UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Analysis of the Spo0A(257V) MUTANT OF Bacillus subtilis Turner, Barbara Marion 2006

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Notice for Google Chrome users:
If you are having trouble viewing or searching the PDF with Google Chrome, please download it here instead.

Item Metadata

Download

Media
831-ubc_2006-0328.pdf [ 10.14MB ]
Metadata
JSON: 831-1.0092656.json
JSON-LD: 831-1.0092656-ld.json
RDF/XML (Pretty): 831-1.0092656-rdf.xml
RDF/JSON: 831-1.0092656-rdf.json
Turtle: 831-1.0092656-turtle.txt
N-Triples: 831-1.0092656-rdf-ntriples.txt
Original Record: 831-1.0092656-source.json
Full Text
831-1.0092656-fulltext.txt
Citation
831-1.0092656.ris

Full Text

ANALYSIS OF THE SPO0A(A257V) MUTANT OF BACILLUS SUBTILIS by B A R B A R A M A R I O N T U R N E R B . S c , University of British Columbia, 2001 A THESIS S U B M I T T E D IN P A R T I A L F U L F I L L M E N T OF T H E R E Q U I R E M E N T S F O R T H E D E G R E E OF M A S T E R OF S C I E N C E in T H E F A C U L T Y OF G R A D U A T E S T U D I E S (Microbiology and Immunology) T H E U N I V E R S I T Y OF B R I T I S H C O L U M B I A Apr i l , 2006 © Barbara Marion Turner, 2006 ABSTRACT In response to a deteriorating environment, Bacillus subtilis cells are capable of several alternate survival strategies including motility, competence development, secretion of proteases and surfactants, and sporulation. Members of the response regulator family of proteins play key roles regulating entry into these alternate states. In the case of sporulation, the master regulator is SpoOA. SpoOA initiates the onset of sporulation by direct or indirect activation or repression of transcription of over 500 genes within the SpoOA regulon. The A 2 5 7 V mutation within SpoOA was previously identified as a mutation which abolishes the ability of B. subtilis cells to sporulate. In vivo, the mutation prevents transcription activation at both the aA-dependent spoIIG operon promoter and the cH-dependent spolIA operon promoter, yet does not affect the ability of SpoOA to repress transcription at the abrB promoter. In this thesis I investigated the biochemical properties of SpoOA(A257V) to determine how the A 2 5 7 V mutation uncouples transcription activation from repression to lead to a sporulation negative phenotype. I demonstrated that the protein is phosphorylated efficiently by a reconstituted phosphorelay in vitro. I showed that Spo0A(A257V) can both repress transcription and activate oA-dependent transcription in vitro, although at a reduced level compared to wi ld type SpoOA. I showed that the A 2 5 7 V mutation did not affect the ability of SpoOA to recognize and bind specific sequences within promoter D N A , but rather that the reduction in transcription activation and repression in vitro could be attributed to a modest decrease in the apparent binding affinity of the mutant protein. While the reduction in apparent binding affinity could explain the in vitro results, it did not account for the complete lack of sporulation in spoOA(A257V) B. subtilis cells. Analysis of SpoOA expression in wild type and mutant strains indicated that SpoOA(A257V) expression was decreased as compared to SpoOA. To discriminate between mechanisms controlling the amount of SpoOA in vivo, strains were constructed which overexpressed wi ld type or mutant SpoOA proteins and used to test activation of a A and oH-dependent promoters in vivo. Results from these experiments were inconclusive; the levels of induced protein may have been insufficient to activate stage II sporulation genes. Ill TABLE OF CONTENTS Abstract i i Table of Contents i i i List of Tables v i List of Figures v i i List of Abbreviations and Symbols ix Acknowledgements x 1. INTRODUCTION 1 1.1. Sporulation in Bacillus subtilis 1 1.1.1. Endospore development 1 1.1.2. Sporulation morphology 1 1.1.3. The sigma factor cascade 4 1.2. Regulation of sporulation 5 1.2.1. SpoOA structure 8 1.2.1.1. The receiver domain of SpoOA 8 1.2.1.2. The DNA-binding domain of SpoOA 10 1.2.2. Sporulation initiation signals 13 1.2.2.1. Nutrient deprivation 13 1.2.2.2. High cell density 13 1.2.2.3. Cell-cycle progression 14 1.2.3. Regulation of SpoOA activation 14 1.2.4. The sporulation regulatory network 15 1.2.4.1. Repression of abrB 15 1.2.4.2. Alleviation of SinR repression 15 1.2.4.3. Antagonism by a negative regulator o f sporulation, Soj 18 1.2.4.4. Induction of a H expression 18 1.3. Transcription 19 1.3.1. R N A polymerase 20 1.3.2. Promoter elements 20 1.3.3. The transcription initiation cycle 21 1.4. Regulation o f transcription initiation 22 1.4.1. Transcriptional activators 23 1.4.2. Transcriptional repressors 23 1.5. Mechanism of activation by positive regulators 24 1.5.1. Catabolite activator protein (CAP) 24 1.5.2. Regulation of transcription by SpoOA 25 1.6. Experimental rationale 26 iv 2 . EXPERIMENTAL PROCEDURES 28 2.1. Bacterial strains and media 28 2.2. Synthesis and cloning of spoOA(A257V) 28 2.3. Expression and purification of SpoOA(A257V) 31 2.4. In vitro phosphorylation reactions 31 2.4.1. In vitro phosphorylation at equilibrium 31 2.4.2. Rate of in vitro phosphorylation 32 2.5. In vitro transcription reactions 32 2.5.1. Preparation of template D N A 32 2.5.2. In vitro transcription reactions 33 2.6. In vitro DNase I footprinting assay 33 2.6.1. Preparation of template D N A 33 2.6.2. In vitro DNasel footprinting reactions 34 2.7. Electrophoretic mobility shift assay ( E M S A ) 35 2.7.1. Preparation of template D N A 35 2.7.2. Electrophoretic mobility shift assay 35 2.8. Cloning of Pspac-5po04f'7-7^6'; 36 2.9. Construction of Vspac-spoOA and Fspac-spoOA(A257V) B. subtilis strains 39 2.10. Determination of sporulation frequency 39 2.11. Immunoblot analysis 40 2.12. P-Galactosidase assay 41 2.12.1 Construction of LacZ reporter strains 41 2.12.2. P-Galactosidase activity 41 3. RESULTS 42 3.1. Examination of the effect of the A 2 5 7 V mutation on SpoOA phosphorylation in vitro 42 3.2. Investigation of the effect of the A 2 5 7 V substitution on in vitro repression of the abrB promoter 43 3.3. Investigation of the effect of the A 2 5 7 V substitution on in vitro stimulation of the spoIIG promoter 47 3.4. Examination of the effect of the A 2 5 7 V mutation of SpoOA on binding to OA boxes encoded within the spoIIG promoter 51 3.5. In vitro examination of the binding affinity of Spo0A(A257V) for consensus OA boxes 52 3.6. Effect of mutations in the OA boxes encoded within the spoIIG promoter on stimulation of transcription by SpoOA~P and SpoOA(A257V)~P 55 3.7. SpoOA(A257V) protein expression in the sporulation negative B. subtilis strain JH695 58 3.8. Construction of B. subtilis strains which over-express wild type and mutant SpoOA proteins 61 V 3.9. Sporulation frequencies of the Vspac-spoOA B. subtilis strains 64 3.10. Investigation of the effect of varying IPTG concentration on expression from Pspac-spoOA(A257V) 66 3.11. Construction of B. subtilis spoilA-lacZ and spoIIG-lacZ reporter strains which overexpress wi ld type and mutant SpoOA 66 3.12. Measurement of spoIIA-lacZ induction in strains encoding inducible wi ld type and mutant SpoOA 68 4. DISCUSSION 74 4.1. SpoOA is the master regulator of the onset of sporulation 74 4.2. The A 2 5 7 V mutation of SpoOA uncouples transcription activation and repression 74 4.3. SpoOA can activate and repress transcription initiation 74 4.3.1 Repression of transcription by SpoOA 74 4.3.2 Activation of oA-dependent transcription by SpoOA 75 4.3.3. Activation of aH-dependent transcription by SpoOA 76 4.4. Objective of thesis 76 4.5. In vitro characterization of Spo0A(A257V) 78 4.6. In vivo investigation of the effect of the A 2 5 7 V mutation of SpoOA 80 4.7. Effects o f residue A257 on SpoOA dimer function 83 4.8. Residue A257 as a part o f a o -SpoOA interaction surface 88 References 90 VI LIST OF TABLES Table 1. Bacterial strains used in this study 29 Table 2. Plasmids used in this study 30 Table 3. Oligonucleotides used in this study 31 Table 4. Activation of spoIIA-lacZ and spoIIG-lacZ transcription in B. subtilis strains which overexpress wi ld type or mutant SpoOA 72 v i i LIST OF FIGURES Figure 1. Sporulation morphology 2 Figure 2. A typical two-component signal transduction system 6 Figure 3. Activation and regulation of SpoOA 7 Figure 4. Structure of the receiver domain of SpoOA 9 Figure 5. Structure of the D N A binding domain of SpoOA 11 Figure 6. SpoOA DNA-bind ing domains bind D N A as a head-to-tail dimer 12 Figure 7. Sporulation regulatory network 17 Figure 8. Cloning of Pspac-spoOA(]-J46) 38 Figure 9. Time course of in vitro phosphorylation of SpoOA and SpoOA(A257V) 44 Figure 10. Phosphorylation of varying amounts of SpoOA and SpoOA(A257V) in vitro 45 Figure 11. Repression of the abrB promoter by SpoOA and Spo0A(A257V) in vitro 46 Figure 12. Time course of in vitro transcription initiation stimulated by SpoOA and SpoOA(A257V) 49 Figure 13. Effect o f SpoOA or Spo0A(A257V) protein concentration on stimulation of spolIG promoter activity in vitro 50 Figure 14. DNase footprint o f SpoOA~P and SpoOA(A257V)~P at the spolIG Promoter 53 Figure 15. E M S A of binding of SpoOA and SpoOA(A257V) 54 Figure 16. W i l d type and mutant SpoOA stimulation of transcription from mutant spolIG promoters in vitro 57 Figure 17. W i l d type and mutant SpoOA protein expression in B. subtilis JH642 and JH695 60 Figure 18. Construction of B. subtilis strains which overexpress wi ld type or mutant SpoOA 63 Figure 19. W i l d type and mutant SpoOA protein expression and sporulation in B. subtilis strains BT2001 and BT2002 in response to IPTG 65 Figure 20. SpoOA(A257V) expression in BT2002 as a function of IPTG concentration 67 Figure 21. Protein expression and sporulation in B. subtilis strains which overexpress wi ld type or mutant SpoOA and encode lacZ translational fusions Figure 22. PspoIlAilacZ activity in B. subtilis strains BT2003 and BT2005 Figure 23. Location of OA boxes within promoters activated and repressed by SpoOA Figure 24. Suppressor mutations of spoOA(A257V) strengthen intermolecular contacts within the SpoOA dimer ix LIST OF ABBREVIATIONS AND SYMBOLS C A P Catabolite activator protein C m R Chloramphenicol resistance C T D C-terminal domain E M S A electrophoretic mobility shift assay E r m R Erythromycin resistance E c R N A polymerase with associated sigma factor ("holoenzyme") H T H helix-turn-helix motif IPTG isopropyl-P-D-thiogalactopyranoside K a n R Kanamycin resistance N T D N-terminal domain OA box specific SpoOA binding site encoded within D N A ( 5 ' T G N C G A A 3 ' ) R N A P R N A polymerase RPci initial closed R N A polymerase-promoter complex R P C 2 final closed R N A polymerase-promoter complex RPo open R N A polymerase-promoter complex RPinit initiated R N A polymerase-promoter complex S A A R sigma-A-activating region within SpoOA SpoOA~P activated, phosphorylated form of SpoOA SpoOA N N-terminal domain of SpoOA o sigma factor X ACKNOWLEDGEMENTS When I began writing this thesis I expected that I would complete it before one of the greatest adventures of my life began. It didn't happen. Instead, I've been busy working on a different kind of experiment, which ( if I may say) is no where near as predictable as the experiments I've done as a student thus far. I 'd like to thank George for having me in his lab as an undergraduate student, employee, and graduate student. Y o u ' v e been a great mentor and I value your guidance and the experience I've had in your lab. I 'd like to thank members of my committee, Rachel Fernandez and Lindsay Eltis, for direction and help throughout the duration of my work and for patiently waiting whilst I finish the never-ending-thesis. I 'd like to thank members of the lab (past and present): Martin Richer for teaching me the basics, Brett McLeod for many discussions over coffee, and lastly my SpoOA-co-conspirator, Steve Seredick, for helping me in countless ways and indulging my chocolate, ice cream, and coffee addictions. I'd like to thank members of the Fernandez lab (past and present), especially Dave Oliver, who was always wi l l ing to answer questions from across the hall. Lastly, I 'd like to thank my family, especially a little monkey named Markus. 1 1. INTRODUCTION 1.1. Sporulation in Bacillus subtilis In bacteria, regulation of gene expression facilitates adaptation to changes in the environment so that growth and division may be optimized (Lodish, 1999). Bacillus subtilis cells are capable o f undergoing several alternate survival strategies in response to an environment with changing nutrients. These include production and secretion of antibiotics, motility and chemotaxis, the development o f competence, and endospore formation. The process of endospore formation is possibly the best understood example of cellular development and differentiation today. Endospores are specialized cell types formed by members of the genus Clostridium and Bacillus in response to prolonged environmental conditions that prevent continued division. Endospores are highly resistant forms that enable the cell to survive environmental stressors such as ionizing radiation, chemical solvents, and hydrolytic enzymes (Nicholson et al, 2000). 1.1.1. Endospore development Following exhaustion of alternate survival strategies and in response to high cell density and nutrient deprivation, B. subtilis cells wi l l enter the sporulation pathway (Freese, 1981; Grossman and Losick, 1988; Hilbert and Piggot, 2004; Sonenshein, 1989). The master regulator of the onset of this process is the response regulator SpoOA. SpoOA integrates information from a complex network of signals from both internal and external environments to initiate a genetic program that divides the cell asymmetrically and ultimately builds the endospore. 1.1.2. Sporulation morphology The development of the endospore can be classified into seven stages based upon cell morphology (Figure 1) (Losick et al, 1986; Ryter, 1965). The first stage is characterized by condensation of duplicate copies of the chromosome and elongation to form an 'axial filament' o f nucleoprotein that stretches longitudinally across the cell (Ben-Yehuda et al., 2 Vegetative _ .„ Cortex ° + u Engulfment Growth Formation Asymmetric Forespore Coat Septation Protoplast Formation Maturation Release K A O o o o o o II, II2 III iv v VI VII spoO spoil spoil spoIII spoIV spoV spoVI Figure 1. Sporulation morphology. The development of the endospore in B. subtilis can be classified into seven stages (0-VII) based upon cell morphology. Vegetative growth is classified as stage "0". Sporulation begins with condensation of the chromosome to form an axial filament along the logitudinalaxis of the cell (not shown). Formation of an asymmetric septum in stage II \ captures one third of the chromosome in the forespore compartment. The forespore is engulfed by the mother cell during stage Il2- The presence of the forespore as a membrane bound vesicle in the mother cell cytoplasm marks stage III. The spore cortex and proteinaceous spore coat are deposited outside the forespore membrane during stages IV and V. Sporulation is completed upon spore maturation and mother cell lysis (stages V I and VII), releasing the endospore into the environment. Sporulation genes are named according to the stage at which mutants stall sporulation (spoO-spoIV). This figure is adapted from Seredick, 2005. 3 2003; Bylund et al, 1993; W u and Errington, 2003). The second morphological event is an atypical cell division in which the cell divides asymmetrically to form a mother cell and forespore. During normal vegetative growth, the cell divides medially to produce two-equally sized daughter cells by forming a structure called a 'Z- r ing ' at the mid-cell. The Z -ring and associated cell division machinery constrict to invaginate the cytoplasmic membrane while at the same time direct synthesis of new cell wall material in the space between the membranes (Errington, 2003). During sporulation, the formation of a single medial Z-ring is inhibited and two Z-rings form, one at each pole (Ben-Yehuda and Losick, 2002). Constriction of one of the two Z-rings and disassembly o f the other leads to formation of a polar septum at one end of the cell, creating the mother cell and forespore (Errington, 2003). Formation of the polar septum captures approximately one-third o f the chromosome in the forespore; the remainder of the chromosome is transferred through the polar septum to the forespore by the D N A transporter protein SpoIIIE (Errington, 2003). In the second half of stage II the forespore is engulfed by the mother cell. During this process the cell wall material in the septum is degraded and the septal membranes migrate around the forespore cytosol, enveloping the forespore in two membranes, one with reversed polarity (Losick et al, 1986). Fusion of septal membranes at the cell apex releases the forespore as a protoplast into a membrane-enclosed vesicle within the mother cell cytoplasm (Errington, 2003), marking stage III of endospore development. The spore cortex and "primordial cell wall" , composed of peptidoglycan, are produced and deposited between the two membranes in stage IV (Losick et al, 1986). Endospore development continues with production of the proteinaceous spore coat from deposition of mother cell structural proteins outside the cortex, creating a tough outer shell (Losick et al, 1986). Throughout maturation the spore acquires properties that confer resistance to several environmental stressors. For example, the chromosome is secured from damage by a coating of protective low-molecular weight proteins, and the spore is protected from heat by dehydration and mineralization through uptake of large amounts of dipicolinic acid and calcium ions from the mother cell (Errington, 2003). In the last stage of development the 4 fully developed spore is released into the environment by lysis of the mother cell (Losick et al, 1986). When the spore is again located in a nutrient-rich environment it wi l l germinate and outgrow into a vegetative cell (Paidhungat and Setlow, 2002). 1.1.3. The sigma factor cascade Expression of genes required for sporulation is regulated by the sequential production of five sporulation-specific sigma factors (Errington, 1993; Piggot and Losick, 2002). The activated, phosphorylated form of SpoOA, SpoOA~P, initiates this "sigma factor cascade" through indirect regulation of transcription of the first "alternate" sigma factor, a H . During vegetative growth, transcription of spoOH, the gene encoding G h , is inhibited by the transition state regulator, A b r B (Weir et al, 1991). A t the onset o f sporulation, transcription of abrB is repressed by SpoOA, facilitating expression of o H and expression of over 87 genes within the o regulon (Britton et al, 2002; Hahn et al., 1995). The next sigma factors produced, a E and o F , are responsible for initiating a specific program of gene expression in the mother cell and forespore, respectively (Dworkin, 2003). Transcription of the spolIG and spoIIA operons, encoding a E and a F , is activated by SpoOA prior to polar septum formation (Gholamhoseinian and Piggot, 1989), although the proteins are held in an inactive state until septum formation is complete (Errington, 2003). The temporal and spatial aspects of a E and c F activation are regulated. Activation of rj F is regulated through the activity of an inhibitory protein that binds to a F whereas activation of c E is regulated by post-translation processing o f a pro-a E form (Errington, 2003). Activation of each sigma factor takes place only in the appropriate compartment. Engulfment of the forespore by the mother cell at the end of stage II leads to activation of a third sporulation-specific sigma factor, a (Sun et al, 1989). Expression of a is limited to the forespore as transcription of the gene encoding o G , spoIIIG, is dependent upon R N A polymerase containing r j F ( R N A P - o F ) (Partridge and Errington, 1993). Activi ty of a G is regulated post-translationally, possibly by an anti-sigma factor, and inhibition is not relieved until engulfment is completed (Partridge and Errington, 1993). Engulfment of the forespore also triggers activation of the last sporulation-specific sigma factor in the cascade, o K . This 5 sigma factor is produced in the mother cell and its activity is regulated at multiple levels. Transcription of sigK occurs specifically in the mother cell under direction of R N A P - o (Kunkel et al, 1988) after excision of a prophage that is integrated at the sigK locus and interrupts the coding region of the sigK gene (Kunkel et al, 1990). a K activity is also regulated post-transcriptionally as it remains inactive until it has been processed proteolytically to remove a short inhibitory N-terminal sequence (Kroos et al, 1989). 1.2. Regulation of sporulation In prokaryotes, detection and response to changes in the surrounding environment is often controlled by the activity of "two-component signal transduction systems". Hundreds o f two-component systems have been identified in archaea and prokaryotes (Lohrmann and Harter, 2002; Stock et al, 2000). There are 34 two-component systems in Bacillus subtilis (Kunst et al, 1997) controlling such diverse processes as phosphate assimilation (Hulett, 1996), secretion of degradative enzymes, the development of competence and endospore formation (Msadek, 1999). The simplest two-component system is composed of a sensor kinase, responsible for detection of extracellular stimuli, and a response regulator which facilitates an appropriate response (Figure 2). Communication between the two proteins occurs using a conserved mechanism involving phosphoryl group transfer. In response to external stimuli, the sensor kinase undergoes ATP-dependent autophosphorylation of a conserved histidine residue within the sensor kinase core. The response regulator catalyzes transfer of the phosphoryl group from the sensor kinase to a conserved aspartate within the response regulator. This prototypical system may be expanded to a "phosphorelay" that includes intermediate phosphotransfer proteins that serve as additional points for regulation. SpoOA is the terminal member of the phosphorelay signal transduction system that governs the commitment of B. subtilis cells to sporulation (Grossman, 1995; Hoch, 1993; Stragier and Losick, 1996). Phosphoryl transfer to activate SpoOA occurs via an elaborate phosphorelay (Figure 3) whose complexity is indicative of the diversity of signals that must be integrated (Burbulys et al, 1991; Perego et al, 1994). There are five histidine kinases ( K i n A , B , C, D , 6 Sensor Kinase N N-Sensing domain Kinase core -( M 1 ^ )-' PO3 ADP ATP aut.ophosphoryla.tion phosphorylation Response Regulator Receiver Output domain domain Asp P O 3 Figure 2. A typical two-component signal transduction system. The simplest two component signal transduction system is composed of a sensor kinase and response regulator. The sensor kinase is usually composed of an N-terminal 'sensing' domain and a C-terminal kinase core. In response to external stimuli, the sensor kinase autophosphorylates by forming a dimer (shown here as a monomer) in which one monomer phosphorylates the other monomer at a conserved histidine residue within the kinase core. The response regulator is able to facilitate a response to the stimuli upon phosphoryl transfer to a conserved aspartate residue within the receiver domain of the response regulator. 7 Signal High cell density, nutrient deprivation, cell cycle progression ATP A D P K i n A K i n B , K i n C , K i n D . K i n E K i n A - P V K i p l V K i p A SpoOF SpoOF~P |-R a p A RapB RapE phr pentapeptides SpoOB SpoOB~P SpoOA SpoOA~P abrBp spoOApv spoOAps spoOFps kinAp +• rap A spolIGp spoIIAp spoIIEp Figure 3. Activation and regulation of SpoOA. Nutrient deprivation, high cell density, and progression o f the cell cycle initiate the sporulation pathway. Phosphate is transferred to SpoOA from a sensor kinase (e.g. K i n A ) , and two phosphotransfer proteins (SpoOF and SpoOB) in a phosphorelay signal transduction system (indicated by curved arrows). Upon activation, Spo0A~P represses transcription from target promoters such as abrB and spoOAv, and activates transcription from spolIGp, spoIIAp, and spoOAps. Transfer o f phosphate from K i n A ~ P and Spo0F~P, and the activity o f SpoOA~P, are all subject to dephosphorylation by phosphatases. Inhibition/transcription repression are indicated by 1 and activation/transcription stimulation are indicated by > . 8 and E) capable of initiating the sporulation phosphorelay (Fabret et al, 1999; Jiang et al, 1999; LeDeaux and Grossman, 1995; Trach et al, 1991). In the case o f at least K i n A , B , and C, the target of each kinase is the phosphotransferase protein, SpoOF, itself a response regulator lacking an output domain (Burbulys et al, 1991; Jiang et al, 2000b; Perego et al, 1989; Stock et al, 1989a; Trach and Hoch, 1993). The phosphoryl group is transferred from SpoOF to another phosphotransfer protein, SpoOB, before reaching the final phosphoacceptor, SpoOA (Burbulys et al, 1991). Once phosphorylated, SpoOA~P initiates a genetic network which culminates in sporulation by direct or indirect regulation of the expression of over 500 genes within the SpoOA regulon (Molle et al, 2003 a). 1.2.1. SpoOA structure SpoOA shares a common architecture with other members of the response regulator family o f proteins. Response regulators generally contain two domains joined by a linker of variable length; an N-terminal receiver domain, and a C-terminal output domain, although some members, such as SpoOF (Stock et al, 1989b), lack an output domain altogether. While the receiver domains of response regulators share a common fold (Stock et al, 1989a), the structures of the output domains vary. With the exception of several members involved in chemotaxis, most response regulators have DNA-binding output domains and function as activators or repressors o f transcription initiation (Stock et al, 2000). 1.2.1.1. The receiver domain of SpoOA The crystal structure of the receiver domain of SpoOA, in an unphosphorylated or phosphorylated monomer form, or in an unphosphorylated dimer form, has been determined (Lewis et al, 1999; Lewis et al, 2000b; Muchova et al, 1998). Like other response regulators it has a three-layer (a(3a) sandwich architecture in a Rossman fold, with five central parallel p-sheets surrounded by five a-helices (Lewis et al, 1999; Muchova et al, 1998) (Figure 4). The site of phosphorylation is located within a pocket located at the C-terminal end of strand p3 (Lewis et al, 1999; Muchova et al, 1998). Phosphorylation of the receiver domain of SpoOA results in a concerted and conserved rearrangement of the receiver domain as in other response regulators (Birck et al, 1999; Kern et al, 1999; Lee et al, 2001; Lewis et al, 1999), which in several cases has been shown to result in N-terminal 9 Figure 4. Structure of the receiver domain of SpoOA. The chain is coloured from the N -terminus (dark blue) to the C-terminus (red) and the secondary structure elements are numbered (ocl-oc5, (51 -(35). The site of phosphorylation (D56) is found at the C-terminal end of (33 (shown with arrow). The structure shown is from Bacillus stearothermophilus, differing from the receiver domain of B.subtilis SpoOA by 24 conservative substitutions (98 conserved residues). The figure is based on P D B file 1QMP deposited by Lewis et al, 1999 in the R C S B protein data bank (http://pdbbeta.rcsb.org/pdbAVelcome.do) and was constructed using P y M O L (Delano Scientific). This figure is adapted from Seredick, 2005. 10 dimerization (Birck et al, 2003; Da Re et al, 1999; Fiedler and Weiss, 1995; McCleary, 1996). Unlike the response regulators PhoB, Nt rC, and FixJ (Birck et al, 2003; Da Re et al, 1999; Fiedler and Weiss, 1995; McCleary, 1996), the structure of the dimer form o f SpoOA indicates that a dimer can be formed by unphosphorylated receiver domains through an exchange of helix oc5 (Lewis et al, 2000b). However, it is suspected that this unusual dimer form may be a consequence of the low p H conditions used to promote crystallization (Lewis et al, 2000b). It is believed that N-terminal interactions after phosphorylation drive dimerization (Lewis et al, 2002). 1.2.1.2. The DNA-binding domain of SpoOA The crystal structure of the C-terminal D N A binding domain o f SpoOA, alone or in complex with D N A , has also been determined (Lewis et al, 2000a; Zhao et al, 2002) (Figure 5). Unlike the receiver domain, the DNA-bind ing domain is conserved only among SpoOA homologues from endospore-forming bacteria (Brown et al, 1994). The crystal structure indicates the DNA-binding domain is composed of six a-helices joined by short segments o f polypeptide and contains a helix-turn-helix (HTH) motif, commonly found in D N A binding proteins (Lewis et al, 2000a). The ocC and a D helices form the H T H motif used for recognition of SpoOA binding sites ("OA boxes") while helix a E composes the S A A R , or "sigma A-activating region" (Lewis et al, 2000a), shown through genetic analysis to be required for activation of aA-dependent promoters (Buckner et al, 1998; Hatt and Youngman, 1998). The crystal structure of the DNA-binding domain in complex with a consensus SpoOA-binding site (from the site of repression of the abrB gene) indicates that SpoOA DNA-binding domains bind tandem OA boxes by forming a head-to-tail dimer (Zhao et al, 2002) (Figure 6). Hydrophobic interactions, salt bridges, and hydrogen bonds hold the monomers together in the dimer, burying 10% of the surface area o f each monomer in the dimer interface (Zhao et al, 2002). The dimer interface is formed by helix aF of the upstream SpoOA monomer and helix aB of the downstream SpoOA monomer (Zhao et al, 2002). The recognition helix, a D , fits perpendicularly into the major groove of the D N A to make three base-specific contacts within the OA box (Zhao et al, 2002). Other contacts with the D N A are non-specific interactions with the D N A backbone (Zhao et al, 2002). 11 Figure 5. Structure of the D N A binding domain of SpoOA. The C-terminal D N A binding domain of SpoOA contains six a-helices connected by short segments of polypeptide and a helix-turn-helix (HTH) motif (helices ocC and aD). Helix a E contains the S A A R , important for activation of o"A-dependent promoters. The chain is coloured from the N-terminus (dark blue) to the C-terminus (red) and the helices are labeled (aA-aF) . The structure shown is from Bacillus stearothermophilus, differing from the D N A binding domain of B.subtilis by 6 conservative substitutions (62 conserved residues). The figure is based on P D B file 1FC3 deposited by Lewis et al., 2000 in the R C S B protein data bank (http://pdbbeta.rcsb.org/pdb/Welcome.do) and was constructed using P y M O L (Delano Scientific). This figure is adapted from Seredick, 2005. 12 Figure 6. SpoOA DNA-bind ing domains bind D N A as a head-to-tail dimer. Tandem molecules of the SpoOA DNA-binding domain bind the consensus SpoOA binding sequence ( 5 - T G N C G A A - 3 ' ) as a head-to-tail dimer. The D N A is shown 5' to 3' and the structure is rotated 90° to indicate the dimer interface. The dimer interface is formed between helices ocB and ocF. Salt bridges, hydrogen bonds and hydrophobic interactions hold the molecules together as a dimer. The recognition helix a D fits perpendicularly into the major groove of the D N A . The S A A R (helix aE) of each monomer is located on the same side of the dimer. The molecules contact both the phosphate backbone and specific base pairs. Helices: a A , blue; ocB, cyan; ocC, green; a D , yellow; aE , gold. The structure is based on P D B file 1LQ1 deposited by Zhao and Varughese, 2002 in the R C S B protein data bank (http://pdbbeta.rcsb.org/pdb/Welcome.do). Figure constructed using P y M O L (Delano Scientific). This figure is adapted from Seredick, 2005. 13 1.2.2. Sporulation initiation signals The initiation of sporulation involves integration of signals from both external and internal environments concerning the nutrient status of the environment, cell density, and progression of the cell cycle (Burkholder and Grossman, 2000; Perego and Hoch, 2002; Trach and Hoch, 1993). 1.2.2.1. Nutrient deprivation One of the main signals for sporulation initiation is nutrient deprivation (Burkholder and Grossman, 2000). The nutrient status of the environment is detected by the transition state regulator C o d Y (Serror and Sonenshein, 1996). C o d Y senses intracellular levels of guanine nucleotides and branched chain amino acids; when bound to G T P or when stimulated by valine, isoleucine or leucine, C o d Y activity is stimulated and represses transcription o f genes involved in sporulation, motility, competence, and genes required for utilization of alternate energy sources (Ratnayake-Lecamwasam et al, 2001; Serror and Sonenshein, 1996). When G T P levels decrease at the onset of stationary phase, repression of sporulation genes, such as phrA, phrE, and kinB, by C o d Y is alleviated (Molle et al., 2003b; Ratnayake-Lecamwasam et al, 2001). The net effect is increased transfer of phosphoryl groups to SpoOA, thus facilitating sporulation initiation. 1.2.2.2. High cell density A second requirement for the initiation of sporulation is high-cell density (Perego and Hoch, 2002). The cell-density of the environment is detected in a mechanism reminiscent of quorum-sensing and involves the secretion and import of short peptides. The peptides are produced from proteolytic processing of the products of the phrA and phrE genes, transcribed with the gene encoding the phosphatases which they inhibit, RapA and RapE (Perego and Hoch, 1996). At low cell density, the RapA and RapE phosphatases dephosphorylate Spo0F~P (Jiang et al., 2000a; Perego et al, 1994). A t the same time, the Phr peptide precursors are exported from the cell and processed into short peptides (Jiang et al, 2000a; Perego et al, 1991a; Rudner et al, 1991). The peptides are re-internalized by means o f the oligopeptide permease (Opp) transport system to inhibit activity of the Rap 14 phosphatases (Bongiorni et al, 2005; Perego et al, 1994), thus allowing phosphoryl transfer to SpoOA and facilitating the onset of sporulation. 1.2.2.3. Cell-cycle progression Sporulation initiation is also dependent upon cell cycle progression. If the chromosome is damaged or i f there is a block in D N A replication, sporulation wi l l not commence due to inhibition of K i n A autophosphorylation by the Sda protein (Ireton and Grossman, 1992, 1994; Lemon et al, 2000; Rowland et al, 2004). Normally, transcription of the sda gene is repressed by the DnaA protein; however, sda repression is alleviated when replication is blocked or D N A is damaged, respectively (Burkholder et al, 2001). 1.2.3. Regulation of SpoOA activation The modular nature of the phosphorelay facilitates many points for regulation of SpoOA activation (Figure 3). The existence of different sensor kinases permits transfer of varying levels of phosphate through the system and permit response to a variety of inputs. Although the activity of K i n C and K i n D produce a level of Spo0A~P sufficient for regulating abrB transcription, the activity of K i n A has proved most critical during sporulation (Antoniewski et al, 1990). Autophosphorylation of K i n A is inhibited by the K i p l protein, itself negatively regulated by K i p A (Wang et al, 1997). Phosphorylation of SpoOF is negatively regulated by three phosphatases, RapA, B , and E (Jiang et al, 2000a) and the activity of each of these is subject to negative regulation by small peptides (Jiang et al, 2000a; Perego, 1999). SpoOA is dephosphorylated by the SpoOE, Yis I and Y n z D phosphatases (Ohlsen et al, 1994; Perego, 2001). Transcription of the kinA, spoOF, spoOA, and rap A genes is activated by Spo0A~P (Errington, 2003; Hilbert and Piggot, 2004; Mol l e et al, 2003a; Piggot and Losick, 2002). In addition, the genes encoding the protein precursors (phr) o f the peptides required to inactivate Rap phosphatase activity are transcribed by R N A P - a H (McQuade et al, 2001), and transcription of the gene encoding a H is indirectly dependent upon activation by Spo0A~P (McQuade et al, 2001). 15 1.2.4. The sporulation regulatory network Activation o f SpoOA results in the modulation of expression of a complex genetic network as shown in Figure 7. During vegetative growth, intracellular levels of SpoOA~P are low due to constitutive expression of spoOA from the aA-dependent spoOApv promoter, SpoOA-mediated repression of the higher-activity spoOAps promoter (Strauch et al, 1992), and reduced phosphorylation of SpoOA by regulation of the phosphorelay as described previously. Upon detection of nutrient deprivation, high cell density, and normal cell cycle progression, there is an increase in transfer of phosphate through the phosphorelay to activate SpoOA. Activated SpoOA initiates two programs of gene expression, one specific for the forespore, the other specific to the mother cell. 1.2.4.1. Repression of abrB A n important consequence of SpoOA activation is repression of the global regulatory protein, A b r B (Strauch et al, 1990). During vegetative growth, A b r B regulates the expression of genes encoding other regulatory proteins, such as ScoC (Perego and Hoch, 1988) and o H (Britton et al, 2002), and sporulation genes such as spoOE (Perego and Hoch, 1991; Strauch et al, 1989), and sinl (Shafikhani et al, 2002). A b r B also represses synthesis of enzymes needed to search for alternate carbon sources (Strauch and Hoch, 1993), thus A b r B is usually thought of as a transition state regulator. Repression of abrB also permits expression of the SpoOE phosphatase (Perego and Hoch, 1991; Strauch et al, 1989), which might limit the level o f Spo0A~P during the time when alternate energy sources are investigated (Phillips and Strauch, 2002). Spo0A~P-mediated repression of abrB inhibits the activity of ScoC, itself a global regulator. ScoC represses transcription of sinl (Kall io et al, 1991) and the opp operon (Koide et al, 1999; Perego and Hoch, 1988). Continued repression of abrB indirectly leads to increased levels of Spo0A~P and thus entry into stationary phase. 1.2.4.2. Alleviation of SinR repression A n additional effect of abrB repression is expression of Sinl , an antagonist of a negative regulator of sporulation, SinR (Bai et al, 1993). The two proteins are encoded in the sinlR operon (Gaur et al, 1988). SinR is expressed from an internal promoter (Gaur et al, 1988) 16 Figure 7. Sporulation regulatory network. A complex genetic network regulated by SpoOA~P governs creation the forespore and mother cell. Genes expressed during vegetative growth are indicated at the top of the diagram and those expressed during later stages of sporulation are indicated progressively from the top. The regulatory inputs affecting transcription of each gene are indicated; positive regulation is indicated by arrow heads while short horizontal bars indicate negative regulation. This figure is adapted from Seredick, 2005. 17 sigA I spoOE J J Z soj v ± ± ± t r T ktnE sinIR - L - L Y t r spoUG 4 4 T spoQApg spoOApy spoOF ^JL-Lj—> - L - t r — spoOH scoC OPP spoIIA rig*: ix i i spoWD spoIUG spoVT Mother Cell Forespore 18 and inhibits transcription from the spoOAps, spolIG, spoIIA, and spoIIE promoters (Louie et al, 1992; Mandic-Mulec et al, 1992; Mandic-Mulec et al, 1995). A t the onset of sporulation, SpoOA~P repression of abrB allows synthesis of o H and the subsequent transcription o f sinl (Shafikhani et al, 2002). This permits expression of SinI which interacts with SinR to relieve SinR inhibition of key sporulation gene promoters (Bai et al, 1993). 1.2.4.3. Antagonism by a negative regulator of sporulation, Soj Another key regulator of the spoOAps, spolIG, spoIIA, and spoIIE promoters is Soj (Cervin et al, 1998; Marston and Errington, 1999; Quisel et al, 1999; Quisel and Grossman, 2000). Soj, and another protein SpoOJ, are thought to play a role in linking chromosome partitioning with sporulation. During chromosome segregation, SpoOJ binds near the oriC and is required for proper chromosome segregation (Lin and Grossman, 1998). However, in the absence of SpoOJ, Soj binds to the spoOAps, spolIG, spoIIA, and spoIIE promoters to repress transcription (Cervin et al, 1998; Marston and Errington, 1999; Quisel et al, 1999; Quisel and Grossman, 2000). Repression is alleviated by SpoOJ, which may antagonize Soj by retaining Soj at the poles of cell (Marston and Errington, 1999; Quisel et al, 1999). 1.2.4.4. Induction of a expression O f particular importance for the initiation of sporulation is de-repression of the spoOH gene which encodes the sigma factor, o H ( W e i r et al, 1991). Expression of spoOH and post-transcriptional stimulation of cH-directed transcription by the C l p X protease (Liu et al, 1999; L i u and Zuber, 2000) permits expression of sporulation genes such as spoVG, required for spore coat synthesis (Rosenbluh et al, 1981), transcription of genes encoding the sensor kinases K i n A and K i n E (Britton et al, 2002; Predich et al, 1992), and genes encoding the phr peptide precursors required for repression of the Rap phosphatases (Britton et al, 2002). In addition, transcription of the spoOF, and ftsAZ genes is increased due to promoter switching to the oH-dependent promoters of these genes (Britton et al, 2002; Predich et al, 1992). 19 The most significant effect of a expression is induction of a positive-feedback loop that leads to increased transcription of spoOA from the a -dependent, SpoOA-dependent promoter of spoOA (spoOAps). Together, the accumulative effects of abrB repression, alleviation o f SinR and soj repression, and oH-dependent transcription are to increase intracellular levels of phosphorylated SpoOA. This increase in SpoOA~P is required for regulation o f "high-threshold SpoOA" genes (Fujita et al, 2005), such as those encoding other sporulation-specific sigma factors, spoIIA and spoIIG (Errington, 1993; Hilbert and Piggot, 2004; Piggot and Losick, 2002), and spoIIE (York et al, 1992), encoding a protein responsible for r j F activation (Arigoni et al, 1996; Duncan et al, 1995; Feucht et al, 1996). Expression of the F E spoIIA and spoIIG operons and subsequent activation of the a and o sigma factors directs compartmentalized gene expression in the forespore and mother cell, respectively (Errington, 1993; Hilbert and Piggot, 2004; Piggot and Losick, 2002), committing the cell to endospore formation. Activation of o~ permits transcription of sigK and spoIIID, which in turn activate transcription of the final mother cell-specific transcription factor, gerE. Similarly, transcription of the final forespore-specific transcription factor, spoVT, is activated following o F-directed transcription of spoIIIG, encoding the a G transcription factor, required for spoVT transcription. 1.3. Transcription The ability to adapt to cellular circumstance through differential gene expression is essential for free-living organisms, whether single- or multi-cellular. In fact, changes in the patterns of gene regulation, in addition to the evolution of new genes, may have played a significant role in generating much of the biological diversity observed today (Ptashne and Gann, 2002). In principle, regulation of gene expression may affect any step leading to a functional gene product. There are four levels of regulation common to both eukaryotes and prokaryotes. These are transcription initiation (how often, when or where a gene is transcribed into m R N A ) , translation (how efficiently m R N A is translated into a protein), m R N A degradation (how long each molecule of m R N A is functional), and protein degradation (how long each protein molecule is functional, i f the functional gene product is protein and not R N A ) . Although each regulatory level impacts the amount of functional gene product ultimately produced, to initiate new metabolic or developmental activities requires induction of 20 transcription of new genes. Thus much of the control over regulation, from bacteria to higher eukaryotes, involves the control o f initiation of transcription by R N A P (Ptashne and Gann, 2002). 1.3.1. RNA polymerase R N A P is a multi-subunit enzyme responsible for R N A synthesis. In prokaryotes, the transcriptionally-competent core of R N A P is a stable non-covalent assembly of four subunits: two a subunits, one P subunit and one P' subunit (Darst, 2001). In vitro, the core wi l l bind and transcribe template D N A , but in a non-specific manner (Borukhov and Nudler, 2003; Murakami and Darst, 2003; Record et al., 1996). Specificity o f initiation is provided by a a subunit which associates more loosely with the core enzyme to form the holoenzyme (Ec) (Ptashne and Gann, 2002). The high-resolution crystal structure o f core R N A P from Thermus aquaticus has revealed that the R N A P is reminiscent of a crab claw, with an internal channel running along its length (Borukhov and Nudler, 2003; Darst, 2001; Zhang et al., 1999). One pincer o f the claw is formed primarily by the p subunit while the P' subunit comprises most of the other pincer. The two a subunits are relatively far removed from the active site, consistent with their roles in promoter recognition and as a scaffold upon which R N A P assembles (Darst, 2001). Organisms such as Escherichia coli and Bacillus subtilis produce multiple sigma factors which recognize different sequences, allowing expression of entire batteries of genes (Gross et al., 1998; Gruber and Gross, 2003; Helmann and Moran, 2002; Paget and Helmann, 2003). In E.coli, a 7 0 is produced during exponential growth, whereas alternate a-factors such as a 3 8 and o~54 are activated during stationary phase and in response to nitrogen limitation, respectively. Although sigma factors confer the ability of R N A P to recognize different classes of promoters, they can be categorized into one of two families, the a 7 0 or a 5 4 families (Gruber and Gross, 2003; Paget and Helmann, 2003). 1.3.2. Promoter elements A s the primary control point for transcription in prokaryotes, the process of initiation has been extensively studied. The basic elements of the transcription cycle have been elucidated through study of E.coli and involve R N A P complexed with the major vegetative sigma 70 factor, a , leading to the mechanism described below. The transcription cycle begins with 21 location of the promoter. The sigma subunit bound to R N A P recognizes two conserved 6-base pair sequences centered approximately 10 and 33 base pairs upstream from the transcription start site (+1), called the "-10" and "-35" hexamers (Record et al, 1996). The 70 regions within o which contact the conserved sequences have been identified from mutational studies and contain H T H motifs responsible for D N A binding (Arthur et al, 2000; Gruber and Gross, 2003; Paget and Helmann, 2003; Zhang et al, 1999). Flanking sequences upstream of the -35 hexamer also contribute to promoter binding. AT- r i ch sequences ("UP elements"), found 40 to 60 base pairs upstream of the transcription start site, stabilize R N A P at the promoter by binding the C-terminal domain of at least one of the a subunits of R N A P (Estrem et al, 1998; Estrem et al, 1999; Record et al, 1996). In the absence o f regulatory factors, promoter strength, the efficiency at which a given promoter is utilized by R N A P , is a function of core promoter sequence and spacing relative to an idealized consensus promoter. Deviations from this ideal reduce the amount of transcription initiation (Record et al, 1996). 1.3.3. The transcription initiation cycle The initiation process for B. subtilis R N A P is generally believed to be similar to that for E.coli (Helmann and Moran, 2002). Structural and kinetic data for initiation of R N A synthesis in E.coli has been recently reviewed (Browning and Busby, 2004; Murakami and Darst, 2003; Record et al, 1996). The basic pathway presented below and the following description is based on these reviews. R + P ^ RPci <-> RPC2 *-> RPoi <-> RP 02 <"> RPinit In the case of E.coli the initial complex formed by R N A P at a strong promoter such as P| a cuv5 is referred to as an initial "closed" complex (RPci) . In this complex, E a 7 0 is bound to only one face of the double helix and the D N A near the start site of transcription is double-stranded or "closed". The initial closed complex is then converted to a secondary closed complex, RPc2, following major conformational changes in R N A P and promoter D N A . In this intermediate form, R N A P makes more extensive contact with the promoter by binding additional D N A upstream and downstream of the transcription start site. Downstream of the 22 -10 element polymerase contacts both strands indicating that the D N A is enveloped by R N A P . Without further changes in the extent o f the R N A P - D N A interface, RPc2 is then reversibly converted to an initial "open" complex (RPoi) in which the D N A strands extending from the -10 element region to immediately adjacent to the start site region (-1) are opened in a 'transcription bubble'. The transcriptionally-competent, or final open complex (RP02), is formed when the denatured region extends past the transcription start site (+1). Formation o f this complex is not observed at all promoters and has been shown to be M g 2 + and/or 70 temperature dependent at E.coli E a promoters. The mechanism behind D N A melting remains unknown despite years of research. Once the final open complex is formed, the template strand is accessible to initiating nucleotide triphosphates (NTPs), and is primed to complete transcription initiation. The N T P complementary to the first base pair o f the template D N A strand binds reversibly to the stable open complex to form the first in a series of ternary initiation complexes (RPjnit). The ternary initiation complex advances by binding subsequent NTPs , the P subunit catalyzing covalent bond formation. R N A P characteristically undergoes a process of "abortive initiation" in which short transcripts are continually synthesized and released to reform RPc2-The transcription cleavage factors GreA and GreB (Borukhov et al, 1993; Sparkowski and Das, 1990) are involved in increasing the efficiency of transcription elongation. The two proteins promote cleavage o f R N A in elongating complexes that are stalled (Borukhov et al., 1993; Feng et al, 1994; Izban and Luse, 1992; Komissarova and Kashlev, 1997a, b; Lee et al, 1994; Nudler et al, 1997; Reines, 1992a; Reines, 1992b). This allows R N A P to reinitiate to create a productive elongation complex (Toulme et al, 2000). Upon formation of a 7-12 nucleotide-long R N A chain, R N A P undergoes "promoter clearance", characterized by release of the a-subunit from the core and synthesis of full-length transcripts; at this point no specific contacts between R N A P and the promoter remain. 23 1.4. Regulation of transcription initiation Regulation of transcription is facilitated by proteins that either activate or repress transcription initiation by binding to specific sites on D N A and having a direct effect on the kinetics or equilibrium o f the individual steps of initiation (Record et al, 1996). These key regulatory proteins, "activators" and "repressors", underlie the ability of cells to turn genes on and off(Lodish, 1999). 1.4.1. Transcriptional activators In prokaryotes, there are four known mechanisms whereby activators stimulate transcription from specific promoters. In the first mechanism, activators regulate recruitment of E G 7 0 to specific promoters. In this scenario the holoenzyme is in a 'constitutively active' state and cooperative binding of holoenzyme with the transcriptional activator, such as C A P , leads to transcription initiation (Busby and Ebright, 1999; Lawson et al, 2004). In the second mechanism, activators such as SpoOA (Bird et al, 1996) and c l (Jain et al, 2004; Nickels et al, 2002) stimulate a conformational change in R N A P after binding which converts the R N A P - D N A complex from a closed to open complex. Conversely, some holoenzymes, such as E r j 5 4 , are not constitutively active. In this case the activator, such as NtrC, activates a holoenzyme that is pre-bound to the D N A by inducing a conformational change in E r j 5 4 such that it becomes capable o f initiating transcription (Ptashne and Gann, 2002; Zhang et al, 2002). A fourth proposed mechanism of transcriptional activation involves binding of an activator, such as MerR, to induce a conformational change in the conformation of promoter D N A , thus leading to initiation of transcription (Heldwein and Brennan, 2001). In this mechanism it is unclear whether MerR binds prior to E o 7 0 or after the D N A occupied by a 70 pre-bound, inactive E a . 1.4.2. Transcriptional repressors A s in activation, there are multiple mechanisms whereby transcriptional repressors can reduce the frequency of transcription initiation. In the simplest case, repressors may physically exclude R N A P from binding to target promoter sequences. For example, in the bacteriophage X, the repressor A.cl and R N A P compete for binding to overlapping sites at the 24 PR promoter (Ptashne and Gann, 2002). In cases where repressor DNA-bind ing sites are adjacent to promoter D N A , cooperative repressor binding causes intervening regions of D N A to loop out, again excluding R N A P from binding to promoter sequences (Ptashne and Gann, 2002). Repressors can also inhibit an activator, either by preventing the activator from binding to operator sequences, as the repressor CytR inhibits C A P binding (Gerlach et al, 1990), or repressors can interfere with activator-RNAP interaction (Ptashne and Gann, 2002). Lastly, transcriptional repressors, such as the Gal repressor and A b r B , inhibit DNA-bound R N A P by inhibiting the conversion from closed to open complex (Choy et al, 1995; Choy et al, 1997). 1.5. Mechanism of activation by positive regulators 1.5.1. Catabolite activator protein (CAP) The mechanism of activation of transcription initiation has been extensively studied in E.coli using the transcriptional activator, C A P . C A P is responsible for activating transcription of over one-hundred genes in the presence of the allosteric effector c A M P by binding as a dimer to specific D N A sites within target promoters and enabling R N A P to initiate transcription (Busby and Ebright, 1999; Lawson et al, 2004). There are three mechanisms whereby C A P stimulates transcription, distinguished by the position of the C A P binding site within the target promoter and whether that promoter requires additional regulatory proteins for activation. A s a "class I activator", C A P can stimulate transcription initiation by simple recruitment of R N A P to promoter D N A . In this mechanism, the binding site for the C A P dimer is located upstream of the conserved promoter elements (typically located between -62 and -93), such as at the lac promoter (Busby and Ebright, 1999). Interactions between the DNA-bound C A P dimer and the C-terminal domain (CTD) of one of the a subunits of R N A P facilitates binding o f a C T D and the remainder o f R N A P to the promoter, thereby permitting formation of an initial closed promoter complex (RPci) and stimulation of transcription initiation (Busby and Ebright, 1999). C A P can also activate transcription from promoters in which the C A P binding site overlaps the conserved -35 promoter element, as in the case at the galPl promoter (Busby and Ebright, 1999). In this mechanism, C A P functions as a "class II activator" and both recruits R N A P to the promoter and stimulates a post-recruitment isomerization of R N A P leading to formation of an open promoter complex (RPo) by making 25 three interactions with R N A P (Busby and Ebright, 1999). A n interaction between the upstream C A P monomer and a C T D functions to recruit R N A P to the promoter while additional interactions between the downstream C A P monomer and the N-terminal domain 70 (NTD) of a and a facilitate isomerization from a closed to open promoter complex (Busby and Ebright, 1999). Lastly, as a "class III activator" C A P can activate transcription at promoters which encode multiple C A P binding sites within the promoter D N A sequence. In this mechanism, C A P may interact with additional regulatory proteins to recruit R N A P to the promoter, or recruit R N A P to the promoter and stimulate closed to open complex isomerization, or facilitate interaction between the additional regulatory proteins and R N A P by bending promoter D N A (Busby and Ebright, 1999). 1.5.2. Regulation of transcription by SpoOA A s a transcription factor, the activated form of SpoOA (SpoOA~P) exerts its regulatory function by binding to specific D N A sequences termed "OA boxes" via its C-terminal domain (Spiegelman et al, 1995; Strauch et al., 1990). A t promoters activated by SpoOA, binding of SpoOA to OA boxes 5' o f the transcription start site increases the rate of transcription initiation (Bird et al, 1996; Rowe-Magnus and Spiegelman, 1998). Promoters activated by SpoOA include the spoIIA, and spoIIG operons, which encode the forespore-specific sigma factor a F , and the mother cell specific transcription factor a E (Hoch and Silhavy, 1995). In contrast, binding of SpoOA to specific D N A sequences 3' o f the transcription start site causes repression of transcription (Spiegelman et al., 1995; Strauch et al, 1990). Promoters repressed by SpoOA include the abrB, spoOF and spoOA promoters, which encode the transition state regulator A b r B , the response regulator SpoOF, and SpoOA itself (Bai et al, 1990; Chibazakura et al, 1991; Hoch and Silhavy, 1995; Strauch et al, 1990; Strauch etal, 1992). SpoOA is a particularly compelling response regulator in that it likely activates transcription by two distinct mechanisms. At the spoIIA promoter, SpoOA interacts with R N A P H A containing a while at the spoIIG promoter, SpoOA interacts with another sigma factor, a (Buckner et al, 1998; Hatt and Youngman, 1998; Kumar et al, 2004). A t spoIIG, SpoOA appears to compensate for overlong spacing between the conserved consensus elements to 26 which R N A polymerase binds (McLeod and Spiegelman, 2005; Seredick and Spiegelman, 2004). The binding sites for SpoOA overlap the upstream -35 element for R N A polymerase. Mutations in both SpoOA and a A specifically affecting transcription from the spolIG promoter have been defined (Baldus et al, 1995; Buckner et al, 1998; Hatt and Youngman, 1998; Schyns et al, 1997). The majority o f the mutations within SpoOA that effect activation are located on the S A A R (Lewis et al, 2000a). In contrast, the role of SpoOA in transcription activation of aH-dependent promoters such as spoIIA is not understood. 1.6. Experimental rationale A large number of mutations within SpoOA have been identified and tested. One SpoOA mutant which contains a substitution of valine for alanine at position 257 within the C-terminal domain, SpoOA(A257V), is particularly unusual (Ferrari et al, 1985; Perego et al, 1991b). Previous in vivo studies indicate that this mutant is incapable of activating transcription initiation at either the spoIIA promoter or the spolIG promoter, yet proficiently represses the abrB promoter (Perego et al, 1991b; Rowe-Magnus et al, 2000). These data suggest that SpoOA(A257V) binds D N A normally, but is somehow defective in stimulating transcription, perhaps due to a faulty interaction between R N A P and SpoOA(A257V). Previous determination of the crystal structure of the isolated C T D (Lewis et al, 2000a) has determined that this mutation lies in helix aF. A more recent structure of the isolated C T D complexed with D N A revealed that when the C T D alone is bound at OA boxes such as those at the abrB gene the A 2 5 7 V mutation would likely indirectly weaken intermolecular contacts between SpoOA monomers (Zhao et al, 2002). The location of two suppressor mutations of SpoOA(A257V) within the dimer structure support this hypothesis. The suv4 (H162R on helix aA) and suv3 (L174F on helix aB) (Perego et al, 1991b) mutations have been proposed to suppress the A 2 5 7 V mutation by strengthening intermolecular interactions between two molecules of SpoOA complexed at promoter D N A (Zhao et al, 2002). However, since Spo0A(A257V) represses transcription o f the abrB gene normally the explanation that the A 2 5 7 V mutation disrupts dimer binding seems improbable. Adding to the confusion is the fact that the OA boxes presumed to be involved do not always occur in pairs, such as at the SpoOA-activated spoIIA and spoOAps promoters (Seredick and 27 Spiegelman, 2001; Spiegelman et al, 1995) or with the same spacing, such as at the spoIIG promoter. In addition, the flexibility in the linker region between the receiver and D N A -binding domains permits the possibility of formation of several orientations upon D N A binding. If SpoOA does activate transcription as a dimer, a recent model of SpoOA activation suggests that dimerization is mediated by interactions between the receiver domains and not by interactions between DNA-binding domains (Lewis et al, 2002), possibly minimizing the role of the A 2 5 7 V mutation in disruption of crucial interactions within a dimer. Another possible explanation for the effect of the A 2 5 7 V mutation on SpoOA function is that it disrupts an interaction with o H required for transcription activation. Rowe-Magnus et. al proposed that the C-terminal region of SpoOA (containing A 2 5 7 V ) is a second area required for transcription stimulation (Rowe-Magnus et al, 2000). Mutations in this region might reduce interaction with a H . Since interaction with o H is needed to increase levels o f Spo0A~P which is required for stimulation of stage II sporulation genes such as spoIIA and spoIIG (Fujita et al, 2005), it seemed possible that SpoOA(A257V) was not defective in transcription initiation of a A dependent genes. To determine i f the A 2 5 7 V mutation specifically effected cH-dependent transcription initiation, I have conducted an in vitro characterization of A 2 5 7 V . Here I have demonstrated that SpoOA(A257V) is phosphorylated normally, can bind D N A , and is able to activate and repress transcription initiation in vitro. However, the mutant protein is expressed at reduced levels as compared to SpoOA. In order to distinguish whether the reduction in Spo0A(A257V) expression was due to a defective interaction between SpoOA and a H or between SpoOA dimers I analyzed activation of a o A - and a aH-dependent promoter in vivo when wi ld type or mutant proteins were overexpressed. Although the results from these experiments were inconclusive and require further investigation, I suggest that the A 2 5 7 V mutation uncouples a positive feedback loop, preventing the accumulation of SpoOA to levels sufficient to activate transcription of stage II genes required for sporulation. 28 2. EXPERIMENTAL PROCEDURES 2.1. Bacterial strains and media Bacterial strains, plasmids, and oligonucleotides used in this study are indicated in Tables 1, 2, and 3. E.coli cultures were grown in Luria Bertani (LB) media supplemented with 100 ug/ml ampicillin and 34 pg/ml chloramphenicol where necessary for plasmid selection. B. subtilis cultures were grown in L B (Sambrook et al, 1989) or Schaeffer's sporulation media ( S S M ; (Schaeffer et al, 1965), First Growth Period Supplemented M G (Hoch, 1991), or Second Growth Period Supplemented M G (Hoch, 1991) with the addition of tryptophan and phenylalanine (10 p:g/ml each), and the antibiotics erythromycin (0.3 ug/ml), chloramphenicol (5 pg/ml), or kanamycin (5 ug/ml) when necessary. Standard genetic techniques, enzymatic reactions and D N A manipulations were performed as described (Sambrook et al, 1989) or as recommended by the manufacturer. Bacillus transformations were performed as described (Hoch, 1991). 2.2. Synthesis and cloning of spoOA(A257V) Chromosomal D N A was isolated from B. subtilis strain JH695 as described previously (Hoch, 1991). The spoOA(A257V) gene was amplified by P C R using the upstream primer OA-5 and the downstream primer OA-4 (Table 3). The resulting 848 bp fragment was directly ligated into the p G E M - T cloning vector, creating plasmid p G E M A 9 V , and transformed into E.coli D H 5 a . The A 2 5 7 V substitution was confirmed by sequencing completed by the Nucleic A c i d and Protein Service Unit, University of British Columbia. To create a plasmid construct for protein expression, p G E M A 9 V was digested with Nco\ and BamHl to yield an 848 bp fragment encoding the full length spoOA(A257V) sequence, including the spoOA(A257V) stop codon and transcription terminator. This fragment was ligated into a NcoI/BamHI digested p E T l 6b (Novagen) expression vector creating plasmid pET16bA9V. pET16bA9V was transformed into E.coli BL21( lDE3)pLysS , creating strain B T A 9 V . 29 Table 1. Bacterial strains used in this study Strain Genotype" Source or Reference E. coli D H 5 a [hsdR\l(x\c,m^+)supE44 Thi-1 Invitrogen recAl gyrA (NaT) relAl A(lacZYA-argF)U169 (cp80lacZAM15) BL21(A,DE3)pLysS F" ompT hsdSB (rB"mB") gal dcm Invitrogen (DE3) pLysS(Cm R ) B T A 9 V BL21(?J)E3)pLysS pET16bA9V This study M C O A BL21(^DE3)pLysS pET16bOA Lab Stock B. subtilis b JH642 trpC2 phe-1 J. Hoch, Scripps Research Institute La Jolla California U S A JH695 trpC2 phe-1 spoOA(A257V) Ferrari et al, 1985 JH16124 trpC2 phe-1 amyE:: (spoil'A- M . Perego, Scripps Research lacZ), C m R Institute L a Jolla California U S A JH16304 trpC2 phe-1 amyE::(spoIIG- M . Perego, Scripps Research lacZ), K a n R Institute La Jolla California U S A BT2001 JH642 s/w&4::(pMNSpoOAN), E r m R This study BT2002 JH695 ^o6»^. : (pMNSpoOAN;, E r m R This study BT2003 BT2001 amyE::(spoIIA-lacZ), C m R This study BT2004 BT2001 amyE::(spoIIG-lacZ), K a n R This study BT2005 BT2002 amyE:.(spoil'A-lacZ), C m R This study BT2006 BT2002 amyE::(spoIIG-lacZ), K a n R This study aspoIIA-lacZ and spoIIG-lacZ are integrated in the amyE gene; p M N S p o O A N is integrated into the spoOA gene; C m R , chloramphenicol resistance; K a n R , kanamycin resistance; E r m R , erythromycin resistance. b all B. subtilis strains were derived from the parent JH642 and contain the trpC2 and phe-1 mutations. 30 Table 2. Plasmids used in this study Plasmid Description Source or Reference p G E M - T p G E M A 9 V pGEMSpoOA p G E M O A N pET16b pET16bOA pET16bA9V p M U T I N - 4 pMNSpoOA p M N O A N pVCIIGtrpA pJM5134 p U C / / G 2 7 down p\JCIIG2.2 down A m p vector used for cloning P C R products p G E M - T with a 848 bp insert bearing the spoOA(A257V) coding sequence, start and stop codons and transcription terminator. p G E M - T with an 831 bp insert bearing the spoOA ribosome binding site and coding sequence up to E263. p G E M - T with a 505 bp insert bearing the spoOA ribosome binding site and coding sequence up to PI46. A m p R , K a n R vector used for protein expression pET16b with a 848 bp Ncol-BamHl insert bearing the spoOA coding sequence, start and stop codons and transcription terminator. pET16b with a 848 bp Ncol-BamHl insert bearing the spoOA(A257V) coding sequence, start and stop codons and transcription terminator. R R A m p , Erm integration vector used for systematic inactivation of coding sequences in B. subtilis. Features a spoVG-lacZ translational fusion, the LacI-repressed/IPTG-inducible Pspac promoter, lad, ermAM, bla, and TO, T l , and T3 transcription terminators. p M U T I N - 4 with a 2025 bp deletion (AspoVG-lacZumA) and a 874 bp insert bearing the spoOA ribosome binding site and coding sequence up to E263. pMNSpoOA with a 855 bp deletion (AspoOA) and a 481 bp insert bearing the spoOA ribosome binding site and coding sequence up to PI46. pUC19 with a 240 bp Hindlll to BamUl D N A fragment bearing the spolIG promoter and 100 bp downstream of the transcription start site encoding the trpA terminator Cloning vector with an 814 bp D N A fragment bearing the -703 to +37 region of abrB, relative to the P2 promoter start site. Includes two abrB transcription initiation sites (PI , P2) and two downstream and one upstream OA box. pXJCIIGtrpA with mutation of the site 2.1 OA box pVCJIGtrpA with mutation of the site 2.2 OA box Promega This study This study This study Novagen Lab stock This study Vagner et al., 1998 This study This study Satola et al., 1991 Perego et al., 1988 Seredick et al., unpublished Seredick et al., unpublished 31 Table 3. Oligonucleotides used in this study Name Sequence OA-5 5' - C G C C A T G G A G A A A A T T A A A G T T T G T G T T G - 3 ' OA-4 5' - C G G G A T C C A A A G A C G T T T G A T - 3 ' abrB-F 5' - A A G G A T T T T G T C G A A T A A T G A C G A A - 3 ' abrB-R 5' - T C T T C G T C A T T A T T C G A C A A A A T C C - 3 ' IIG 2 X 5 ' - G G G G A T C C C T C G A G G T C A - 3 ' M 1 3 R 5' -C A G G A A A C A G C T A T G A C C - 3 ' B T O A R B S 5 ' - A A G C T T G G T G A A T C C T G T T A - 3 ' OAEco 5 ' - T C T A A C C T C A G C T T A T C C G C - 3 ' BTOAlinker 5 ' - G T C G A C A G G C T G G C T G C T G C G T A T A A T - 3 ' 2.3. Expression and purification of SpoOA(A257V) SpoOA and SpoOA(A257V) were expressed and purified from strains M C O A and B T A 9 V as described previously (Seredick et al, 2003) up to and including concentration of protein following heparin-agarose affinity purification. Concentrated protein was dialyzed overnight at 4°C against 2 L o f buffer C (20mM sodium phosphate (pH 8.0), 1 m M ethylenediaminetetraacetic acid ( E D T A ) , and 1 m M phenylmethylsulfonyl fluoride (PMSF)) + 50mM N a C l . Following dialysis the protein was loaded directly onto a 30 ml D N A -cellulose column equilibrated with buffer C + 50 m M N a C l and eluted with a 150 ml linear gradient of 50-850 m M N a C l in buffer C. Samples were analyzed by S D S - P A G E and those containing SpoOA or Spo0A(A257V) were pooled and concentrated by placing in dialysis tubing and surrounding with PEG2o,ooo at 4°C for approximately 4 hours. The concentrated protein was dialyzed overnight at 4°C against 2 L of buffer C containing 150 m M N a C l , 0.1 m M dithiothreitol (DTT) and 30% glycerol. Purified SpoOA and SpoOA(A257V) were aliquoted and stored at -20°C. The final products were approximately 95-98% pure as determined by spot densitometry of silver stained S D S - P A G E gels and was used directly for in vitro studies. 2.4. In Vitro phosphorylation reactions 2.4.1. In vitro phosphorylation at equilibrium SpoOA and Spo0A(A257V) were activated as previously described (Bird et al, 1993). Briefly, 4 u M SpoOA and SpoOA(A257V) were incubated with 1.56 u M K i n A , 0.1 p M 32 SpoOB, 2 u M SpoOF, 10 u M A T P in l x transcription buffer (Bird et al, 1993) for 2 hours at 25°C. The phosphorelay proteins used in activation of wi ld type and mutant SpoOA proteins were isolated as described previously (Grimshaw et al, 1998; Zapf et al, 1996; Zhou et al, 1997). In vitro phosphorylation of SpoOA and SpoOA(A257V) was compared by incubating 0.5, 1.0, or 2.0 u M SpoOA or Spo0A(A257V) with 0.75 u M K i n A , 0.1 u M SpoOB, 0.5 u M SpoOF, 25 u M A T P , and 30 uCi of [y- 3 2 P]ATP (6000Ci/mM; Amersham Biosciences) in l x transcription buffer in a final volume of 10 ul for 4.5 hours at 25°C. Labeled proteins were separated by electrophoresis through 15% S D S - P A G E and detected using a Molecular Dynamics Phosphorlmager SI. The level o f protein phosphorylation was quantified using ImageQuant 5.2 software. 2.4.2. Rate of in vitro phosphorylation The in vitro phosphorylation rates of wi ld type and mutant SpoOA proteins were determined by incubating a large reaction (140 ul) o f phosphorelay components (0.75 u M K i n A , 0.1 u M SpoOB, 0.5 u M SpoOF, 25 u M A T P and 25 uCi of [y - 3 2 P]ATP (6000Ci/mM; Amersham Biosciences) in l x transcription buffer at 25°C. Aliquots (10 ul) were removed at 10 seconds and 1, 2, 4, 6, 8, 10, and 12 minutes following addition of 0.5 u M SpoOA or Spo0A(A257V) and were added directly to 5 ul of 2x S D S - P A G E buffer (100 m M Tr is -Cl , pH 6.8, 4 % SDS, 0.2 % bromophenol blue, 20% glycerol, 2 m M P-mercaptoethanol). Labeled proteins were separated by electrophoresis through 15% S D S - P A G E and detected using a Molecular Dynamics Phosphorlmager SI system. The incorporation of radioactive phosphorus was quantified using ImageQuant 5.2 software. 2.5. In vitro transcription reactions 2.5.1. Preparation of template DNA A 600 bp D N A fragment generated from .PvuII digestion of either pUCIIGtrpA, p\JCIIG2.1 down, or p\JCIIG2.2down plasmids were used as D N A templates for spolIG in vitro transcription reactions. The Pvull fragments were separated by electrophoresis though an 8% polyacrylamide gel in l x T B E buffer and recovered using a QIAquick Gel Extraction 33 K i t (Qiagen Inc, Mississauga Ontario). D N A template encoding the abrB promoter used for in vitro transcription reactions was a 804 bp fragment recovered as described above from EcoRVHindm digest of pJM5134 (Greene and Spiegelman, 1996; Perego et al, 1988). This fragment includes both abrB transcription initiation sites (PI , P2) in addition to a pair of downstream OA boxes and one upstream OA box, but lacks a transcription terminator. 2.5.2. In vitro transcription reactions In vitro transcription assays to test the stimulatory or inhibitory effects of different forms of SpoOA were completed as described previously (Bird et al, 1993; Greene and Spiegelman, 1996). For spoIIG transcription, template D N A (4 nM) and 0-1200 n M unphosphorylated or phosphorylated SpoOA and SpoOA(A257V) were incubated with the initiating nucleotides, A T P (0.4 nM) and G T P (5 uM), in l x transcription buffer for two minutes at 37°C. R N A P containing a A (Dobinson and Spiegelman, 1987) (20nM) was added to the reaction to permit transcription initiation. After two minutes (for equilibrium assays) or 0-90 seconds (for kinetic assays), U T P , C T P (0.4 n M each) and heparin (5 ug/ml final concentration) were added to allow elongation of initiated complexes. For abrB transcription, template D N A (4 nM) and unphosphorylated or phosphorylated SpoOA and SpoOA(A257V) was incubated with A T P , G T P and U T P in l x transcription buffer for three minutes at 37°C. R N A P - c j A (20nM) was added to the reaction and the mixture allowed to equilibrate for 2 minutes before the addition of C T P and heparin. The reactions were terminated after five minutes by the addition of 5 pi of stop buffer (7 M urea, 0.1% bromophenol blue, and 0.1 % xylene cyanol in 0.5x T B E (Sambrook et al, 1989). Transcripts were separated by electrophoresis through an 8% polyacrylamide gel containing 7 M urea and detected using a Molecular Dynamics Phosphorlmager SI system. The amount of transcripts produced was quantified using ImageQuant 5.2 software. 2.6. In Vitro DNase I footprinting assay 2.6.1. Preparation of template DNA Forty micrograms of pUCIIGtrpA plasmid D N A was incubated with 30 U BamHl to linearize the vector 135 bp downstream of the +1 transcription start site o f the spoIIG promoter. The linearized vector was dephosphorylated by two sequential 45 minute 34 incubations with 20 U of calf intestinal alkaline phosphatase (CIAP) . The dephosphorylation reaction was terminated with the addition of 0.1% SDS and 20 m M ethyleneglycol-bis(P-aminoethyl)-N,N,N',N'-tetraacetic A c i d ( E G T A ) . C I A P was removed from the reaction by phenol-chloroform extraction and the D N A was ethanol precipitated. Dephosphorylated template D N A was end-labeled with [y P ] A T P by resuspension of the precipitated D N A in l x forward kinase buffer (0.5 M Tr i s -HCl , p H 7.6; 0.1 M M g C l 2 ; 50 m M D T T ; 1 m M spermidine; 1 m M E D T A , pH 8.0) and incubation with 1333 uCi [y 3 2 P]ATP (6000 Ci/mmol) ( ICN Biomedicals) and 12 U T4 kinase (Invitrogen) at 37°C. Following 45 minutes incubation, the reaction was stopped by incubation at 60°C for 15 minutes and the end-labeled D N A was treated with 80 U Pvull for 3 hours to generate 111 bp, 411 bp and 2394 bp fragments from cleavage of pUCIIGtrpA at sites within the vector backbone. The D N A fragments were separated by electrophoresis through a 5 % non-denaturing gel and located by autoradiography. The piece of the acrylamide gel containing the 411 bp D N A fragment with the spoIIG promoter was excised from the gel and incubated in 800 pi of passive elution buffer (500 m M N H 4 A c ; 10 m M M g A c ; 1 0 m M Tris, p H 7.9; 1 m M E D T A , p H 8.0; 0.1 % SDS) at room temperature. After 12 hours incubation, the labeled D N A fragment was recovered by ethanol precipitation and the total radioactivity in the re-dissolved D N A sample was determined by measuring the Cerenkov radiation in an aliquot. 2.6.2. In vitro DNasel footprinting reactions DNase 1 footprinting assays to determine the location o f Spo0A~P and Spo0A(A257V)~P binding were carried out as described previously (Bird et al., 1996). Briefly, 2.0 x 10 5 C P M [y 3 2 P]ATP end-labeled spoIIG promoter D N A was incubated with 0-600 n M Spo0A~P or SpoOA(A257V)~P in l x T2 transcription buffer (40 m M Hepes, pH 8.0; 10 m M M g A c ; 1 m M E D T A , pH 8.0; 1 m M D T T ; 1 mg/ml acetylated B S A ) at 37°C. Control reactions contained only spoIIG promoter D N A in l x T2 transcription buffer. After two minutes incubation, DNasel was added to the reactions at a final concentration o f 4 pg/ml and allowed to cleave the D N A for 10 seconds before the reaction was terminated with the addition of 75 pi of DNase stop buffer (0.1 % SDS; 4 m M E D T A , p H 8.0; 270 m M N a C l ; 40 pg/ml salmon sperm D N A ) . The reactions were ethanol precipitated and resuspended in 5 pi of formamide loading buffer (80 % formamide; 10 m M E D T A , pH 8.0; 1 mg/ml xylene 35 cyanol; 1 mg/ml bromophenol blue) and the activity of labeled D N A in each reaction was estimated from Cerenkov radiation in the sample. Approximately 1.13 x 10 5 C P M o f labeled fragments from each reaction was separated by electrophoresis through a 6% sequencing polyacrylamide gel. Following 3 hours electrophoresis at 2000 V , the gel was dried at 80°C for 1 hour and the DNasel protection patterns of SpoOA~P and SpoOA(A257V)~P were detected by autoradiography. 2.7. Electrophoretic mobility shift assay (EMSA) 2.7.1. Preparation of template DNA To assess binding o f phosphorylated and unphosphorylated SpoOA and SpoOA(A257V) to consensus OA boxes, template D N A was prepared by annealing complementary oligonucleotides abrB-F and abrB-R (Table 3) (Alpha D N A , Montreal Quebec) to form a 23 bp duplex D N A with two-base pair overhangs at both 5' and 3' ends. The duplex sequence matched that of abrB gene from +3 to +29 and contained two OA boxes which exactly matched the consensus OA binding site. Duplex D N A (10 pmol) was labeled by fil l ing in the two base overhangs by incubation with 0.016 U Klenow Fragment (Invitrogen), 16.6 u M dGTP, 16.6 u M d A T P , 20 u M dTTP (Amersham Biosciences), and 30 uCi [a- 3 2 P]dTTP (3000Ci/mmol) in l x React 2 buffer (Invitrogen) in a final volume of 30 ul for 30 minutes at 25°C. The labeled oligonucleotide duplex was precipitated, re-suspended in 10 m M Tris, 0.1 m M E D T A (pH 7.6) and the amount of incorporated radioactivity was estimated from the Cerenkov radiation in an aliquot. 2.7.2. Electrophoretic mobility shift assay Electrophoretic mobility shift assays were performed as described previously (Seredick et al, 2003). To test binding to consensus OA boxes encoded within abrB D N A , unphosphorylated and phosphorylated SpoOA and Spo0A(A257) were incubated with 5 x 10 4 C P M (8 nM) of labeled duplex D N A in l x transcription buffer at 37°C. After two minutes, 3.3 ul of loading buffer (0.3 mg/ml sonicated calf thymus D N A and 20% glycerol in l x transcription buffer) was added to each sample and the reactions were loaded onto a running 8% polyacrylamide gel (40% acrylamide:1.38% bisacrylamide) in 0.8x T A E (Sambrook et al, 1989) containing 2% glycerol. Protein-DNA complexes were separated from unbound 36 duplex D N A by electrophoresis at 12 V cm" 1 for 1.5 hr and detected using a Molecular Dynamics Phosphorlmager SI system. The fractional saturation of D N A binding was determined at each protein concentration. The dissociation constant (Kd) for binding o f phosphorylated and unphosphorylated SpoOA and SpoOA(A257V) was determined by fitting the normalized percent duplex D N A bound into a one-site ligand binding model (y = B m a x x / k d + x) using the program SigmaPlot version 8.0 (SPSS). This calculation assumes that SpoOA binds as a dimer to the two OA boxes which act as a single site on the duplex. 2.8. C lon ing of Yspac-spoOA(l-146) Plasmid p M N O A N is the integrative vector used to create strains of B. subtilis which encode a copy ofspoOA or spoOA(A257V) under control of the LacI/IPTG-inducible Pspac promoter. This plasmid was created in a two-step process which involved removing most of the spoVG-lacZ translational fusion from the vector p M U T I N 4 in the first step and sub-cloning the N -terminal sequence of spoOA into this construct in the second step. p M U T I N 4 is an integrative Bacillus vector designed to separate a coding sequence from its natural regulatory regions (Figure 8A). The plasmid contains a multiple cloning site upstream of the Pspac promoter, an origin of replication for growth and maintenance in E.coli {priE.coii), selectable markers for both E.coli and Bacillus (bla and ermAM), a lacl cassette, a spoVG-lacZ translational fusion to follow expression via P-galactosidase activity, and the transcription termination sequences To, T] and T 2 . To remove the spoVG ribosome binding site and create a truncated, non-functional copy of the lacZ gene (Welply et al, 1980), p M U T I N 4 was treated with Hindlll and Sad to remove 2025 bp from the vector. The 6585 bp vector backbone was ligated directly with an 874 bp fragment generated from a similarly digested plasmid, pGEMSpoOA, to create the integrative vector pMNSpoOA (Figure 8B, C). The plasmid pGEMSpoOA had been created by ligation of the cloning vector p G E M - T with an 831 bp P C R product generated by amplification o f the spoOA locus from isolated B. subtilis JH642 chromosomal D N A using primers B T O A R B S and OAEco (Table 3). pGEMSpoOA encodes the first 263 amino acids of the spoOA coding sequence and 37 bp of sequence upstream of the G T G start codon which encodes the spoOA ribosome binding site. 37 Figure 8. Construction of VspaQ-spoOA(l-l46). (A) p M U T J N 4 is an integrative Bacillus vector designed to separate a coding sequence from its natural regulatory regions. The plasmid contains a multiple cloning site upstream of the LacI-repressed/IPTG-inducible PSp a ( promoter, an origin of replication {oriE.Coii) for growth and maintenance in E.coli, selectable markers for both E.coli and Bacillus (bla and ermAM), a lad cassette,, a spoVG-lacZ translational fusion to follow expression via (3-galactosidase activity, and the transcription termination sequences To, T i and T 2 . The Hindlll-Sacl region of pGEMSpoOA (B) was inserted into Hind\\l-Sac\ digested p M U T I N 4 to create pMNSpoOA (C), replacing the spoVG-lacZ translational fusion in the parent vector with sequences encoding a truncated spoOA gene and the spoOA ribosome binding site in pMNSpoOA. To construct the P s p a c-spoOA strains, the HinAWl-Sall region of pMNSpoOA was replaced with the Hindlll-Sall region of p G E M O A N (D) to create p M N O A N (E). This plasmid contains the same elements as p M U T I N 4 but the spoVG-lacZ translational fusion has been replaced by sequences encoding the N-terminal domain of spoOA (amino acids 1 - 146) and the spoOA ribosome binding site. 38 39 To create an integrative vector construct that encoded only the N-terminal domain of spoOA under the control of Pspac, JH642 chromosomal D N A was used to amplify the first 146 amino acids of spoOA using the primers B T O A R B S and BTOAlinker (Table 3). The 481 bp P C R product was ligated directly into p G E M - T to create p G E M O A N . Hindllll Sail digest of p G E M O A N (Figure 8D) generated a D N A fragment encoding the N-terminal domain of spoOA. This fragment was ligated with the 6604 bp Hindlll-Sall fragment from pMNSpoOA. The resulting construct, p M N O A N (Figure 8E), encodes all the same elements as the parent vector p M U T I N 4 but has the spoVG-lacZ translational fusion replaced by the spoOA ribosome binding site and sequences encoding the N-terminal domain of spoOA (amino acids 1- 146). The sequence of the spoOA gene was confirmed to be free of errors by sequencing completed by the Nucleic A c i d and Protein Service Unit, University of British Columbia. 2.9. Construction of Yspac-spoOA and T*spac-spoOA(A257V) B. subtilis strains To create strains of B. subtilis which encoded a copy of spoOA or spoOA(A257V) under control of the LacI-repressed/IPTG-inducible Pspac promoter, JH642 and JH695 were transformed with p M N O A N as described previously (Hoch, 1991). Briefly, JH642 and JH695 were grown in First Growth Period Supplemented M G media (Hoch, 1991) until stationary phase. The cultures were induced to develop competence by causing amino acid starvation through dilution of the cultures with Second Growth Period Supplemented M G media (Hoch, 1991) and incubation with 2.5 ug p M N O A N . Transformants were selected by erythromycin resistance (0.3 ug/ml) and screened by western blot for the ability to express SpoOA or SpoOA(A257V) in response to IPTG induction using 0, 1, and 4 m M IPTG. 2.10. Determination of sporulation frequency Sporulation frequency was determined as described previously (Hoch, 1991). Briefly, cultures of B. subtilis were grown at 37°C in S S M supplemented with 10 ug/ml tryptophan and phenylalanine, the appropriate antibiotic, and IPTG when required. After 28 hours growth the cultures were sampled and the total number of viable cells per ml of culture was determined by serial dilution and plating on L B agar supplemented with antibiotic ( if required). The number of spores was determined by treating each dilution with 0.1 volume of chloroform and plating onto L B agar containing the appropriate antibiotic ( if required). 40 After 24 hours incubation, the number of colonies on plates containing between 30 and 300 colonies were counted and the sporulation frequency determined. Sporulation frequency is defined as the number of colony forming units before and after chloroform treatment. 2.11. Immunoblot analysis B. subtilis cultures were grown in S S M at 37°C and 10 ml samples were collected at selected times and harvested by centrifugation. The cell pellets were rinsed with wash buffer (50 m M Tr i s -HCl , p H 8.0; 10 m M E D T A , p H 8.0; 10% glycerol; 1 M KC1; 1.7 m M P M S F ) and resuspended in 1 ml of lysis buffer (20 m M Tr i s -HCl , p H 8.0; 1 m M E D T A , pH 8.0; 300 m M KC1; 100 m M M g C l 2 ; 1.7 m M P M S F ; 0.1 m M D T T ) prior to breaking open the cells by sonication (3 x 30 sec, level 4, Sonicator® Ultrasonic Liquid Processor Model XL2020 , Misonix Inc). The protein concentration of whole-cell extracts was determined by absorbance at 595 nm in a Bradford Assay (BioRad Inc) using bovine serum albumin as a standard. Purified recombinant SpoOA or SpoOA(A257V) (11-50 ng used as standards) and equivalent amounts of extracts (10-25 ul) were separated by electrophoresis through 12% S D S - P A G E and transferred onto nitrocellulose membranes (BioRad) by electrophoresis. The membranes were blocked overnight at 4°C in phosphate-buffered saline (PBS) containing 0.1 % Tween-20 (PBST) and 5 % skim milk. The membranes were washed four times in P B S T for 8 minutes each before incubation with a 1:5000 dilution of rabbit-anti-SpoOA antiserum in P B S T containing 2% skim milk at room temperature with gentle agitation. The anti-SpoOA antiserum was generously provided by the lab of C . P. Moran, Jr (Emory University, Atlanta, Georgia). After 1 hour incubation, unbound anti-SpoOA antibodies were removed by washing the membranes four times in P B S T for 8 minutes each. The membranes were then probed with a 1:10 000 dilution of secondary antibody (donkey anti-rabbit IgG Horseradish Peroxidase-linked whole antibody, G E Healthcare) in P B S T containing 2 % skim milk for 30 minutes at room temperature with gentle agitation. Unbound antibodies were removed by washing twice with P B S T and twice with P B S for 8 minutes each. The SpoOA and SpoOA(A257V) proteins were then detected using E C L Western blotting detection reagents (GE Healthcare) as described by the manufacturer and exposure to X-ray film. 41 2.12. P-Galactosidase assay 2.12.1. Construction of LacZ reporter strains The spoIIG and spoIIA promoter fusions were used as reporters of SpoOA and SpoOA(A257V) activity in strains encoding mutant or wi ld type proteins under transcriptional control of the Pspac promoter. To create reporter strains, chromosomal D N A was isolated from B. subtilis strains 16124 and 16304 containing the spoIIG-lacZ and spoIIA-lacZ fusions, respectively (Perego et al., 1991b; Rowe-Magnus et al., 2000). BT2001 and BT2002 were transformed with 2.5 ng chromosomal D N A as described previously and clones containing the fusions were selected for by plating on L B supplemented with erythromycin (0.3 pg/ml) and 5 pg/ml kanamycin (for spoIIG-lacZ) or chloramphenicol (for spolIA-lacZ). Transformants were screened by sporulation assay and by western blot for the ability to express SpoOA or SpoOA(A257V) in response to IPTG induction. 2.12.2. P-Galactosidase activity To assay for P-galactosidase activity, 50 ml of S S M supplemented with the appropriate antibiotic was inoculated with a 1/100 dilution of an overnight culture of the appropriate strain. Four millimolar I P T G was either added to the cultures at the time of inoculation or was added to the cultures at various time points during growth, as indicated. The growth of each culture at 37°C was followed by measurement of optical density at 525 nm. At various times during logarithmic growth and stationary phase, 1 ml samples of each culture were collected in triplicate and harvested by centrifugation. The cell pellets were frozen at -20°C until assayed for P-galactosidase activity. Cel l pellets were thawed by resuspension in 730 pi of Z-buffer (60 m M N a 2 H P 0 4 ; 40 m M N a H 2 P 0 4 ; 10 m M KC1; 1 m M M g S 0 4 ; 50 m M p-Mercaptoethanol). Cells were permeabilized by addition of 0.1 % Triton X following 5 minutes incubation with 0.1 mg/ml lysozyme. O-nitrophenol-P-D-galactoside (ONPG) , a chromogenic p-galactosidase substrate, was added to a final concentration of 0.45 mg/ml and incubated with the permeabilized cells at 28°C. After 15 minutes incubation the reactions were terminated with the addition of N a 2 C 0 3 tol 80 m M . The absorbance at 420 nm was measured for each reaction and the enzyme specific activity was determined using the calculation: specific activity (Miller Units) = (A 4 2 o nm x 6 6 . 7 ) / O D 5 2 5 n m O f the culture. 42 3. RESULTS In this study I have investigated the in vitro and in vivo characteristics of a substitution mutant of the response regulator SpoOA. I was interested in how changes in the D N A binding domain affect the ability of SpoOA to activate transcription initiation. It was previously reported that a single amino acid substitution made within the D N A binding domain of SpoOA, A257V, resulted in a sporulation-deficient phenotype (Ferrari et al., 1985). Although the substitution was associated with loss of activation of transcription initiation in vivo from both a A-and cH-dependent promoters by SpoOA (Perego et al., 1991b; Rowe-Magnus et al., 2000), it did not affect inhibition of transcription initiation by SpoOA at the abrB promoter (Perego et al., 1991b; Rowe-Magnus et al., 2000), shown to depend on the D N A binding properties of SpoOA. In this thesis I have investigated the uncoupling o f the ability of SpoOA to activate and repress transcription initiation in vitro by testing for potential defects in SpoOA(A257V). First, did the A 2 5 7 V mutation interfere with the ability of SpoOA to be phosphorylated? Second, did the mutation affect the ability to repress transcription initiation? Third, did the mutation specifically eliminate the ability to stimulate transcription initiation at a oA-dependent promoter or bind D N A from that promoter? Finally, did the A 2 5 7 V mutation interfere with a positive feedback mechanism in vivo which leads to upregulation of spoOA transcription? 3.1. Examination of the effect of the A257V mutation on SpoOA phosphorylation in vitro The ability of SpoOA to stimulate transcription is dramatically increased by transfer of a phosphoryl group through a phosphorelay signal transduction system to D56 located within the N-terminal domain of SpoOA (Bird et al., 1993). Although the A257 residue is distant from the site of phosphorylation and is located in the C T D of SpoOA, it was possible that the valine substitution could alter the structure of the protein such that it became a poor substrate for the phosphotransferase protein SpoOB, and that decreased phosphorylation could explain the lack of transcription activation observed in vivo (Perego et al, 1991b; Rowe-Magnus et al, 2000). I expressed and purified wild type and mutant SpoOA proteins and compared both the rate and level of protein phosphorylation using an in vitro phosphorylation assay composed of reconstituted phosphorelay components. The rate of phosphorylation was 43 determined by incubation of SpoOA and Spo0A(A257V) with phosphorelay components K i n A , SpoOF, SpoOB and [y 3 2 P]ATP for various times prior to separation by S D S - P A G E and quantification o f phosphorylation by Phosphorlmager analysis (Figure 9A) . The initial rates o f phosphorylation of wi ld type and mutant SpoOA proteins were the same (Figure 9B). Phosphorylation o f SpoOA and SpoOA(A257V) increased linearly until maximum phosphorylation was reached after six minutes incubation. This suggested that the A 2 5 7 V mutation did not influence phosphotransfer from SpoOB. A s a second test to compare phosphorylation, excess concentrations of wi ld type and mutant proteins were incubated in a phosphorylation reaction for 4.5 hours prior to S D S - P A G E separation and quantification by Phosphorlmager analysis (Figure 10A). Both SpoOA and SpoOA(A257V) were phosphorylated to the same level at each concentration tested (Figure 10B), suggesting that decreased phosphorylation of SpoOA(A257V) could not explain the lack o f transcription activation in vivo in the mutant strain (Perego et al., 1991b; Rowe-Magnus et al., 2000). 3.2. Investigation of the effect of the A257V substitution on in vitro repression of the abrB promoter SpoOA represses transcription of the abrB promoter by preventing R N A polymerase from inducing D N A strand separation during transcription initiation (Greene and Spiegelman, 1996). Previous studies have shown that both SpoOA and SpoOA(A257V) are capable of repressing abrB transcription to similar levels in vivo (Perego et al, 1991b; Rowe-Magnus et al, 2000). In order to validate these results in vitro, I compared the abilities of both phosphorylated and unphosphorylated wi ld type and mutant proteins to repress abrB transcription in an in vitro transcription assay. Unphosphorylated and phosphorylated wi ld type and mutant proteins were incubated with a linear D N A fragment encoding both the PI and P2 transcription initiation sites of the abrB promoter (abrBp), initiating nucleotides (ATP, U T P and GTP) , and R N A P - a A . After two minutes incubation, transcripts were permitted to elongate with the addition of C T P and heparin. Transcripts resulting from a single round of transcription were separated by electrophoresis and quantified by Phosphorlmager analysis (Figure 11 A ) . 44 A Time (min) 0:10 1 2 4 6 8 10 12 SpoOA Spo0A(A257V) Figure 9. Time course of in vitro phosphorylation of SpoOA and SpoOA(A257V). The phosphorelay components KinA, SpoOF and SpoOB were incubated with [y^^P]ATP prior to addition of0.5 u M SpoOA or Spo0A(A257V). Samples were removed at various intervals and the transfer reaction was terminated by addition of SDS-PAGE loading buffer. Following separation of phosphorylated proteins by 15% SDS-PAGE, 32p labeled SpoOA or SpoOA(A257V) were detected by exposure to a phosphor screen (A) and the level of phosphorylation was quantified by Phosphorlmager analysis (B). Symbols: filled circles, SpoOA~P; filled squares, SpoOA(A257V)~P. A representative Phosphorlmage is shown and values are representative of multiple experiments. 45 A [Protein](nM) 500 1000 2000 SpoOA mm mm Spo()A(A257V) B 100 500 1000 2000 |Protein| (nM) Figure 10. Phosphorylation of varying amounts of SpoOA and SpoOA(A257V) in vitro. SpoOA and SpoOA(A257V) were incubated with the phosphorelay components K i n A , SpoOF and SpoOB, and [y32p]ATP for 4.5 hours before the transfer reaction was terminated by the addition o f S D S - P A G E loading buffer. The radiolabeled components were separated by 15% S D S - P A G E and ( A ) 3 2 P labeled SpoOA or S p o 0 A ( A 2 5 7 V ) were detected by exposure to a phosphor screen. The extent o f phosphorylation was determined by Phosphorlmager analysis (B). White bars, S p o 0 A ~ P ; grey bars, Spo0A(A257V) . A representative Phosphorlmage is shown. The values reflect the average o f three independent experiments and their standard deviations. 46 A SpoOA ' SpoOA(A257V)fc SpoOA~P SpoOA(A257V)~P IProtein] (nM) 0 200 400 800 1200 [Protein] (nM) 0 200 400 800 1200 Figure 11. Repression of the abrB promoter by SpoOA and SpoOA(A257V) in vitro. Phosphorylated or unphosphorylated mutant and w i l d type SpoOA proteins were incubated with initiating nucleotides ATP. UTP, and G T P and a linear D N A fragment encoding both the PI and P2 transcription initiation sites o f the abrB promoter (abrBp). C T P and heparin were added three minutes after addition o f 0 ^ - R N A polymerase to permit transcript elongation. The reactions were terminated after five-minutes and the transcripts were separated by electrophoresis through an 8% denaturing polyacrylamide gel. (A) J 2 p _ i a D e j e t j abrB transcripts were detected by autoradiography and (B) the level o f transcripts produced from the abrB promoter was determined by Phosphorlmager analysis. Symbols: open circles, SpoOA; filled circles, Spo0A~P; open squares, S p o 0 A ( A 2 5 7 V ) ; filled squares, SpoOA(A257V)~P. Representative films are shown. Values reflect the average of three independent experiments and their standard deviations. 47 Consistent with earlier results, increasing amounts of SpoOA reduced the amount of run-off transcript produced from the abrB promoter, and SpoOA(A257V) was capable of repressing transcription from abrB (Figure 1 IB) . A s measured by the decrease in transcript levels, SpoOA(A257V)~P did not inhibit transcription as effectively as SpoOA~P; at 800 n M , repression by SpoOA~P was 2-fold more effective and at 1200 n M , repression by Spo0A~P was 5-fold more effective. Phosphorylation enhanced the ability of SpoOA to repress abrB transcription by 3.3-fold, whereas phosphorylation of SpoOA(A257V) only enhanced repression by 1.3-fold. This may be a consequence of the unexpected effectiveness o f unphosphorylated SpoOA(A257V) in repressing transcription. The significance of the apparently effective repression by SpoOA(A257V) is uncertain as in vivo experiments have indicated that the temporal pattern of repression of the abrB gene in a strain with SpoOA(A257V) mimics that in the wi ld type strain (Perego et al, 1991b). Critically, these data confirmed that the A 2 5 7 V substitution did not substantially affect the ability of SpoOA to repress abrB transcription. 3.3. Investigation of the effect of the A257V substitution on in vitro stimulation of the spolIG promoter Gene expression during sporulation is directed by five different sigma factors produced at various times during endospore formation. Among the first sigma factors produced are the E F a and a sigma factors which direct gene expression in the mother cell and forespore, respectively (Dworkin, 2003). The o E sigma factor is transcribed from the oA-dependent F H spolIG operon whereas o is transcribed from the a -dependent spoIIA operon. Both the spolIG and spoIIA operons are dependent upon activation by Spo0A~P. The spoIIA promoter is directly dependent on a H since it is transcribed by R N A polymerase containing this sigma factor. The spolIG promoter is indirectly dependent on a H as it is known to be transcribed by R N A polymerase containing a A , but is not transcribed in a spoOH mutant. For both the spolIG and spoIIA operons, transcription requires that Spo0A~P reaches a threshold sufficient for activation (Fujita et al, 2005). This increase in concentration of phosphorylated SpoOA appears to require increased transcription of spoOA and the phosphorelay components kinA and spoOF. 48 Previous studies have indicated that the A 2 5 7 V eliminates activation of transcription initiation from the spolIG promoter in vivo. However, because activation of transcription initiation at this promoter in vivo is not only dependent upon SpoOA~P, but also upon a a H -dependent increase in concentration of SpoOA~P, it was possible that the A 2 5 7 V mutation did not change the ability of the protein to stimulate aA-dependent transcription initiation. I tested whether the A 2 5 7 V mutation affected the ability of SpoOA to activate transcription initiation in vitro testing both the initial rate of transcription and the effect of increasing SpoOA~P concentrations (Figures 12, 13). Phosphorylated and unphosphorylated SpoOA and SpoOA(A257V) were incubated with template D N A and the initiating nucleotides A T P and G T P . R N A P - a A was added to the reactions and the proteins were allowed to form initiated complexes for 0-120 seconds before elongation was permitted with the addition of heparin and the remaining nucleotides. Phosphorylated SpoOA was able to stimulate the rate of spolIG transcription initiation to a greater extent than the other proteins tested (Figure 12A, B) . The number of transcripts increased rapidly in the presence of SpoOA~P and reached a maximum after 90 seconds incubation. A s noted in previous results (Rowe-Magnus and Spiegelman, 1998), both unphosphorylated and phosphorylated SpoOA were capable of stimulation o f spolIG transcription initiation, although the unphosphorylated protein stimulates the rate at a much reduced level. Similarly, in the presence of phosphorylated SpoOA(A257V) there was an increase in the number of spolIG transcripts, although at a rate approximately four times lower than that found in the presence o f SpoOA~P. In the presence o f unphosphorylated mutant protein the rate of transcription initiation was approximately ten times slower than stimulation by SpoOA~P. The effect of varying the concentration of unphosphorylated and phosphorylated wi ld type and mutant proteins on spolIG transcription was determined (Figure 13). Unphosphorylated and phosphorylated wild type and mutant proteins were incubated with promoter D N A and R N A P - a A for two minutes to allow formation of initiated complexes. Transcription was limited to a single-round from initiated complexes by the addition of the C T P , U T P and heparin (Figure 13A). 49 A Time (s) o 51015.30 45 60 a 90 120 SpoOA >• m «•% •» SpoOA-P • • mm mm SpoOA(A257V) • - - « • SpoOA(A257V)~P * * Figure 12. Time course of in vitro transcription initiation stimulated by SpoOA and SpoOA(A257V). Phosphorylated or unphosphorylated SpoOA or S p o O A ( A 2 5 7 V ) (800 n M ) were incubated wi th a linear D N A fragment encoding the spolIG operon promoter (spolIGp) and initiating nucleotides A T P and GTP. r j A - R N A P was added to the mixture at varying times, the remaining nucleotides and heparin were added. Reactions were terminated by the addition o f transcription stop buffer at the times indicated. Elongated 32p-]abelled spolIG transcripts were separated by electrophoresis through an 8% denaturing polyacrylamide gel and (A) detected by autoradiography. The level o f transcripts produced in a given time interval from the spolIGp promoter were determined by Phosphorlmager analysis (B) . Symbols: open circles, SpoOA; filled circles, SpoOA~P; open squares, SpoOA(A257V) ; fi l led squares, SpoOA(A257V)~P. Representative films are shown. Values reflect the averages o f three independent experiments and their standard deviations. 50 A SpoOA SpoOA(A257V ; SpoOA~P SpoOA(A257V)~P [ProteinR nM) 0 200 400 800 1200 [Protein] (nM) 0 200 400 800 1200 B 0 200 400 600 800 1000 1200 [Protein] (nM) Figure 13. Effect of SpoOA or SpoOA(A257V) protein concentration on sti m ii latin of spoIIG promoter activity in vitro. Phosphorylated or unphosphorylated SpoOA or S p o 0 A ( A 2 5 7 V ) were incubated with a linear D N A fragment encoding the spoIIG operon promoter (spoIIGp) and initiating nucleotides A T P and G T P . a ^ - R N A P was added to the mixture and allowed to bind D N A for 2 minutes prior to addition o f the remaining nucleotides and heparin. Fol lowing a live-minute incubation, elongated transcripts were separated by electrophoresis through an 8% denaturing polyacrylamide gel. (A) 32p_i a beled spoIIG transcripts were detected by autoradiography and (B) the level o f transcripts produced from the spoIIG promoter were determined by Phosphorlmager analysis. Symbols: open circles, SpoOA; filled circles, SpoOAHP; open squares. S p o 0 A ( A 2 5 7 V ) ; filled squares. Spo0A(A257V)~P . Representative films are shown. Values reflect the average o f three independent experiments and their standard deviations. 51 Increasing amounts of SpoOA~P led to increasing amounts of transcription over the range of 200 to 800 n M protein at which point the level o f transcription was maximal (Figure 13B). Overall SpoOA~P stimulated transcription 23-fold relative to the number of transcripts produced in the absence of SpoOA. Both phosphorylated and unphosphorylated SpoOA(A257V) were capable of stimulating transcription initiation, but only to levels approximately 50% of those stimulated by wi ld type SpoOA. Over the range of concentration at which Spo0A(A257V) was tested, transcription increased linearly and at 1200 n M the amount of transcription was 14-fold greater than that obtained in the absence o f SpoOA. In addition, both SpoOA and SpoOA(A257V) exhibited the same fold-increase in transcription stimulation upon phosphorylation at each protein concentration tested. For example, at 800 n M the phosphorylated form of both proteins was four-fold more effective in stimulating transcription than was the unphosphorylated protein. These data demonstrate that Spo0A(A257V) was capable o f stimulating transcription from a aA-dependent promoter in vitro. 3.4. Examination of the effect of the A257V mutation of SpoOA on binding to OA boxes encoded within the spoIIG promoter SpoOA binds to specific sites (OA boxes) located upstream or downstream o f the transcription start site at promoters activated or repressed by SpoOA. At these sites SpoOA makes both base-specific contacts and contacts with the D N A backbone (Zhao et al, 2002). At the spoIIG promoter there are four OA boxes at two different sites (Baldus et al., 1994). The site 1 OA boxes, centered 90 bp upstream of the +1 transcription start site, were previously shown to be high affinity SpoOA binding sites (Baldus et al, 1994). In contrast the site 2 OA boxes, centered approximately 45 bp upstream of the +1 transcription start site, are low-affinity SpoOA binding sites and are bound only by phosphorylated SpoOA (Baldus et al, 1994). I tested whether the two fold reduction in spoIIG transcription stimulation observed in this study was due to lack of binding or inappropriate binding o f SpoOA(A257V) to OA boxes encoded within promoter D N A using an in vitro DNasel footprinting assay. Phosphorylated SpoOA and SpoOA(A257V) were incubated with a [y 3 2 P]ATP end-labeled fragment of the spoIIG promoter and partially digested with DNasel . The labeled fragments 52 were separated by electrophoresis and the protection patterns were detected by autoradiography (Figure 14). Consistent with previous findings, phosphorylated SpoOA bound to both site 1 and site 2 OA boxes protecting a large region of the promoter (Baldus et al, 1994). Phosphorylated SpoOA(A257V)~P also bound to site 1 and site 2 OA boxes within promoter D N A , although with lower affinity than SpoOA~P. The pattern of protected sites was not changed as far as could be observed. These data suggested that the A 2 5 7 V mutation reduced spolIG binding to some degree but did not eliminate it. The reduction could be the cause of lower transcription stimulation. 3.5. In vitro examination of the binding affinity of SpoOA(A257V) for consensus OA boxes Although the A 2 5 7 V mutation did not cause inappropriate binding to the spolIG promoter, results from the DNasel footprinting assay suggested that the mutation did reduce the affinity of the protein for its D N A binding sites. I tested this possibility using a different approach by measuring the ability of both phosphorylated and unphosphorylated SpoOA and SpoOA(A257V) to bind D N A fragments containing OA boxes in an electrophoretic mobility shift assay ( E M S A ) . Various concentrations of unphosphorylated and phosphorylated wild type and mutant proteins were incubated with [ a 3 2 P ] N T P end-labeled duplex D N A encoding the two OA boxes found at the abrB promoter. After two minutes incubation, binding reactions were loaded onto a running polyacrylamide gel and complexes were separated by electrophoresis. The extent of binding was determined using Phosphorlmager analysis (Figure 15A). Both SpoOA(A257V) and SpoOA(A257V)~P were able to bind to the duplex D N A (Figure 15B). A single complex was observed at all protein concentrations for both protein forms. Dissociation constants were determined for both phosphorylated and unphosphorylated proteins using the Program SigmaPlot 8.0 (SPSS) as described in Experimental Procedures. Phosphorylated SpoOA bound with approximately 2-fold greater affinity than phosphorylated SpoOA(A257V) and had an apparent dissociation constant o f 1 x 10"7 M (standard error = 0.22) compared to an apparent dissociation constant of 3 x 10"7 M (standard error = 0.44) for SpoOA(A257V)~P. Unphosphorylated SpoOA bound D N A with a reduced affinity (about 53 Figure 14. DNase footprint of SpoOA and SpoOA(A257V) at the spoIIG promoter. Phosphorylated SpoOA and SpoOA(A257V) (200. 400, 600 n M ) were incubated with a 454 bp Pvull-BamHl D N A fragment from the spoIIG operon promoter (spoIIGp) end labeled with [y32p]ATP. Control reactions contained only spoIIGp D N A . After two minutes, D N A s e l was added and allowed to cleave the D N A for 10 seconds before terminating cleavage with addition o f DNase stop buffer. 32p Labeled fragments were separated by electrophoresis through an 6% denaturing polyacrylamide gel and (A) the D N A s e l protection patterns were detected by autoradiography. The positions o f the nucleotides relative to the +1 start site are indicated to the left o f diagram. (B) Location o f the site 1 and site 2 OA boxes relative to the conserved -10 and -35 elements o f the spoIIG promoter. 54 A B Figure 15. E M S A of binding of SpoOA and SpoOA(A257V). Phosphorylated and unphosphorylated SpoOA and SpoOA(A257V) were incubated with double-stranded a^2p-labeled oligonucleotides that contain two consensus OA boxes ( 5 ' - T G A C G A A - 3 ' ) and had the same sequences as the +3 to +29 region o f the abrB gene. The proteins were allowed to bind D N A for two minutes prior to challenge with calf thymus D N A in glycerol and loading on a running 8% non-denaturing polyacrylamide gel. (A) 32p labeled D N A was detected by autoradiography. (B) The percentage o f free and bound D N A was quantified by Phosphorlmager analysis. Symbols: open circles, SpoOA; filled circles, SpoOA~P; open squares, SpoOA(A257V) ; filled squares, SpoOA(A257V)~P. Representative films are shown. Values reflect the average o f three independent experiments and their standard deviations. 55 one-third the amount bound by SpoOA~P at 800 n M ) and had an apparent dissociation constant of 1 x 10"6 M . The binding o f SpoOA(A257V) to the duplex D N A was negligible, precluding an accurate assessment of its dissociation constant. These data indicated that SpoOA(A257V)~P was able to bind to duplex D N A with approximately half the apparent affinity of SpoOA~P and suggested that the two-fold reduction in in vitro transcription activation and repression described in this study could be explained by a reduction in the binding activity of the mutant protein. 3.6. Effect of mutations in the OA boxes encoded within the spolIG promoter on stimulation of transcription by SpoOA~P and SpoOA(A257V)~P A recent model of SpoOA activation suggests that the primary consequence o f N T D phosphorylation is to facilitate interactions between the domains leading to dimerization (Lewis et al., 2002). The implication of this model is that the active form of SpoOA is a dimer. The crystal structures of the D N A binding domain alone and in complex with D N A containing tandem OA box sequences derived from the abrB promoter show the two domains in a head-to-tail orientation with each member making specific D N A backbone and base contacts within the OA box sequence (Lewis et al., 2000a; Zhao et al., 2002). At the spolIG promoter the site 2 OA boxes are found between positions -53 to -37 bp with the downstream OA box (site 2.2, positions -43 to -37) overlapping the -35 sequence of the spolIG promoter. When the site 2 OA boxes are occupied by a dimer of Spo0A~P, only the downstream monomer is expected to be close to the sigma subunit of the polymerase. Genetic evidence (Baldus et al, 1995; Buckner et al, 1998; Hatt and Youngman, 1998; Schyns et al, 1997) and recent structural modeling (Kumar et al, 2004) have indicated that SpoOA contacts the sigma subunit to activate transcription at the spolIG. While A257 is distinct from the predicted a A activation surface, the helix within which the A257 residue is located (helix aF) forms part of the interface between the two SpoOA monomers in the crystal structures. Furthermore, the structures predict that the A 2 5 7 V mutation would prevent appropriate packing between residues forming this interface by influencing the flexibility and orientation of aF (Zhao et al, 2002). 56 It seems likely that the strength o f binding o f a SpoOA~P dimer to the tandem OA boxes in its D N A binding site would reflect both the strength of interaction between each monomer and one OA box and the strength of interaction between the two monomers. I f dimer-binding conditions were compromised, so that simultaneous contact of the monomers and OA boxes was altered, then potentially one of the monomers would be able to make favorable contacts with the D N A while the second protein might not be unable to make the expected contacts. This situation might well explain why the SpoOA(A257V) bound D N A with less affinity and was both less effective at repressing abrB transcription and at stimulating spoIIG transcription. Since the interaction of SpoOA~P with the sigma subunit is critical to transcription activation at the spoIIG promoter, it should be more affected by binding of the downstream monomer of the SpoOA dimer than binding of the upstream monomer. To test this idea I altered the spoIIG promoter through mutation of each o f the site 2 OA boxes separately and tested whether the effects of these mutations would be exaggerated for SpoOA(A257V). In the case of wi ld type SpoOA, where the protein-protein interactions are most favorable, mutation of either OA box should have roughly the same effect, reducing the sum of contacts stabilizing dimer binding to the D N A . On the other hand, in the case of SpoOA(A257V), potentially only one of the binding domains could make the appropriate contacts with the D N A at any one time since dimer packing may be disrupted. If contacts with the downstream 2.2 OA box are essential to position SpoOA for subsequent sigma contacts, the mutation of the downstream 2.2 OA box might have a more deleterious effect on transcription than mutation of the upstream 2.1 OA box. Thus I measured the effects of mutations in each of the two OA boxes on transcription activation by wild type SpoOA and SpoOA(A257V). Two mutant promoter D N A templates were used in this experiment that encoded versions of the site 2 OA boxes with decreased similarity to the consensus OA box sequence (Figure 16A). The "2.1 down" template encodes a mutant 2.1 OA box in which the sequence 5'-C T C A A C A - 3 ' was changed to 5 ' - C A G A A G A - 3 ' , eliminating a single base-specific D N A contact for SpoOA on both the template and non-template strands that have been identified from the crystal structure (Zhao et al, 2002). To mutate the 2.2 OA box (creating the "2.2-down" template), the sequence was changed from 5 ' - A T T G A C A - 3 ' to 5 ' - A C T G A G A - 3 ' , 57 A spoIIG 2.1 down 2.2 down B spoIIG 2.1 down c -50 -40 C C T C T C A A C A T T A A 7 T G A C A G A C G G A G A G T T G T A ATTA 4CYGICJG CCTCagAAgATTAA7TGACAGAC GGAGtcTTcTAATTA/f(7r(rTCTG CCTCTCAACATTAAcTGAgAGAC G G AG AGTTGTA KTTeA CTcTCTG SpoOA~P SpoOA(A257V)~P 6000000, 200 400 600 800 1000 1200 [Protein] (nM) 2.2 down Figure 16. Wi ld type and mutant SpoOA stimulation of transcription from mutant spoIIG promoters in vitro. Phosphorylated or unphosphorylated SpoOA or SpoOA(A257V) were incubated with a linear D N A fragment encoding either w i ld type or mutant spoIIG operon promoters (spoIIG, 2.1 down, 2.2 down) and the initiating nucleotides A T P and G T P . r j A - R N A polymerase was added to the mixture and allowed to bind D N A for 2 minutes prior to addition o f the remaining nucleotides and heparin. Fo l lowing a five-minute incubation, elongated transcripts were separated by electrophoresis through an 8% denaturing polyacrylamide gel. (A) The -34 to -56 region o f the spoIIG promoter. SpoOA binding sites (OA boxes, concensus sequence 5 ' - T G A C G A A - 3 ' ) are underlined. Nucleotides thought to be specifically contacted by SpoOA are shown in bold and those expected to be contacted by the a A subunit o f R N A polymerase are italicized. Mutated bases are shown in lower case. (B) 32p-labeled transcripts were detected by autoradiography and (C) the level o f transcripts produced were determined by Phosphorlmager analysis. Symbols: black circles, S p o 0 A ~ P (spoIIGp): black squares, SpoOA(A257V)~P) (spoIIGp); grey circles, S p o 0 A ~ P (promoter templates: 2.1 down, — ; 2.2 down, ); grey squares, S p o 0 A ( A 2 5 7 V ) ~ P (promoter templates: 2.1 down, ; 2.2 down ). Representative films are shown. Values reflect the average o f three independent experiments and their standard deviations. 58 mutating one base contacted for both SpoOA and the rj subunit of R N A P and eliminating one c-specific contact, as predicted from the crystal structure (Campbell et al, 2002). The ability of SpoOA~P and SpoOA(A257V)~P to stimulate transcription initiation from wild type and mutant promoter templates was determined using an in vitro transcription assay (Figure 16B). Mutation of either the 2.1 or 2.2 OA boxes decreased, but did not abolish, the ability of phosphorylated SpoOA or phosphorylated SpoOA(A257V) to stimulate transcription (Figure 16C). Overall the pattern of transcription stimulation as a function of activator input was similar to that seen with the wi ld type spolIG promoter; transcription in the presence of SpoOA~P reached a maximum at 800 n M whereas stimulation by SpoOA(A257V)~P increased in a roughly linear manner over the range tested (up to 1200nM). The 2.1 OA box mutation led to a decrease in the maximum level of transcription stimulation by both phosphorylated SpoOA and Spo0A(A257V) as would be expected i f the binding affinity of the dimer was reduced by eliminating critical contacts. For both SpoOA~P and SpoOA(A257V)~P there was a 3-fold reduction in transcription from the mutant 2.1 down promoter compared to transcription from the wild type spolIG promoter. The reduction was similar to the decrease in SpoOA~P binding in vitro and transcription in vivo observed when the guanine and thymine residues at positions -47 and -51, relative to the +1 transcription start site, are mutated independently (Baldus et al, 1994; Satola et al, 1991; Satola et al, 1992). In the case of the 2.2 down template, stimulation by SpoOA~P was reduced 3-fold and stimulation by SpoOA(A257V)~P was reduced 4.5-fold. Thus it appeared that the A 2 5 7 V mutation did make transcription stimulation slightly more sensitive to changes in the 2.2 OA box. While the difference between the effects of the OA box mutations on stimulation by the two types o f activator proteins was not dramatic, the combination of the A 2 5 7 V mutation and loss of one of the OA boxes lead to very low levels of transcription. 3.7. SpoOA(A257V) protein expression in the sporulation negative B. subtilis strain JH695 Previous studies have indicated that genes within the SpoOA regulon respond differentially to high and low doses of SpoOA and that sporulation requires a high level of SpoOA (Chung et 59 al, 1994; Fujita et al, 2005). Genes which require low-levels of SpoOA for repression ('low-threshold repressed genes') include the gene for the transition state regulator A b r B (abrB) and the gene for the positive regulator o f competence, C o m K (comK) (Chung et al., 1994; Fujita et al, 2005). Other genes, such as the spoIIE and sinl genes and the spolIG and spoIIA operons, require a higher level of SpoOA for activation and are referred to as 'high-threshold activated genes' of the SpoOA regulon (Chung et al, 1994; Fujita et al, 2005). One possible explanation for the uncoupling of transcription stimulation from transcription repression in B. subtilis JH695 is that Spo0A(A257V) is expressed to lower levels than SpoOA. Reduced expression of the mutant protein would be sufficient to repress low-threshold genes such as abrB, but would not be o f sufficient threshold to activate high-threshold genes such as the spolIG and spoIIA operons nor be of sufficient concentration to permit sporulation. To test this idea, we observed expression o f wi ld type and mutant SpoOA proteins over time in an immunoblot analysis. Lysates of whole cell-extracts were collected from wild type and spoOA(A257V) B. subtilis strains JH642 and JH695 at 1 hour intervals during late-logarithmic growth through stationary phase. The protein samples were subject to immunoblot analysis and probed for expression o f SpoOA and SpoOA(A257V) using anti-SpoOA antibodies (Figure 17A). Expression of both wild type and mutant SpoOA proteins increased over time and reached a maximum level approximately two hours after the onset of stationary phase (T+2). However, whereas previous reports suggested that similar amounts of the SpoOA protein were observed in spo0A+ and spoOA(A257V) B. subtilis strains (Perego et al, 1991b), SpoOA(A257V) expression was estimated to be two-thirds that of SpoOA expression (35ng SpoOA(A257V) vs.52 ng SpoOA in samples taken at T + 2 ) (Figure 17A). To correlate the reduction in mutant SpoOA expression with the ability to sporulate, strains JH642 and JH695 were tested for the ability to sporulate (Figure 17B). A s expected, the wild type strain (JH642) had a sporulation frequency of 5.2 x 10"', whereas the mutant strain (JH695) did not sporulate at all . This correlation suggested that reduced protein expression and lower transcription stimulation efficiency could account for the lack of sporulation in spoOA(A257V) B. subtilis strains. 60 A JH642 purified protein JH695 T , T 0 T + T+2 T + 3 T+4 1 lng 33ng B Strain No. C F U No. Spores Sporulation (ml 1 ) (ml 1 ) Frequency JH642 6.3 x 10 x 3.3 x 10' 5.2 x 10"' JH695 6.2 x 10 8 0 <1.0x 10"8 Figure 17. Wi ld type and mutant SpoOA protein expression B. subtilis JH642 and JH695. B. subtilis strains JH642 and JH695 were grown in S S M and samples were collected and harvested at 1 hour intervals from the end o f exponential growth ( T . i ) into stationary phase ( T + i to T+4). TQ indicates the end o f exponential growth. Samples containing 40.5 ug of total protein from whole cell extracts and samples containing 11 and 33 ng o f purified recombinant SpoOA and SpoOA(A257V) were separated by electrophoresis through 12 % S D S - P A G E . Proteins were transferred onto nitrocellulose membranes and (A) subject to immunoblot analysis using anti-SpoOA polyclonal antibodies. (B) After 28 hours o f growth, the strains were assayed for sporulation frequency. Sporulation frequency is defined as the number o f colony forming units before and after chloroform treatment. 61 3.8. Construction of B. subtilis strains which over-express wild type and mutant SpoOA proteins During the onset o f sporulation, both SpoOA synthesis and activity are ultimately dependent upon transcription by R N A P - o H . The spoOA gene can be transcribed from one o f two promoters, spoOApv or spoOAps (Strauch et al, 1992). During vegetative growth, transcription from the cA-dependent vegetative promoter (spoOApv) results in low-level expression of SpoOA. Increased expression of SpoOA required for sporulation occurs during the onset of sporulation as a consequence of a positive feedback mechanism in which SpoOA stimulates transcription of its own gene from the aH-dependent, sporulation specific promoter (spoOAps). Similarly, an increase in SpoOA activity could be achieved by increased phosphorylation as a result of greater synthesis of the phosphorelay components spoOF and kinA whose transcription also depends on c H (Britton et al, 2002; Predich et al., 1992). One possible explanation for the reduction in SpoOA protein expression observed in B. subtilis strain JH695 is that A257 or the region around it is required for specific interaction with o H - R N A polymerase. Strains containing the spoOA(A257V) mutation would not only be unable to sporulate because of lack of activation of SpoOA-dependent, oH-dependent promoters but also because the threshold level of SpoOA would never increase sufficiently to permit activation of GH-independent, high threshold SpoOA activated promoters, such as the aA-dependent spolIG promoter. One way to test this hypothesis in vivo was to create B. subtilis strains which over-expressed either wi ld type or mutant SpoOA proteins and monitor transcription activation of both a aH-dependent and a rjA-dependent promoter in vivo. To test this hypothesis I created strains of B. subtilis in which expression of wi ld type or mutant SpoOA protein was placed under the control of the LacI-repressed/IPTG-inducible Pspac promoter (Vagner et al, 1998). The plasmid construct used to create these strains, p M N O A N (Figure 8), encodes the first 146 amino acids of the spoOA gene downstream of Pspac and was created from the integrative B. subtilis vector p M U T I N - 4 as outlined in Experimental Procedures. p M N O A N was transformed into B. subtilis strains JH642 and JH695 and integrated into the spoOA locus by a single recombination event (Figure 18A). 62 Figure 18. Construction of B. subtilis strains which overexpress wild type or mutant SpoOA. Construction of B. subtilis strains encoding Vspac-spoOA and P s p a c-spoOA(A257V). Plasmid p M N O A N encodes the spoOA ribosome-binding site and the N-terminal receiver domain (amino acids 1-146) of spoOA downstream of the LacI-repressed/IPTG-inducible Pspac promoter. The plasmid contains an origin of replication (oriE.coii) for growth and maintenance in E.coli, selectable markers for both E.coli and Bacillus (bla and ermAM), a lad cassette, sequences encoding the terminal 644-1019 amino acids of the lacZ gene, and the transcription termination sequences To, T i and T 2 . p M N O A N was transformed into B. subtilis strains JH642 and JH695 and integrated into the spoOA locus of the chromosome by a single recombination event. The resulting strains, BT2001 and BT2002, had the genotypes JH642 s/wO4.v(pMNSpo0AN ermAM) and JH695 s/?oO4.:(pMNSpo0AN ermAM), as shown in the schematic in panel B . Both recombinant strains encode the N-terminal receiver domain of SpoOA transcribed from the native sporulation and vegetative promoters spoOApv and spoOAps, in addition to either the full length spoOA or spoOA(A257V) gene transcribed from the Vspac promoter. 0\ 64 The strains created, BT2001 and BT2002, encoded both a full length copy of either the wi ld type or mutant spoOA gene under control of the Pspac promoter in addition to a truncated spoOA gene encoding the first 146 amino acids of the N-terminal domain downstream of the two spoOA promoters (spoOApv and spoOAps) (Figure 18B). 3.9. Sporulation frequencies of the Vspac-spoOA B. subtilis strains A s an initial experiment, strains BT2001 and BT2002 were assessed for the ability to produce wi ld type or mutant SpoOA proteins and sporulate in the presence of inducer. Strains BT2001 and BT2002 were grown in S S M supplemented with 0, 1 or 4 m M IPTG. After 16 hours o f growth a sample of each culture was removed and tested for expression of wild type or mutant SpoOA proteins using an immunoblot analysis (Figure 19A). Lysates of whole-cell extracts collected from cultures of BT2001 and BT2002 were probed for presence of mutant or wi ld type SpoOA proteins using anti-SpoOA antibodies. A s expected, the cultures grown in the absence of inducer did not produce detectable amounts of wi ld type or mutant SpoOA whereas both BT2001 and BT2002 expressed protein reacting with the a-SpoOA antibody in response to IPTG. Under these conditions it was estimated that both samples contained approximately the same amount of mutant and wi ld type protein: 36 ng SpoOA in BT2001 and 32 ng SpoOA(A257V) in BT2002 when either strain was induced with 1 m M IPTG. After 28 hours of growth strains BT2001 and BT2002 were tested for the ability to sporulate. Only strain BT2001, which encodes the wi ld type spoOA gene under the control of Pspac (Vspac-spoOA), was able to sporulate, albeit poorly, with the addition of IPTG (Figure 19B). Strain BT2002, which encodes Vspac-spoOA (A25 7 V), was unable to sporulate at both IPTG concentrations tested. Strain BT2001 was also able to sporulate at an extremely low frequency (3.4 x 10"6 spores/ml) in the absence of IPTG. Addition of 1 m M IPTG increased the ability of BT2001 to sporulate by a factor of 100 while addition of 4 m M IPTG caused a 1000-fold increase in sporulation frequency. However, while higher levels of IPTG caused an increase in the ability of strain BT2001 to sporulate, there was no obvious difference in expression of mutant or wild type proteins from Pspac at 4 m M IPTG (Figure 19A). Moreover, although IPTG induction of Pspac resulted in an increased ability o f strain 65 A [IPTG](mM) 0 1 4 0 1 4 50 ng BT2001 BT2002 purified SpoOA B Strain [IPTG] (mM) No. C F U No. Spores Sporulation (ml 1 ) (ml 1 ) Frequency BT2001 0 3.5 x 10* 1.2 x 10 3 3.4 x 10* 1 7.9 x 10 s 5.6 x 10 5 7.0 x 10"4 4 7.1 x 10 8 1.5 x 10 6 2.0 x 10"3 BT2002 0 1.5 x 10* 0 <1 .0x 1 0 s 1 6.3 x 10 s 0 <1 .0x 10~8 4 5.7 x 10 8 0 <1 .0x 10"8 Figure 19. Wi ld type and mutant SpoOA protein expression and sporulation in B. subtilis strains BT2001 and BT2002 in response to I P T G . B. subtilis strains BT2001 and B T 2 0 0 2 were grown in S S M supplemented with 0, or 4 m M I P T G . After 16 hrs o f growth at 3 7 ° C , a portion o f each culture was harvested and samples o f whole cell extracts containing 50 ug o f total protein were separated by electrophoresis through 12 % S D S - P A G E . A 50 ng sample o f purified recombinant SpoOA was also separated by electrophoresis to serve as a control. The protein samples were transferred onto a nitrocellulose membrane and ( A ) subject to immunoblot analysis using anti-SpoOA polyclonal antibodies. After 28 hours o f growth, the remaining culture grown in each condition was assayed for sporulation frequency (B). Sporulation frequency is defined as the number o f colony forming units before and after chloroform treatment. 66 BT2001 to sporulate, the maximum sporulation frequency observed was 100-fold lower than the sporulation frequency typically observed in wi ld type B. subtilis strains. 3.10. Investigation of the effect of varying IPTG concentration on expression from Pspac-spoOA (A25 7V) The goal of constructing the inducible B. subtilis strains was to alleviate the requirement o f SpoOA-a H interaction for increased expression of SpoOA. However, it appeared that protein expression from the Pspac promoter was decreased in strains BT2001 and BT2002 as compared to protein expression from the endogenous spoOAps promoter in JH642 (Figures 17 and 19). Thus it was possible that the lack of sporulation in strain BT2002 relative to strain BT2001 was due to decreased expression from the Pspac promoter and not due to the A 2 5 7 V mutation. I tested this hypothesis by monitoring Spo0A(A257V) expression and the ability to sporulate in response to increasing concentrations of IPTG. Cultures were grown in S S M supplemented with 0-16mM IPTG for 16 hours prior to harvest for immunoblot analysis or for 28 hours before sporulation assay. Lysates of whole cell extracts were probed for SpoOA(A257V) using anti-SpoOA antibodies. In the absence of any IPTG, no protein reacting with the anti-SpoOA antibody was detected (Figure 20A, lane 2). Addition o f 1 m M IPTG induced expression of SpoOA(A257V) (Figure 20A, lane 3) and increasing concentrations did not appear to cause an additional increase in protein accumulation (Figure 20A, lanes 4-7). However, although SpoOA(A257V) was expressed in strain BT2002, no concentration of IPTG lead to induction of sporulation (Figure 20B). 3.11. Construction of B. subtilis spoIIA-lacZ and spoIIG-lacZ reporter strains which overexpress wild type and mutant SpoOA While the data presented in this study have suggested that the A 2 5 7 V mutation does not inhibit the ability of the mutant protein to be phosphorylated, the mutation did alter binding to OA boxes, transcription repression and activation of aA-dependent transcription. A n unanswered question is the effect of spoOA(A257V) transcription directed by o H . One way to test this interaction is to compare the ability of strains expressing equal levels of wild type or A H mutant SpoOA to activate a -dependent or o -dependent transcription in vivo. 67 lane 3 4 5 6 7 B [IPTG] No. C F U No. Spores Sporulation (mM) (ml 1 ) (ml 1 ) Frequency 0 1.1 x 10* 0 < 1 . 0 x l ( P 1 4.0 x 10 s 0 <1.0x 10"8 2 3.3 x 10 8 0 <1.0x 10~8 4 4.2 x 10 8 0 <1.0x 10"8 s 3.4 x l O 8 0 <1.0x 10~8 16 3.2 x 10 s 0 <1.0x 10"8 Figure 20. Spo0A(A257V) expression in BT2002 as a function of I P T G induction. Samples o f B. subtilis strain BT2002 were collected and harvested from cultures grown in S S M supplemented with or without I P T G supplementation (0-16 m M ) for 16 hours at 3 7 ° C . Purified recombinant Spo0A( A 2 5 7 V ) and samples containing 50 ug o f total protein from whole cell extracts were separated by electrophoresis through 12 % S D S - P A G E . The protein samples were transferred onto a nitrocellulose membrane and (A) subject to immunoblot analysis using anti-SpoOA polyclonal antibodies. Lane 1: 33 ng purified recombinant SpoOA(A257V) ; lane 2-7: lysates from BT2002 cultures supplemented with 0, 1, 2. 4, 8, or 16 m M I P T G . (B) After 28 hours o f growth, the remaining culture grown in each condition was assayed for sporulation frequency. Sporulation frequency is defined as the number o f colony forming units before and after chloroform treatment. 68 To create B. subtilis strains encoding cA-dependent and oH-dependent lacZ reporter constructs, strains BT2001 and BT2002 were transformed with chromosomal D N A encoding either a spoIIA-lacZ or a spoIIG-lacZ translational fusion integrated in the amyE locus of the chromosome. A double recombination event resulted in integration of the lacZ translational reporter into the amyE locus of the chromosome of strain BT2001 and BT2002, creating strains BT2003, BT2004, BT2005, and BT2006. I first tested the ability of the strains to sporulate in the absence and presence of inducer (Figure 21 A ) . Cultures were grown in S S M supplemented with 0 or 4 m M IPTG for 28 hours prior to sampling for sporulation assays. A s expected, strains BT2003 and BT2004 were able to sporulate to extremely low levels in the absence o f IPTG and the addition of 4 m M IPTG increased sporulation frequency by at least 100-fold, although this frequency was still 100-fold lower than wi ld type B. subtilis strain JH642. In contrast, strains BT2005 and BT2006, encoding the spoIIA-lacZ and spoIIG-lacZ fusions in a ?spac-spoOA(A257V) background, did not sporulate with or without IPTG. Analysis of protein expression in strains BT2003, BT2004, BT2005 and BT2006 with and without IPTG revealed that all strains expressed either the mutant or wild type proteins to similar levels (Figure 2 IB) . 3.12.Measurement of spoIIA-lacZ induction in strains encoding inducible wild type and mutant SpoOA To determine whether the A 2 5 7 V mutation caused a faulty interaction between o H and SpoOA, we tested the ability o f strains encoding an IPTG-inducible spoOA or spoOA(A257V) gene to activate spoilA-lacZ transcription with and without induction. Strains JH16124, BT2003 and BT2005 were grown in S S M supplemented with 0 or 4 m M IPTG at the time of inoculation. Samples were collected from each culture over half-hour intervals from mid-log growth through stationary phase and tested for P-galactosidase activity (Figure 22). A s expected, spoIIA-lacZ activation in strain JH16124, which encodes a spoIIA-lacZ fusion in a wi ld type B. subtilis background, increased linearly beginning 1.5 hours before the onset o f stationary phase (T.1.5) and reached a maximum three hours later ( T + 1 5 ) . In contrast, activation of spoIIA-lacZ transcription in strains BT2003 and BT2005 was negligible, even with the addition of 4 m M IPTG. In general, there was little or no difference in the (3-galactosidase activity measured in strains grown with or IPTG, although the activities 69 A lane 1 2 3 4 5 6 7 8 9 m — mm -B Strain [IPTG] No. C F U No. Spores Sporulation (mM) (ml 1 ) (ml 1 ) Frequency BT2003 0 5.5 x 10' 4.0 x 10" 7.2 x 10"6 4 7.2 x 1()8 7.1 x I0 5 9.9 x 10"4 B T 2 0 0 4 0 2.1 x 10 8 2.0 x 10" 9.5 x 10"' 4 6.8 x 10 s 8.4 x 10 5 1.2 x 10~3 BT2005 0 4.6 x 10 8 0 <1.0x 10"8 4 8.0 x 10 8 0 <1.0x 10"8 BT2006 0 1.3 x 10 8 0 <1 .0x 10"8 4 1.7 x 10 8 0 <1.0x 10"8 Figure 21. Protein expression and sporulation in B.subtilis strains which overexpress wild type or mutant SpoOA and encode lacZ translational fusions. B.subtilis strains BT2003 and B T 2 0 0 4 encode Fsp&c-spoOA and strains BT2005 and B T 2 0 0 6 encode Pspac-spoOA(A257V). Samples o f B. subtilis strains BT2003-BT2006 were collected and harvested from cultures grown in S S M supplemented with or without I P T G supplementation for 16 hours at 3 7 ° C . Purified recombinant SpoOA and samples containing 50 ug o f total protein from whole cell extracts were separated by electrophoresis through 12 % S D S - P A G E . The protein samples were transferred onto a nitrocellulose membrane and (A) subject to immunoblot analysis using anti-SpoOA polyclonal antibodies. Lane 1: 50 ng purified recombinant SpoOA; lane 2: BT2003 ; lane 3: BT2003 + 4 m M I P T G ; lane 4: BT2005 ; lane 5: BT2005 + 4 m M I P T G ; lane 6: BT2004; lane 7: BT2004 + 4 m M I P T G ; lane 8: BT2006; lane 9: BT2006 + 4 m M I P T G . (B) After 28 hours o f growth, the remaining culture grown in each condition was assayed for sporulation frequency. Sporulation frequency is defined as the number o f colony forming units before and after chloroform treatment. 70 100 -2.5 -2.0 -1.5 -1.0 0 0.5 1.0 1.5 2.0 2.5 3.0 Time (hi) Figure 22. VspoIJAAacZ activity in B.subtilis strains BT2003 and BT2005 . B. subtilis strains JH16124, BT2003 and BT2005 were grown in S S M +/- 4 m M I P T G and samples were collected in half-hour intervals from mid exponential growth and assayed for (3-galactosidase activity. Time '0' indicates the end o f exponential growth. Symbols: closed triangles, JH16124, open circles. BT2003 (no IPTG) ; filled circles, BT2003 + 4 m M I P T G ; open squares, BT2005 (no IPTG) ; filled squares, BT2005 + 4 m M I P T G . Values reflect the average o f three independent experiments and their standard deviations. 71 measured in strain BT2003, which encode Pspac-spoOA, were marginally greater at all time points than the (3-galactosidase activity measured in strain BT2005. A t maximum, spoIIA-lacZ activation in strains BT2003 and BT2005, with or without induction, was over 10-fold lower than the maximum level reached in strain JH16124. It was possible that the inability of strains BT2003 and BT2005 to activate spoIIA-lacZ transcription was due to inappropriate timing of induction. If spoOA or spoOA(A257V) were expressed early in growth and i f IPTG induction decreased over time, then it would be possible that the protein produced could have been degraded prior to entry into sporulation. A s a result, the concentration o f wi ld type or mutant SpoOA would be below the threshold required for activation of stage II sporulation genes such as spoIIA and spoIIG. To address this potential problem I attempted to optimize the timing of IPTG induction in strains harboring either spoIIA-lacZ or spoIIG-lacZ fusions in Yspac-spoOA and Pspac-spoOA(A257V) backgrounds. Cultures were grown in S S M and were supplemented with 4 m M IPTG at half hour intervals during exponential growth in hopes of inducing wi ld type and mutant SpoOA protein expression coordinately with an increase in phosphorelay component expression. Samples of each culture were harvested 3 hours after the onset of stationary phase and tested for P-galactosidase activity (Table 4). Altering the time of IPTG induction did not lead to a significant increase in the p-galactosidase activity measured in induced cultures of BT2003, BT2004, BT2005 and BT2006. Addition of IPTG at different time points during exponential growth had little effect upon activation of spoIIG-lacZ transcription in strains BT2004 and BT2006. The greatest P-galactosidase activity measured for both o f these strains was only 2-fold greater than that of the uninduced cultures and occurred when IPTG was added 1.5 hours prior to the onset of stationary phase. Activation of spoIIA-lacZ transcription was greatest upon IPTG addition during exponential growth approximately 2 to 2.5 hours before the onset of stationary phase in strain BT2003. In these cultures, P-galactosidase activity was increased 5-fold over the uninduced culture. Similarly, spoIIA-lacZ transcription was greatest when strain BT2005 was induced between 1.5 and 2 hours before the onset of stationary phase, although the maximum P-galactosidase activity measured for this strain was only 2-fold 72 Table 4. Activation of spoIIA-lacZ and spoIIG-lacZ transcription in B. subtilis strains which overexpress wild type or mutant SpoOA. Genotype3 Induction [IPTG] P-galactosidase Activity Time (hr) (mM) (Miller Units)b spoIIA-lacZ spoIIG-lacZ JH642 spoOA::(pMNspoOAN uninduced 0 0 4.05 2.96 ermAM) T-2.5 4 20.18 3.63 T-2.0 4 21.00 4.85 T-1.5 4 15.47 6.35 T-1.0 4 8.76 4.77 TO 4 6.7 4.79 JH695 spoOA::(pMNspoOAN uninduced 0 0 4.13 1.85 ermAM) T-2.5 4 7.65 2.09 T-2.0 4 7.87 3.04 T-1.5 4 8.01 4.30 T-1.0 4 7.75 4.07 TO 4 6.56 3.77 aB. subtilis strains used in this experiment also encode amyE: :(spoIIA-lacZ) C m R or amyE': :(spoIIG-lacZ) K a n R . bB. subtilis strains BT2003-BT2006 were grown in S S M and supplemented with 4 m M IPTG at half hour intervals during exponential growth through the onset of stationary phase (T-2.5 to TO). Samples of each culture were harvested 3 hours after the onset of stationary phase and tested for P-galactosidase activity. Values shown are the average of three independent experiments. C A culture of each B. subtilis strain was grown in S S M without addition of IPTG. Samples of each culture were harvested and tested for P-galactosidase activity similarly to the test cultures. Values shown are the average of three independent experiments. 73 greater than the uninduced culture. However, although there were marginal increases in spoIIA and spoIIG promoter activities when induced at different times during exponential growth, the (3-galactosidase activities measured in each condition were well below the levels normally reported in the literature (Perego et al, 1991b; Rowe-Magnus et al, 2000). This indicates that both the spoIIA-lacZ and spoIIG-lacZ promoters are not activated in strains encoding Vspac-spoOA or ?spac-spoOA(A257V) and suggest that the concentration of phosphorylated wi ld type or mutant protein is not of sufficient threshold concentration to activate transcription from either promoter. A s a consequence, the data from this experiment did not provide insight into the specific question of whether the A 2 5 7 V mutation causes a faulty interaction with o H . 74 4. DISCUSSION 4.1. SpoOA is the master regulator of the onset of sporulation In response to nutrient deprivation and high cell density, B. subtilis cells w i l l undergo the developmental process of sporulation. Sporulation is a tightly regulated process and initiation of sporulation is ultimately controlled by the response regulator SpoOA. SpoOA directly controls the expression of 121 genes by repressing the expression of genes required for alternate stationary phase phenomena and activating genes required for sporulation (Molle et al, 2003a). A n early step in the process begins with the SpoOA-dependent repression of the abrB gene which encodes a transition state regulator. During vegetative growth, A b r B represses transcription of many stationary phase genes and some genes required for sporulation (Strauch and Hoch, .1993). Repression of abrB permits expression of the spoOH gene, encoding o H , an alternate o factor that together with SpoOA regulates transcription during the onset of sporulation. Together the two proteins cooperate to increase transcription of the spoOA gene itself as the result of a positive feedback mechanism. o H is also responsible for increasing the concentration o f activated, phosphorylated SpoOA by increasing transcription of the genes of the phosphorelay components, kinA and spoOF. 4.2. The A257V mutation of SpoOA uncouples transcription activation and repression I was interested in how changes in the DNA-bind ing domain of SpoOA affect the ability of SpoOA to activate transcription. One substitution mutation in SpoOA, A257V, was found to differentially affect the ability of SpoOA to activate and repress transcription. The A 2 5 7 V mutation abolishes the ability of B. subtilis cells to sporulate but does not affect the ability of SpoOA to repress transcription at the abrB promoter. Instead, the mutation eliminates the ability of SpoOA to activate both oA-dependent transcription of the spoIIG operon promoter and oH-dependent transcription of the spoIIA operon promoter (Perego et al, 1991b). 4.3. SpoOA can activate and repress transcription initiation 4.3.1. Repression of transcription by SpoOA 75 The mechanism by which SpoOA represses transcription initiation has been investigated at the abrB promoter. The abrB promoter contains two transcription initiation start sites, PI and P2 (Perego et al, 1988), and two consensus OA boxes located downstream of the second transcription start site (Strauch et al, 1990). SpoOA~P binds to the OA boxes at abrB by interacting with only one side of the D N A by binding across the minor groove (Greene and Spiegelman, 1996; Strauch et al, 1990). SpoOA~P represses transcription initiation at P2 by preventing D N A strand separation without blocking the initial interaction of R N A P with the abrB promoter (Greene and Spiegelman, 1996). It has been presumed, although not directly proven, that the formation of a blocked ternary RNAP-SpoOA~P-a£>ri? P2 complex inhibits transcription from abrB PI by steric occlusion (Greene and Spiegelman, 1996). 4.3.2. Activation of aA-dependent transcription by SpoOA How SpoOA stimulates transcription is best understood at the aA-dependent spoIIG promoter. Previous work has demonstrated that R N A P can bind to the spoIIG promoter but is transcriptionally inactive (Bird et al, 1993, 1996). A t spoIIG, SpoOA functions as a class II transcriptional activator (Busby and Ebright, 1997, 1999), binding to a pair of OA boxes lying immediately upstream of and overlapping with the -35 element, placing it in a position to interact with the 4.1 region of o~A. Importantly, the distance separating the conserved -35 and -10 elements of this promoter and another SpoOA-dependent, oA-dependent promoter, spoIIE, are 22 and 21 base pairs respectively, instead of the consensus 17 base pairs (Spiegelman et al, 1995). Recently it has been demonstrated that reducing the spacer length at spoIIG eliminates the requirement for SpoOA during transcription initiation, suggesting that SpoOA stimulates transcription by compensating for the overlong spacing of these promoters (McLeod and Spiegelman, 2005). The requirement for Spo0A~P during transcription initiation at spoIIG can also be bypassed by artificially denaturing the -10 element (Seredick and Spiegelman, 2004; Seredick, 2005), demonstrating that activated SpoOA and R N A P cooperate to separate the D N A strands around the -10 element. A recent study has also shown that Spo0A~P bound to the OA box overlapping with the -35 element appears to re-position R N A P to facilitate the interaction of the a A subunit of R N A P with the non-template strand of the -10 region of heteroduplex spoIIG promoters (Seredick and Spiegelman, 2004). From these data, one would predict that SpoOA re-positions o A for recognition of the double-stranded -10 element and for nucleation of promoter melting. 76 Thus, transcription initiation at spoIIG involves at least three steps: formation of an inactive KNAP-spoIIG complex; activation of the RNAP-spoIIG complex by SpoOA, including open complex formation; and initiation of transcription and promoter clearance. However, it is not clear whether the binding of SpoOA~P to OA boxes overlapping the -35 element and the contact with the a A subunit stimulates release o f a A contacts at the -35 element while tethering R N A P close to the -10 element, or whether SpoOA~P passively retains R N A P that has diffused downstream (Seredick and Spiegelman, 2004). Recent work suggests that R N A P maintains contact with the upstream promoter and recruits SpoOA~P as the downstream OA boxes are exposed when a slides off the -35 element (Seredick & Spiegelman, unpublished data). 4.3.3. Activation of o -dependent transcription by SpoOA Little is known of the role SpoOA plays in transcription initiation at promoters transcribed by R N A P - a H . Genetic studies suggest that SpoOA contacts homologous regions in o H and o A (Baldus et al, 1995). However, the arrangement and orientation of OA boxes and the spacing of the -35 and -10 elements at aH-dependent promoters differs from those at a A -dependent promoters (Spiegelman et al., 1995) (Figure 23), suggesting that SpoOA uses a different surface to contact a H and a different mechanism to activate transcription (Seredick and Spiegelman, 2001). Studies investigating SpoOA-dependent transcription activation of the spoIIA promoter indicate that deletion of sequences 64 bases 5' to the transcription initiation site completely abolish promoter activity in vivo (Wu et al., 1991), suggesting that the OA box located upstream is critical for activation. While a crA contact surface on SpoOA has been defined (Buckner et al., 1998; Hatt and Youngman, 1998; Kumar et al., 2004; Schyns et al., 1997), the region responsible for interaction with o H has not been identified. Genetic screens designed to identify the a H contact surface (Hatt and Youngman, 1998) may have been unsuccessful because of the complexity of the genetic regulatory network controlling SpoOA synthesis and activation, or for other technical reasons. 4.4. Objective of thesis The objective of this study was to resolve how a single amino acid substitution within the D N A binding domain of SpoOA could prevent sporulation while differentially affecting the 77 -100 SO -60 -40 -20 +1 +20 +40 spoIIGp ' ^ ^ 1 spoLJAp spoOApsL^ spoOApvl— abrBp L— + oH + oH + aA -Figure 23. Location of OA boxes within promoters activated and repressed by SpoOA. SpoOA binds to OA boxes ( 5 ' - T G N C G A A - 3 ' ) encoded upstream and downstream of the +1 transcription start site of target promoters to activate and repress transcription, respectively. Examples of promoters activated by SpoOA ('+', to right of promoter) include the spoIIG, spoIIA promoters and the sporulation promoter of spoOA. Two promoters repressed by SpoOA ('-', to right of promoter) include the vegetative promoter of spoOA and the abrB promoter. The number, location (bp relative to +1 indicated above promoters) and orientation of OA boxes within target promoters is not conserved. OA boxes are indicated by arrows, and the direction of the arrow indicates orientation. Promoters recognized by R N A P complexed with and are indicated. 78 ability of the protein to activate and repress transcription (Perego et al, 1991b; Rowe-Magnus et al, 2000). I used biochemical methods to explore how the A 2 5 7 V mutation affected SpoOA function to prevent activation without compromising abrB repression. 4.5. In vitro characterization of SpoOA(A257V) SpoOA(A257V) was phosphorylated at the same rate and to the same extent as SpoOA (Figures 9, 10), indicating that the mutation did not alter the structure of the protein to compromise it as a substrate for the phosphotransferase protein SpoOB. This indicated that insufficient phosphorylation of SpoOA(A257V) could not account for the absence o f sporulation in vivo. Similarly, Spo0A(A257V) was able to repress transcription from the abrB promoter in vitro, although to levels approximately half those achieved by the wi ld type protein (Figure 11). This suggested that the A 2 5 7 V mutation did affect D N A binding. Previous research determined that the A 2 5 7 V mutation does not affect abrB repression in vivo (Perego et al, 1991b), indicating that in vivo the lowered binding efficiency is not crucial for repression. I showed for the first time that the A 2 5 7 V mutation did not abolish the ability of SpoOA to activate cA-dependent transcription in vitro. SpoOA(A257V) stimulated spolIG transcription, although at a reduced rate (Figure 12) and to levels approximately half those observed for SpoOA (Figure 13). This finding was novel since in vivo research had indicated that the A 2 5 7 V mutation rendered SpoOA unable to activate both the oA-dependent spolIG promoter and the a -dependent spoIIA promoter (Rowe-Magnus et al, 2000). This indicated that the A 2 5 7 V mutation did not obliterate the ability of SpoOA to stimulate transcription activation but suggested the mutation could possibly represent a defect in activation of cH-dependent transcription. One possible explanation for the reduced ability of SpoOA(A257V) to activate spolIG and repress abrB in vitro was that the A 2 5 7 V mutation may have caused SpoOA to bind inappropriately to OA boxes located at these promoters . However, DNasel footprinting analysis revealed that SpoOA(A257V) interacted with both site 1 and site 2 boxes o f the spolIG promoter in a seemingly identical fashion, although with reduced affinity relative to 79 the wi ld type protein (Figure 14). This indicated that the A 2 5 7 V mutation of SpoOA did not alter the specificity o f binding or alter the interaction of the mutant protein with D N A bases or the D N A backbone. To quantitatively measure the effect of the A 2 5 7 V mutation on the binding affinity of SpoOA for OA boxes I used an E M S A to compare the ability o f wi ld type and mutant proteins to bind consensus OA boxes encoded within duplex D N A . Analysis of D N A binding revealed that the affinity of SpoOA(A257V) for a consensus binding site was reduced relative to SpoOA (Figure 15). The observed 2-fold reduction in binding correlated with the 2-fold reduction in transcription stimulation and repression. Thus a common modest defect in D N A binding likely underlies the modest reduction observed for in vitro transcription activation and repression. However, the twofold reduction in D N A binding is associated with a 10 fold reduction in sporulation in spoOA(A257V) B. subtilis strains (Perego et al, 1991b). Previous research has indicated that mutations within SpoOA at residues crucial for D N A contact (E213, R214) render SpoOA incapable o f stimulation of both o A - and oH-dependent promoters in vivo and either decrease or eliminate D N A binding in vitro (Hatt and Youngman, 2000). Unlike the A 2 5 7 V substitution, mutation of these residues compromises the ability of SpoOA to repress abrB in vivo as well , arguing against the possibility that A257 might play a direct role in D N A binding. To further investigate how the A 2 5 7 V mutation decreased the apparent D N A binding affinity of SpoOA for OA boxes, I assessed whether the binding interactions by two SpoOA monomers in the dimer was equivalent. I found that the A 2 5 7 V mutation had a minor effect on dimer packing at the spolIG promoter as assessed by productive interactions with R N A P leading to transcription (Figure 16), suggesting that the binding interactions with D N A by the downstream monomer are more critical than the binding interactions of the upstream monomer. This indicated that altered packing of the D N A binding domain, as a consequence of the A 2 5 7 V substitution, reduced the affinity of the mutant proteins for the OA boxes. However, this effect was relatively modest and seemed unlikely to be able to account for the loss of sporulation in vivo. In sum, these results indicated that the A 2 5 7 V mutation did not significantly effect on the ability of SpoOA to stimulate transcription activation. The lack of 80 a dramatic effect on transcription activation by R N A P - a A points to the possibility that the mutation could possibly represent a defect in activation of aH-dependent transcription. 4.6. In vivo investigation of the effect of the A257V mutation of SpoOA Results from this study have revealed that the A 2 5 7 V substitution reduced but did not eliminate the ability of the SpoOA to activate oA-dependent transcription. SpoOA(A257V) was able to both activate and repress transcription in vitro, albeit to levels approximately half that achieved by wi ld type SpoOA. SpoOA(A257V) did not impair phosphorylation, or cause inappropriate binding to OA boxes within the spoIIG promoter. The mutation modestly reduced the affinity of SpoOA for OA boxes and appeared to have a negligible effect on the packing of SpoOA dimers against R N A P . The data from this in vitro investigation indicated noticeable but slight effects of the A 2 5 7 V mutation contrasted with the dramatic changes in the phenotype in vivo. Studies have indicated that genes within the SpoOA regulon respond differentially to high and low concentrations of SpoOA (Chung et al, 1994; Fujita et al, 2005). One possible explanation for the lack o f sporulation observed in vivo in the spoOA(A257V) mutant was that the mutant protein was not expressed to the same level as the wild type protein. A s a consequence, genes that are activated by high levels o f SpoOA and required for sporulation, such as the spoIIA and spoIIG operons would not be transcribed and the cells would not sporulate. I tested this idea and found that the mutant SpoOA protein was not expressed to the same level as wi ld type SpoOA (Figure 17). This result was unexpected since previous work had suggested that both wi ld type and mutant proteins were expressed to the same extent (Perego et al, 1991b). This suggested that the lack of sporulation in B. subtilis strains encoding spoOA(A257V) could be attributed to insufficient mutant SpoOA protein expression. SpoOA expression increases at the onset of stationary phase as a consequence of a positive feedback mechanism in which transcription of the spoOA gene is increased due to activation of a second promoter for the spoOA gene transcribed by R N A P - c r H . A similar regulatory mechanism exists controlling expression of the master regulator of competence in B. subtilis, comK. L ike SpoOA, there are multiple regulatory inputs affecting transcription from the 81 comK promoter that determine the threshold level of comK expression (Hahn et al, 1996; Smits et al, 2005; van Sinderen and Venema, 1994). A t some point, the concentration of C o m K becomes sufficient to activate an auto-stimulatory loop (Maamar and Dubnau, 2005; Smits et al, 2005). In competence development, this auto-stimulatory loop is essential and sufficient to establish the "bistable response" typical of biological systems such as competence and sporulation (Hofer et al, 2002; Smits et al, 2005). The reduction in expression of Spo0A(A257V) observed in this study could be attributed to a deficiency in the positive feedback loop leading to upregulation of spoOA(A257V) expression. There are two possible explanations to explain this defect. Firstly, the A 2 5 7 V mutation could interfere with a specific interaction between SpoOA and o H leading to lack of, or inefficient, expression from spoOApv. Alternatively, since SpoOA activates transcription as a dimer, small changes in dimer properties could lead to low levels of SpoOA protein and failure to activate essential promoters. In order to differentiate between these two possibilities in vivo, I attempted to create a system in which the expression of wild type and mutant proteins was inducible and then tested the ability of cells over-expressing wi ld type or mutant protein to activate transcription from both a aA-dependent and a oH-dependent promoter in vivo. I predicted that i f the A 2 5 7 V mutation caused a defective interaction between SpoOA and a H , cells expressing the mutant protein would activate oA-dependent promoters, but not oH-dependent promoters whereas cells expressing the wild type protein would be able to activate both aA-dependent and aH-dependent transcription. If the A 2 5 7 V mutation caused a defective interaction between SpoOA monomers leading to low levels of SpoOA in vivo, over-expression of SpoOA(A257V) would overcome the defective interaction and permit activation of both o A - and oH-dependent transcription. Although wild type and mutant SpoOA proteins were expressed in response to IPTG induction, the levels of expression of either protein were low and were insufficient to permit wild type levels of sporulation or drive activation of either o A - or oH-dependent transcription. Increasing the concentration of IPTG used to induce expression revealed that expression from the Pspac promoter construct could not be increased. Similarly, induction at various cell densities eliminated the possibility that the timing of SpoOA induction was not 82 synchronized with the expression of other proteins required for sporulation as induction at various points during growth did not activate either aA-dependent or aH-dependent transcription. There are two possible explanations for the decreased levels o f sporulation and lack of activation of both spoIIG and spoIIA promoters in the inducible-SpoOA B. subtilis strains used in this study. Fujita and Losick (2005) induced a constitutively active form of SpoOA during mid-exponential growth and demonstrated that sudden high level expression of SpoOA exerts a dominant-negative effect on sporulation. From their study, the authors postulated that a gradual increase in the threshold concentration of activated SpoOA is required for sporulation. Alternatively, decreased levels of sporulation and lack of activation of stage II promoters in the inducible-SpoOA B. subtilis strains used in this study may have been observed because there were insufficient levels of phosphorylated wild type or mutant protein available for stimulation of transcription and subsequent sporulation. The low concentration of phosphorylated protein available may have been a consequence of the genotype of the strains created. For example, the strains created here encode a wi ld type or mutant spoOA gene under transcriptional control of Pspac and one or more copies of the N -terminal receiver domain of SpoOA (SpoOA N ) transcribed under the control of the native vegetative- and sporulation-specific promoters of spoOA. The spoOA gene duplication arose as a result of the recombination event that inserted the Pspac-controlled spoOA allele into the chromosomal spoOA locus. It is conceivable that i f SpoOA N was expressed it would compete for phosphorylation by the phosphorelay in vivo. Furthermore, i f multiple copies of p M N S p o O A N were integrated into the spoOA locus, there would be many duplications of the truncated spoOA gene encoding SpoOA N and only a single copy of the full length wild type or mutant spoOA under Pspac transcriptional control. In this genetic background there is an even greater possibility for competition for phosphorylation by the phosphorelay between SpoOA N and the full length wild type or mutant SpoOA proteins. This may be sufficient to reduce the concentration of phosphorylated full length wi ld type or mutant SpoOA below the level required for activation and repression of high-threshold SpoOA-dependent promoters and prevent sporulation. The hypothesis has some support from previous research that has shown that SpoOA N can successfully compete with the full length protein for phosphorylation 83 in vitro, and when SpoOA is over-expressed in the mother cell sporulation is impaired prior to polar septum formation (Fujita and Losick, 2003). In Western blot analyses performed in this study a low molecular weight band corresponding to a protein of approximately 14 K D a was observed which could represent SpoOA N (data not shown). However, since the polyclonal antibody used in the analyses reacted with other proteins in cell lysates, it was unclear i f this band represented SpoOA N or another cellular protein. There are several methods by which one might increase the low levels of SpoOA and SpoOA(A257V) expression in the reporter strains. One alternative is to eliminate competition by SpoOA N for phosphorylation. This can be accomplished by reconstructing the Pspac-spoOA and Pspac-spoOA(A257V) strains encoding spoIIA-lacZ and spoIIG-lacZ translational fusions in either a AspoOA background, or by removing the native spoOA promoters from the existing strains. Alternatively, expression of the full length wi ld type or mutant SpoOA proteins could be increased in vivo without removing the spoOAN sequence. One method to achieve this is to introduce a second copy of Vspac-spoOA or Yspac-spoOA(A257V) into a site on the chromosome separate from the spoOA locus. Alternatively wi ld type and mutant SpoOA protein expression could be increased in spoOA and spoOA(A257V) strains by expressing a a A mutant, crsA, which permits high-level transcription of spoOA from the vegetative promoter (Dixon and Spiegelman, 2002; Yamashita et al, 1989). The latter approaches wi l l increase the concentration of wi ld type or mutant SpoOA protein but do not eliminate the possibility of competition for phosphorylation by SpoOA N . Repetition of the sporulation and p-galactosidase assays using true over-expression strains should provide a more definitive answer to whether the A 2 5 7 V substitution interferes with the interaction of SpoOA and o H or whether the results are more consistent with the A 2 5 7 V mutation interfering with SpoOA dimerization. 4.7. Effect of residue A257 on SpoOA dimer function It has been suggested that the effect of the A 2 5 7 V mutation within SpoOA is to weaken or disrupt crucial interactions within a SpoOA dimer (Lewis et al, 2002). The crystal structure of the D N A binding domain of SpoOA complexed with D N A indicates that two C T D bind tandem OA boxes in a head-to-tail orientation (Zhao et al, 2002). In this orientation two 84 adjacent D N A binding domains are able to interact with each other through a network of salt bridges, hydrogen bonds and hydrophobic patches. The dimer interface in the crystal structure is formed by helix ccF of the upstream SpoOA monomer and helix aB of the downstream SpoOA monomer (Zhao et al, 2002) (Figure 6). Predictions o f the effect o f substitution o f valine for alanine at position 257 within the dimer interface suggest that the mutation would weaken interactions between the monomers by altering the orientation and flexibility of the helix within which A257 is located (Zhao et al, 2002). It has been suggested that two suppressor mutations of spoOA(A257V), H162R and L174F, repress the effects of the A 2 5 7 V mutation by facilitating compensatory interactions between adjacent monomers (Seredick and Spiegelman, 2001; Zhao et al, 2002). Head-to-tail dimerization places the suppressor mutations and the A 2 5 7 V mutation together at the dimer interface as both suppressor mutations are located at the opposite face o f the C-terminal domain from residue A257 (Zhao et al, 2002) (Figure 24). This orientation strengthens hydrophobic interactions between monomers and permits formation of a new hydrogen bond between H162R and F236 (Zhao et al, 2002). L ike the A 2 5 7 V mutation, deletion of the last 10 amino acids of SpoOA and the D258V and L260V mutations abolish the ability of SpoOA to activate both c H - and aA-dependent transcription but do not effect the ability of the protein to repress transcription (Rowe-Magnus et al, 2000). These residues are also located at the dimer interface and as such may also affect SpoOA activity because of disrupted or weakened contacts within a SpoOA dimer. Residue D258 in one monomer forms an intermolecular salt bridge with R177 in the other monomer and mutation would eliminate this interaction, weakening dimerization (Zhao et al, 2002). Similarly, the predicted effect of the L260V mutation is similar to that of the A 2 5 7 V mutation in that it likely weakens intermolecular interactions by decreasing the flexibility of helix aF (Zhao et al, 2002). Similarly to SpoOA, members of the OmpR-PhoB family of response regulators bind D N A in a head-to-tail orientation (Blanco et al, 2002). The C-terminal domains of SpoOA and PhoB lack structural homology outside their D N A binding motifs and consequently the dimer interface differs between the two. Stable interactions between monomers may be crucial in 85 A V Figure 24. Suppressor mutations of spoOA(A257V) strengthen intermolecular contacts within the SpoOA dimer. Tandem SpoOA D N A binding domains bind two concensus OA boxes as a head-to-tail dimer. Head-to-tail dimerization places the suppressor mutations (H162R on a A and L174F on aB) and the A257V mutation (aF) together at the dimer interface, strengthening hydrophobic interactions between monomers and permiting formation of a new hydrogen bond between. HI 62, L I 74 and A257 are indicated in space fdling model. The D N A is shown 5' to 3' and the structure is rotated 90° to indicate the dimer interface. Helices: a A , blue; a B , cyan; a C , green; a D , yellow; a E , gold.The structure is based on P D B fde 1LQ1 deposited by Zhao and Varughese, 2002 in the R C S B protein data bank (http://pdbbeta.rcsb.org/pdb/Welcome.do). Figure constructed using P y M O L (Delano Scientific). 86 sequential monomer binding or cooperative binding of response regulators such as SpoOA and PhoB to D N A . L ike SpoOA(A257V), mutations within PhoB have been isolated that map to the dimer interface which reduce or completely abolish D N A binding (Makino et al, 1996). However all known D N A binding sequences of all members of the OmpR-PhoB family are direct repeats with identical orientation with respect to the direction of transcription (Blanco et al, 2002). In contrast, OA boxes do not always occur in the same orientation. Dimerization facilitated by interactions between adjacent DNA-bind ing domains is also suggested from the crystal structure of another response regulator, NarL, although NarL dimers are oriented in an antiparallel orientation (Maris et al, 2002). Like the spoOAAW allele in which the last 10 amino acids (residues 253 to 263) have been removed (Rowe-Magnus et al, 2000), deletion of the last 7 amino acids of the NarL homolog U h p A abolishes the ability o f the regulator to activate transcription (Webber and Kadner, 1995). Based on the NarL structure bound to D N A , the last 7 amino acids in UhpA would also be predicted to form part of the dimer interface. While the C-terminal domain structures of SpoOA, PhoB/OmpR and U h p A / N a r L are different, the dimer interface of the output domain clearly makes a contribution to response regulator function. There is evidence in opposition to the hypothesis that the A 2 5 7 V mutation within the C-terminal domain o f SpoOA weakens interactions required for dimer stabilization. A recent model suggests that the primary effect of receiver phosphorylation in SpoOA is dimerization (Lewis et al, 2002); that is, the response regulator receiver domains act as inducible dimerization domains (Fiedler and Weiss, 1995). Receiver domain dimerization has been observed in the response regulators NtrC, PhoB, F ixJ , and PhoP, and speculated in NarL (Baikalov et al, 1996) in response to phosphorylation (Birck et al, 2003; Da Re et al, 1999; Fiedler and Weiss, 1995; McCleary, 1996). In both FixJ and PhoP mutations have been isolated within the receiver domain which do not dimerize and have decreased D N A binding activity (Chen et al, 2003; Da Re et al, 1999). While the DNA-binding domain interactions revealed from the crystal structures should not be ignored, such interactions would seem to be redundant in light of the ability of the receivers to dimerize (Lewis et al, 2002) and the ability of the SpoOA(A257V) mutant to bind D N A (Figures 14,15). In addition, while A 2 5 7 V does reduce transcription activation by half, repression of the abrB promoter, which 87 also depends on D N A binding and therefore SpoOA dimerization is unaffected by the spoOA(A257V) mutation in vivo (Perego et al., 1991b). If SpoOA is active as a dimer, it is conceivable that stabilizing interactions play an important role in increasing the local concentration of SpoOA at 'high-threshold SpoOA-activated' promoters but not at promoters responding to low concentrations of SpoOA. This prediction is consistent with the lack of activation of the 'high-threshold SpoOA-activated' (Fujita et al, 2005) spoIIA and spolIG operon promoters in vivo (Perego et al., 1991b) and repression of the 'low-threshold SpoOA-repressed' (Fujita et al, 2005) abrB promoter in vivo (Perego et al, 1991b) by SpoOA(A257V). One serious problem for this threshold model is the lack of induction o f the 'low-threshold SpoOA-activated' (Fujita et al, 2005) aH-dependent, sporulation-specific promoter of spoOA (Chibazakura et al, 1995) in vivo and the subsequent decrease in SpoOA(A257V) expression observed in this study. This promoter is key to the autostimulatory loop proposed in vivo. One hypothesis that may account for these observations is that at low concentrations of activated protein, SpoOA may dimerize on the D N A instead of in solution. Under these conditions, monomers of Spo0A~P may bind D N A cooperatively with intermolecular interactions between the C-terminal domains playing a crucial role. Following the initial interactions between the SpoOA dimers and the OA boxes and between the C-terminal domains of the monomers, interaction of the phosphorylated receiver domains would serve to stabilize the S p o 0 A ~ P - D N A complex. The stability of the complex would be a function of the sum of interdomain interactions between N-terminal and C-terminal domains, and the interactions of the C-terminal domains with the D N A . If the A 2 5 7 V mutation affected dimerization, then at protein concentrations or conditions too low to allow receiver dimerization in solution SpoOA and Spo0A(A257V) would be expected to bind D N A differently. Specifically, SpoOA would be expected to bind cooperatively, and SpoOA(A257V) would be expected to bind with a lesser degree of cooperativity. None of the binding experiments carried out so far show any indication of cooperativity with either Spo0A~P or SpoOA(A257)~P. This may reflect a technical issue that the phosphorylation reactions were carried out at relatively high concentrations of protein. The choice of protein 88 concentration was dictated by the desire to keep the conditions for binding similar to those used for transcription analysis. In the transcription analysis it was necessary to add small volumes of activator to the reactions to minimize the effects of ionic and organic solvent (glycerol). Thus, the concentrations of SpoOA or SpoOA(A257) were kept high. However, it is also interesting to note that in the experiments with binding to the oligonucleotide containing the OA boxes from the abrB promoter, no evidence o f binding o f a single SpoOA monomer to the D N A fragment was ever detected and only a single species with an altered electrophoretic mobility was ever detected. 4.8. Residue A257 as a part of a oH-SpoOA interaction surface The second possible explanation for the effect o f the A 2 5 7 V mutation on SpoOA function is that residue A257 is important for contact with the a H subunit of R N A P and that the A 2 5 7 V mutation diminishes this interaction. This hypothesis would explain the lack of activation of the o -dependent spoIIA promoter (Perego et al, 1991b) and the lack of transcript from the o -dependent sporulation-specific promoter of spoOA (Chibazakura et al, 1995) observed in vivo. Furthermore, this hypothesis accounts for the lack of activation o f the 'high-threshold SpoOA-activated' aA-dependent spoIIG promoter in vivo since increased expression of spoOA, which is needed to increase SpoOA levels, is dependent upon successful interaction between SpoOA and a H during stimulation o f spoOAps transcription. This hypothesis could also explain the lack of transcription activation of promoters that do not contain tandem OA boxes and presumably bind monomelic SpoOA~P, such as spoOAps (Figure 23). Moreover, because the direction of the OA boxes at aH-dependent promoters (eg. spoIIA, spoOAps) differs from the direction o f OA boxes at aA-dependent promoters (eg. spoIIG), the face of the DNA-bind ing domain of SpoOA(A257V) encoding A 2 5 7 V would be oriented downstream in a position to contact R N A P bound downstream (Figure 23). Although the hypothesis that A257 and/or the region around it represents that a H contact region within SpoOA accounts for the in vivo and in vitro characteristics of SpoOA(A257V) thus far, the structural data available run counter to this hypothesis. The region within SpoOA that contacts o A during transcription activation (the S A A R ) is located within and around the aE helix of the D N A binding domain of SpoOA (Lewis et al, 2000a) (Figure 5). 89 Head-to-tail binding of a dimer of C-terminal domains of SpoOA to tandem OA boxes places the aE helix of both monomers on the same side o f the dimer in proximity to a A during transcription activation (Zhao et al, 2002). In comparison, efforts to locate a similar region within SpoOA that contacts a H during stimulation of transcription initiation have been unsuccessful, although previous research have found regions within a H contacted by SpoOA during transcription activation (Baldus et al, 1995). If A257 was located in a region within SpoOA contacted by c H , we would expect that suppressor mutations of A 2 5 7 V would be located near this residue. This is not observed as two of three A 2 5 7 V suppressor mutations are located far from A257 on the opposite face o f the D N A binding domain of SpoOA. The third A 2 5 7 V suppressor mutation, sofll4 (a D 9 2 Y substitution), was isolated as a suppressor of a spoOF deletion mutant (Spiegelman et al, 1990). D92 is located in the receiver domain of SpoOA and because the structure of the full length SpoOA protein has yet to be determined, it is possible that the orientation of the two domains places D 9 2 Y near A 2 5 7 V . However, a more likely explanation for suppression of A 2 5 7 V by D 9 2 Y is that intermolecular contacts between the receiver domains within a SpoOA dimer are strengthened in SpoOA(D92Y, A257V) since previous work demonstrated that the D 9 2 Y mutation enhanced stability o f SpoOA-RNAP complexes (Cervin and Spiegelman, 1999). Further research could determine i f residue A257 and/or the region around it identifies the o H interaction region within SpoOA. A direct test of this hypothesis in vitro would be to include Spo0A(A257V)~P and R N A P - o H in an in vitro transcription reaction and assess the stimulation of either the spoIIA or the spoOAps promoters. From this experiment I would expect that SpoOA(A257V)~P would be unable to stimulate either spoIIA or spoOAps transcription at any protein concentration whereas SpoOA~P would be able to stimulate transcription activation. Similarly, whether A257 represented a residue within SpoOA contacted by a could be tested in vivo utilizing the experiments attempted in this work involving inducible-SpoOA expression and activation o f spoIIA-lacZ and spoIIG-lacZ translational fusions. 90 REFERENCES Antoniewski, C , Savelli, B . , and Stragier, P. (1990) The spoIIJ gene, which regulates early developmental steps in Bacillus subtilis, belongs to a class of environmentally responsive genes. JBacteriol 172: 86-93. Arigoni , F., Duncan, L . , Alper, S., Losick, R., and Stragier, P. (1996) SpoIIE governs the phosphorylation state o f a protein regulating transcription factor sigma F during sporulation in Bacillus subtilis. Proc Natl Acad Sci USA 93: 3238-3242. Arthur, T . M . , Anthony, L . C . , and Burgess, R .R . (2000) Mutational analysis of beta '260-309, a sigma 70 binding site located on Escherichia coli core R N A polymerase. J Biol Chem 275:23113-23119. Bai , U . , Lewandoski, M . , Dubnau, E . , and Smith, I. (1990) Temporal regulation o f the Bacillus subtilis early sporulation gene spoOF. J Bacteriol 172: 5432-5439. Ba i , U . , Mandic-Mulec, I., and Smith, I. (1993) Sin l modulates the activity o f SinR, a developmental switch protein of Bacillus subtilis, by protein-protein interaction. Genes Devi: 139-148. Baikalov, I., Schroder, I., Kaczor-Grzeskowiak, M . , Grzeskowiak, K . , Gunsalus, R.P. , and Dickerson, R . E . (1996) Structure of the Escherichia coli response regulator NarL. Biochemistry 35: 11053-11061. Baldus, J . M . , Green, B . D . , Youngman, P., and Moran, C.P. , Jr. (1994) Phosphorylation of Bacillus subtilis transcription factor SpoOA stimulates transcription from the spoIIG promoter by enhancing binding to weak OA boxes. J Bacteriol 176: 296-306. Baldus, J . M . , Buckner, C . M . , and Moran, C.P. , Jr. (1995) Evidence that the transcriptional activator SpoOA interacts with two sigma factors in Bacillus subtilis. Mol Microbiol 17: 281-290. Ben-Yehuda, S., and Losick, R. (2002) Asymmetric cell division in B . subtilis involves a spiral-like intermediate o f the cytokinetic protein FtsZ. Cell 109: 257-266. Ben-Yehuda, S., Rudner, D .Z . , and Losick, R. (2003) RacA, a bacterial protein that anchors chromosomes to the cell poles. Science 299: 532-536. Birck, C. , Mourey, L . , Gouet, P., Fabry, B . , Schumacher, J. , Rousseau, P., Kahn, D. , and Samama, J.P. (1999) Conformational changes induced by phosphorylation of the F ixJ receiver domain. Structure Fold Des 7: 1505-1515. Birck, C , Chen, Y . , Hulett, F . M . , and Samama, J.P. (2003) The crystal structure of the phosphorylation domain in PhoP reveals a functional tandem association mediated by an asymmetric interface. J Bacteriol 185: 254-261. Bird , T . H . , Grimsley, J .K. , Hoch, J .A. , and Spiegelman, G . B . (1993) Phosphorylation of SpoOA activates its stimulation of in vitro transcription from the Bacillus subtilis spoIIG operon. Mol Microbiol 9: 741-749. Bird , T . H . , Grimsley, J .K. , Hoch, J .A. , and Spiegelman, G . B . (1996) The Bacillus subtilis response regulator SpoOA stimulates transcription of the spoIIG operon through modification of R N A polymerase promoter complexes. J Mol Biol 256: 436-448. Blanco, A . G . , Sola, M . , Gomis-Ruth, F . X . , and C o l l , M . (2002) Tandem D N A recognition by PhoB, a two-component signal transduction transcriptional activator. Structure (Camb) 10: 701-713. Bongiorni, C , Ishikawa, S., Stephenson, S., Ogasawara, N . , and Perego, M . (2005) Synergistic regulation of competence development in Bacillus subtilis by two Rap-Phr systems. J Bacteriol 187: 4353-4361. 91 Borukhov, S., and Nudler, E . (2003) R N A polymerase holoenzyme: structure, function and biological implications. Curr Opin Microbiol 6: 93-100. Britton, R . A . , Eichenberger, P., Gonzalez-Pastor, J.E., Fawcett, P., Monson, R., Losick, R., and Grossman, A . D . (2002) Genome-wide analysis of the stationary-phase sigma factor (sigma-H) regulon of Bacillus subtilis. J Bacteriol 184: 4881-4890. Brown, D.P. , Ganova-Raeva, L . , Green, B . D . , Wilkinson, S.R., Young, M . , and Youngman, P. (1994) Characterization of spoOA homologues in diverse Bacillus and Clostridium species identifies a probable DNA-binding domain. Mol Microbiol 14: 411-426. Browning, D.F . , and Busby, S.J. (2004) The regulation of bacterial transcription initiation. Nat Rev Microbiol 2: 57-65. Buckner, C M . , Schyns, G . , and Moran, C P . , Jr. (1998) A region in the Bacillus subtilis transcription factor SpoOA that is important for spolIG promoter activation. J Bacteriol 180: 3578-3583. Burbulys, D . , Trach, K . A . , and Hoch, J .A. (1991) Initiation o f sporulation in B . subtilis is controlled by a multicomponent phosphorelay. Cell 64: 545-552. Burkholder, W.F . , and Grossman, A . D . (2000) Regulation of the initiation of endospore formation in Bacillus subtilis. In Prokaryotic development. Brun, Y . V . and Shimkets, L . J . (eds). Washington, D C : American Society for Microbiology, pp. 151-166. Burkholder, W.F. , Kurtser, I., and Grossman, A . D . (2001) Replication initiation proteins regulate a developmental checkpoint in Bacillus subtilis. Cell 104: 269-279. Busby, S., and Ebright, R . H . (1997) Transcription activation at class II CAP-dependent promoters. Mol Microbiol 23: 853-859. Busby, S., and Ebright, R . H . (1999) Transcription activation by catabolite activator protein (CAP) . J Mol Biol 293: 199-213. Bylund, J.E., Haines, M . A . , Piggot, P.J., and Higgins, M . L . (1993) A x i a l filament formation in Bacillus subtilis: induction of nucleoids of increasing length after addition of chloramphenicol to exponential-phase cultures approaching stationary phase. J Bacteriol 175: 1886-1890. Campbell, E . A . , Muzz in , O., Chlenov, M . , Sun, J.L., Olson, C . A . , Weinman, O., Trester-Zedlitz, M . L . , and Darst, S A . (2002) Structure of the bacterial R N A polymerase promoter specificity sigma subunit. Mol Cell 9: 527-539. Cervin, M . A . , Spiegelman, G .B . , Raether, B . , Ohlsen, K . , Perego, M . , and Hoch, J .A. (1998) A negative regulator linking chromosome segregation to developmental transcription in Bacillus subtilis. Mol Microbiol 29: 85-95. Cervin, M . A . , and Spiegelman, G .B . (1999) The SpoOA sof mutations reveal regions o f the regulatory domain that interact with a sensor kinase and R N A polymerase. Mol Microbiol 31: 597-607. Chen, Y . , Birck, C , Samama, J.P., and Hulett, F . M . (2003) Residue R l 13 is essential for PhoP dimerization and function: a residue buried in the asymmetric PhoP dimer interface determined in the PhoPN three-dimensional crystal structure. J Bacteriol 185: 262-273. Chibazakura, T., Kawamura, F., and Takahashi, H . (1991) Differential regulation of spoOA transcription in Bacillus subtilis: glucose represses promoter switching at the initiation of sporulation. J Bacteriol 173: 2625-2632. 92 Chibazakura, T., Kawamura, F., Asai , K . , and Takahashi, H . (1995) Effects of spoO mutations on spoOA promoter switching at the initiation of sporulation in Bacillus subtilis. J Bacteriol 111: 4520-4523. Choy, H .E . , Park, S.W., A k i , T., Parrack, P., Fujita, N . , Ishihama, A . , and Adhya, S. (1995) Repression and activation of transcription by Gal and Lac repressors: involvement o f alpha subunit of R N A polymerase. Embo J14: 4523-4529. Choy, H .E . , Hanger, R.R. , A k i , T., Mahoney, M . , Murakami, K . , Ishihama, A . , and Adhya, S. (1997) Repression and activation of promoter-bound R N A polymerase activity by Gal repressor. J Mol Biol 111: 293-300. Chung, J.D., Stephanopoulos, G . , Ireton, K . , and Grossman, A . D . (1994) Gene expression in single cells of Bacillus subtilis: evidence that a threshold mechanism controls the initiation of sporulation. J Bacteriol 176: 1977-1984. Da Re, S., Schumacher, J., Rousseau, P., Fourment, J., Ebel, C , and Kahn, D . (1999) Phosphorylation-induced dimerization of the FixJ receiver domain. Mol Microbiol 34: 504-511. Darst, S.A. (2001) Bacterial R N A polymerase. Curr Opin Struct Biol 11: 155-162. Dixon, L . G . , and Spiegelman, G . B . (2002) Glucose-resistant sporulation in Bacillus subtilis crsA47 mutants does not depend on promoter switching at the spoOA gene. J Bacteriol 184: 1458-1461. Dobinson, K . F . , and Spiegelman, G . B . (1987) Effect of the delta subunit of Bacillus subtilis R N A polymerase on initiation of R N A synthesis at two bacteriophage phi 29 promoters. Biochemistry 26: 8206-8213. Duncan, L . , Alper, S., Arigoni , F., Losick, R., and Stragier, P. (1995) Activation of cell-specific transcription by a serine phosphatase at the site of asymmetric division. Science 270: 641-644. Dworkin, J. (2003) Transient genetic asymmetry and cell fate in a bacterium. Trends Genet 19: 107-112. Errington, J. (1993) Bacillus subtilis sporulation: regulation of gene expression and control of morphogenesis. Microbiol Rev 57: 1 -33. Errington, J. (2003) Regulation of endospore formation in Bacillus subtilis. Nat Rev Microbiol 1: 117-126. Estrem, S.T., Gaal, T., Ross, W. , and Gourse, R . L . (1998) Identification of an U P element consensus sequence for bacterial promoters. Proc Natl Acad Sci USA 95: 9761-9766. Estrem, S.T., Ross, W. , Gaal, T., Chen, Z .W. , N i u , W. , Ebright, R . H . , and Gourse, R . L . (1999) Bacterial promoter architecture: subsite structure of U P elements and interactions with the carboxy-terminal domain of the R N A polymerase alpha subunit. Genes Dev 13: 2134-2147. Fabret, C , Feher, V . A . , and Hoch, J .A. (1999) Two-component signal transduction in Bacillus subtilis: how one organism sees its world. J Bacteriol 181: 1975-1983. Ferrari, F . A . , Trach, K . , LeCoq, D . , Spence, J., Ferrari, E . , and Hoch, J .A. (1985) Characterization of the spoOA locus and its deduced product. Proc Natl Acad Sci U S A 82: 2647-2651. Feucht, A . , Magnin, T., Yudkin , M . D . , and Errington, J. (1996) Bifunctional protein required for asymmetric cell division and cell-specific transcription in Bacillus subtilis. Genes Dev 10: 794-803. 93 Fiedler, U . , and Weiss, V . (1995) A common switch in activation of the response regulators NtrC and PhoB: phosphorylation induces dimerization of the receiver modules. Embo J14: 3696-3705. Freese, E . (1981) Initiation of bacterial sporulation. In Sporulation and germination. Levinson, H.S. , Sonenshein, A . L . and D.J. , T. (eds). Washington, D C : American Society for Microbiology, pp. 1-12. Fujita, M . , and Losick, R. (2003) The master regulator for entry into sporulation in Bacillus subtilis becomes a cell-specific transcription factor after asymmetric division. Genes Devil: 1166-1174. Fujita, M . , Gonzalez-Pastor, J.E., and Losick, R. (2005) High- and low-threshold genes in the SpoOA regulon of Bacillus subtilis. J Bacteriol 187: 1357-1368. Gaur, N . K . , Cabane, K . , and Smith, I. (1988) Structure and expression of the Bacillus subtilis sin operon. J Bacteriol 170: 1046-1053. Gerlach, P., Valentin-Hansen, P., and Bremer, E . (1990) Transcriptional regulation of the cytR repressor gene o f Escherichia coli : autoregulation and positive control by the c A M P / C A P complex. Mol Microbiol 4: 479-488. Gholamhoseinian, A . , and Piggot, P.J. (1989) Timing of spoil gene expression relative to septum formation during sporulation of Bacillus subtilis. J Bacteriol 171: 5747-5749. Greene, E . A . , and Spiegelman, G . B . (1996) The SpoOA protein of Bacillus subtilis inhibits transcription of the abrB gene without preventing binding of the polymerase to the promoter. J Biol Chem 271: 11455-11461. Grimshaw, C .E . , Huang, S., Hanstein, C . G . , Strauch, M . A . , Burbulys, D . , Wang, L . , Hoch, J .A. , and Whiteley, J . M . (1998) Synergistic kinetic interactions between components of the phosphorelay controlling sporulation in Bacillus subtilis. Biochemistry 37: 1365-1375. Gross, C . A . , Chan, C , Dombroski, A . , Gruber, T., Sharp, M . , Tupy, J., and Young, B . (1998) The functional and regulatory roles of sigma factors in transcription. Cold Spring Harb Symp Quant Biol 63: 141 -155. Grossman, A . D . , and Losick, R. (1988) Extracellular control of spore formation in Bacillus subtilis. Proc Natl Acad Sci USA 85: 4369-4373. Grossman, A . D . (1995) Genetic networks controlling the initiation of sporulation and the development of genetic competence in Bacillus subtilis. Annu Rev Genet 29: 477-508. Gruber, T . M . , and Gross, C A . (2003) Multiple sigma subunits and the partitioning of bacterial transcription space. Annu Rev Microbiol 57: 441-466. Hahn, J., Roggiani, M . , and Dubnau, D . (1995) The major role of SpoOA in genetic competence is to downregulate abrB, an essential competence gene. J Bacteriol 111: 3601-3605. Hahn, J., Luttinger, A . , and Dubnau, D . (1996) Regulatory inputs for the synthesis of C o m K , the competence transcription factor of Bacillus subtilis. Mol Microbiol 21: 763-775. Hatt, J .K. , and Youngman, P. (1998) SpoOA mutants of Bacillus subtilis with sigma factor-specific defects in transcription activation. J Bacteriol 180: 3584-3591. Hatt, J .K. , and Youngman, P. (2000) Mutational analysis of conserved residues in the putative DNA-bind ing domain of the response regulator SpoOA of Bacillus subtilis. J Bacteriol 182: 6975-6982. 94 Heldwein, E .E . , and Brennan, R . G . (2001) Crystal structure of the transcription activator BmrR bound to D N A and a drug. Nature 409: 378-382. Helmann, J.D., and Moran, C P . , Jr. (2002) R N A Polymerase and Sigma Factors. In Bacillus subtilis and Its Closest Relatives: from Genes to Cells. Sonenshein, A . L . , Hoch, J .A. and Losick, R. (eds). Washington, D . C . : A S M Press, pp. 289-312. Hilbert, D .W. , and Piggot, P.J. (2004) Compartmentalization of gene expression during Bacillus subtilis spore formation. Microbiol Mol Biol Rev 68: 234-262. Hoch, J .A. (1991) Genetic analysis in Bacillus subtilis. In Methods in Enzymology. V o l . 204. Mi l le r , H . (ed). San Diego, C A : Academic Press, Inc., pp. 305-320. Hoch, J .A. (1993) Regulation of the phosphorelay and the initiation of sporulation in Bacillus subtilis. Annu Rev Microbiol 47: 441-465. Hoch, J .A. , and Silhavy, T.J. (1995) Two-component signal transduction. Washington, D . C . : A S M Press. Hofer, T., Nathansen, H . , Lohning, M . , Radbruch, A . , and Heinrich, R. (2002) G A T A - 3 transcriptional imprinting in Th2 lymphocytes: a mathematical model. Proc Natl Acad Sci USA 99: 9364-9368. Hulett, F . M . (1996) The signal-transduction network for Pho regulation in Bacillus subtilis. Mol Microbiol 19: 933-939. Ireton, K . , and Grossman, A . D . (1992) Coupling between gene expression and D N A synthesis early during development in Bacillus subtilis. Proc Natl Acad Sci USA 89: 8808-8812. Ireton, K . , and Grossman, A . D . (1994) DNA-related conditions controlling the initiation of sporulation in Bacillus subtilis. Cell Mol Biol Res 40: 193-198. Jain, D. , Nickels, B . E . , Sun, L . , Hochschild, A . , and Darst, S.A. (2004) Structure of a ternary transcription activation complex. Mol Cell 13: 45-53. Jiang, M . , Tzeng, Y . L . , Feher, V . A . , Perego, M . , and Hoch, J .A. (1999) Alanine mutants of the SpoOF response regulator modifying specificity for sensor kinases in sporulation initiation. Mol Microbiol 33: 389-395. Jiang, M . , Grau, R., and Perego, M . (2000a) Differential processing of propeptide inhibitors of Rap phosphatases in Bacillus subtilis. J Bacteriol 182: 303-310. Jiang, M . , Shao, W. , Perego, M . , and Hoch, J .A. (2000b) Multiple histidine kinases regulate entry into stationary phase and sporulation in Bacillus subtilis. Mol Microbiol 38: 535-542. Kal l io , P.T., Fagelson, J.E., Hoch, J .A. , and Strauch, M . A . (1991) The transition state regulator Hpr of Bacillus subtilis is a DNA-binding protein. J Biol Chem 266: 13411-13417. Kern, D . , Volkman, B .F . , Luginbuhl, P., Nohaile, M . J . , Kustu, S., and Wemmer, D . E . (1999) Structure of a transiently phosphorylated switch in bacterial signal transduction. Nature 402: 894-898. Koide, A . , Perego, M . , and Hoch, J .A . (1999) ScoC regulates peptide transport and sporulation initiation in Bacillus subtilis. J Bacteriol 181: 4114-4117. Kroos, L . , Kunkel , B . , and Losick, R. (1989) Switch protein alters specificity of R N A polymerase containing a compartment-specific sigma factor. Science 243: 526-529. Kumar, A . , Buckner Starke, C , DeZalia, M . , and Moran, C P . , Jr. (2004) Surfaces of SpoOA and R N A polymerase sigma factor A that interact at the spoIIG promoter in Bacillus subtilis. J Bacteriol 186: 200-206. 95 Kunkel , B . , Sandman, K . , Panzer, S., Youngman, P., and Losick, R. (1988) The promoter for a sporulation gene in the spoIVC locus o f Bacillus subtilis and its use in studies of temporal and spatial control of gene expression. J Bacteriol 170: 3513-3522. Kunkel , B . , Losick, R., and Stragier, P. (1990) The Bacillus subtilis gene for the development transcription factor sigma K is generated by excision of a dispensable D N A element containing a sporulation recombinase gene. Genes Dev 4: 525-535. Kunst, F., Ogasawara, N . , Moszer, I., Albertini, A . M . , A l l o n i , G . , Azevedo, V . , Bertero, M . G . , Bessieres, P., Bolotin, A . , Borchert, S., Borriss, R., Boursier, L . , Brans, A . , Braun, M . , Brignell, S.C., Bron, S., Brouillet, S., Bruschi, C . V . , Caldwell, B . , Capuano, V . , Carter, N . M . , Choi , S.K., Codani, J.J., Connerton, I.F., Danchin, A . , and et al. (1997) The complete genome sequence of the gram-positive bacterium Bacillus subtilis. Nature 390: 249-256. Lawson, C . L . , Swigon, D . , Murakami, K . S . , Darst, S.A., Berman, H . M . , and Ebright, R . H . (2004) Catabolite activator protein: D N A binding and transcription activation. Curr Opin Struct Biol 14: 10-20. LeDeaux, J.R., and Grossman, A . D . (1995) Isolation and characterization of k inC, a gene that encodes a sensor kinase homologous to the sporulation sensor kinases K i n A and K i n B in Bacillus subtilis. J Bacteriol 111: 166-175. Lee, S.Y. , Cho, H.S. , Pelton, J .G., Yan , D . , Henderson, R . K . , K i n g , D.S. , Huang, L . , Kustu, S., Berry, E . A . , and Wemmer, D . E . (2001) Crystal structure of an activated response regulator bound to its target. Nat Struct Biol 8: 52-56. Lemon, K . P . , Kurtser, I., W u , J., and Grossman, A . D . (2000) Control of initiation of sporulation by replication initiation genes in Bacillus subtilis. J Bacteriol 182: 2989-2991. Lewis, R.J . , Brannigan, J .A. , Muchova, K . , Barak, I., and Wilkinson, A . J . (1999) Phosphorylated aspartate in the structure of a response regulator protein. J Mol Biol 294: 9-15. Lewis, R.J . , Krzywda, S., Brannigan, J .A. , Turkenburg, J.P., Muchova, K . , Dodson, E.J . , Barak, I., and Wilkinson, A . J . (2000a) The trans-activation domain of the sporulation response regulator SpoOA revealed by X-ray crystallography. Mol Microbiol 38: 198-212. Lewis, R.J . , Muchova, K . , Brannigan, J .A. , Barak, I., Leonard, G . , and Wilkinson, A . J . (2000b) Domain swapping in the sporulation response regulator SpoOA. J Mol Biol 291: 757-770. Lewis, R.J . , Scott, D.J . , Brannigan, J .A. , Ladds, J.C., Cervin, M . A . , Spiegelman, G .B . , Hoggett, J .G. , Barak, I., and Wilkinson, A . J . (2002) Dimer formation and transcription activation in the sporulation response regulator SpoOA. J Mol Biol 316: 235-245. L i n , D .C . , and Grossman, A . D . (1998) Identification and characterization of abacterial chromosome partitioning site. Cell 92: 675-685. L i u , J., Cosby, W . M . , and Zuber, P. (1999) Role of Ion and C l p X in the post-translational regulation of a sigma subunit of R N A polymerase required for cellular differentiation in Bacillus subtilis. Mol Microbiol 33: 415-428. L i u , J., and Zuber, P. (2000) The C l p X protein of Bacillus subtilis indirectly influences R N A polymerase holoenzyme composition and directly stimulates sigma-dependent transcription. Mol Microbiol 37: 885-897. 96 Lodish, H . B . , Arnold; Zipursky, S. Lawrence; Matsudaira, Paul; Baltimore, David; Darnell, James E . (1999) Molecular Cell Biology. New York : W H Freeman & Company. Lohrmann, J., and Harter, K . (2002) Plant two-component signaling systems and the role of response regulators. Plant Physiol 128: 363-369. Losick, R., Youngman, P., and Piggot, P.J. (1986) Genetics of endospore formation in Bacillus subtilis. Annu Rev Genet 20: 625-669. Louie, P., Lee, A . , Stansmore, K . , Grant, R., Ginther, C , and Leighton, T. (1992) Roles of rpoD, spoIIF, spoIIJ, spoIIN, and sin in regulation of Bacillus subtilis stage II sporulation-specific transcription. J Bacteriol 174: 3570-3576. Maamar, H . , and Dubnau, D . (2005) Bistability in the Bacillus subtilis K-state (competence) system requires a positive feedback loop. Mol Microbiol 56: 615-624. Makino, K . , Amemura, M . , Kawamoto, T., Kimura, S., Shinagawa, H . , Nakata, A . , and Suzuki, M . (1996) D N A binding of PhoB and its interaction with R N A polymerase. J Mol Biol 259: 15-26. Mandic-Mulec, I., Gaur, N . , Bai , U . , and Smith, I. (1992) Sin, a stage-specific repressor of cellular differentiation. J Bacteriol 174: 3561-3569. Mandic-Mulec, I., Doukhan, L . , and Smith, I. (1995) The Bacillus subtilis SinR protein is a repressor of the key sporulation gene spoOA. J Bacteriol 111: 4619-4627. Maris, A . E . , Sawaya, M . R . , Kaczor-Grzeskowiak, M . , Jarvis, M . R . , Bearson, S .M. , Kopka, M . L . , Schroder, I., Gunsalus, R.P. , and Dickerson, R . E . (2002) Dimerization allows D N A target site recognition by the NarL response regulator. Nat Struct Biol 9: 771-778. Marston, A . L . , and Errington, J. (1999) Dynamic movement of the ParA-like Soj protein of B . subtilis and its dual role in nucleoid organization and developmental regulation. Mol Cell 4: 673-682. McCleary, W . R . (1996) The activation of PhoB by acetylphosphate. Mol Microbiol 20: 1155-1163. McLeod , B . N . , and Spiegelman, G . B . (2005) Soj antagonizes SpoOA activation of transcription in Bacillus subtilis. J Bacteriol 187: 2532-2536. McQuade, R.S. , Cornelia, N . , and Grossman, A . D . (2001) Control of a family of phosphatase regulatory genes (phr) by the alternate sigma factor sigma-H of Bacillus subtilis. J Bacteriol 183: 4905-4909. Mol le , V . , Fujita, M . , Jensen, S.T., Eichenberger, P., Gonzalez-Pastor, J.E., L i u , J.S., and Losick, R. (2003a) The SpoOA regulon of Bacillus subtilis. Mol Microbiol 50: 1683-1701. Mol le , V . , Nakaura, Y . , Shivers, R.P. , Yamaguchi, H . , Losick, R., Fujita, Y . , and Sonenshein, A . L . (2003b) Additional targets of the Bacillus subtilis global regulator C o d Y identified by chromatin immunoprecipitation and genome-wide transcript analysis. J Bacteriol 185: 1911-1922. Msadek, T. (1999) When the going gets tough: survival strategies and environmental signaling networks in Bacillus subtilis. Trends Microbiol 7: 201-207. Muchova, K . , Lewis, R.J . , Brannigan, J .A. , Schmeisser, F., Wilkinson, A . J . , and Barak, I. (1998) Primary sporulation response regulator SpoOA. Gen Physiol Biophys 17 Suppl 1:34-36. Murakami, K . S . , and Darst, S .A. (2003) Bacterial R N A polymerases: the wholo story. Curr Opin Struct Biol 13: 31-39. 97 Nicholson, W . L . , Munakata, N . , Horneck, G . , Melosh, H.J . , and Setlow, P. (2000) Resistance of Bacillus endospores to extreme terrestrial and extraterrestrial environments. Microbiol Mol Biol Rev 64: 548-572. Nickels, B . E . , Dove, S.L., Murakami, K . S . , Darst, S.A., and Hochschild, A . (2002) Protein-protein and protein-DNA interactions of sigma70 region 4 involved in transcription activation by lambdacl. J Mol Biol 324: 17-34. Ohlsen, K . L . , Grimsley, J .K. , and Hoch, J .A. (1994) Deactivation of the sporulation transcription factor SpoOA by the SpoOE protein phosphatase. Proc Natl Acad Sci U S A91: 1756-1760. Paget, M . S . , and Helmann, J.D. (2003) The sigma70 family of sigma factors. Genome Biol 4: ' 203. Paidhungat, M . , and Setlow, P. (2002) Spore germination and outgrowth. In Bacillus subtilis and its closest relatives: from genes to cells. Sonenshein, A . L . , Hoch, J .A. and Losick, R. (eds). Washington, D C : American Society for Microbiology. Partridge, S.R., and Errington, J. (1993) The importance o f morphological events and intercellular interactions in the regulation o f prespore-specific gene expression during sporulation in Bacillus subtilis. Mol Microbiol 8: 945-955. Perego, M . , and Hoch, J .A. (1988) Sequence analysis and regulation of the hpr locus, a regulatory gene for protease production and sporulation in Bacillus subtilis. J Bacteriol 170: 2560-2567. Perego, M . , Spiegelman, G .B . , and Hoch, J .A. (1988) Structure of the gene for the transition state regulator, abrB: regulator synthesis is controlled by the spoOA sporulation gene in Bacillus subtilis. Mol Microbiol 2: 689-699. Perego, M . , Cole, S.P., Burbulys, D . , Trach, K . , and Hoch, J .A. (1989) Characterization of the gene for a protein kinase which phosphorylates the sporulation-regulatory proteins SpoOA and SpoOF of Bacillus subtilis. J Bacteriol 171: 6187-6196. Perego, M . , Higgins, C.F. , Pearce, S.R., Gallagher, M . P . , and Hoch, J .A. (1991a) The oligopeptide transport system of Bacillus subtilis plays a role in the initiation of sporulation. Mol Microbiol 5: 173-185. Perego, M . , and Hoch, J .A. (1991) Negative regulation of Bacillus subtilis sporulation by the spoOE gene product. J Bacteriol 173: 2514-2520. Perego, M . , W u , J.J., Spiegelman, G .B . , and Hoch, J .A. (1991b) Mutational dissociation of the positive and negative regulatory properties of the SpoOA sporulation transcription factor of Bacillus subtilis. Gene 100: 207-212. Perego, M . , Hanstein, C , Welsh, K . M . , Djavakhishvili, T., Glaser, P., and Hoch, J .A. (1994) Multiple protein-aspartate phosphatases provide a mechanism for the integration of diverse signals in the control of development in B . subtilis. Cell 79: 1047-1055. Perego, M . , and Hoch, J .A. (1996) Cell-cell communication regulates the effects of protein aspartate phosphatases on the phosphorelay controlling development in Bacillus subtilis. Proc Natl Acad Sci U SA 93: 1549-1553. Perego, M . (1999) Self-signaling by Phr peptides modulates Bacillus subtilis development. In Cell-cell signaling in bacteria. Dunny, G . M . and Winans, S . C (eds). Washington, D C : American Society for Microbiology, pp. 243-258. Perego, M . (2001) A new family of aspartyl phosphate phosphatases targeting the sporulation transcription factor SpoOA of Bacillus subtilis. Mol Microbiol 42: 133-143. 98 Perego, M . , and Hoch, J .A. (2002) Two-Component systems, phosphorelays, and regulation of thir activites by phosphatases. In Bacillus subtilis and its closest relatives: from genes to cells. Sonenshein, A . L . , Hoch, J .A. and Losick, R. (eds). Washington, D C : American Society for Microbiology, pp. 473-782. Phillips, Z .E . , and Strauch, M . A . (2002) Bacillus subtilis sporulation and stationary phase gene expression. Cell Mol Life Sci 59: 392-402. Piggot, P.J., and Losick, R. (2002) Sporulation Genes and Intercompartmental Regulation. In Bacillus subtilis and Its Closest Relatives: from Genes to Cells. Sonenshein, A . L . , Hoch, J .A. and Losick, R . M . (eds). Washington, D . C . : A S M Press. Predich, M . , Nair, G. , and Smith, I. (1992) Bacillus subtilis early sporulation genes k inA, spoOF, and spoOA are transcribed by the R N A polymerase containing sigma H . J Bacteriol \14: 2771-2778. Ptashne, M . , and Gann, A . (2002) Genes & signals. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press. Quisel, J.D., L i n , D .C . , and Grossman, A . D . (1999) Control o f development by altered localization o f a transcription factor in B . subtilis. Mol Cell 4: 665-672. Quisel, J.D., and Grossman, A . D . (2000) Control of sporulation gene expression in Bacillus subtilis by the chromosome partitioning proteins Soj (ParA) and SpoOJ (ParB). J Bacteriol 182: 3446-3451. Ratnayake-Lecamwasam, M . , Serror, P., Wong, K . W . , and Sonenshein, A . L . (2001) Bacillus subtilis C o d Y represses early-stationary-phase genes by sensing G T P levels. Genes Dev 15: 1093-1103. Record, M . T . , Jr., Reznikoff, W.S. , Craig, M . L . , McQuade, K . L . , and Schlax, P.J. (1996) Escherichia coli and R N A Polymerase, Promoters, and the Kinetics of the Steps of Transcription Initiation. In Escherichia coli and Salmonella: cellular and molecular biology. V o l . 2. Neidhardt, F .C . and Curtiss, R. (eds). Washington, D . C . : A S M Press, pp. 792-821. Rosenbluh, A . , Banner, C D . , Losick, R., and Fitz-James, P .C. (1981) Identification of a new developmental locus in Bacillus subtilis by construction of a deletion mutation in a cloned gene under sporulation control. J Bacteriol 148: 341-351. Rowe-Magnus, D . A . , and Spiegelman, G . B . (1998) Contributions of the domains of the Bacillus subtilis response regulator SpoOA to transcription stimulation of the spoIIG operon. J Biol Chem 273: 25818-25824. Rowe-Magnus, D . A . , Richer, M . J . , and Spiegelman, G . B . (2000) Identification of a second region of the SpoOA response regulator o f Bacillus subtilis required for transcription activation. J Bacteriol 182: 4352-4355. Rowland, S.L., Burkholder, W.F . , Cunningham, K . A . , Maciejewski, M . W . , Grossman, A . D . , and King , G.F. (2004) Structure and mechanism of action o f Sda, an inhibitor o f the histidine kinases that regulate initiation of sporulation in Bacillus subtilis. Mol Cell 13:689-701. Rudner, D .Z . , LeDeaux, J.R., Ireton, K . , and Grossman, A . D . (1991) The spoOK locus of Bacillus subtilis is homologous to the oligopeptide permease locus and is required for sporulation and competence. J Bacteriol 173: 1388-1398. Ryter, A . (1965) [Morphologic Study of the Sporulation of Bacillus Subtilis.]. Ann Inst Pasteur (Paris) 108: 40-60. 99 Sambrook, J., Maniatis, T., and Fritsch, E .F . (1989) Molecular cloning : a laboratory manual. Cold Spring Harbor, N . Y . : Cold Spring Harbor Laboratory. Satola, S., Kirchman, P .A . , and Moran, C P . , Jr. (1991) SpoOA binds to a promoter used by sigma A R N A polymerase during sporulation in Bacillus subtilis. Proc Natl Acad Sci ( 7 5 ^ 88:4533-4537. Satola, S.W., Baldus, J . M . , and Moran, C P . , Jr. (1992) Binding of SpoOA stimulates spolIG promoter activity in Bacillus subtilis. J Bacteriol 174: 1448-1453. Schaeffer, P., Mil le t , J., and Aubert, J.P. (1965) Catabolic repression of bacterial sporulation. Proc Natl Acad Sci U S A 54: 704-711. Schyns, G . , Buckner, C M . , and Moran, C P . , Jr. (1997) Activation of the Bacillus subtilis spolIG promoter requires interaction of SpoOA and the sigma subunit of R N A polymerase. J Bacteriol 179: 5605-5608. Seredick, S., and Spiegelman, G . B . (2001) Lessons and questions from the structure of the SpoOA activation domain. Trends Microbiol 9: 148-151. Seredick, S.D., Turner, B . M . , and Spiegelman, G . B . (2003) Assay of transcription modulation by SpoOA of Bacillus subtilis. Methods Enzymol 370: 312-323. Seredick, S.D., and Spiegelman, G . B . (2004) The Bacillus subtilis response regulator SpoOA stimulates sigmaA-dependent transcription prior to the major energetic barrier. J Biol Chem 279: 17397-17403. Seredick, S.D. (2005) SpoOA-Stimulated Transcription Initiation at the Bacillus subtilis spolIG promoter. In Department of Microbiology and Immunology, UBC Vancouver, B C : U B C . Serror, P., and Sonenshein, A . L . (1996) C o d Y is required for nutritional repression of Bacillus subtilis genetic competence. J Bacteriol 178: 5910-5915. Shafikhani, S.H., Mandic-Mulec, I., Strauch, M . A . , Smith, I., and Leighton, T. (2002) Postexponential regulation of sin operon expression in Bacillus subtilis. J Bacteriol 184: 564-571. Smits, W . K . , Eschevins, C .C . , Susanna, K . A . , Bron, S., Kuipers, O.P., and Hamoen, L . W . (2005) Stripping Bacillus: C o m K auto-stimulation is responsible for the bistable response in competence development. Mol Microbiol 56: 604-614. Sonenshein, A . L . (1989) Metabolic regulation of sporulation and other stationary-phase phenomena. In Regulation ofprokaryotic development: a structural and functional analysisof sporulation and germination. Smith, I., Slepecky, R A . and Setlow, P. (eds). Washington, D C : American Society for Microbiology, pp. 109-130. Spiegelman, G . , Van Hoy, B . , Perego, M . , Day, J., Trach, K . , and Hoch, J .A. (1990) Structural alterations in the Bacillus subtilis SpoOA regulatory protein which suppress mutations at several spoO loci. J Bacteriol 172: 5011-5019. Spiegelman, G .B . , Bi rd , T . H . , and Voon, V . (1995) Transcription Regulation by the Bacillus subtilis Response Regulator SpoOA. In Two-Component Signal Transduction. Hoch, J .A. and Silhavy, T.J. (eds). Washington, D . C . : American Society for Microbiology, pp. 159-179. Stock, A . M . , Mottonen, J . M . , Stock, J.B., and Schutt, C . E . (1989a) Three-dimensional structure of CheY, the response regulator of bacterial chemotaxis. Nature 337: 745-749. Stock, A . M . , Robinson, V . L . , and Goudreau, P . N . (2000) Two-component signal transduction. Annu Rev Biochem 69: 183-215. 100 Stock, J .B. , Ninfa, A . J . , and Stock, A . M . (1989b) Protein phosphorylation and regulation o f adaptive responses in bacteria. Microbiol Rev 53: 450-490. Stragier, P., and Losick, R. (1996) Molecular genetics of sporulation in Bacillus subtilis. Annu Rev Genet 30: 297-241. Strauch, M . , Webb, V . , Spiegelman, G . , and Hoch, J .A. (1990) The SpoOA protein o f Bacillus subtilis is a repressor of the abrB gene. Proc Natl Acad Sci USA 87: 1801-1805. Strauch, M . A . , Spiegelman, G .B . , Perego, M . , Johnson, W . C . , Burbulys, D . , and Hoch, J .A. (1989) The transition state transcription regulator abrB of Bacillus subtilis is a D N A binding protein. Embo J8: 1615-1621. Strauch, M . A . , Trach, K . A . , Day, J., and Hoch, J .A. (1992) SpoOA activates and represses its own synthesis by binding at its dual promoters. Biochimie 74: 619-626. Strauch, M . A . , and Hoch, J .A . (1993) Transition-state regulators: sentinels o f Bacillus subtilis post-exponential gene expression. Mol Microbiol 7: 337-342. Sun, D . X . , Stragier, P., and Setlow, P. (1989) Identification of a new sigma-factor involved in compartmentalized gene expression during sporulation of Bacillus subtilis. Genes Dev 3: 141-149. Trach, K . , Burbulys, D. , Strauch, M . , W u , J.J., Dhillon, N . , Jonas, R., Hanstein, C. , Ka l l io , P., Perego, M . , Bird , T., and et al. (1991) Control of the initiation of sporulation in Bacillus subtilis by a phosphorelay. Res Microbiol 142: 815-823. Trach, K . A . , and Hoch, J .A. (1993) Multisensory activation of the phosphorelay initiating sporulation in Bacillus subtilis: identification and sequence of the protein kinase of the alternate pathway. Mol Microbiol 8: 69-79. Vagner, V . , Dervyn, E . , and Ehrlich, S.D. (1998) A vector for systematic gene inactivation in Bacillus subtilis. Microbiology 144 ( Pt 11): 3097-3104. van Sinderen, D . , and Venema, G . (1994) comK acts as an autoregulatory control switch in the signal transduction route to competence in Bacillus subtilis. J Bacteriol 176: 5762-5770. Wang, L . , Grau, R., Perego, M . , and Hoch, J .A. (1997) A novel histidine kinase inhibitor regulating development in Bacillus subtilis. Genes Dev 11: 2569-2579. Webber, C . A . , and Kadner, R .J . (1995) Action of receiver and activator modules of U h p A in transcriptional control of the Escherichia coli sugar phosphate transport system. Mol Microbiol 15: 883-893. Weir, J., Predich, M . , Dubnau, E . , Nair, G . , and Smith, I. (1991) Regulation of spoOH, a gene coding for the Bacillus subtilis sigma H factor. J Bacteriol 173: 521-529. Welply, J .K. , Fowler, A . V . , Beckwith, J.R., and Zabin, I. (1980) Positions of early nonsense and deletion mutations in lacZ. J Bacteriol 142: 732-734. W u , J.J., Piggot, P.J., Tatti, K . M . , and Moran, C P . , Jr. (1991) Transcription of the Bacillus subtilis spoIIA locus. Gene 101: 113-116. W u , L . J . , and Errington, J. (2003) RacA and the Soj-SpoOJ system combine to effect polar chromosome segregation in sporulating Bacillus subtilis. Mol Microbiol 49: 1463-1475. Yamashita, S., Kawamura, F. , Yoshikawa, H . , Takahashi, H . , Kobayashi, Y . , and Saito, H . (1989) Dissection of the expression signals of the spoA gene of Bacillus subtilis: glucose represses sporulation-specific expression. J Gen Microbiol 135: 1335-1345. 101 York , K . , Kenney, T.J. , Satola, S., Moran, C P . , Jr., Poth, H . , and Youngman, P. (1992) SpoOA controls the sigma A-dependent activation of Bacillus subtilis sporulation-specific transcription unit spoIIE. J Bacteriol 174: 2648-2658. Zapf, J.W., Hoch, J .A. , and Whiteley, J . M . (1996) A phosphotransferase activity of the Bacillus subtilis sporulation protein SpoOF that employs phosphoramidate substrates. Biochemistry 35: 2926-2933. Zhang, G . , Campbell, E . A . , Minakhin, L . , Richter, C , Severinov, K . , and Darst, S.A. (1999) Crystal structure of Thermus aquaticus core R N A polymerase at 3.3 A resolution. CW/98: 811-824. Zhang, X . , Chaney, M . , Wigneshweraraj, S.R., Schumacher, J., Bordes, P., Cannon, W. , and Buck, M . (2002) Mechanochemical ATPases and transcriptional activation. Mol Microbiol 45: 895-903. Zhao, H . , Msadek, T., Zapf, J., Madhusudan, Hoch, J .A. , and Varughese, K . I . (2002) D N A complexed structure of the key transcription factor initiating development in sporulating bacteria. Structure (Camb) 10: 1041-1050. Zhou, X . Z . , Madhusudan, Whiteley, J . M . , Hoch, J .A. , and Varughese, K . I . (1997) Purification and preliminary crystallographic studies on the sporulation response regulatory phosphotransferase protein, SpoOB, from Bacillus subtilis. Proteins 27: 597-600. 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            data-media="{[{embed.selectedMedia}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0092656/manifest

Comment

Related Items