that one copy of the cdhA gene (MA3860) is more highly expressed than the other cdhA copy (MA1016) when cells are grown on acetate (65-67). In contrast, a study by Grahame (42) found that only one of the carbon monoxide dehydrogenase/acetyl CoA synthase complexes was present, leading the author to conclude that this species had only one copy of the gene, which is the ortholog of the highly expressed MA3860. 12 1.4. Transcription in Archaea 1.4.1. Archaeal transcriptional machinery and mechanisms The components involved in transcription in Archaea are very similar to those found in Eukarya. The core machinery of archaeal transcription involves a RNA polymerase that shares many properties with the RNA polymerase II from Eukarya (63). Archaeal RNA polymerase is a multisubunit protein whose very structure is similar to that of the eukaryotic RNA polymerase. The gene sequences of the individual subunits are also highly conserved between Eukarya and Archaea. The archaeal RNA polymerase cannot initiate transcription on its own, but requires the presence of two transcription factors described below. Transcription initiation in Archaea involves the recognition of an AT-rich promoter region, also known as the TATA-box. This region is recognized by a TATA-binding protein (TBP), which is functionally interchangeable with the TBP from yeast and humans (126). TBP binds to the promoter, and the protein TFB, a functional analog of eukaryotic TFIIB, is recruited to the complex. This transcription factor recognizes the B recognition element (BRE), a sequence located upstream of the TATA box. The new complex, containing both the TBP and TFB, recruits the RNA polymerase. The archaeal RNA polymerase structure resembles that of eukaryotic RNA polymerases. Archaea also contain a homolog of eukaryal TFIIE, which is called TFE. In Eukaryotes, this transcription factor is used to recruit TFIIH. A homolog for TFIIH has not been found in Archaea. TFE appears to promote transcription in vitro from 13 weak promoters in Archaea. Another transcription factor present in Archaea is TFS, which is homologous to the C-terminal part of TFIIS in Eukaryotes. This protein induces cleavage of paused RNA polymerase and also gives the polymerase proofreading activity (62). 1.4.2. Regulation of transcription in Archaea In Archaea, there are many examples of transcriptional activators and repressors that control transcription. Activators of transcription have been described previously such as Ptr2, an activator found in Methanocaldococcus jannaschii, which binds upstream of the target gene and recruits TBP to activate transcription (87). This protein, which is a member of the Lrp family of regulators, regulates transcription of ferredoxin A and rubredoxin 2. The Lrp family of regulators are found within the Bacteria and Archaea and are mostly involved in the regulation of amino acid metabolism-related genes (23). Another member of the Lrp family, LysM, activates transcription of genes involved in lysine biosynthesis in the absence of lysine in Sulfolobus solfataricus (22). In Halobacterium, the transcriptional regulator Bat activates transcription of multiple genes involved in the synthesis of purple membranes, and this regulator is responsive to light intensity and oxygen tension (9). Repressor proteins that inhibit transcription have also been identified in Archaea. One example of a repressor is LrpA from Pyrococcus furiosus. Pyrococcus furiosus LrpA represses transcription of its own gene by preventing RNA polymerase from binding to the promoter (28), and it is similar to other bacterial Lrp family transcriptional regulators. Other Archaea, such as S. solfataricus and M. jannaschii, 14 also have Lrp family proteins that act as repressors (15, 85). The Archaeoglobus fulgidus mdr1 (metal dependent repressor) gene codes for a protein that can bind to DNA to prevent polymerase recruitment. This binding depends on the presence of bivalent cations. The protein, Mdr1, is in the DtxR family of bacterial transcriptional regulators (14). Mdr1 controls expression of its own gene, as well as three genes encoding an iron-importing ABC transporter that are cotranscribed with mdr1. Another example involves a repressor protein, NrpR, from Methanococcus maripaludis (68). This repressor is a helix-turn-helix protein that binds upstream of nif and glnA promoter regions to prevent transcription depending on nitrogen availability. When levels of ammonia are high, NprR represses transcription of nif. NrpR has two operator sequences to which it can bind, OR1 and OR2 (69). Derepression is achieved through binding of 2-oxoglutarate, which is an indicator of low nitrogen availability. Although this protein appears to be unique to Euryarchaeotes, the cooperative binding to two operators is similar to other repression mechanisms found in bacteria. Archaeal-specific regulators of transcription have also been described. These transcriptional control mechanisms are unique to the Archaea, and are not found in the other domains of life, to date. One example of an activator that appears to be unique to Archaea is GvpE. The GvpE protein from Haloarchaea sp. activates the transcription of gas vesicle formation genes (59). This protein has a motif that is similar to bZIP eukaryotic transcriptional regulators, but it has no sequence similarity to eukaryal transcriptional regulators. GvpE is one protein partner in a positive- 15 negative regulation pair. While GvpE is the activator protein, the repressor protein GvpD inhibits the activity of GvpE (134). Another example of archaeal-specific transcription regulation involves sugar transport systems. In Pyrococcus there are two distinct ABC transporters that control uptake of the sugars maltose/trehalose (mal genes) and malodextrin (mdx genes) (64). The expression of the genes encoding these transporters is controlled by the global transcriptional regulator TrmB. TrmB binds to the promoter regions of these genes and inhibits transcription. The sequence to which it binds at the promoters is different depending on which sugar transport system is being regulated. When TrmB is bound to the promoter of the maltose/trehalose transporter genes, the inhibition can be reversed with the addition of either maltose or trehalose. TrmB also binds to the promoter of the malodextrin transporter genes. This binding is reversed by addition of malodextrin. However, only the presence of the sugar whose transport is being inhibited can cause TrmB to release from the promoter. For example, the addition of maltose or trehalose causes the depression of mal gene, but not of mdx genes. This regulator responds in a different way to two different ligands, depending on the DNA sequence. This type of regulatory mechanism appears to be present in only Archaea. 1.4.3. Role of 5? untranslated leader regions in transcriptional regulation In bacterial systems, long 5? untranslated leader regions (UTRs) are involved in transcriptional regulation via multiple mechanisms. Usually, these mechanisms involve the formation of secondary structures within the RNA. One example of this type of regulation is the attenuation mechanism, which involves the formation of 16 different RNA secondary structures that either inhibit or promote transcription elongation (133). This type of regulation employs stem loop structures, along with a long stretch of uridines immediately following one of the structures. Another mechanism found in bacteria involves intrinsic terminators such as riboswitches. In this type of regulatory motif, found in many bacterial systems, the RNA forms different secondary structures that either permit or terminate transcriptional elongation (103). There are multiple permutations of this system present in both eukaryotes and prokaryotes. One of the most well described systems involves regulation of tryptophan biosynthesis by a conformational change in mRNA secondary structure that causes early transcription termination of tryptophan- producing genes in the presence of tryptophan (132). Other systems involve a regulatory protein (103) or ligands (78, 80, 117, 118, 129) that bind to the RNA, which creates a conformational change that affects elongation of the transcript. Early termination of transcriptional elongation in sequence distal to the regulatory UTR is consistent with regulatory mechanisms involving changes in secondary structure, such as attenuation or riboswitches. There have been reports of 5? UTRs in Archaea. For example, 5? UTRs have been found in the gene for monomethylamine methyltransferase in M. barkeri (26), methanol methyltransferase genes from M. barkeri (98), and carbon monoxide dehydrogenase genes from M. thermophila (114). Although not all of these 5? UTRs have been determined to play a role in regulation in gene expression, there have been a few reports of regulatory 5? UTRs in methanogenic Archaea. One report of a potential regulatory 5? UTR in Archaea is the 113 bp UTR identified upstream of the 17 gene of a DEAD-box RNA helicase in the Antarctic methanogen Methanococcoides burtonii (70). This 5?UTR of deaD, which contains cold-box elements similar to those found in bacteria, is regulated in response to cold shock. There is also a 5? UTR in Escherichia coli cspA, which is a cold induced gene. This 5? UTR has been implicated in transcriptional stability at cold temperatures and instability at higher temperatures (79). The mechanisms of regulation from this 5? UTR are unclear. However, an analogous region in M. burtonii has not been confirmed to have a role in regulation. Another example of a 5? UTR in methanogenic Archaea is the methanol transferase genes mtaCB1, mtaCB2, and mtaCB3. These genes all appear to have long 5?UTRs (18). Deletion analysis of these regions showed that they had an effect on gene expression, indicating that they are important in regulation. However, the mechanism of this regulation was not determined. Carbon monoxide dehydrogenase/acetyl Co-A synthase (cdh) from M. thermophila has a 371bp 5? leader region (114). An earlier study involving northern analysis showed that this gene is differentially transcribed in response to different substrates, when growth on acetate and methylotrophic substrates was compared (114). These results were confirmed in a more recent study (5) that employed a translational fusion of the cdh promoter region to lacZ. In this study gene expression was up to 54-fold and 31-fold greater when cells were grown on acetate as opposed to methanol or trimethylamine, respectively. However, the role of the 5? leader region in regulation was not investigated. 18 Although there is evidence that suggests 5? UTRs are involved in regulation in methanogenic Archaea, the exact mechanism of their involvement has yet to be determined. 1.5. Objectives of research The overall goal of this work was to further investigate transcriptional regulation in Methanosarcina species, focusing specifically on carbon monoxide dehydrogenase/acetyl Co-A synthase as a model to study the mechanisms of catabolic gene regulation in the Archaea. My hypothesis is that catabolic carbon monoxide dehydrogenase/acetyl Co-A synthase is transcriptionally regulated in response to substrate by a mechanism or mechanisms that involve the 5? leader region. 1.5.1. Objective 1: Functional analysis of an archaeal Rad25 homolog in Methanosarcina thermophila TM1 The first objective was to identify putative transcription factors in the genome of Methanosarcina thermophila TM1. A putative helicase was identified with homology to rad25 helicase. Genome sequence analysis was done to characterize the gene, including confirming its expression and mapping the transcriptional start site. Further experiments were also conducted to determine if 1) the protein was functional in vitro and 2) had any role in transcription. The results of these experiments are presented in Chapter 2. 19 1.5.2. Objective 2: Identification of cis regions and trans factors involved in the regulation of carbon monoxide dehydrogenase/acetyl CoA synthase from Methanosarcina species The second objective of this work was to study regulation of gene expression of a gene involved in the catabolic fermentation of acetate to methane. The gene that was studied was cdhABCDE (MA3860), which encodes the five subunit carbon monoxide dehydrogenase/acetyl co-A synthase. This enzyme, as was discussed earlier, is responsible for a critical step in the conversion of acetate to methane. Previous work had indicated that this gene was regulated at the level of transcription (114). The region upstream of the start of transcription of cdhA, the first gene in the operon, was investigated for the presence of cis elements and trans factors that might be responsible for the regulation of this operon. Using the sequence located upstream of the transcription start site, electrophoretic gel shift assays were used to determine if differential protein binding was occurring in this region. Two dimensional gel electrophoresis was used to identify any proteins that might differentially bind to this sequence in the presence of different substrates. These proteins were tested for their ability to bind to the sequence upstream of the transcriptional start site. The results of this research are presented in Chapter 3. 20 1.5.3. Objective 3: The role of the 5? leader region of carbon monoxide dehydrogenase/acetyl CoA synthase The third objective was to examine the 5? leader region located downstream of the transcriptional start site of cdhABCDE (MA3860). This 5? leader region is highly conserved among Methanosarcina species. 5? leader regions are associated with transcriptional and translation regulation in Bacteria, and they have been shown to play a role in regulation in Archaea. However, the mechanism of this regulation in Archaea has not yet been determined. The goal of the research was to determine if this 5? leader region did play a role in regulation of gene expression, and if so, by what mechanism the regulation was occurring. Deletion analysis of the 5? leader sequence was used to determine if this region was involved in transcriptional regulation. In vitro transcription assays were also conducted to study the affects of the 5? leader region on transcription. Quantitative reverse transcriptase PCR reactions were completed to study transcript abundance and to determine where in the sequence the regulation was occurring downstream of the transcriptional start site. Results of this work are presented in Chapter 4. 21 Chapter 2: Characterization of a rad25 helicase homolog in Methanosarcina thermophila 2.1. Abstract Helicases play an essential role in many cellular functions, including DNA replication, recombination, repair, and gene transcription. A homolog of Rad25, a transcriptional helicase protein, was identified in Methanosarcina thermophila. Although eukaryotic Rad25 is a component of the TFIIH complex, which functions in both transcription initiation and nucleotide excision repair, there is no evidence to indicate that the archaeal homolog is required for transcription initiation in Archaea. M. thermophila Rad25 has 30% amino acid sequence identity with yeast RAD25 and has high similarity in all conserved helicase domains. Analysis of the M. thermophila Rad25 protein showed divergent sequence at the C-terminus in a region normally required for eukaryal nucleotide excision repair, suggesting that this archaeal protein functions either in transcription or as part of a modified repair mechanism. Further analysis indicated that rad25 was expressed in M. thermophila. Using in vitro helicase assays, it was also shown that this protein may be capable of 3? ? 5? helicase activity. 2.2. Introduction Helicases are essential in DNA replication and transcription. These proteins couple nucleoside triphosphate hydrolysis to the separation and unwinding of two complementary nucleic acid strands. The Rad25 protein is an important member of 22 the helicase superfamily of proteins. In yeast, RAD25 was shown to be involved in both transcription and DNA repair, coupling with RAD3 to form part of the TFIIH complex (45). RAD3 helicase functions in a 5? to 3? direction. RAD25 functions in a 3? to 5? direction, which is necessary for DNA replication and transcription. The combination of these two proteins make the TFIIH complex a bi-directional helicase, which is necessary for nucleotide excision repair (NER) (88, 119). Defects in either of the helicases lead to severe genetic diseases in humans such as xeroderma pigmentosum and trichothiodistrophy (33). In eukaryal transcription, TFIIH is recruited to the pre-initiation complex by TFIIE, and in the presence of ATP catalyzes unwinding of the promoter to form a transcription bubble (120). The Rad25 helicase is needed both for unwinding the double stranded DNA and for release of RNA polymerase II from the pre-initiation complex to allow productive elongation (84). The role of TFIIH seems to be to move along the DNA, suppressing abortive transcription until the elongation complex has cleared the transcriptional start site (116). In addition to its role in transcription, the yeast RAD25 protein also functions in nucleotide excision repair (NER). During eukaryotic NER, TFIIH and several NER specific proteins are recruited to damaged DNA, making two single-strand incisions. The damaged piece is removed as part of an oligonucleotide fragment (12). NER requires both Rad3 and Rad25 for the process to occur (45, 108). In the archaeon Methanothermobacter thermoautotrophicus NER activity is more similar to that of the bacterial system (82). However, this may not be the case for all Archaea, as homologues of the eukaryal NER system are detected in other species (6). 23 There have been reports of functional helicase activity in Archaea, such as the replicative helicase MCM in M. thermoautotrophicus and Sulfolobus solfataricus (27, 56). However, activity from other types of helicases such as transcriptional or repair helicases has not been reported in the Archaea. A homolog of the eukaryotic transcription factor TFIIH, which contains Rad25, has not been detected in Archaea. However, sequencing of a number of archaeal genomes has revealed proteins with similarity to eukaryal Rad25 (90). In an effort to identify the functional role of these putative archaeal Rad25 homologs the gene encoding M. thermophila Rad25 was identified. This protein was overexpressed to determine if it functioned as a 3? to 5? helicase in vitro. The similarity of helicase domains between Rad25 from M. thermophila and eukaryal Rad25, combined with the activity of the archaeal protein suggest that this enzyme may have a role in archaeal transcription or DNA repair in vivo. 2.3. Materials and Methods 2.3.1. Nucleotide sequence accession numbers The nucleotide sequence of M. thermophila rad25 (accession number AF265295) has been submitted to the GenBank database. Methanosarcina acetivorans rad25 (accession number: NP617309), Methanosarcina mazei gene MM3000 (NP635024), Archaeoglobus fulgidus gene AF0358 (NP069194), Pyrococcus horikoshii gene PH0450 (NP142430), Thermoplasma acidophylum gene Ta1016 (NP394476), S. solfataricus gene SSO0473 (NP342006), Halobacterium NRC-1 gene H1047 (NP046038), Saccharomyces cerevisiae gene SSL2 (NP012123), 24 Arabidopsis thaliana gene MYC6 (BAB08508), Drosophila melanogaster gene hay (AAF50150) and Homo sapiens gene ERCC3 (NP000113), were used for analysis. 2.3.2. Strains and plasmids M. acetivorans C2A (=DSM 2834) and M. thermophila TM-1 (=DSM 1825), were obtained from sources described previously and maintained as frozen stocks (115). Escherichia coli strain SURE (Stratagene) was used for recombinant plasmid construction, E. coli strain XL1-blue (Stratagene) was used for phage growth experiments and site-specific mutagenesis constructs, and E. coli strain BL21 (DE3) was used for overexpression of Rad25. M. thermophila TM-1 DNA, prepared as described previously (116), was used to construct a genomic library with Lambda FIX (Stratagene) according to manufacturers directions. Plasmid pCR2.1 (Invitrogen) was used for cloning PCR amplified products, and plasmid pWM315 was used to subclone M. thermophila DNA for sequencing (77). 2.3.3. Media and growth Artificial marine mineral medium was prepared for growth of M. thermophila with 100 mM trimethylamine as growth substrate by methods described previously (112). The medium was prepared anaerobically in a N2/CO2 (4:1) atmosphere using a modification of the Hungate technique (7). LB liquid and agar-solidified plates were prepared for growth of E. coli (96). Ampicillin (100 ?g/ml) or kanamycin (100 25 ?g/ml) was added to the media for selection and maintenance of E. coli transformants. M. thermophila was incubated at 35?C; E. coli was incubated at 37?C. 2.3.4. Cloning and sequence analysis of DNA Degenerate PCR primers 68 and 69 (Table S2.1) were designed for domains I and V, respectively, of conserved helicase sequences. PCR amplification products (1 min at 94?C, 1 min at 48?C and 2 min at 72?C for 30 cycles using 100 ng of each primer, 3 mM MgCl2, 0.8 mM dNTP's, 1x Taq buffer and 1.5 units Taq polymerase in a volume of 50 ?l) from 1 ?g total genomic DNA were cloned into the pCR2.1 vector (Invitrogen) and sequenced by dye terminator cycle sequencing on an ABI 373 automated sequencer (PE Applied Biosystems, Foster City, CA). DNA sequence was analyzed using DNAMAN (Version 3.0, Lynnon BioSoft, 1994) and the Wisconsin GCG program (Genetics Computer Group, 1991, Madison, WI). 2.3.5. Southern analysis Library screening was performed as described in Molecular Cloning: a Laboratory Manual with 100-fold genome coverage (96) to detect rad25 homolog in M. thermophila. Southern analysis was performed as described using 10 ?g total genomic DNA digested overnight with EcoRI (96). Samples were hybridized overnight at 42?C and washed twice for 15 min each in 2 x SSC/0.1% SDS at 42?C. The cloned PCR products were digoxigenin (DIG) labeled for use as hybridization probes. Hybridization products were visualized by CSPD luminescence following manufacturers? directions (Boehringer Mannheim). 26 2.3.6. RNA isolation, Northern hybridization and primer extension analysis M. thermophila cultures were grown at 35?C in anaerobic minimal marine medium supplemented with 100 mM trimethylamine or 100 mM acetate as growth substrate (3). Cells were harvested during exponential growth (O.D.550=0.6 for TMA- grown cells, O.D.550=0.16 for acetate-grown cells) by rapid chilling in a dry ice/ethanol bath to 0?C. Cultures were transferred to chilled centrifuge bottles and pelleted at 4?C for 5 min at 9,000 x g. Cells were lysed by resuspension in ice-chilled 30 mM NaOAc (pH 5.2) followed by addition of SDS to a final concentration of 2% and immediate boiling for 30 sec. RNA was isolated by acidic phenol extraction and ethanol precipitation (83, 116). Northern hybridization was performed with 10 ?g or 20 ?g total RNA as described previously (116). DIG-labeled probe was made from a gel-purified PCR generated 1358 bp fragment of the M. thermophila rad25 gene. Samples were hybridized overnight at 42?C in ULTRAhyb buffer (Ambion). Blots were washed twice for 5 min each in 2 x SSC/0.1% SDS at room temperature and twice for 15 min in 0.1 x SSC/0.1% SDS at 42?C. Hybridization blots were developed using CSPD luminescence as described above. Primer extension analysis was performed on freshly prepared total RNA. Primer 92 complementary to nucleotides 53-30 and primer 93 complementary to nucleotides 171-148 of the rad25 gene (Table S2.1) were end-labeled with [?- 32P]ATP using T4 polynucleotide kinase (New England Biolabs) and subsequently purified on a NENSORB 20 column (Dupont-NEN) following manufacturer?s directions. Labeled primer was annealed to either 5 ?g or 20 ?g of M. thermophila 27 total RNA. Controls included 5 ?g M. acetivorans total RNA and no RNA. cDNA was synthesized using the Thermoscript RT-PCR System (Gibco-BRL) following manufacturer directions. Primer extension reactions were compared to sequencing products prepared with primers 92 or 93 and labeled using [?-32P] dATP. Template pRad326 DNA for sequencing reactions was prepared and sequenced according to Del Sal et al (29). 2.3.7. Protein purification Rad25 protein from M. thermophila was over-expressed using E. coli BL21 (DE3) codon plus cells (Stratagene). To overexpress the protein, the rad25 gene from M. thermophila was labeled with N-terminal His6-tag, and this protein was expressed using the plasmid pJK107. Cells were grown at 37?C in 0.5 liters of LB broth with Kanamycin until reaching an O.D.600 of 0.3, induced by addition of 0.5 mM IPTG for 3 hr, and harvested. Bacterial lysates were prepared by sonication in 0.5 M NaCl, 20 mM Tris, pH 8.0 (buffer A), containing 10 mM imidazole and 20 mg lysozyme. Lysates were centrifuged for 15 min at 10,000 x g, supernatant was extracted and centrifuged for 90 min at 100,000 x g. Protein (7.5 mg) was mixed with 2 ml of Ni chelate resin (Qiagen) for 2 hr at 4?C with gentle shaking. The mixture was loaded onto a column, washed with 40 ml buffer A containing 20 mM imidazole, and eluted with 10 ml buffer A containing 500 mM imidazole. Protein fractions purified to near homogeneity were stored in elution buffer with 20% glycerol at -80?C. 28 2.3.8. Site directed mutagenesis A single base pair mutation was introduced into the Walker A box in rad25 from M. thermophila using the QuikChange Site-Directed Mutagenesis Kit following the manufacturer?s directions (Stratagene). PCR primers 130 and 131 (Table S2.1) were used to introduce the mutation to change Lys 106 to Glu. 2.3.9. Helicase activity assay The recombinant Rad25 protein fractions were gel filtered prior to using in the assay. The oligonucleotide used in preparation of the helicase substrate was an 18- mer, 5?-CCCGACTGGAAAGCGGGC-3?, referred to as Hel-18. After labeling with [?-32P]ATP (GE Healthcare) using T4 polynucleotide kinase, the oligonucleotide was annealed to 100 nucleotides of the longer oligonucleotide 5?- CTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGC TGC(TTTG)15 -3? in a ratio of 1:2, and the annealed product was gel-purified as described previously (10). DNA helicase activity was measured as previously described (56) in a reaction mixture (15 ?l) containing 20 mM Tris-HCl (pH 8.5), 10 mM MgCl2, 2mM DTT, 100 ?g/ml BSA, 5 mM ATP or AMP-PNP (Roche Molecular Biochemicals), 5 fmol of 32P-labeled DNA substrate (3,000/ cpm fmol) and M. thermophila rad25 protein. After incubation at 37?C for 1 h, the reaction was stopped by adding 5 ?l of 5? loading buffer (100 mM EDTA, 1.0 % SDS, 0.1 % xylene cyanol, 0.1 % bromophenol blue, and 50 % glycerol). Aliquots were loaded onto an 8 % 29 polyacrylamide gel in 0.5? TBE (90 mM Tris, 90 mM Boric acid, 1 mM EDTA) and electrophoresed for 1 h at 200 V at 4?C. 2.3.10. Disruption of rad25 from M. thermophila To disrupt M. thermophila rad25, pJK107 was digested with EcoRI to produce a 1.9 kb DNA fragment containing rad25. The partial rad25 fragment was cloned into pCR2.1 (Invitrogen) creating pKA1. The pac cassette, which confers resistance to puromycin, was PCR amplified from pEA103 using primers 157 and 158 (Table S2.1), generating MfeI sites flanking the product. Both pKA1 and pac PCR product were digested with MfeI and ligated to create pKA2. All products were confirmed by sequencing. pKA2 was digested with EcoRI to generate a linear fragment that was subsequently transformed into M. thermophila as described by Metcalf et al (77). To confirm that the transformation procedure worked, pWM313 was transformed into M. thermophila. Transformants were selected for puromycin resistance. 30 2.4. Results 2.4.1. Isolation of archaeal M. thermophila rad25 To identify helicase genes in M. thermophila, degenerate PCR primers were designed for domain I, the Walker A type nucleotide binding domain, and for domain V of conserved helicase sequences from mammals and yeast (40). PCR amplification products from total genomic DNA were cloned and sequenced. Multiple products were identified and searched against sequences in GenBank (3). The largest PCR product was approximately 2kb, and the sequence of this fragment showed a strong identity (55%) to the nucleotide sequence from Archaeoglobus fulgidus AF0358 (NP069194), previously identified as Rad25 through genome sequencing annotation. Five positive plaques were detected by Southern analysis of the M. thermophila ? phage genomic library with the 2 kb PCR product. Restriction analysis followed by Southern hybridization of one of these clones revealed a single 6kb EcoRI fragment with homology to the DNA probe. This fragment was cloned into a low copy number shuttle vector pWM315 to create pRad326. The ends of the insert were sequenced. The insert extended 418 nucleotides and approximately 4 kb from the 3? and 5? flanking region of the PCR amplified fragment, respectively. Sequencing upstream from the 3' end, a gene of 1473 nucleotides was identified that encoded a putative open reading frame (ORF) of 489 amino acids (predicted molecular mass 58kDa) with 25% amino acid identity to RAD25 from S. cerevisiae. Alignment of the predicted translated protein to other Rad25 proteins showed averages of 31% identity between domains I and VI to Rad25 genes from Eukarya, 31 and 52% to annotated genes identified as Rad25 in archaeal genome sequences (Fig. 2.1). Two DNA helicase sequence motifs GxGK(T/S) and DExH (M. thermophila amino acids (aa) 103-107 and 185-188, respectively) are present in M. thermophila Rad25, and a bipartite motif for nuclear transport KRx10RKK (aa 410-424) is present at the C-terminus of the protein. The six domains identified in S. cerevisiae (40), indicating helicase function, are also present in M. thermophila. Domains I, II, III and VI show the strongest conservation between Eukarya and Archaea (Fig. 2.1). Seven imperfect repeats of a 4 amino acid element QSKS occur at the C-terminus. Repetitive elements of this nature are also found in Arg/Ser rich splicing factors. Interestingly, none of the other identified Rad25 genes contain such a repetitive element. 32 Mt 94 KWGVLVLPTGSGKTLVGIRAI ... 172 EILGNRFGLLIFDEVHHLPAVGY Ph 87 -r-ii---------iiameim ... 163 -l--dk-m-i----c-----ea- Af 89 -r-ci---------h-ama-- ... 167 -k-----m------------es- Hb 100 -r-sv---------flavq-- ... 182 ney-dq----vv--------pt- Hs 334 rs--i---c-a--s---vt-a ... 429 wlktqew--m-l----ti--kmf Sc 380 rs-ii---c-a-------t-a ... 476 flt-rew-fi-l----vv--amf Mt 204 PCRLGLTATYEREDGLHTELNRLAG ... 353 TFFIPAITYRTPSKERNSIL Ph 195 -y------fp--s-n--el-pd-i- ... 344 k-l-----hk-dk---se-- Af 199 -f-------f-----r-ei-kevv- ... 348 v-l-----h--sre--ee-- Hb 214 -y----------t--e-nv-ed-i- ... 363 e-iv-c--hq-qtd--te-- Hs 461 h-k------lv---dkivd--f-i- ... 574 rlnk-y-ygp-sqg--mq-- Sc 508 hak------lv---dkigd--f-i- ... 621 kmgk-f-ygs--qq--mn-- Mt 373 EKFRIG.SYRAVVTSKVLDEGIDVPEANVGV.IVSGTGSKRAYVQRLGRILRKKEG Ph 364 k---k-.i-k---s-q---------d-s---.-i-----p-eli--------sap- Af 368 -g--t-.rf--i-s-q---------d-----.-m--s--a-e-i--------psk- Hb 383 -r--t-.e-sml---q---------a----i.-l--sa---q-a--------pt-d Hs 594 qn-khnpkintifi---g-tsf-l-----liq-s-hg--r-qea-----v--a-k- Sc 641 qn-qyndqintifl---g-ts--l---tcliq-s-hy--r-qea--------a-r. Figure 2.1. Alignment of Rad25 protein domains. An alignment of the six helicase- defining domains identified in S. cerevisiae shows strong conservation in Archaea and Eukarya. The M. thermophila Rad25 protein sequence from M. thermophila (Mt) is aligned with Rad25 protein sequences from Pyrococcus horikoshii gene PH0450 (Ph), Archeaoglobus fulgidius gene AF0358 (Af), Halobacterium NRC-1 gene H1047 (Hb), Homo sapiens gene ERCC3 (Hs), and Saccharomyces cerevisiae gene SSL2 (Sc). Amino acid sequence identical to M. thermophila is indicated by a dash, differences are shown in lower case and gaps are shown by periods. Numerical positions within protein sequences are shown prior to each domain. Domain I Domain IV Domain VI Domain V Domain II Domain III 33 All archaeal Rad25-like proteins are truncated compared with the eukaryal proteins, primarily at the N-terminus, but they retain the core elements for helicase function (Fig. 2.2). In M. thermophila and the other archaeal species, the residues involved in nucleotide binding (domain I) are conserved, while the C-terminus is both truncated and divergent. These characteristics suggest that the archaeal protein is not involved in the typical eukaryal type of NER. Figure 2.2. Alignment of helicase protein sequences. Sequences shown are Methanosarcina thermophila, Methanosarcina acetivorans, Methanosarcina mazei, Archaeoglobus fulgidus, Pyrococcus furiosus, Thermoplasma acidophylum, Halobacterium NRC-1, Sulfolobus solfataricus, Pyrobaculum aerophilum, Saccharomyces cerevisiae, Arabidopsis thaliana, Drosophila melanogaster, and Homo sapiens. Representations are to scale and protein sequence length is indicated. Domains: I, diagonal stripe; II, black; III, vertical stripe; IV, horizontal stripe; V, gray; VI, checkered. Regions: QSKS in M. thermophila, dots; region necessary for nucleotide excision repair in S. cerevisiae, wavy lines. M. thermophila H. sapiens D. melanogaster A. thaliana S. cerevisiae Halobacterium S. solfataricus T. acidophylum P. horikoshii A. fulgidius M. mazei M. acetivorans 1 1 1 1 1 1 1 1 1 1 1 1 489 481 474 449 798 782 453 444 469 754 843 452 Ar ch ae a Eu ka ry a 34 2.4.2. Phylogenetic analysis of M. thermophila rad25. The OMNIOME genomic database was searched using BLAST with the predicted M. thermophila rad25 protein sequence to determine the distribution of this helicase among the Archaea. Protein sequences with significant identities were found in the euryarchaeotes M. acetivorans, M. mazei, A. fulgidus, Halobacterium sp NRC- 1, Pyrococcus horikoshii and Pyrococcus abyssi, and in the crenarchaeotes Pyrobaculum aerophilum, Sulfolobus solfataricus and Thermoplasma acidiphilum. These species represent mesophilic methanogens, methanogenic and non- methanogenic thermophiles, and extreme halophiles. The widespread presence of this protein among physiologically disparate groups of Archaea suggests that it has a critical function. Closely related genes are also found in non-archaeal species including the Bacteria Mycobacterium tuberculosis and Treponema pallidium, and the Eukarya Saccharomyces cerevisiae and Homo sapiens. These genes showed strong similarity in conserved DNA helicase domains, while showing greater divergence at amino and carboxyl termini (Fig. 2.2). The eukaryal sequences show a long amino- terminus extension and a shorter carboxyl-terminus extension. Deletion of the carboxyl-terminal 45 amino acids in the yeast RAD25 protein confers UV sensitivity by disrupting nucleotide excision repair functions of the protein. The archaeal Rad25 helicases do not have this carboxyl-terminus region, suggesting that an alternative repair mechanism exists in the Archaea. 35 2.4.3. Expression analysis of the M. thermophila rad25 gene. Northern analysis was used to determine whether the rad25 gene from M. thermophila was expressed during exponential growth. RNA was hybridized with a DIG labeled 1358 bp probe derived from the coding region of M. thermophila rad25 gene. Northern hybridization detected two RNA species of approximately 2.7 kb and 1.5 kb in size in M. thermophila strain TM-1 (Figure 2.3, lanes 1 and 2). The 2.7kb mRNA is 1.2 kb larger than the expected size for a transcription product consisting of only Mtrad25 gene, indicating that M. thermophila rad25 is part of an operon and is cotranscribed with the ORF located directly downstream of rad25. The function of this ORF is currently unknown. The 2.7 kb mRNA is expressed when cells are grown on both methylotrophic (Fig. 2.3, lane 1) and on aceticlastic substrates (Fig. 2.3, lane 3). Figure 2.3. Northern analysis of M. thermophila rad25 expression. A. M. thermophila rad25 probe, 57 min exposure lanes 1 and 2, and 20 min exposure lane 3. Lane 1, 20 ?g and lane 2, 10 ?g RNA from TMA-grown cells; lane 3, 10 ?g TM1 RNA from acetate-grown cells; M, RNA molecular weight markers, from top to bottom: 9.4 kb, 7.5 kb, 4.4 kb, 2.4 kb, 1.4 kb, 0.24 kb. Figure courtesy of Karin Jackson. 36 2.4.4. Identification of transcription initiation site. Primer extension analysis was performed to identify the 5' end of the M. thermophila rad25 transcript (Fig. 2.4A and B). Labeled primer 92 (Fig. 2.4A) or 93 (Fig. 2.4B) was annealed to either 5 ?g (lane 4) or 20 ?g (lane 3) of M. thermophila total RNA, and controls containing 5 ?g M. acetivorans control RNA (lane 1) or no RNA (lane 2), and cDNA was synthesized. Primer extension reactions were compared to sequencing products prepared using the same primers 92 or 93. This comparison revealed that the principal start site is the A of an ATG codon at the 5' end of rad25. Both primers 92 and 93 demonstrated the same start site in independent experiments. A canonical TATA box element and the B recognition element are both present 23 to 38 nucleotides upstream of the start site of the rad25 gene. The distance from these elements to the transcription start site conforms to the typical distance in Archaea (Fig. 2.4C) (39, 49, 95). A putative ribosomal binding site is present 78 to 83 nucleotides downstream of the transcription initiation site. A secondary weaker transcriptional start site is 78 nucleotides upstream. The secondary start site does not correlate with any known ORF or tRNA encoding region, nor is there any RNA polymerase recognition sequence upstream of this weaker start site. 37 C. -80 -70 -60 -50 -40 -30 -20 -10 | ? | | | | | | | AAAAAAATTAACTCATGCAATAAACTCCAGCCTGAGCCTGCTGATAACTGAATTATTATATATCATGTCCCGGCTCTTAGTAAACA ????? ATGATCAAAATCAGCTTCAAGCAGGGAACTATTCTCATAAAGGGAAATGTAAGGGTCCCGAACTCCATATGGGATGAAAGGAGT M I K I S F K Q G T I L I K G N V R V P N S I W D E R S GGAAGCTTCAGGGCTCCTGCAATGTATTACAGGGAAATTATCAACTACATGAGAGAGTCCAGAATTGATTTTGAAGATAGCGTC G S F R A P A M Y Y R E I I N Y M R E S R I D F E D S V CTTGACTTCCTT L D F L Figure 2.4. Primer extension analysis of the 5? terminus of rad25 mRNA from M. thermophila. Panels A and B: The 5? terminus of M. thermophila TM1 Mtrad25 mRNA was mapped to the same sites (arrows) using primer 92 (panel A) and primer 93 (panel B) Lanes:1, M. acetivorans mRNA control; 2, no RNA control; 3, 20 ?g TM1 mRNA; 4, 5 ?g TM1 mRNA; A,C,G,T, antisense DNA sequencing reactions. Panel C shows the sequence of the 5? end of rad25. The putative promoter sequence is in bold. Single arrows indicate a putative ribosomal binding site. Sequences are numbered relative to the transcription initiation sites as determined by primer extension analysis. Figure courtesy of Karin Jackson. 38 2.4.5. Detection of helicase activity in M. thermophila rad25 Recombinant Rad25 was overexpressed in E. coli, purified, and assayed to determine whether there was DNA helicase activity. The protein was incubated with a DNA helicase substrate in the presence of ATP or non-hydrolyzable analogue (AMP-PNP) (Fig. 2.5). M. thermophila Rad25 unwound the duplex DNA in the presence of ATP (Fig. 2.5, lanes 3-5) but not in the presence of non-hydrolyzable ATP (Fig, 2.5, lane 6). The helicase was shown to be 3? to 5?, and no other NTP or dNTP was able to substitute for ATP in the helicase reaction (data not shown). Figure 2.5. DNA helicase activity of Rad25 from M. thermophila. Helicase activity is indicated as primer release in the presence of Mtrad25 protein. DNA helicase activity assay was carried out with 25ng (lane 3), 75ng (lane 4), and 225ng (lane 5) Rad25 protein in the presence of 5 fmol of substrate and either 5 mM ATP (lane 3 ? 5) or AMP-PNP (lane 6). Lane 1 is substrate alone and lane 2 is boiled substrate. Assay done in collaboration with Jae- Ho Shin and Zvi Kelman. 39 A single base pair change was introduced into the Walker A motif in rad25, to determine if this motif was indeed present in the expressed protein, and if this motif was essential, as it is in other helicase proteins. This mutation changed aa 106 from lysine to glutamic acid. If this motif is present and essential for function, the mutation would cause the protein not to function as a helicase. However, it was noted during the purification that there appeared to be other proteins from the recombinant E. coli extract within the extract (Fig. 2.6A and B). The purified protein fractions from both pJK107 (Fig. 2.6A), which encoded the recombinant wild type Rad25, and pKH1 (Fig. 2.6B), which encoded the recombinant mutant Rad25, had other proteins in the extract, along with the overexpressed Rad25. These proteins could be responsible for the helicase activity, therefore the results were inconclusive. 2.4.6. Knockout of M. thermophila rad25 Gene disruption of rad25 in M. thermophila was attempted using the pac cassette as a selectable marker, which confers resistance to puromycin for mutant selection. The rad25 gene disrupted with pac was used in a homologous recombination in an attempt to disrupt genomic rad25. Unfortunately, two attempts at disrupting rad25 yielded only colonies that appeared to be inherently resistant to puromycin, as the pac cassette could not be detected in the genome of M. thermophila using PCR. The positive control strains, which were transformed with pWM313, were successful both times, indicating that the lack of colonies was not due to procedural error. 40 Figure 2.6. Polyacrylamide gel electrophoresis of purified Rad25. Fractions were collected after purification of His-tagged wildtype Rad25 (panel A) and Rad25 with a single basepair change in the 106th amino acid, changing this from lysine to glutamic acid (panel B). 10 ?l of each fraction were run on a polyacrylamide gel. The band marked by the arrow represents Rad25. Numbers at the top of the gel represent the fractions of the purified protein extract. M, protein marker. 41 2.5. Discussion The Rad25 protein sequence of M. thermophila shows strong similarity to RAD25 protein from yeast, particularly in domains I, II, III and VI. In the yeast protein, mutation of the ATP binding domain results in a lethal phenotype through inhibition of the transcription-related functions of RAD25 (88). This domain is conserved in all archaeal sequences analyzed, indicating that the ATP binding and hydrolysis functions of Rad25 are conserved between the eukaryal and archaeal domains. In the Eukarya, the Rad25 helicase participates in transcription as a component of eukaryal transcription factor TFIIH. Several components of the eukaryal transcription complex such as the TATA binding protein (TBP), TFIIB, and RNA polymerase have homologs in the Archaea (39, 47). Of the human transcription factors, a functional archaeal homolog has been identified for TBP (85, 95), TFIIB (50), TFIIS (48), and TFIIE? (46). The archaeal TFIIE? lacks the C-terminal region, which is associated with the binding of TFIIH (46, 83). This observation combined with the lack of evidence for a TFIIH complex suggests that either archaeal Rad25 is recruited to the transcription initiation complex by an alternative mechanism or the helicase has an alternative function in the Archaea. The C-terminal region, whose deletion results in a loss of nucleotide excision repair function in yeast RAD25 (88), is not present in any of the archaeal Rad25 proteins identified by genomic sequence analysis. Deletion and divergence at the C- terminus of M. thermophila Rad25 and other Rad25 homologs in Archaea suggest that transcription and nucleotide excision repair, which are linked in eukaryotes, are 42 not linked in Archaea. In M. thermophila a unique QSKS imperfectly hepta-repeated sequence is substituted. This repetitive element was not found in any other Rad25 gene, even those from archaeal species, suggesting a unique function of this protein in M. thermophila. Rad25 from M. thermophila does not contain the conserved elements for RNA helicase proteins (40, 89, 99), but it does contain the conserved elements involved in DNA helicase activity. Since Archaea lack a homolog of Eukaryotic TFIIH, which contains Rad25, it is likely that M. thermophila Rad25 is not involved in transcription, but is possibly involved in DNA replication. Genes which are similar to those found in bacterial DNA damage repair (uvrABC) have been found in Archaea, mostly in the genomes of organisms growing below 55?C (82, 106). There are also uncharacterized proteins in Archaea that contain features suggesting that they comprise a DNA repair system similar to the UmuC-DinB-Rad30-Rev1 superfamily (75). It is unclear from these results whether the Archaea follow a bacterial-like or a eukaryal-like system for DNA repair. However, it is clear that a function for Rad25 in eukaryal-type nucleotide excision repair in Archaea is not supported by analysis of the M. thermophila rad25 gene. While the promoter element preceding the M thermophila rad25 shares considerable nucleotide identity with the canonical archaeal promoter, there appears to be an unidentified open reading frame downstream of M. thermophila rad25. Analysis of the expression of M. thermophila rad25 identified an mRNA fragment larger than the expected translation product. These data indicate that rad25 from M. thermophila is co-transcribed as an operon with the downstream gene, a process common in the 43 Archaea and Bacteria (14). Future work to elucidate the function of a putative ORF downstream of rad25 may identify transcriptional components unique to the Archaea. Partially purified Methanosarcina Rad25 protein exhibits DNA helicase activity in vitro. Although rad25 was tested with both 3? to 5? and 5? to 3? specific substrates for DNA unwinding, helicase activity was detected only in the 3? to 5? direction. However, when the partially purified fractions of both the wildtype and single basepair mutant Rad25 were run on polyacrylamide genes, it was noted that there were other products that co-eluted with this His-tagged Rad25. Therefore, it cannot be ruled out that one of those proteins could contain the helicase activity that was detected in the assays. Further experiments need to be done to purify the putative M. thermophila Rad25 and to characterize its activity. Multiple attempts to disrupt gene expression of rad25 in M. thermophila were not successful. In yeast, mutation of the ATP binding domain results in a lethal phenotype through inhibition of transcription-related functions of RAD25 (88). This domain is conserved among the archaeal sequences analyzed in this paper. Because of this, it could be possible that disrupting rad25 causes a lethal mutation, which is why a disruption mutant has not been obtained. Further experiments need to be done to try and disrupt this gene, including a partial disruption of the carboxyl terminus. Since fully disrupting this gene may result in a lethal phenotype, a partial disruption may not be lethal to the organism, and the effects of the deletion on the physiology of the cells could be studied. Genome sequence analysis identified helicases in many archaeal species encompassing members of all branches of Euryarchaeota and Crenarchaeota. These 44 helicases also contain conserved domains that were found in other organisms. It is clear from these analyses that rad25 helicase genes have a broad presence in Archaea. This observation combined with experimental evidence that the gene is expressed and it deletion is lethal supports the conclusion that this gene has a critical role in cell function. Since this gene appears to be conserved among different members of the Archaea, its function is likely to be important for cellular processes. Future experimentation will be directed at identifying the specific role of this helicase in M. thermophila. One approach to determining the function of this protein would be to delete different regions of the protein, such as the carboxyl terminus, to determine what the effect was of the deletion. Another approach is to use quantitative reverse transcriptase PCR (qRT-PCR). Cells would be exposed to ionizing radiation, and the mRNA levels of rad25 transcript would be compared to wild type cells to see if this gene is upregulated when cells are exposed to radiation. The current work described in this chapter affirms that this gene is indeed expressed. M. thermophila Rad25 does not contain the conserved elements for RNA helicase activity. M. thermophila do not contain a homolog of TFIIH, the transcription factor which contains Rad25 in Eukaryotes. These two pieces of evidence indicate that Rad25 is most likely not involved in transcription. It does, however, contain conserved elements for a DNA helicase, indicating that it may have a role in DNA replication. Further work, however, needs to be done in order to confirm the role this protein has in Archaea. 45 Chapter 3: Identification of trans factors involved in the regulation of carbon monoxide dehydrogenase in Methanosarcina species 3.1. Abstract Expression of the archaeal CO dehydrogenase/acetyl-coenzyme A synthase (CODH/ACS) operon (cdhABCDE), which catalyzes the dismutation of acetyl CoA in the aceticlastic methanogenic pathway, is highly regulated by Methanosarcina spp. in response to growth on acetate versus the methylotrophic substrates methanol and trimethylamine. However, the mechanism of this regulation is unknown. Many of the known mechanisms of regulation within the Archaea involve repressor or activator proteins that bind upstream of the start of transcription to either inhibit or activate transcription. Using the sequence upstream of the transcriptional start site from -256 to -76, electrophoretic mobility shift assays revealed differential protein binding between extracts of Methanosarcina acetivorans grown with trimethylamine or acetate. This same sequence was used as a substrate in affinity chromatography, which confirmed the differential binding. Affinity chromatography, coupled with two-dimensional gel electrophoresis, allowed for the identification of differentially binding proteins. CdhA, CdhB, and CdhD were all identified as binding to the sequence upstream of the transcriptional start site. Competition assays to test the binding of a mostly purified extract containing the Cdh complex indicated that this extract contained proteins able to bind to DNA, but the binding was not specific. Using a LacZ expression strain, it was shown that deletion of the region upstream of 46 the transcriptional start site did not have an effect on expression. Based on the results of these studies, it was concluded that cdhABCDE is not regulated via trans factors located upstream of the transcriptional start site. In contrast to other archaeal genes that are regulated by binding of either a repressor or activator protein to DNA in proximity of the promoter, the CODH/ACS operon does not appear to be regulated by mechanisms that require activators or repressors. 3.2. Introduction Approximately 70% of the biogenic methane in the environment is derived from acetate (71) and the regulation of the genes involved in the aceticlastic pathway likely has a significant impact on anaerobic degradation processes and the global biogenesis of methane. There are only two genera reported to date whose members are capable of using acetate as a substrate to produce methane: Methanosarcina and Methanosaeta (111). While Methanosaeta species are obligately aceticlastic, Methanosarcina species are able to grow on a variety of substrates, including CO2 with hydrogen, methylamines, methanol, and acetate. Catabolic genes in Methanosarcina spp. are highly regulated in response to substrate. However, when given a choice between two substrates, Methanosarcina species will preferentially use the substrate with the higher free energy, like the methylotrophic substrates trimethylamine and methanol, before using acetate (17, 34, 60). The high metabolic diversity, combined with the availability of a tractable genetic system and complete genome sequence from three species, makes members of the genus Methanosarcina 47 spp. ideal models to study the regulation of catabolic gene expression in the methanogenic Archaea. There are several enzymes involved in aceticlastic methanogenesis, including carbon monoxide dehydrogenase/acetyl CoA synthase (CODH/ACS) (1, 2, 43, 61, 72). This enzyme catalyzes the dismutation of acetyl CoA. The genes encoding this subunit include an operon of 5 genes, with a 5? leader region located downstream of the transcriptional start site. Previous studies have shown that this gene, cdhABCDE, is regulated in response to substrate (5, 114, 121). It has also been demonstrated that this gene is regulated at the level of transcription (114). However, the mechanism of this regulation is not understood. Transcriptional regulation in Archaea has been a topic of many studies. For example, within the literature, there are descriptions of transcriptional activators. Some of these activators include Methanocaldococcus jannaschii Ptr2 (87), Halobacterium Bat (9), and Sulfolobus solfataricus LysM (22). There are also examples of repressor proteins, such as Pyrococcus furiosus LrpA (15), Methanococcus maripaludis NrpR (68), and Archaeoglobus fulgidus Mdr1 (86). All of these transcriptional regulators bind to DNA upstream of the transcriptional start site to affect transcription. The purpose of this study was to identify trans elements that mediate expression of the catabolic CODH/ACS complex in the aceticlastic Methanosarcina. The regions both upstream and downstream of the transcriptional start site from Methanosarcina acetivorans C2A cdhABCDE were amplified with PCR and used as a template to determine if differential protein binding was occurring upstream of the 48 transcriptional start site. Differential binding was detected and further analyzed to identify and characterize the proteins that appeared to be capable of binding. 3.3. Materials and Methods 3.3.1. Methanogen strains and media Methanosarcina acetivorans C2A (=DSM 2834), Methanosarcina thermophila TM-1 (=DSM 1825), and Methanosarcina barkeri Fusaro were obtained from sources described previously and maintained as frozen stocks (115). Artificial marine mineral medium was prepared for growth of methanogenic Archaea by methods described previously (113). Growth substrates used were sodium acetate or trimethylamine-HCl (TMA) at a final concentration of 0.05 M. Methanogen medium was prepared anaerobically in a N2-CO2 (4:1) atmosphere by a modified Hungate technique (110). M. acetivorans strains were incubated at 35 ?C. 3.3.2. Electrophoretic Mobility Shift Assay Electrophoretic mobility shift assays (EMSA) involved three different 100bp double stranded DNA oligonucleotides (dsDNA) generated by annealing two complimentary oligonucleotides. The synthesized oligos were used as substrates for binding of proteins from cell free extract. The sequence of the first oligonucleotide was 5?- CTATATAATTATTTGTTTTTAATATGAAAAAAAGAAATAATAAATAACAA ATATTAAAATCGACGAAAAAAAATATAGAGGCTGAAGAAGGTCAAATCA A-3?, and the second oligonucleotide sequence was 5?- 49 GCTGAAGAAGGTCAAATCAAAAAAACGGCAACATAAAGCCTATAGACTA TTTTTCACGAAATTGATCTTCGAGATTTTTTTTACGAATAGTGTGCCTCTA- 3?. These two oligos were from a region -256 to -156 and -176 to -76 bp upstream of the transcriptional start site. A third oligonucleotide from the coding region of MA1017 was used as a control for nonspecific binding of proteins: 5?- CGTCTTTGCGACCTTCTCCAGATCGAGCAGAATGAGGCTGCTGACCGGAT GGGAATATCCCGAAAAACTTTCTGGAGCGACCTCCAAAGGGCACGGCAA A-3?. This oligo was used to look at nonspecific binding of proteins, to rule out any proteins that were able to bind to both this substrate and the specific substrate. DNA labeling with [?-32P]ATP and purification were performed as described previously (92). Cell-free extracts of acetate- and trimethylamine-grown M. acetivorans in exponential phase were made by resuspending 2 g of cells in 4 ml 10 mM 4-(2- hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.0, containing 1 mM phenylmethylsulphonylfluoride. The cells were lysed by passing cell suspension through a French pressure cell at 140 mPa and lysate was centrifuged at 10,000 x g for 20 min to obtain cell-free extract. Complexes formed between the proteins in the cell-free extract and dsDNA were detected by a gel mobility shift assay in reaction mixtures (15 ?l) containing 20 mM HEPES-NaOH (pH 7.5), 10 mM MgCl2, 1 mM dithiothreitol, 100 ?g/ml bovine serum albumin, 2 ?L (60-100 fmol) of one of three 32P-labeled oligonucleotides mentioned above (3000-6000 cpm/fmol), and 1 ?L (20 ?g) of cell-free extract from either acetate-grown or trimethylamine-grown cells. Samples were incubated at either 4?C or 37 ?C for 30 min. This was done to inhibit any nuclease activity in the 50 extract. 5 ?l of 5x loading buffer (0.1% xylene cyanol, 0.1% bromphenol blue, 50% glycerol) was added to the reaction. Aliquots of the reaction mixture were electrophoresed for 4 h at 150 V through an 8 % polyacrylamide gel containing 5 % glycerol in 0.5x TBE (45 mM Tris, 45 mM boric acid, 1 mM EDTA). The images were visualized using a Storm phosphoimager (Molecular Dynamics). 3.3.3. Affinity chromatography of DNA-binding proteins. Both the sequence upstream of the transcriptional start site and the 371 bp leader region were amplified by PCR. The 204 bp sequence upstream of the promoter was amplified from M. acetivorans genomic DNA with primers EMSA1- bio and EMSA2 (Table S3.1), which created a 204 bp product labeled with biotin. The 371bp sequence immediately downstream of the promoter was PCR amplified from both M. acetivorans and M. thermophila with primers UTR1-bio and UTR2 (Table S3.1), which created a biotin labeled product. The biotin labeled-PCR products (1 ?g) were bound to streptavidin-coated paramagnetic beads (Promega). The DNA-bead complex was incubated for 30 minutes at room temperature with 400 mg cell free extract from either acetate- or TMA-grown M. acetivorans, which were made by resuspending 2 g of cells in 4 ml 10 mM HEPES, pH 7.0, containing 1 mM phenylmethylsulphonylfluoride. The cells were lysed by passing cell suspension through a French pressure cell at 140 mPa and lysate was centrifuged at 10,000 x g for 20 min to obtain cell-free extract. The DNA-bead complexes were washed to remove any unbound protein, and DNA-binding proteins were eluted in 0.1% SDS. The eluted proteins were run on a 10% SDS-PAGE and were silver stained. 51 3.3.4. Two-Dimensional analysis of DNA binding proteins. The DNA region used as a substrate for the gel mobility shift assay was also used to purify potential DNA binding proteins. The 204 bp region was amplified by PCR from M. acetivorans and labeled with biotin using primers EMSA1-biotin and EMSA2 (Table S3.1). The biotin labeled-PCR product was bound to streptavidin- coated paramagnetic beads (Promega), and this was incubated with 900 mg cell free extract from either acetate- or TMA-grown M. acetivorans. The DNA-bead complexes were washed to remove any unbound protein, and DNA-binding proteins were eluted in 0.1%SDS. 9 mg of eluted proteins were run on using 2-dimensional gel electrophoresis, the gels were silver stained and analyzed to determine differential binding. Two dimensional electrophoresis was conducted according to the principles of O?Farrell (81) and as outlined by Gorg et al. (41) and Brady, et al. (19). A total of 14 spots were chosen for identification by the Mass Spectrometry Lab at the University of Texas Medical Branch using peptide mass fingerprinting. 3.3.5. Competition assays Competition assays were used to determine the specificity of binding of Cdh complex. This method used one 204 bp oligo that was generated by PCR using primers EMSA1 and EMSA2 (Table S3.1) using genomic DNA from M. acetivorans. This oligo was located beginning at -251 bp upstream of the start of transcription. Unlabeled specific competitor DNA was generated using the same PCR primers. Unlabeled nonspecific DNA was generated from M. thermophila DNA using primers 292 and 293 (Table S3.1), which amplified a 200bp region of rad25 within the 52 structural gene. The assay was performed as described above, except partially purified Cdh was substituted for cell-free extract. 16?g of partially purified carbon monoxide dehydrogenase complex (Cdh) from M. thermophila, a gift from David A. Grahame, was incubated with 50ng labeled oligo in the presence of increasing amounts of unlabeled specific and nonspecific competitor DNA to confirm the binding ability of this protein. Excesses of 50X, 100X, and 200X of both specific and nonspecific competitor DNA were used. This method was also used to test for binding of the high molecular weight extract to the same 200bp region of M. thermophila and M. barkeri. The 200 bp templates for these reactions were located at -251 bp upstream of the start of transcription and were generated using primers TM1up1 and TM1up2 for M. thermophila and Barkeri-up1 and Barkeri-up2 for M. barkeri (Table S3.1). 3.3.6. Construction of cdh?::lacZ deletion plasmids Deletions upstream of the transcriptional start site were generated from the pEA64 (Table 3.1) template using reverse primer 60 (Table S3.1), which hybridized within the plasmid with forward primers that generated ClaI restriction sites at different locations upstream of the cdh promoter. Forward primer 62 generated a ClaI restriction site 262 bp upstream of the transcriptional start site to create ?62. Forward primer 63 generated a ClaI restriction site 71 bp upstream of the transcriptional start site to create ?63. Forward primer 64 generated ClaI restriction site 29 bp upstream of the transcriptional start site to create ?64, which deleted part of the cdh promoter TATA box. PCR generated DNA fragments were cloned directly into pCR2.1 53 (Invitrogen), then digested with NsiI/SalI and cloned into the NsiI/SalI restriction sites of plasmid pcdhp::lacZ. The latter plasmid was digested with XhoI/BamHI and the fragment containing the cdh-lacZ fusion was ligated into the lacZ reporter shuttle vector pEA103 replacing wildtype cdh sequence. Once the plasmids were transformed into M. acetivorans, the sequence of each plasmid was verified by extracting the plasmid and sequencing for confirmation. Table 3.1. E. coli and M. acetivorans plasmid constructs. Plasmids Parent Characteristics or description of construct Source pCR2.1 --------- Cloning vector, AmpR, PurR, lacZ ? Invitrogen pCDH1.3 --------- cdhA?, AmpR (114) pcdhp::lacZ --------- cdhA?::lacZ, AmpR, Ori pBR322 (5) pEA64 pEA61 Cdh?::CAT, Ori pC2A, AmpR, PurR , Ori R6K This study pEA103 pWM307 Cdh?::lacZ, Ori pC2A, AmpR, PurR , Ori R6K (5) pEA108 pEA103 Cdh??62::lacZ, Ori pC2A, AmpR, PurR , Ori R6K This study pEA109 pEA103 Cdh??63::lacZ, Ori pC2A, AmpR, PurR , Ori R6K This study pEA110 pEA103 Cdh??64::lacZ, Ori pC2A, AmpR, PurR , Ori R6K This study 3.3.7. Reporter gene expression analysis. M. acetivorans transformed with either the wild-type reporter plasmid pEA103 or deletion plasmids were inoculated (10% v/v) in triplicate into liquid medium containing 0.1 M sodium acetate, TMA, or methanol and incubated to mid- exponential growth phase. Cultures were sampled (1.0 ml) to perform ?- galactosidase assays as described previously (5). 1 ml samples were centrifuged at 13,000 rpm for 5 minutes. Cell pellets were resuspended in 1 ml Z Buffer (60 mM Na2HPO4?7?2?, 40 mM NaH2PO4?2H2O, 10 mM KCl, 1 mM MgSO4?7H2O, pH 7.0). Cells were sonicated and centrifuged at 13,000 rpm for 15 minutes. The 54 supernatant was transferred to a new tube. The protein concentration of the supernatant was determined using the Bradford assay. Triplicate samples of the supernatant (100 ?l) were combined with 700 ?l Z Buffer + ?-mercaptoethanol (27 ?l/ 10 ml Z Buffer). The mixture was incubated at 30 ?C. 160 ?l ONPG (4 mg/ml) was added to each mixture, and the time for the color to change to yellow was measured. The reaction was stopped with the addition of 400 ?l 1 M Na2CO3, and the reactions were assayed for absorbance at 420 and 550 nm. 55 3.4. Results 3.4.1. Identification of putative protein binding sites using electrophoretic mobility shift assays. In four methanosarcinal species, the region located downstream of the transcriptional start site of cdhABCDE is highly conserved, with similarity between 80-90%. In contrast, the overall sequence upstream of the transcriptional start site was less conserved between the four methanosarcinal orthologs, and the formation of secondary structures was not evident. However, short lengths of sequence were conserved among species, which suggested that protein binding might occur in these regions (Fig. 3.1). Electrophoretic mobility shift assays (EMSA) were conducted to identify potential regions of protein binding. Two radiolabelled 100 bp oligonucleotides, oligo #1 and oligo #2, whose sequences were located at ?256 to ? 156 bp and ?176 to ?76 bp, respectively, were incubated with cell-free extract of M. acetivorans grown with acetate and trimethylamine to determine if the catabolic substrates caused differential protein binding to the oligonucleotides (Fig. 3.2). Differential mobility shifts were observed between the acetate-grown cell-free extract (extract A) and the trimethylamine-grown cell-free extract (extract T) with both oligonucleotides. Incubation of the reaction mixture at both 4?C and 35?C did not have a significant effect on the banding patterns. The two different temperatures were used to control for nuclease activity within the cell free extracts. 56 M. thermophila TATGAAGAATCTTTAAAATGAGTTGTGGTTAGTATGAAAAAATGAAAAAAATAATAACAA -208 M. acetivorans --g---a-----a--t---t-t----tt---a----------a----t--ta-------- -208 M. mazei --a---a--------gggct-t---cac---------g-----att-g------------ -207 M. barkeri --ac--at--t-ac-t---a-t----t--g-ac-c--gt---aagcgt--t-gt---t-- -205 M. thermophila ATATTAAAATCAATGGAAAAAACTATTGGGAATCAAGAATACTCATTCAAAAAAACCAGT -148 M. acetivorans -----------g-c-a------a---a-a-gc-g-----ggtca-a----------ggca -148 M. mazei -------------ata------a-----a--tc---t-t-----tcatt-------g-ta -147 M. barkeri t--a--gc-ca--g..--gt---aca-ca-tg-t-ta-tggt-tt-a-tg-------cac -147 M. thermophila ATCTACAGGCATTGAGGGCTTTTCTTTCGGAGTAGATCTTCAAGA.TTTTTTTATGAAGA -89 M. acetivorans -ca--a-.--c-at--acta---t-ca--a-a-t-------g---t--------c---t- -89 M. mazei tg--ttgacatagagtat-----t-gatca-aa--tct--g-g-tt------a-ga-ga- -87 M. barkeri -----------c-a-tct-----t-g-accta--at-acaat-a-c--g-aaag-c--ct -87 M. thermophila AGGTATCACTAAATTCCATTTTACCCATTGCGAAATAGGGTGTTACAAATTAGAAAATGT -29 M. acetivorans gt--gc-t---c------c---------ca------------------------------ -29 M. mazei c--gtctc-ac-t-c-at-------tg--cg-..---a---------------------- -29 M. barkeri gat-t-g-ac-ttcc-ac---ac--atc-cga--tgc..-------------------a- -29 M. thermophila TATATAGATTTGGTACATTTAGACTTTTAATTAGTGTTTATTATCCATCGGTAGCGACCT +32 M. acetivorans ------------------------------------------------------------ +32 M. mazei ----------------------t-ac---------------------------------- +32 M. barkeri ----------------g--a----a----------------------------------- +32 M. thermophila CTGCTCAAGATTAAGGTCCCAATCGTTGGTAAAAACGGTTTTTTTGAGACATAGGGCGTA +92 M. acetivorans ------------------t----------c---------------a---t---------- +92 M. mazei ------t----------------------c-------------------a-a-------- +92 M. barkeri -----t-g----------t----------c---------------------a-------- +92 M. thermophila AAAAGGGATTTGGAAGAGAAATAAGGCCCTAAAAACGACTTTTACAAG.AATATATTCTC +151 M. acetivorans ----a-a----a-------t--t-a------------------gg---.g-a------c- +151 M. mazei ----a------t------c-t-g-t-------------t---------a--a------c- +152 M. barkeri ----t--c---.----t-tg-at-a-------------------t-t-.g-a-----tc- +151 M. thermophila TATAAAAACGTTCTTATTTGAAAAAAACGTTGGTATAACGTTTAATGTCCAAATTGAGAG +211 M. acetivorans ----------gct-------.------t--------g------g-g--t-----c----- +210 M. mazei --c-------c-t-------.--------c-----a---c--c---a-t-----a----- +211 M. barkeri -c--------act---gc--g-----cgc-g-ta-a-tat--gttat-tg---aaa--t- +211 M. thermophila CTATGAACGTTTTACCATAAAACGTCAT...TTAAACGGTTGTAACTTTGAAGCAGACGG +268 M. acetivorans ---aa--------g---g----t-c---...------------------a-g--g----- +267 M. mazei ---a--gt-----g----g---------...--------------a---a---------- +268 M. barkeri g-gaat-gtg---ta-cag-tg---at-tac---c--------------a-.-------- +270 M. thermophila TAAGATATAAAATGAATAATCATTAACAATATGCCGCTT..ATCAGGATTACAATCGAAT +326 M. acetivorans ----g----t----t-----------g----------a-..-a....--ag--gc--cca +321 M. mazei c--a-----t---t------------g---------aag..--a--.---g------tc- +325 M. barkeri c---gag.....--tt-gg-a----ca---------aagaat-a-aat--g------tc- +325 M. thermophila TGGCGAAGTCGACTTTAAACAAATTAAGGAGGTAAAGCTCACATGAGCAAACTAACTACC +386 M. acetivorans -------------------------t---------------a------------------ +361 M. mazei ---------t---------------t---------------a------------------ +365 M. barkeri --at-------------t-------t---------------a------------------ +365 M. barkeri --------------------a- Figure 3.1. Alignment of cdhA promoter region from four Methanosarcina species. Numbering in right column is based on the transcription start site for cdhABCDE (114). The start of transcription is marked with an arrow and +1. The start of translation (ATG) is underlined. 57 Figure 3.2. Electrophoretic mobility shift assay of DNA fragments from the region upstream of the cdhA promoter. Each band represents a dsDNA fragment with bound protein. Minichromosome maintenance (M) protein and HmtB histone (H) from Methanothermobacter thermoautrotrophicum were used as positive controls. Oligonucleotides 1 and 2 are 100bp fragments beginning 256bp and 176bp upstream of the start of transcription, respectively. Oligonucleotide NS is a 100bp fragment from a structural gene (MA1017), used as a non-specific sequence. 20?g of either acetate-grown cell free extract (A) or TMA-grown cell free extract were added to each mixture. Reaction mixtures were incubated at 4?C and 35?C for 30 minutes. Lanes labeled with (?) for extract represent samples in which no extract was added. Arrows indicate some of the differentially shifted bands. #1 #2 NS - A T - A T - A T A T A T A T #1 #2 NS Extract Oligo Temp 4?C 35?C M H A=acetate-grown cell extract T=TMA-grown cell extract 58 3.4.2. Isolation and sequence analysis of DNA binding proteins using affinity chromatography. It was investigated whether differential protein binding could be the mechanism of regulation within the 371 bp untranslated region using modified affinity columns. Proteins that were able to bind to this DNA region were separated by running extracts over streptavidin-labeled paramagnetic beads bound to biotin labeled DNA. The DNA used for this experiment was PCR generated fragment of the 5? leader sequence located downstream of the transcriptional start site. The proteins that could bind were eluted and visualized to determine if there was differential protein binding. Differential protein binding patterns were observed between the acetate and trimethylamine grown cell free extract, but the binding patterns for each extract were the same as those present within the negative control columns (lanes 1 and 2) that contained affinity beads with no DNA attached (Figure 3.3). This indicates that the binding detected was nonspecific in nature, and that differential protein binding to DNA in this region is most likely not the cause of the difference in expression. 59 Figure 3.3. Polyacrylamide gel electrophoresis of proteins isolated from affinity chromatography with the 5? 371 bp leader sequence DNA of the cdh operon bound to biotin beads (lanes 3 and 4). 400 mg of cell free extracts from acetate-grown cells (A) or TMA-grown cells (T) were passed over the columns. Proteins that bound were eluted and run on the gel. Lanes 1 and 2 were negative control columns, without bound DNA. To identify which DNA binding proteins that bound upstream of the transcriptional start site might be involved in differential expression of cdh, DNA sequence that was used in the EMSA was labeled with biotin and bound to streptavidin-coated paramagnetic beads. Proteins that bound to this DNA region were eluted and analyzed using two-dimensional gel electrophoresis (2DGE) (Fig. 3.4). 60 There were several proteins that differentially bound to the DNA, both in the acetate and the TMA-grown cell-free extract. Fourteen of these proteins were eluted from the gel for identification by peptide mass fingerprinting (Table 3.2). Some of the eluted proteins could not be identified, either due to low quantity of the protein or because the protein sequence was not in the databases. Protein spots 1, 2, and 3 from acetate-grown extract were identified as three products of the cdh operon, CdhA (alpha subunit), CdhB (beta subunit), and CdhD (delta subunit). Protein spot 14 from trimethylamine grown extract was identified as a corrinoid protein associated with trimethylamine methyltransferase. 61 Figure 3.4. Two-dimensional gel electrophoresis (2DGE) of DNA-binding proteins from acetate- and TMA-grown cell free extract. 1 ?g of 200 bp DNA template, which was located -251 bp upstream of the transcriptional start site of cdhA, was bound to strepavadin paramagnetic beads and incubated with 900 mg of either acetate-grown or TMA-grown cell free extract. 9 mg of eluted proteins were run on 2DGE. Panel A, cells grown with acetate. Panel B, cells grown with trimethylamine. Circles and numbers refer to spots that were identified through peptide mass fingerprinting. 62 Table 3.2. Identification of protein spots from two-dimensional gel electrophoresis. Protein Spot Identification Acetate CFE1 1 Acetyl-CoA decarbonylase/synthase complex alpha subunit 1 2 Acetyl-CoA decarbonylase/synthase complex beta subunit 1 3 Acetyl-CoA decarbonylase/synthase complex delta subunit 1 4 ND3 5 ND 6 Methyl coenzyme-M reductase, gamma subunit (MA4847) TMA CFE2 7 DNA topoisomerase VI subunit A 8 Thiamine biosynthesis protein ThiC (MA4239) 9 Thiamine biosynthesis protein ThiC (MA4239) 10 Elongation factor Tu (MA1256) 11 Elongation factor Tu (MA1256) 12 ND 13 ND 14 Trimethylamine corrinoid protein (MA0529) 1Acetate CFE = cell free extract from acetate grown M. acetivorans 2TMA CFE = cell free extract from trimethylamine grown M. acetivorans 3ND = unable to determine identity 3.4.3. Specificity of Cdh binding To confirm the DNA-binding abilities of Cdh, partially purified Cdh complex from M. thermophila was used in a gel mobility shift assay (Fig. 3.5). The substrate for this assay was the DNA sequence from M. acetivorans used previously for affinity chromatography. A competition assay was also conducted to determine if the binding of Cdh was specific. An excess of unlabeled specific or nonspecific competitor DNA was added to the reactions. Figure 3.5 shows the results of the competition assay. As the amount of specific competitor increased, the bands representing protein binding 63 decreased in intensity. When nonspecific competitor was used, the band intensity decreased for only one band; however, the intensity decrease did not seem to be significant enough to indicate the binding was specific. These results confirm the binding ability of a protein in the Cdh extract, but do not confirm the specificity of binding. Experiments were also conducted to determine if Cdh was able to bind to the same 200bp region in other Methanosarcina species (Fig. 3.5). This region was PCR amplified from the genomes of M. thermophila and M. barkeri. The DNA was then incubated with the Cdh extract to determine if binding occurred with these sequences as well. The Cdh extract was also able to bind to these DNA sequences. As was the case with the DNA from M. acetivorans, the binding of this Cdh extract did not appear to be specific. Figure 3.5. Electrophoretic mobility shift assay with 16 ?g partially purified Cdh complex binding to region upstream of cdh transcriptional start site. Mobility shift using 50 ng of 200 bp oligonucleotide from flanking region 266 bp upstream of cdh promoter. Lanes 1-8, DNA from M. acetivorans. Lane 1, labeled DNA probe only. Lane 2, labeled DNA probe and Cdh only. Lanes 3-5, 50X, 100X, and 200X specific competitor DNA, respectively. Lanes 6-8, 50X, 100X, and 200X nonspecific competitor DNA, respectively. Lanes 9-12, DNA from M. thermophila. Lanes 9-10, 50X and 200X specific competitor DNA, respectively. Lanes 11-12, 50X and 200X nonspecific competitor DNA, respectively. Lanes 13-16, DNA from M. barkeri. Lanes 13-14, 50X and 200X specific competitor DNA, respectively. Lanes 15-16, 50X and 200X nonspecific competitor DNA, respectively. Nonspecific competitor DNA was a 200 bp fragment from within the structural M. thermophila rad25. 65 3.4.4. Effect of sequence deletions on the regulation of cdh by different substrates. In a prior report that employed a translational fusion of the cdh promoter to lacZ, CODH/ACS from M. thermophila TM1 was shown to be up-regulated up to 54- fold during growth on acetate, compared with growth on methanol or trimethylamine (5). To determine whether sequences located upstream of the transcriptional start site might be involved in CODH/ACS regulation, a series of deletions were generated upstream of the promoter (Fig. 3.6A). The cdh deletion sequences were ligated into the lacZ reporter plasmid as translational fusions and transformed into M. acetivorans. The effects of these deletions on the expression of ?-galactosidase in response to different substrates are shown in Fig. 3.6B. Deletion of DNA sequences upstream of the promoter (?62 or ?63) did not have a significant effect on expression of ?-galactosidase compared with wild type sequence in cells grown with acetate, trimethylamine or methanol. These data indicate that this region is not involved in regulation of expression based on substrate. 66 A. B. 0 20 40 60 80 100 WT ?62 ?63 ?64 Deletion construct Fo ld dif fer en ce Acetate versus Methanol Acetate versus TMA Figure 3.6. Map of deletions upstream of the Methanosarcina thermophila TM1 cdhA transcriptional start site and the effects of those deletions on regulation by different substrates. Panel A shows the location of the upstream deletions. Position numbers above relative to start of transcription indicated by arrow. RBS is the location of the predicted ribosomal binding site. Panel B shows the fold difference in LacZ expression for either acetate vs. methanol or acetate vs. TMA grown cells. +1 +371 -751 cdh? lacZ -262 -71 -29 +335 RBS ?62 ?63 ?64 C2A/pEA103 67 3.5. Discussion When grown on acetate, members of the genus Methanosarcina generate CODH/ACS, a key enzyme complex that catalyzes the dismutation of acetate and acetylation of coenzyme CoA (43, 61). This gene was previously shown to be regulated at the level of transcription (114). Since the majority of known mechanisms of transcriptional regulation in Archaea involve trans factors such as activators or repressors, cdhABCDE was studied to determine if trans factors might be involved in regulation either upstream or downstream of the transcriptional start site. Downstream of the transcriptional start site of cdhABCDE there is a 371 bp 5? leader region. Long 5? UTRs are involved in transcriptional regulation in bacteria via multiple mechanisms. Usually, these mechanisms involve the formation of secondary structures within the RNA (128, 131). Some of these mechanisms also involve trans factors that bind to cause conformational shifts in the RNA. Using affinity chromatography, it was concluded that, although there was differential binding between substrates, the binding was the same as the negative control, indicating that the binding was nonspecific. Therefore, the results indicate that there are no apparent trans factors binding to this region downstream of the transcriptional start site under the conditions that were tested. Further experiments would need to be conducted to determine if there are any substrate specific DNA-binding proteins that can bind to this region under different conditions. The region within 232 bp upstream of the cdhABCDE transcriptional start site was also studied to determine if this region contained the binding site for DNA- binding proteins. Gene regulation involving both repressors and activators binding 68 upstream of transcriptional start sites has been reported in the Archaea (13, 38). Within the methanogenic Archaea nitrogen regulation is controlled by a helix-turn- helix DNA-binding repressor protein, NrpR (68, 69). The binding of this protein to DNA directly interferes with the binding of RNA polymerase, repressing expression of genes involved in nitrogen fixation. Since the 100 bp sequences used in this study were located 256 bp and 176 bp upstream of the transcriptional start site, it is unlikely that regulation of CODH/ACS expression occurred by occlusion of RNA polymerase binding to the promoter, which has been shown to bind to DNA around 29 bp upstream of the start of transcription in DNA footprinting studies in Methanococcus vannielii (122). Although homologs to a number of repressor type proteins encoding helix-turn-helix motifs have been identified in the annotated sequences of methanosarcinal genomes, palindromic sequence typically associated with their binding to DNA were not detected upstream of the CODH/ACS promoter. However, analysis of this region revealed sequences that are similar to known eukaryotic activator protein binding sites, including N-Oct-3, which regulates transcription of neuronal cells, NIT2, a global-acting positive regulator of nitrogen-utilizing genes, and GCN4, a yeast transcriptional activator protein (101). These proteins, however, have no known homologs in Methanosarcina spp. Several proteins were shown to bind differentially to this region in response to aceticlastic (CdhA, CdhB, and CdhD) or methylotrophic (MttB) growth. However, further analysis of a partially purified extract of Cdh complex did not prove this binding was specific. The binding of MttB is intriguing as this suggests that binding 69 of this protein may have a role in suppressing cdh expression during methylotrophic growth, but studies with purified MttB are required to confirm a role for this protein. The results of this study indicate that the region between -256 and -78 is most likely not important in the regulation of cdhABCDE. The results of these experiments did not detect any regulatory proteins, either putative or confirmed, that were binding to this region upstream of the cdhA transcriptional start site. Analysis of the sequence upstream of the transcriptional start site did not detect any motifs that are usually associated with DNA-binding proteins, such as direct repeats. Using a lacZ translational fusion to determine the effects of different substrates on expression, it was shown that the region upstream of the start of transcription was not involved in regulation. There also does not appear to be specific sequences for DNA-binding proteins within the 5? leader region located downstream of the transcriptional start site. The majority of regulatory mechanisms found in Archaea involve DNA- binding proteins. The results presented in this chapter indicate that trans acting elements are not involved in the regulation of expression of CODH under the conditions described in this chapter. Deletions made within the region located upstream of the transcriptional start site indicate that this region is not involved in regulation. However, further experiments are needed to determine if there are any trans acting elements capable of regulation the expression of CODH under condition other than those used in this chapter. These experiments should focus on the 5? leader region and should encompass putative trans elements that may act on the DNA or the RNA sequence. 70 Chapter 4: A 5? leader region regulates expression of Methanosarcinal CO dehydrogenase/acetyl-coenzyme A synthase 4.1. Abstract Expression of the archaeal CO dehydrogenase/acetyl-coenzyme A synthase (CODH/ACS) operon (cdhABCDE), which catalyzes the cleavage of the acetyl C?C bond of acetyl CoA in the aceticlastic pathway, is highly regulated by Methanosarcina spp. in response to growth on different substrates. However, the mechanism of CODH/ACS regulation is unknown. A highly conserved 371 base pair untranslated leader region (UTR) was identified as a cis regulatory element of this gene in vivo by deletion analysis using translational fusions. Deletions within the UTR significantly reduced the differential expression of the operon between aceticlastic and methylotrophic growth observed in wild type cells. One apparent regulatory region located within the leader region contains several putative mRNA secondary stem-loop structures, one of which appears to have a poly-U region typical of mechanisms associated with differential transcription termination. Deletions of these putative structures also had an effect on expression under methylotrophic substrates. Comparison of LacZ amounts and mRNA amounts, as calculated by quantitative reverse transcriptase PCR (qRTPCR) showed that protein and mRNA levels were not significantly different, indicating that translational regulation was most likely not a factor. QRTPCR along different lengths of the UTR showed a 15 fold difference in transcript levels 358 bp downstream of the 5? end, indicating that 71 approximately 22% of cdh regulation was the result of differential transcription initiation; the remaining difference in cdh expression observed was the result of differential transcript levels near the 3? end of the UTR. Transcript stability studies confirmed that differential transcript levels observed near the 3? end resulted from elongation termination rather than differential message degradation. In contrast to other archaeal genes that are regulated by a repressor or activator binding near the promoter, the CODH/ACS operon appears to be attenuated by a novel mechanism located within the conserved leader region that has not been described previously in the Archaea. The results indicate that methanosarcinal CODH/ACS expression is controlled by multiple mechanisms as part of the regulatory strategy employed by these methanogenic Archaea to efficiently direct carbon and electron flow in anaerobic consortia during fermentative processes. 4.2. Introduction Methanogenic species in the genus Methanosarcina are the most metabolically diverse among methanogens, with the ability to grow by CO2 reduction with H2, methyl reduction with H2, aceticlastic fermentation of acetate, or methylotrophic catabolism of methanol, methylated amines, and dimethylsulfide (125). Methanosarcina acetivorans is also reported to grow with CO (93). These organisms preferentially use substrates with higher free energy, such as methanol and trimethylamine, before utilizing acetate, exhibiting diauxic growth in the presence of both types of substrates (17, 34, 60, 74, 107). Despite their preference for non- aceticlastic substrates and the relatively lower growth rates and yields of 72 Methanosarcina spp. with acetate, approximately 70% of the biogenic methane produced globally is generated from the catabolism of acetate (71). Therefore, regulation of genes involved in the aceticlastic pathway is likely to have a significant impact on anaerobic degradation processes and the global biogenesis of methane. Several enzymes associated with aceticlastic methanogenesis, including acetate kinase, phosphotransacetylase, carbonic anhydrase, and carbon monoxide dehydrogenase/acetyl CoA synthase (CODH/ACS), are regulated in response to substrates (52, 54, 65). Expression of the CODH/ACS operon (cdhABCDE), which catalyzes the catabolism of acetyl CoA in the aceticlastic pathway, is highly regulated in response to growth on acetate compared with methanol and trimethylamine (TMA), but the mechanism(s) of this regulation is not known (114, 121). Two CODH/ACS operons are detected in the annotated genome sequences of M. acetivorans (37), Methanosarcina barkeri (73) and Methanonsarcin mazei (30). In contrast, only one cdhABCDE operon has been detected in M. thermophila (42). In M. acetivorans, both of these operons appear to be regulated in response to substrate, although the extent of the regulation is greater in one (MA3680) than the other (MA1016) (65). Northern analysis of CODH/ACS mRNA from Methanosarcina thermophila indicates that the regulation of this CODH/ACS ortholog in response to substrate occurs, at least in part, at the level of transcription (114). The regulation of this ortholog was corroborated with translational fusions of the M. thermophila CODH/ACS promoter to lacZ (5) and by peptide fragment analysis of a CODH/ACS component, CdhA, in M. mazei (32). This CODH/ACS operon has an unusually long 371 bp 5? leader region (114). In contrast, the 5? leader region of CODH/ACS in the 73 obligately aceticlastic Methanosaeta concilii (formerly ?Methanothrix soehgenii?) is less than 100 bases long and has no significant sequence similarity to the methanosarcinal 5? leader regions (31). These observations suggest that CODH/ACS 5? leader region has a regulatory role, possibly at the transcriptional and/or post- transcriptional level. The purpose of this study was to localize regulatory regions that mediate expression of the catabolic CODH/ACS complex in the aceticlastic Methanosarcina to determine whether the 5? leader region is involved in expression. The promoter and leader sequence from M. thermophila TM1 CODH/ACS operon was fused to lacZ as a translational reporter to study differential gene expression on different substrates. Deletion analyses of sequences adjacent to the promoter combined with quantitative reverse transcriptase PCR (qRT-PCR) analyses of the transcript indicate that CODH/ACS expression is regulated in part by a sequence located downstream of the CODH/ACS promoter in the 5? leader in response to acetate and methylotrophic substrates. 4.3. Materials and Methods 4.3.1. Archaeal and Bacterial strains. Methanosarcina acetivorans C2A (=DSM 2834) and Methanosarcina thermophila TM-1 (=DSM 1825) were obtained from sources described previously 74 and maintained as frozen stocks (115). Escherichia coli SURE was obtained from Stratagene. Escherichia coli strain DH5?/?pir was obtained from W. Metcalf (77). 4.3.2. Media and cell growth. Artificial marine mineral medium was prepared for growth of M. acetivorans by methods described previously (113). Growth substrates used were sodium acetate, methanol or trimethylamine-HCl (TMA) at a final concentration of 0.05 M. Methanogen medium was prepared anaerobically in a N2-CO2 (4:1) atmosphere by a modified Hungate technique (110). Plating on solidified medium was performed in an anaerobic glove box as described previously by Apolinario et al (4). LB liquid or agar-solidified plates were used for E. coli (96). Puromycin (2.0 ?g/mL) and ampicillin (100 ?g/ml) were added to media for selection and maintenance of M. acetivorans and E. coli transformants, respectively. M. acetivorans strains were incubated at 35?C; E. coli strains were incubated at 37?C. 4.3.3. Sequence analysis. Sequences within the cdh mRNA leader sequence from the transcriptional start site to the beginning of the cdhA structural gene (ATG) from four Methanosarcina species were aligned using DNAMAN PC software [DNAMAN version 3.0, Lynnon BioSoft]. M. thermophila sequence was obtained from Genbank Accession No. L20952 from 961 to 1390, M. acetivorans sequence was obtained from Genbank Accession No. AE010299 from region 4741963 to 4742326, M. 75 barkeri sequence was obtained from Genbank Accession No. NC007355 from region 240857 to 241493, and M. mazei sequence was obtained from Genbank Accession No. NC007355 from 819301 to 819937. 4.3.4. Construction of cdh?::lacZ deletion plasmids. Primers were designed to delete sequences either downstream or within the promoter upstream of cdhA (Table S4.1), which is the first gene in the cdhABCDE operon encoding catabolic CODH/ACS. The transcriptional start site was previously determined (76, 114). The reporter plasmid pEA64, which contained the M. thermophila cdh promoter from pCDH1.3 (114), was used as a PCR template for deletions in the lacZ reporter described in Table 4.1. Table 4.1. E. coli and M. acetivorans plasmid constructs. Plasmids Parent Characteristics or description of construct Source pCR2.1 --------- Cloning vector, AmpR, PurR, lacZ ? Invitrogen pWM315 --------- Ori R6K, Ori pC2A, AmpR, PurR , lacZ ? (77) pCDH1.3 --------- cdhA?, AmpR (114) pcdhp::lacZ --------- cdhA?::lacZ, AmpR, Ori pBR322 (5) pEA61 pWM315 Cdh?, Ori PC2A, AmpR, PurR , Ori R6K This study pEA64 pEA61 Cdh?::CAT, Ori pC2A, AmpR, PurR , Ori R6K This study pEA103 pWM307 Cdh?::lacZ, Ori pC2A, AmpR, PurR , Ori R6K (5) pEA110 pEA103 Cdh??64::lacZ, Ori pC2A, AmpR, PurR , Ori R6K This study pEA111 pEA103 Cdh??65::lacZ, Ori pC2A, AmpR, PurR, Ori R6K This study pEA112 pEA103 Cdh??66::lacZ, Ori pC2A, AmpR, PurR , Ori R6K This study pEA113 pEA103 Cdh??67::lacZ, Ori pC2A, AmpR, PurR , Ori R6K This study pKA6 pEA103 TBP-1?::lacZ, Ori pC2A, AmpR, PurR , Ori R6K This study pKA7 pEA103 TBP-2?::lacZ, Ori pC2A, AmpR, PurR , Ori R6K This study pKA8 pEA103 TBP-3?::lacZ, Ori pC2A, AmpR, PurR , Ori R6K This study 76 Deletion 64, located upstream of the transcriptional start site, was generated from the pEA64 template using reverse primer 60 (Table S4.1), which hybridized within the plasmid with forward primers that generated ClaI restriction sites at different locations upstream of the cdh promoter. Forward primer 64 generated ClaI restriction site 29 bp upstream of the transcriptional start site to create ?64, which deleted part of the cdh promoter TATA box. The PCR generated DNA fragment was cloned directly into pCR2.1 (Invitrogen), then digested with NsiI/SalI and cloned into the NsiI/SalI restriction sites of plasmid pcdhp::lacZ. The latter plasmid was digested with XhoI/BamHI and the fragment containing the cdh-lacZ fusion was ligated into the lacZ reporter shuttle vector pEA103 replacing wildtype cdh sequence. Deletions downstream of the transcriptional start site were created by PCR using pCDH1.3 (Table 4.1). The same forward primer 53 was used with reverse primer 65 to generate deletion ?65 with a SalI restriction site 211 bp downstream of the CODH/ACS transcriptional start site. Likewise, reverse primers 66 and 67 were used to generate a SalI restriction site 133 bp (?66) and 28 bp (?67), respectively, downstream of the transcriptional start site of cdhA. PCR fragments were ligated directly into pCR2.1 (Invitrogen), then digested with PstI and SalI and ligated into pcdhp::lacZ. Finally, the latter plasmid was digested again with XhoI /BamHI and ligated into pEA103 replacing wildtype cdh sequence. After transformation, each deletion plasmid was extracted from M. acetivorans and confirmed for the correct sequence. 77 4.3.5. Identification and deletion of putative mRNA secondary structures. A PCR-based overlap procedure was used to create deletions in DNA encoding putative mRNA loop structures in the region downstream of the transcriptional start site (102). Primers B1 and C1 were used to create the loop 1 deletion 28 to 50 bp downstream of the transcriptional start site (Table S4.1). Primers B2 and C2 were used to create the loop 2 deletion 56 to 69 downstream of the transcriptional start site. Primers B3 and C3 were used to create the loop 3 deletion 84 to 123 bp downstream of the transcriptional start site. Primers B4 and C4 were used to create the polyU deletion 71 to 77 bp downstream of the transcriptional start site. The 1st PCR product (A1 & B primers) and 2nd PCR product (C & D1 primers) were amplified from pEA103 template in a 50 ?L PCR mixture using the following cycle parameters: 25 cycles of denaturation at 95oC for 30 sec, annealing at 55oC for 30 sec, and elongation at 72oC for 1 min. The final PCR product that included the subdeletion was generated using A1 and D1 primers with the first two PCR products as DNA templates in a 50 ?L PCR mixture using the following cycle parameters: 25 cycles of denaturation at 95oC for 1 min, annealing at 60oC for 2 min, and elongation at 72oC for 3 min. Correct size and yield of PCR products were confirmed by electrophoresis in 0.8% (v/v) agarose (Fisher Biotech) in TAE (Tris base, 40 mM; acetic acid, 20 mM; EDTA, 1 mM; pH 8.5). PCR fragments were digested with XhoI and BamHI restriction endonucleases and ligated into linear pEA103 digested with the same restriction enzymes to remove the wild-type cdh promoter fragment. After transformation, each deletion plasmid was extracted from M. acetivorans and confirmed for the correct sequence. 78 4.3.6. Reporter Gene Expression Analysis. M. acetivorans transformed with either the wild-type reporter plasmid pEA103, deletion plasmids or the control plasmid pWM315 (no cdh promoter) were inoculated (10% v/v) in triplicate into liquid medium containing 0.1 M sodium acetate, TMA, or methanol and incubated to mid-exponential growth phase. Cultures were sampled (1.0 ml) to perform ?-galactosidase assays as described previously (5). 1 ml samples were centrifuged at 13,000 rpm for 5 minutes. Cell pellets were resuspended in 1 ml Z Buffer (60 mM Na2HPO4?7?2?, 40 mM NaH2PO4?2H2O, 10 mM KCl, 1 mM MgSO4?7H2O, pH 7.0). Cells were sonicated and centrifuged at 13,000 rpm for 15 minutes. The supernatant was transferred to a new tube. The protein concentration of the supernatant was determined using the Bradford assay. Triplicate samples of the supernatant (100 ?l) were combined with 700 ?l Z Buffer + ?-mercaptoethanol (27 ?l/ 10 ml Z Buffer). The mixture was incubated at 30 ?C. 160 ?l ONPG (4 mg/ml) was added to each mixture, and the time for the color to change to yellow was measured. The reaction was stopped with the addition of 400 ?l 1 M Na2CO3, and the reactions were assayed for absorbance at 420 and 550 nm. 4.3.7. In vitro transcription assays. In vitro transcription assays were performed as previously described with some modifications. The templates for the reaction were PCR products amplified from pEA103. The first product was generated with primers Sowers1 and Sowers2 (Table S4.1). This product began 210bp upstream of the transcriptional start site of cdhA and terminated 200 bp into the lacZ structural gene. The second template was 79 made using primers Sowers3 and Sowers2. This product contained the Methanothermobacter thermoautotrophicum hmtB promoter for initiating transcription of the 5? leader region of cdhA. The product also terminated 200 bp into lacZ. The in vitro transcription assay was performed as described previously (97), with the modification that the limiting nucleotide was CTP, which was added with the other three nucleotides as the chase after elongation had stalled. In vitro transcription assays were done at varying temperatures to account for variability. 4.3.8. RNA extraction and quantitative reverse transcriptase PCR. RNA was extracted from M. acetivorans exponential phase cells grown on either acetate or methanol with the RNeasy kit (Qiagen) following the manufacturer?s instructions. RNA (50 ng) was used as template for each qRTPCR using the iScript one-step RT-PCR kit (Bio-Rad) following the manufacturer?s instructions. The primers used in the qRTPCR reactions are listed in Table S4.1. To compare the fold differences in expression, the C(t) values for each reaction were normalized to the C(t) value for the gene product of MA4504 (124). The fold differences were calculated using the formula (100): Fold difference = 2(??(C(t)) where ??C(t) is the difference in the normalized C(t) values of acetate- versus methanol-grown cultures. 80 4.3.9. Transcript stability studies. Transcript stability was determined by quantitating cdh mRNA in exponentially growing cultures of M. acetivorans grown with either acetate or methanol after inhibition of transcription with Actinomycin D. Samples were taken 0, 2, 5, 10, 15, and 30 minutes after addition of the 100 ug/ml Actinomycin D, and RNA was extracted from these samples as described above. The RNA was quantified and used as a template for qRTPCR using primers 221 and 315 (Table S4.1). These primers were specific for the entire 5? leader region as well as 40 bp of the structural gene, generating a product that was 405 bp in length. 4.3.10. Construction of TBP::lacZ reporter plasmids Plasmid reporter constructs for genes encoding methanosarcinal TATA binding proteins (TBPs) are shown in Table 4.1. M. acetivorans C2A genomic DNA was used as a template for PCR to generate the promoter sequences for ligation with lacZ using primers specific for the three genes encoding TATA binding proteins (TBPs) in M. acetivorans (Table S4.1). For TBP-1 (MA4331), forward primer #161 and reverse primer #162 were used to PCR amplify the region 599bp upstream of the start of translation. These primers introduced an XhoI site 595bp upstream and a BamHI site 37bp downstream of the translational start site. TBP-2 (MA0179) was made using forward primer #167 and reverse primer #168, amplifying the region 550bp upstream of the start of translation with an XhoI site 543bp upstream and a BamHI site 2bp downstream of the translational start site. TBP-3 (MA0278) was made using forward primer #165 and reverse primer #166, creating a product that 81 started 590bp upstream of the translational start site with an XhoI site 582bp upstream and a BamHI site 3bp downstream of the translational start site. Once the PCR- generated promoter fragments were made, they were digested with XhoI/BamHI in order to make the fusion. To construct the fusion plasmid, pEA103 was used as the backbone. This plasmid was digested with XhoI/BamHI to remove the cdh promoter region, and the remaining backbone of the plasmid was ligated with the PCR-generated TBP promoter fragments to form the reporter plasmids pKA6, pKA7, and pKA8 containing the promoters of tbp-1, tbp-2, and tbp-3, respectively. 82 4.4. Results 4.4.1. Identification of conserved sequence in carbon monoxide dehydrogenase/acetyl CoA synthase (CODH/ACS) orthologs. Members of the genus Methanosarcina contain multiple copies of several catabolic genes including two copies of the cdhABCDE operon that encodes carbon monoxide dehydrogenase/acetyl CoA synthase (CODH/ACS) (30, 37, 73). However, only one cdhABCDE operon has been detected in M. thermophila (42). The current study focused on the CODH/ACS ortholog in M. thermophila (Genbank Accession No. L20952, = M. acetivorans MA3860, = M. barkeri MbarA0204, = M. mazei MM0684), which had been reported previously to be highly regulated in response to substrate (5, 32, 114). To identify conserved regions of DNA that might be critical for cdh regulation, sequences upstream of the translation start site from CODH/ACS orthologs in M. thermophila, M. acetivorans, M. mazei, and M. barkeri were aligned (Fig. 4.1). The alignments revealed that the orthologous cdh 5? UTR regions between Methanosarcina. spp. were highly conserved ranging from 73% to 87% sequence similarity, which is similar to the sequence conservation of 77% to 86% similarity observed between methanosarcinal cdhA encoding orthologs. In contrast, only 45% and 62% similarity is observed within the region 256 bp upstream of the transcription start site. These regions were evaluated further by deletion analysis to determine whether the highly conserved UTR has a role in CODH/ACS regulation. 83 M. thermophila TATGAAGAATCTTTAAAATGAGTTGTGGTTAGTATGAAAAAATGAAAAAAATAATAACAA -208 M. acetivorans --g---a-----a--t---t-t----tt---a----------a----t--ta-------- -208 M. mazei --a---a--------gggct-t---cac---------g-----att-g------------ -207 M. barkeri --ac--at--t-ac-t---a-t----t--g-ac-c--gt---aagcgt--t-gt---t-- -205 M. thermophila ATATTAAAATCAATGGAAAAAACTATTGGGAATCAAGAATACTCATTCAAAAAAACCAGT -148 M. acetivorans -----------g-c-a------a---a-a-gc-g-----ggtca-a----------ggca -148 M. mazei -------------ata------a-----a--tc---t-t-----tcatt-------g-ta -147 M. barkeri t--a--gc-ca--g..--gt---aca-ca-tg-t-ta-tggt-tt-a-tg-------cac -147 M. thermophila ATCTACAGGCATTGAGGGCTTTTCTTTCGGAGTAGATCTTCAAGA.TTTTTTTATGAAGA -89 M. acetivorans -ca--a-.--c-at--acta---t-ca--a-a-t-------g---t--------c---t- -89 M. mazei tg--ttgacatagagtat-----t-gatca-aa--tct--g-g-tt------a-ga-ga- -87 M. barkeri -----------c-a-tct-----t-g-accta--at-acaat-a-c--g-aaag-c--ct -87 M. thermophila AGGTATCACTAAATTCCATTTTACCCATTGCGAAATAGGGTGTTACAAATTAGAAAATGT -29 M. acetivorans gt--gc-t---c------c---------ca------------------------------ -29 M. mazei c--gtctc-ac-t-c-at-------tg--cg-..---a---------------------- -29 M. barkeri gat-t-g-ac-ttcc-ac---ac--atc-cga--tgc..-------------------a- -29 M. thermophila TATATAGATTTGGTACATTTAGACTTTTAATTAGTGTTTATTATCCATCGGTAGCGACCT +32 M. acetivorans ------------------------------------------------------------ +32 M. mazei ----------------------t-ac---------------------------------- +32 M. barkeri ----------------g--a----a----------------------------------- +32 M. thermophila CTGCTCAAGATTAAGGTCCCAATCGTTGGTAAAAACGGTTTTTTTGAGACATAGGGCGTA +92 M. acetivorans ------------------t----------c---------------a---t---------- +92 M. mazei ------t----------------------c-------------------a-a-------- +92 M. barkeri -----t-g----------t----------c---------------------a-------- +92 M. thermophila AAAAGGGATTTGGAAGAGAAATAAGGCCCTAAAAACGACTTTTACAAG.AATATATTCTC +151 M. acetivorans ----a-a----a-------t--t-a------------------gg---.g-a------c- +151 M. mazei ----a------t------c-t-g-t-------------t---------a--a------c- +152 M. barkeri ----t--c---.----t-tg-at-a-------------------t-t-.g-a-----tc- +151 M. thermophila TATAAAAACGTTCTTATTTGAAAAAAACGTTGGTATAACGTTTAATGTCCAAATTGAGAG +211 M. acetivorans ----------gct-------.------t--------g------g-g--t-----c----- +210 M. mazei --c-------c-t-------.--------c-----a---c--c---a-t-----a----- +211 M. barkeri -c--------act---gc--g-----cgc-g-ta-a-tat--gttat-tg---aaa--t- +211 M. thermophila CTATGAACGTTTTACCATAAAACGTCAT...TTAAACGGTTGTAACTTTGAAGCAGACGG +268 M. acetivorans ---aa--------g---g----t-c---...------------------a-g--g----- +267 M. mazei ---a--gt-----g----g---------...--------------a---a---------- +268 M. barkeri g-gaat-gtg---ta-cag-tg---at-tac---c--------------a-.-------- +270 M. thermophila TAAGATATAAAATGAATAATCATTAACAATATGCCGCTT..ATCAGGATTACAATCGAAT +326 M. acetivorans ----g----t----t-----------g----------a-..-a....--ag--gc--cca +321 M. mazei c--a-----t---t------------g---------aag..--a--.---g------tc- +325 M. barkeri c---gag.....--tt-gg-a----ca---------aagaat-a-aat--g------tc- +325 M. thermophila TGGCGAAGTCGACTTTAAACAAATTAAGGAGGTAAAGCTCACATGAGCAAACTAACTACC +386 M. acetivorans -------------------------t---------------a------------------ +361 M. mazei ---------t---------------t---------------a------------------ +365 M. barkeri --at-------------t-------t---------------a------------------ +365 Figure 4.1. Alignment of cdhA promoter region from four different Methanosarcina species. Numbering in right column is based on the transcription start site for cdhABCDE (114). The start of transcription is marked with an arrow and +1. The start of translation (ATG) is underlined. 84 4.4.2. Effect of sequence deletions on the regulation of cdh by different substrates. In a prior report that employed a translational fusion of the cdh promoter to lacZ, CODH/ACS from M. thermophila TM1 was shown to be up-regulated up to 54- fold during growth on acetate, compared with growth on methanol or trimethylamine (5). To identify sequences involved in CODH/ACS regulation a series of deletions were generated downstream in the 371 bp 5? leader region between the transcriptional start site and the predicted ribosomal binding site of the cdhA encoding gene (Fig. 4.2). The cdh deletion sequences were ligated into the lacZ reporter plasmid as translational fusions and transformed into M. acetivorans. The effects of these deletions on the expression of ?-galactosidase in response to different substrates are shown in Fig. 4.3. Figure 4.2. Map of deletions within the 5? leader region of M. thermophila TM1 cdhA. Position numbers above relative to start of transcription indicated by arrow. RBS is the location of the predicted ribosomal binding site. +1 +371 -751 cdh? lacZ +211 +133 +28 -29 +335 RBS ?64 ?65 ?66 ?67 C2A/pEA103 85 Some deletions within the 5? UTR did have an effect on expression during methylotrophic growth. Figure 4.3 shows that ?66 and ?67 had only a 4- and 13-fold difference in expression, respectively, between acetate and methanol grown cells compared to the 61-fold difference seen in wild type constructs. The same was observed for cells grown on TMA, where ?66 and ?67 were 7- and 5-fold higher, respectively, compared to 20-fold difference for the wild type construct. These data indicate that this region is involved in regulation of expression based on substrate. Differences were also observed between expression of all constructs when grown on methanol and TMA. The methanogenic pathway for these substrates is the same, so it is not clear why expression under methanol and TMA is different. It is possible that there is some difference in the mechanism of regulation between growth on the two substrates that could account for this difference. 86 0 20 40 60 80 WT ?64 ?65 ?66 ?67 Deletion construct Fo ld dif fer en ce Acetate versus Methanol Acetate versus TMA Figure 4.3. Effects of deletions downstream of the transcriptional start site on regulation by different substrates. The graph shows the fold difference in expression for either acetate vs. methanol or acetate vs. TMA grown cells. 4.4.3. Effect of deletions in sequence within the 5? leader region of cdhA. Based on the results mentioned above, the 5? leader region was investigated further. Putative secondary structures within the 5? leader region mRNA were found in all four Methanosarcina species that consist of three stem-loop structures (Figure 4.4A). The ?G values for these structures were calculated to be -4.10 kcal/mol, -1.5 kcal/mol, and -6.90 kcal/mol for loops 1, 2, and 3, respectively. In all four species a poly-U sequence was located directly downstream of the second putative loop structure. To investigate whether these potential secondary structures have any effect on the regulation of cdh, promoter sequence with deletions of putative secondary structures was fused to lacZ and assayed for ?-galactosidase expression. 87 The deletion of the putative structures, as well as the poly-U sequence, had a significant effect on the expression of ?-galactosidase (Figure 4.4B). During methylotrophic growth on trimethylamine, the deletion of each of the putative secondary structures significantly increased expression of ?-galactosidase from 3- to 8-fold. Deletion strains grown with methanol showed slightly greater ?-galactosidase expression, ranging from 4- to 11-fold. In contrast deletion strains grown aceticlastically showed different effects on expression. When putative loops 1 (?loop1) and 2 (?loop2) were deleted, the expression of ?-galactosidase decreased by half, while the deletions of the poly-U sequence (?polyU) and loop 3 (?loop3) had no effect on expression during aceticlastic growth. 88 A. B. Figure 4.4. Effects of deletions in putative mRNA hairpin loops in UTR of M. thermophila TM1 cdhA. A) Predicted stem loop structures located within the 5? untranslated leader sequence based on algorithms described in material and methods. B) Effects of deletions of putative stem loop structures and poly-U region. Numbers are reported as ?-galactosidase specific activity in ?g/mL of protein. Numbers and arrows above each bar represent the fold increase ( ) or decrease ( ) in activity between the wild-type strain and the mutant strains. Values are means and standard deviations for 3 replicate cultures. Growth substrate ?-gal specific activity unit ?g/mL protein A G G G C GU A A A A A G G G A U U U G G A A G A G A A A U AA GG C C C U A A 90 100 110 12 3 80 U70C G U U G G U A A A A A C G 60 U A A CC A C C UC U G C U C A A G A U U AA G G U C 30 40 50 1 C GGA 2 U U U U U UU G A G A C A G AU A U A UUUU C A U C GC GA U U A G U A G wildtype ? Loop1 ? Loop2 ? Poly U ? Loop3 Acetate 24.18 ? 2.83 13.86 ? 0.18 10.62 ? 1.64 26.35 ? 2.17 25.02 ? 1.86 (0.6 ) (0.4 ) (1.1 ) (1.0) Methanol 0.45 ? 0.03 2.56 ? 0.00 1.59 ? 0.08 4.48 ? 0.00 2.97 ?0.12 (5.7 ) (3.5 ) (10 ) (6.6 ) Trimethylamine 0.51 ? 0.08 3.93 ? 0.03 1.70 ? 0.04 2.08 ? 0.02 1.30 ? 0.04 (7.7 ) (3.3 ) (4.1 ) (2.5 ) 89 4.4.4. Comparison of Cdh::LacZ fusion protein and cdh transcript levels with different substrates. RNA was extracted from M. acetivorans wild-type cells to determine the difference between cdh transcript levels in acetate- and methanol-grown cells. The fold difference of transcript level was determined by qRTPCR and compared to the fold difference of ?-galactosidase level expressed by the cdhA?::lacZ reporter in cells grow with acetate and methanol. The fold difference of transcript in cells grown with acetate and methanol determined by qRT-PCR was 68 ? 20 compared with 62 ? 6 fold difference of ?-galactosidase level expressed by the cdhA?::lacZ reporter. The fold difference between transcript and protein levels was not significant, indicating translation is not a factor in the regulation of CODH/ACS expression. 4.4.5. In vitro transcription of 5? leader region The region located upstream of the transcriptional start site as well as the 5? leader region was used as a template (Sowers1/2 template) for in vitro transcription assays to determine if there was a definitive sequence element that was stopping transcription. Another template was also used (Sowers3/2 template), this one containing the definitive promoter from M. thermoautotrophicum htmB, since it was known where transcription occurred from this promoter. The results of the in vitro transcription assay are shown in Figure 4.5. Transcription was initiated from both templates, and temperature was not a factor, as each template had the same pattern of transcription termination. 90 Interestingly, transcription occurred as expected from the template with the htmB promoter. However, transcription from the native cdh promoter initiated further downstream than the determined start site. Even taking into account this discrepancy, both templates appeared to have the same pattern. Regardless of the promoter recognized, the pattern of transcriptional elongation, pausing, and termination remained the same. Most of the transcripts that initiated from either template did not reach the end of the template, indicating that transcription was not efficient throughout this 5? leader region. Each band on the gel represents a length of transcript, where the transcription machinery either paused (P) or completely stopped (S) and detached from the template. The transcripts in the pausing lanes were more abundant than those in the complete stoppage lanes. The large numbers of transcripts that were stopped along the entire length of the 5? leader region indicate that transcription of this sequence is difficult with the basal components used in this assay. 91 Figure 4.5. In vitro transcription assay of cdhA promoter and 5? leader region. Each band represents transcripts where elongation has paused (P) or terminated and the RNA has come loose (S). The reactions were done at multiple temperatures, shown at the tops of the lanes. The templates either contained the native cdhA promoter or Methanothermobacter thermoautotrophicum htmB promoter. Assay done by Tom Santangelo at the Ohio State University. 1 2 3 4 5 6 7 8 9 10 1112 1314 150 170 190 160 180 10 5 15 20 25 30 35 40 P P P P P P P P P P S S S S S S S S S S +16 cdhA promoter htmB promoter 23? 37? 45? 60? +16 23? 37? 45? 60? Temp (?C) of 5 min. 200?M NTP chase 92 Interestingly, transcription occurred as expected from the template with the htmB promoter. However, transcription from the native cdh promoter appeared to be initiated further downstream than the determined start site. Even taking into account this discrepancy, both templates appeared to have the same pattern. This means that, regardless of the promoter recognized, the pattern of transcriptional elongation, pausing, and termination remained the same. Most of the transcripts that initiated from either template did not reach the end of the template, indicating that transcription was not efficient throughout this 5? leader region. Each band on the gel represents a length of transcript, where the transcription machinery either paused (P) or completely stopped (S) and detached from the template. The transcripts in the pausing lanes were more abundant than those in the complete stoppage lanes. The large numbers of transcripts that were stopped along the entire length of the 5? leader region indicate that transcription of this sequence is difficult with the basal components used in this assay. 4.4.6. Transcript stability of cdh during growth with different substrates. Actinomycin D has been shown to inhibit transcription in Archaea (16, 55), and we confirmed that actinomycin D effectively inhibited transcription in M. acetivorans by adding actinomycin D to exponentially growing cultures containing 3H-uridine and monitoring the incorporation of the labeled compound over time. The results indicated that actinomycin D inhibited incorporation of the 3H-uridine as a result of transcription inhibition, while cells without actinomycin D continued to show 3H-uridine incorporation. Actinomycin D was added to M. acetivorans grown 93 on either acetate or methanol, and cdh mRNA was amplified at multiple timepoints after transcription inhibition using qRTPCR. Figure 4.6 shows the fold difference of cdh transcript between acetate- and methanol- grown cells. No significant difference (p>0.05) was observed between the ratio of C(t) values of acetate- versus methanol- grown cells at any of the time points, indicating that the relative stability of the message isolated from acetate- and methanol-grown cells was similar. 0 20 40 60 80 0 2 5 10 15 30 Time (min) Fo ld dif fer en ce Figure 4.6. Differences in transcript levels after addition of actinomycin D. Fold differences were calculated from the C(t) values for RNA from acetate- versus methanol-grown cells after the addition of actinomycin D (100 ?g/ml) at 0, 2, 5, 10, 15, and 30 minutes. One way analysis of variance (ANOVA) showed no significant difference (p>0.05) between the fold differences at the different time points. 94 4.4.7. Effect of substrates on elongation of cdh transcript. To determine whether differential elongation occurred within the 5? UTR as a possible mechanism of regulation, nine primers that hybridized along the 5? length of the transcript were used with qRTPCR to detect any changes in transcript length between acetate- and methanol-grown cells (Figure 4.7). A 15-fold difference in transcript level was observed within 358 bases of the 5? end in cells grown on acetate and methanol. However, transcript levels 405 bases downstream of the 5? end showed a significantly greater difference, increasing to 68-fold difference between acetate and methanol-grown cells. These results suggested that either early termination of elongation or differential post-transcriptional mRNA processing occurs between 358 and 405 bases downstream of the 5? end of the cdh transcript during methylotrophic growth. 95 Figure 4.7. Differences in levels of transcripts of varying lengths demonstrated by qRTPCR. Fold differences were calculated from the C(t) values for RNA from acetate- versus methanol-grown cells. Arrows above the graph represent the position of primers used to generate templates of different lengths. 4.4.8. Regulation of different TATA-binding proteins by different substrates Using primers to generate transcripts of different lengths, it was determined that the majority of the regulation within the 5? leader region was at the level of transcription elongation. A small portion this regulation, however, appeared to be at the level of transcription initiation. Since trans factors acting upstream of the transcriptional start site were not found, components involved in transcription initiation were investigated. It has been theorized that use of different TBPs could be involved in regulation at the level of transcription initiation. To determine whether differential transcription factor pairing was a possible mechanism of regulation for P cdh A +1 +70 +99 +140 +195 +227 +265 +304 +358 +405 F 96 cdh, M. acetivorans with translational fusions of TATA-binding protein (TBP) promoters ligated to lacZ assayed for ?-galactosidase activities during growth with different substrates. The results of these experiments revealed that neither tbp-2::lacZ or tbp-3::lacZ were expressed under any substrate conditions, as the specific activities ranged from 0 to 0.04 per ?g/ml of protein. tbp-1::lacZ, however, was expressed when cells were grown with any of the three substrates. It was also observed that tbp- 1::lacZ had a higher level of expression when grown in the presence of acetate with a specific activity of 44.4 ? 4.2 per ?g/ml of protein. In contrast, the specific activities in cells grown on methanol and TMA were 10.93 ? 0.9 and 11.5 ? 0.4 per ?g/ml of protein, respectively. 97 4.5. Discussion When grown on acetate, members of the genus Methanosarcina express CODH/ACS, a key enzyme complex that catalyzes the cleavage of the acetyl C?C bond of coenzyme CoA (43, 61). As the CODH/ACS operon is highly expressed during aceticlastic growth, the effects of deletions in sequences adjacent to the transcriptional start site were studied to identify cis elements critical for regulation. Deleting sequence upstream of the TSS had no significant effect on regulation indicating that upstream trans elements were not involved in regulation of transcription initiation. In contrast, downstream deletions in sequence within the highly conserved 371 base pair 5? leader region prior to the ribosomal binding site were shown to have a significant affect on the regulation of CODH/ACS expression. Further deletion of putative secondary structures within the 5? leader sequence were also shown to have a significant effect on regulation. Differential expression of transcript within the 358 bp downstream of the 5? end accounted for approximately 22% of the difference in cdh expression observed between acetate and methanol grown cells. However, when sequence within the 5? leader region 133 bases downstream of the promoter was deleted, gene expression was up-regulated relative to wild type during methylotrophic growth of M. acetivorans with TMA or methanol. Trans-acting DNA binding factors such as repressors typically bind immediately adjacent to the promoter to cause disruption of RNA polymerase binding. The observations in this study suggest that the observed regulation of transcriptional initiation results from a factor that either binds to the 98 promoter or interacts with the basal transcription initiation components. Possible mechanisms might include differential expression of a TATA binding protein or TFB. Multiple genes encoding for multiple copies of TBPs and transcription factor B (TFB) have been observed in the genomes of several Archaea. This includes, among the methanogens, three copies of TBP in M. acetivorans, M. barkeri and M. mazei, two copies of TBP in Methanospirillum hungatei and two copies of transcription initiation factor B (TFB) in Methanopyrus kandleri. We also tested for the possibility that the three TBPs detected in methanosarcinal genomes could form different pairings with TFB in response to different growth substrates to regulate gene expression, but only TBP-1 was expressed at detectable levels. However, some role in cdh regulation by TBP-1 cannot be ruled out as it was expressed at greater levels during growth on acetate. Up-regulation of TBP and TFB have been reported for other Archaea in response to UV irradiation (8) and heat shock (104, 123), but a clear association between TBP/TFB expression and regulation of a target promoter has yet to be established. The results also indicate that the 5? leader region has a role in post- transcriptional regulation of CODH/ACS via early termination of elongation during methylotrophic growth. This conclusion is supported by several observations. First, post-transcriptional regulation by differential translation was ruled out, as the fold difference of the protein levels and the transcript levels were not significantly different. Second, cdh transcript stability was similar in cells grown aceticlastically and methylotrophically, ruling out differential mRNA degradation as a possible mechanism. Finally, a significant difference in transcript levels was observed 405 99 bases downstream of the 5? end of the transcript. Deletion of the leader region encoding sequence between +211 and +335 had no effect on expression in the lacZ reporter fusion and regulation and qRT-PCR analysis of cdh transcript showed differential transcript levels occurred between 358 and 405 bp downstream of the 5? end. This observation is consistent with Northern analysis of CODH/ACS transcript that showed a rapid decay of signal after acetate-grown cells were spiked with methanol using a probe that hybridized 34 bases into the 3? leader region (114). Deletions upstream of cdhA within UTR sequence between +28 to +211 also had a significant effect on expression, which suggests that this region has a role in the differential termination of elongation downstream. However, the exact role this sequence plays has yet to be determined. The results support the hypothesis that the CODH/ACS operon is regulated by two mechanisms: differential transcription initiation that accounts for approximately a quarter of the differential expression and early termination of elongation that accounts for the balance of the differential expression observed between acetate and methanol-grown cells. In bacterial systems, long 5? leader regions are involved in transcriptional regulation via multiple mechanisms. Usually, these mechanisms involve the formation of secondary structures within the RNA. One example of this type of regulation is the attenuation mechanism, which involves the formation of different RNA secondary structures that either inhibit or promote transcription elongation (133). This type of regulation involves the formation of stem loop structures, along with a long stretch of uridines immediately following one of the structures. Interestingly, a conserved stretch of predicted uridines occurs in positions +71 to +77 100 in 5? leader regions of all four methanosarcinal CODH/ACS orthologs. Another mechanism found in bacteria involves intrinsic terminators such as riboswitches. This type of regulation motif has been found in many bacterial systems in which the RNA forms different secondary structures to either allow or terminate transcription (103). There are multiple permutations of this system present in both eukaryotes and prokaryotes. One of the most well known systems involves regulation of tryptophan biosynthesis by a conformational change in mRNA secondary structure that causes early transcription termination in the presence of tryptophan (132). Other systems involve a regulatory protein (103) or ligands (78, 80, 117, 118, 129) that bind to the RNA, which creates a conformational change that either terminates or allows transcription to occur. Early termination of transcriptional elongation in sequence distal to the regulatory UTR is consistent with regulatory mechanisms involving changes in secondary structure, such as attenuation or riboswitches. Although the experimental evidence indicates that the 5? leader region of methanosarcinal CODH/ACS has a role in controlling early termination of elongation, the precise regulatory mechanism is currently unknown. In the Archaea, there are several examples of transcriptional regulation mechanisms. Most of these examples involve activator or repressor proteins which bind upstream of the transcriptional start site and either induce or inhibit transcription. The difference between these known systems and the one described here is that the previous studies involve transcriptional regulation occurring prior to the start of transcription. 5? UTRs identified within the Archaea include the 113 bp UTR identified upstream of a DEAD-box RNA helicase in the Antarctic methanogen 101 Methanococcoides burtonii (70) and the methyltransferase genes in Methanosarcina spp. (18). In both of these examples, the 5? UTR was implicated in regulation, although the role of the 5? UTR in regulation was not confirmed. The system described in this paper appears to involve sequences downstream of the start of transcription, well into the 5? leader region. To the best of our knowledge this is the first evidence of regulation of transcriptional elongation by a 5? leader region as a mechanism for gene regulation in the Archaea. Further studies to identify putative trans-acting elements and secondary structures are necessary to characterize the paradigm for catabolic CODH/ACS regulation in these Archaea. 102 Chapter 5: Discussion and Future Perspectives The current knowledge of how transcription occurs in Archaea has indicated that the machinery involved in transcription appears to be homologous to eukaryotic systems. Some transcription factors, such as TBP and TFB, have been identified. In vitro transcription systems have demonstrated that transcription in Archaea can be achieved with relatively few transcription factors when compared to eukaryotic systems. However, this is not to say that there are not other proteins that facilitate transcription in Archaea. With the sequencing of multiple Archaeal genomes, multiple putative transcription factors and regulators have been annotated. Further experiments are needed to characterize the function of these putative transcription factors. With the development of in vitro transcription systems, gene expression systems, and gene deletion systems, proteins of unknown function can be studied to verify their annotation and to determine their role in vivo in cellular processes. Understanding how catabolic gene regulation occurs in methanogenic Archaea is essential to understanding how these organisms function in fermentative processes, yet little is known about this regulation. Carbon monoxide dehydrogenase/acetyl CoA synthase is a key enzyme in aceticlastic methanogenesis, and this gene is regulated at the transcriptional level in response to substrate (65). The work presented here shows for the first time that the 5? leader region located directly downstream of the transcriptional start site of cdhA is involved in transcriptional 103 regulation (Chapter 4). Other examples of 5? untranslated leader regions involved in regulation are present in the literature (18, 70). There are also studies in haloarchaea that have characterized the abundance of leadered transcripts. Brenneis et al demonstrated that, of 40 genes studied, the majority were leaderless. Other studies on haloarchaea have indicated that these 5? UTRs have a role in translational regulation (20, 21). Overall, it appears as though most archaeal transcripts do not have long 5? UTRs. As stated above some archaeal 5? UTRs are involved in regulation, but a mechanism(s) for this regulation has not yet been identified. This study is the first to show that transcriptional regulation of archaeal CODH/ACS occurs, for the most part, at the level of transcription elongation from within the 5? leader region. A smaller fraction of the regulation appears to occur at the level of transcription initiation, although this result needs to be investigated further. Based on the results presented in this dissertation, I propose a model for the regulation of expression of CODH/ACS (Figure 5.1). When Methanosarcina species are grown on methylotrophic substrates, such as methanol or trimethylamine, a terminator protein binds to the end of the transcribed RNA and translocates along the length of the RNA. This protein would function similar to Rho-dependent termination in bacteria, involving the helicase protein Rho. Once the protein reached the DNA-RNA complex, it would unwind the RNA, causing termination of transcription. However, when cells are grown in acetate, a trans factor would be activated that could bind to the RNA, generating secondary structures in the RNA. These structures would inhibit the progress of the terminator protein, preventing it 104 from interfering with transcription, and allowing transcription to continue. The trans factor protein would be inhibited in the presence of methylotrophic substrates, to the point that it would only be active when there was only acetate present. In the genome of M. acetivorans, there is no annotated homolog of Rho present. However, there are homologs of other factors involved in Rho-dependent termination, such as NusG. There is an annotated homolog of NusG in M. acetivorans (MA4273), M. mazei (MM_1010), and M .barkeri (Mbar_A0616). It is possible that there is a homolog of Rho present, but it has not been correctly annotated. Carefully analysis of the genome, along with functional studies, would help to determine if a homolog of this transcription factor is present. But the model presented in the previous paragraph is supported by the data in this dissertation. I have shown that regulation is at the level of transcription elongation, and that this regulation is occurring within the 5? leader region. Putative stem loop structures and a conserved polyU sequence appear to be present in the 5? leader region and possibly play a role in regulation. Based on the in vitro transcription assay, there are numerous site along the 5? leader region where pausing of the RNA polymerase occurs during transcription. The work in Chapter 3 failed to identify any trans factors that bind to the 5? leader region, but it is still possible that the conditions tested were not ideal for isolation of the protein. Further research is needed to determine if there indeed are any trans factors that can possibly bind to this region and are involved in transcriptional regulation. Figure 5.1. Proposed model for the regulation of cdhABCDE. The steps on the left illustrate what occurs during methylotrophic growth. Under these conditions, a terminator protein binds to the newly synthesized RNA, interfering with the RNA polymerase and terminating transcription. The steps on the right illustrate what occurs during aceticlastic growth. Under this condition, the terminator protein still bind, but secondary structures which form within the RNA, due to a regulator protein, prevent termination of transcription. 106 The model presented in the previous paragraph is not the only possible mechanism for regulation. There are multiple mechanisms present in other organisms that involve 5? leader regions. Usually, these mechanisms involve the formation of secondary structures within the RNA. One example of this type of regulation is the attenuation mechanism, which involves the formation of different RNA secondary structures that either inhibit or promote transcription elongation (133). These types of regulatory mechanisms are usually characterized by a region of poly-Us located downstream of one of the putative loop structures. Interestingly, one of the predicted loop structures found in this dissertation also had a stretch of poly-Us downstream. This type of regulatory mechanism can involve the intrinsic formation of secondary structure, or the formation of the structures can be mediated by a regulatory protein. This type of mechanism could also be involved here, as the poly-U region would allow the RNA polymerase to pause during transcription. Under growth on methylotrophic substrates, the terminator protein could catch up to the elongation complex and cause the termination of transcription. For growth under acetate, a regulatory protein could interfere with the terminator protein, preventing transcription termination. Another mechanism found in bacteria involves intrinsic terminators such as riboswitches. This type of mechanism involves ligands (78, 80, 117, 118, 129) that bind to the RNA, which creates a conformational change that either terminates or allows transcription to occur. While this mechanism could be feasible for regulation 107 of cdhABCDE, further research would be necessary to determine what ligand could be controlling the regulation. The 5? leader region of cdhABCDE needs to be further characterized to determine its specific role in regulation of the gene. As stated previously, 5? leader regions have been implicated in transcription and translation regulation in bacteria. It is feasible that these regions in Archaea function in a similar way, involving putative secondary structures. This type of mechanism would allow the cells to respond quickly to changes in the environment. Since acetate is not the favored substrate for these organisms, this type of mechanism would allow the organisms to quickly shut off expression of CODH/ACS, which when expressed accounts for up to 20% of the total cell proteins, when in the presence of preferred substrates, such as methylotrophic substrates. Future work should focus on investigating this region for specific mechanisms, such as riboswitches and attenuators. Using in vitro transcription assays would be essential for these studies, in order to determine the exact sequence that is important for the regulation. Unfortunately, there is no in vitro transcription assay available at the moment that uses the components from M. acetivorans, although this system will hopefully be developed in the near future. Currently, the only system available for use with methanogenic Archaea involves components from Methanothermobacter thermoautotrophicum. A M. acetivorans in vitro transcription system will allow for the use of native transcriptional components to study transcription, which combined with site-directed mutagenesis is essential for identifying mechanisms at both the DNA and RNA levels. 108 Further work is needed to characterize the exact mechanism by which the regulation is occurring, particularly at the level of transcription initiation. More investigation into the putative promoter region is necessary to identify factors involved in differential transcription initiation. Methanosarcina species often contain multiple copies of some transcription factors, such as TATA-binding protein (TBP) and transcription factor B (TFB). Other Archaea have also been predicted to contain multiple copies of TBPs and TFBs. It has been speculated that differential transcription factor pairing could play a role in transcriptional regulation, although this theory has not been proven. Previous work in our lab in which the putative promoter regions of the three TBPs present in the genome of Methanosarcina acetivorans were fused to lacZ to make translational fusions demonstrated that TBP-1 appears to be regulated in response to substrate. Further analysis is needed to evaluate if this gene could be playing a role in the transcriptional initiation regulation that is seen with cdhABCDE. Overexpression of the putative TBP proteins would allow for other experiments, such as DNA-binding assays, to determine if different TBPs have varying affinities for promoters under different conditions. More characterization of transcription initiation is also necessary to further determine which transcription factors are present, and to identify the role of these factors in transcription. Development of an in vitro transcription system that uses Methanosarcina components is essential for identifying other factors. Since genome annotation has discovered multiple putative transcription factors, these factors could be overexpressed and purified and used in in vitro transcription assays to determine their effects on transcription. The availability of gene disruption systems also allows 109 investigators to disrupt the genes of interest to determine their effect on transcription in vivo. This is the first study to elucidate the mechanism of regulation involving a 5? leader sequence in Archaea. Understanding the mechanism of regulation from this 5? leader will allow greater understanding of catabolic gene regulation, as well as increasing the knowledge of how the aceticlastic pathway is regulated. Since the majority of the biologically produced methane in methanogenic consortia of microorganisms is produced from acetate, which is also a rate-limiting step, understanding how this process is regulated is essential for understanding how carbon flow is effectively regulated throughout the consortia for optimal conversion of biomass to methane and carbon dioxide. Biomass conversion is directly dependent on the interaction of three groups of microorganisms, and one of the rate limiting steps involves aceticlastic methanogenesis. Because methanogenic Archaea are important for many industries, including waste treatment and processing, understanding more about how catabolic pathways function and are regulated can lead to more efficient processes. This is also important, because methane is both a greenhouse gas and an energy source, thus being able to control methane production and substrate utilization would help increase production of biogas as an alternative form of energy. Thoroughly understanding how transcriptional regulation of cdhABCDE occurs will also help to give insight to other possible catabolic gene regulation mechanisms within the methanogenic Archaea. Since the only known regulation mechanisms in Archaea involve DNA-binding proteins that bind near the start of 110 transcription, this mechanism would be the first of this type to be described for Archaea. It would present a new mechanism by which Archaea are regulating gene expression, and it would open the field to the study of other 5? leader regions, which may be acting in the same fashion. This understanding of the regulation of methanogenic processes, in particular aceticlastic methanogenesis, would have global importance. The conversion of biomass by a methanogenic consortia provides a renewable energy resource in the form of methane, and is also functional as waste treatment processes. Although 5? leader regions identified for other archaeal genes have been postulated to be involved in regulation, this was the first study to demonstrate a regulatory role by an archaeal leader sequence for differential transcription elongation. Based upon the results described in this dissertation, a model can be proposed for the regulation of catabolic CODH/ACS. This model involves a Rho- dependent like mechanism, similar to that found in bacteria. When grown on methylotrophic substrates, transcription elongation would be terminated by a trans element that binds to the RNA. When grown under acetate, the terminator protein would be inhibited by another trans element, which would allow transcription to continue. Further testing is necessary to confirm the proposed model. Identifying the regulatory mechanism of catabolic genes such as CODH/ACS is critical for understanding the regulatory strategies employed by methanogenic consortia for efficient conversion of biomass to methane. 111 Appendix A: Supplemental Information Table S2.1. Primers used in Chapter 2 for characterization of rad25 from M. thermophila. Primer Primer sequence Purpose #68 5? GANATGCCNTCNGGNACNGGNAA-3? Cloning helicase sequences #69 5?-CAAANTCNATNCCTTCNGANACN-3? Cloning helicase sequences #92 5?-CTTACATTTCCCTTTATGAGAATA-3? Primer extension #93 5?-AAGGACGCTATCTTCAAAATCAAT-3? Primer extension #130 5?-CAGGAAGTGGAGAGACCCTTGTTGG-3? Site-directed mutagenesis #131 5?-CCAACAAGGGTCTCTCCACTTCCTG-3? Site-directed mutagenesis #157 5?-CGCCCAATTGGTAATATACTATTC-3? PCR for pac cassette #158 5?-CACAGGAACAATTGACGGCTG-3? PCR for pac cassette Table S3.1. Primers used in Chapter 3. Primer Sequence UTR1-bio 5?-bio-AATTAGTGTTTATTATCCATCGGTAGCG-3? UTR2 5?-CATGTGAGCTTTACCTCCTTAATTTG-3? EMSA1-bio 5?-bio-GGAAAAATCTATATAATTATTTGTTTTTAATATGAA-3? EMSA2 5?-GGGTAAAGTGGAATGTAGAGGCACACTATTC-3? EMSA1 5?-GGAAAAATCTATATAATTATTTGTTTTTAATATGAA-3? #292 5?-GGCTTCAAAAACCGCTTCTCTGGC-3? #293 5?-GAGCTTGCAGGGCTCCTTATTGAAACC-3? TM1up1 5?-TGAAGAATCTTTAAAATGAGTTGTGGTTAGTATGAA-3? TM1up2 5?-GGGTAAAATGGAATTTAGTGATACCTTCTTC-3? Barkeri-up1 5?-ACAAATATTTACATAATAATTTGTTGTGAACACGAC-3? Barkeri-up2 5?-TGGGTAAAGTGGGAATGTTCAAAATC-3? #60 5?-GCCATTGGGATATATCAACGG-3? #62 5?-AGAATCGATAAAATGAGTTGT-3? #63 5?-ATTTTATCGATTGCGAAATAG-3? #64 5?-TTATATCGATTTGGTACATTT-3? 112 Table S4.1. Primers used to construct deletion mutants, TBP constructs and qRTPCR products in Chapter 4. Primers Name Location in reference to Transcriptional start site Deletion constructs TGAAGAAGTATCGATAATCAA #53 ? GCCATTGGGATATATCAACGG # 60 ? TTATATCGATTTGGTACATTT # 64 - 29 bp CTCAATGTCGACATTAAACGT # 65 + 211 bp AAGTCGACTTTAGGGCCTTAT # 66 + 133 bp CGCTACCGGTCGACAATAAAC # 67 + 28 bp AGGAAACAGCTATGACCATGATTACGGTA A1 ? AACCGTTTTTACCAACGATTGG/GCTACCGATGGATAATAAAC B1 + 51 bp / + 8 bp CCAATCGTTGGTAAAAACGGTT C1 + 51 bp TCATCACCGGATCCAGAGC D1 + 365 bp TACGCCCTATGTCTCAAAAAAACATTGGGACCT B2 + 46 bp GTTTTTTTGAGACATAGGGCGTA C2 + 70 bp CTTGTAAAAGTCGTTTTTGTCTCAAAAAAAC B3 + 70 bp GAGACAAAAACGACTTTTACAAG C3 + 78 bp CCTTTTTACGCCCTATGTCTCCCGTTTTTACCAACG B4 + 56 bp GAGACATAGGGCGTAAAAAGG C4 + 78 bp TBP constructs CGAAATCCTCGAGGTGCGGTC #161 ? GGTGGATCCAACCACGTTTTC #162 ? GGGGAGACTCGAGAAAACTCAGG #165 ? CTCTATAGTTATTGTGGATCCCATGCC #166 ? CCCCGTCCTCGAGATACAGTCTC #167 ? 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