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

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ANALYSIS OF THE SPO0A(A257V) MUTANT OF BACILLUS SUBTILIS by BARBARA MARION TURNER B . S c , University o f 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 THE REQUIREMENTS FOR T H E D E G R E E OF  M A S T E R OF SCIENCE in  THE F A C U L T Y OF G R A D U A T E STUDIES  (Microbiology and Immunology)  THE UNIVERSITY OF BRITISH C O L U M B I A A p r i l , 2006 © Barbara Marion Turner, 2006  ABSTRACT In response to a deteriorating environment, Bacillus subtilis cells are capable o f several alternate survival strategies including motility, competence development, secretion o f proteases and surfactants, and sporulation. Members o f the response regulator family o f proteins play key roles regulating entry into these alternate states. In the case o f sporulation, the master regulator is SpoOA. SpoOA initiates the onset o f sporulation by direct or indirect activation or repression o f transcription o f 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 o f B. subtilis cells to sporulate. In vivo, the mutation prevents transcription activation at both the a -dependent spoIIG operon promoter and the c -dependent spolIA A  H  operon promoter, yet does not affect the ability o f SpoOA to repress transcription at the abrB promoter. In this thesis I investigated the biochemical properties o f 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 S p o 0 A ( A 2 5 7 V ) can both repress transcription and activate o -dependent transcription in vitro, although at a reduced A  level compared to w i l d type SpoOA. I showed that the A 2 5 7 V mutation did not affect the ability o f 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 o f the mutant protein. W h i l e the reduction in apparent binding affinity could explain the in vitro results, it did not account for the complete lack o f sporulation in spoOA(A257V) B. subtilis cells. Analysis o f 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 o f SpoOA in vivo, strains were constructed which overexpressed w i l d type or mutant SpoOA proteins and used to test activation o f a  A  and o -dependent promoters in vivo. H  Results from these experiments were inconclusive; the levels o f induced protein may have been insufficient to activate stage II sporulation genes.  Ill  TABLE OF CONTENTS Abstract  ii  Table of Contents  iii  List of Tables  vi  List of Figures  vii  List of Abbreviations and Symbols  ix  Acknowledgements  x  1. INTRODUCTION  1  1.1.  Sporulation in Bacillus subtilis  1.1.1. Endospore development 1.1.2. Sporulation morphology 1.1.3. The sigma factor cascade 1.2. Regulation o f sporulation 1.2.1. SpoOA structure 1.2.1.1. The receiver domain o f SpoOA 1.2.1.2. The D N A - b i n d i n g domain o f SpoOA 1.2.2. Sporulation initiation signals 1.2.2.1. Nutrient deprivation 1.2.2.2. High cell density 1.2.2.3. Cell-cycle progression 1.2.3. Regulation o f SpoOA activation 1.2.4. The sporulation regulatory network 1.2.4.1. Repression o f abrB 1.2.4.2. Alleviation o f SinR repression 1.2.4.3. Antagonism by a negative regulator o f sporulation, Soj 1.2.4.4. Induction o f a expression 1.3. Transcription 1.3.1. R N A polymerase 1.3.2. Promoter elements 1.3.3. The transcription initiation cycle 1.4. Regulation o f transcription initiation 1.4.1. Transcriptional activators 1.4.2. Transcriptional repressors 1.5. Mechanism o f activation by positive regulators 1.5.1. Catabolite activator protein ( C A P ) 1.5.2. Regulation o f transcription by SpoOA 1.6. Experimental rationale H  1 1 1 4 5 8 8 10 13 13 13 14 14 15 15 15 18 18 19 20 20 21 22 23 23 24 24 25 26  iv  2. EXPERIMENTAL PROCEDURES Bacterial strains and media Synthesis and cloning o f spoOA(A257V) Expression and purification o f SpoOA(A257V) In vitro phosphorylation reactions 2.4.1. In vitro phosphorylation at equilibrium 2.4.2. Rate o f in vitro phosphorylation 2.5. In vitro transcription reactions 2.5.1. Preparation o f template D N A 2.5.2. In vitro transcription reactions 2.6. In vitro DNase I footprinting assay 2.6.1. Preparation o f template D N A 2.6.2. In vitro DNasel footprinting reactions 2.7. Electrophoretic mobility shift assay ( E M S A ) 2.7.1. Preparation o f template D N A 2.7.2. Electrophoretic mobility shift assay 2.8. Cloning o f Pspac-5po04f'7-7^6'; 2.9. Construction o f Vspac-spoOA and Fspac-spoOA(A257V) strains 2.10. Determination o f sporulation frequency 2.11. Immunoblot analysis 2.12. P-Galactosidase assay 2.12.1 Construction o f L a c Z reporter strains 2.12.2. P-Galactosidase activity  28  2.1. 2.2. 2.3. 2.4.  28 28 31 31 31 32 32 32 33 33 33 34 35 35 35 36 B. subtilis  3. RESULTS 3.1. 3.2. 3.3. 3.4. 3.5. 3.6.  3.7. 3.8.  Examination o f the effect o f the A 2 5 7 V mutation on SpoOA phosphorylation in vitro Investigation o f the effect o f the A 2 5 7 V substitution on in vitro repression o f the abrB promoter Investigation o f the effect o f the A 2 5 7 V substitution on in vitro stimulation o f the spoIIG promoter Examination o f the effect o f the A 2 5 7 V mutation o f SpoOA on binding to OA boxes encoded within the spoIIG promoter In vitro examination o f the binding affinity o f S p o 0 A ( A 2 5 7 V ) for consensus OA boxes Effect o f mutations in the OA boxes encoded within the spoIIG promoter on stimulation o f transcription by SpoOA~P and SpoOA(A257V)~P SpoOA(A257V) protein expression in the sporulation negative B. subtilis strain JH695 Construction o f B. subtilis strains which over-express wild type and mutant SpoOA proteins  39 39 40 41 41 41  42 42 43 47 51 52  55 58 61  V  3.9. 3.10. 3.11. 3.12.  Sporulation frequencies o f the Vspac-spoOA B. subtilis strains Investigation o f the effect o f varying I P T G concentration on expression from Pspac-spoOA(A257V) Construction o f B. subtilis spoilA-lacZ and spoIIG-lacZ reporter strains which overexpress w i l d type and mutant SpoOA Measurement o f spoIIA-lacZ induction i n strains encoding inducible w i l d type and mutant SpoOA  4. DISCUSSION  66 66 68  74  4.1. 4.2.  SpoOA is the master regulator o f the onset o f sporulation The A 2 5 7 V mutation o f SpoOA uncouples transcription activation and repression 4.3. SpoOA can activate and repress transcription initiation 4.3.1 Repression o f transcription by SpoOA 4.3.2 Activation o f o -dependent transcription by SpoOA 4.3.3. Activation o f a -dependent transcription by SpoOA 4.4. Objective o f thesis 4.5. In vitro characterization o f S p o 0 A ( A 2 5 7 V ) 4.6. In vivo investigation o f the effect o f the A 2 5 7 V mutation o f SpoOA 4.7. Effects o f residue A 2 5 7 on SpoOA dimer function 4.8. Residue A257 as a part o f a o -SpoOA interaction surface A  H  References  64  74 74 74 74 75 76 76 78 80 83 88  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 o f spoIIA-lacZ and spoIIG-lacZ transcription in B. subtilis strains which overexpress w i l d type or mutant SpoOA  72  vii  LIST OF FIGURES F i g u r e 1. Sporulation morphology  2  F i g u r e 2. A typical two-component signal transduction system  6  F i g u r e 3. Activation and regulation o f SpoOA  7  F i g u r e 4. Structure o f the receiver domain o f SpoOA  9  F i g u r e 5. Structure o f the D N A binding domain o f SpoOA  11  F i g u r e 6. SpoOA D N A - b i n d i n g domains bind D N A as a head-to-tail dimer  12  Figure 7. Sporulation regulatory network  17  Figure 8. Cloning o f Pspac-spoOA(]-J46)  38  F i g u r e 9. Time course o f in vitro phosphorylation o f SpoOA and SpoOA(A257V)  44  F i g u r e 10. Phosphorylation of varying amounts o f SpoOA and SpoOA(A257V) in vitro  45  F i g u r e 11. Repression o f the abrB promoter by SpoOA and S p o 0 A ( A 2 5 7 V ) in vitro  46  F i g u r e 12. Time course o f in vitro transcription initiation stimulated by SpoOA and SpoOA(A257V)  49  F i g u r e 13. Effect o f SpoOA or S p o 0 A ( A 2 5 7 V ) protein concentration on stimulation o f spolIG promoter activity in vitro  50  F i g u r e 14. DNase footprint o f SpoOA~P and SpoOA(A257V)~P at the spolIG Promoter  53  F i g u r e 15. E M S A of binding o f SpoOA and SpoOA(A257V)  54  F i g u r e 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 o f B. subtilis strains which overexpress w i l d 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 I P T G  65  F i g u r e 20. SpoOA(A257V) expression i n BT2002 as a function o f IPTG concentration  67  Figure 21. Protein expression and sporulation in B. subtilis strains which overexpress w i l d type or mutant SpoOA and encode lacZ translational fusions Figure 22. PspoIlAilacZ  activity in B. subtilis strains BT2003 and BT2005  Figure 23. Location o f OA boxes within promoters activated and repressed b y SpoOA Figure 24. Suppressor mutations o f spoOA(A257V) strengthen intermolecular contacts within the SpoOA dimer  ix  LIST OF ABBREVIATIONS AND SYMBOLS CAP  Catabolite activator protein  Cm  Chloramphenicol resistance  R  CTD  C-terminal domain  EMSA  electrophoretic mobility shift assay  Erm  Erythromycin resistance  R  Ec  R N A polymerase with associated sigma factor ("holoenzyme")  HTH  helix-turn-helix motif  IPTG  isopropyl-P-D-thiogalactopyranoside  Kan  Kanamycin resistance  R  NTD  N-terminal domain  OA box  specific SpoOA binding site encoded within D N A ( 5 ' T G N C G A A 3 ' )  RNAP  R N A polymerase  RPci  initial closed R N A polymerase-promoter complex  RP 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  SAAR  sigma-A-activating region within SpoOA  SpoOA~P  activated, phosphorylated form o f SpoOA  SpoOA  N-terminal domain o f SpoOA  C  o  N  sigma factor  X  ACKNOWLEDGEMENTS When I began writing this thesis I expected that I would complete it before one o f the greatest adventures o f m y life began. It didn't happen. Instead, I've been busy working on a different kind o f experiment, which ( i f 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 o f my committee, Rachel Fernandez and Lindsay Eltis, for direction and help throughout the duration o f m y work and for patiently waiting whilst I finish the never-ending-thesis. I'd like to thank members o f the lab (past and present): Martin Richer for teaching me the basics, Brett M c L e o d for many discussions over coffee, and lastly m y SpoOA-co-conspirator, Steve Seredick, for helping me in countless ways and indulging m y chocolate, ice cream, and coffee addictions. I'd like to thank members o f the Fernandez lab (past and present), especially Dave Oliver, who was always w i l l i n g to answer questions from across the hall.  Lastly, I'd like to thank m y family, especially a little monkey named Markus.  1  1. INTRODUCTION 1.1. Sporulation in Bacillus subtilis In bacteria, regulation o f 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 o f antibiotics, motility and chemotaxis, the development o f competence, and endospore formation.  The process o f endospore formation is possibly the best understood example o f cellular development and differentiation today. Endospores are specialized cell types formed by members o f 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 o f alternate survival strategies and in response to high cell density and nutrient deprivation, B. subtilis cells w i l l enter the sporulation pathway (Freese, 1981; Grossman and Losick, 1988; Hilbert and Piggot, 2004; Sonenshein, 1989). The master regulator o f the onset o f this process is the response regulator SpoOA. SpoOA integrates information from a complex network o f 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 o f 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 o f duplicate copies o f the chromosome and elongation to form an 'axial filament' o f nucleoprotein that stretches longitudinally across the cell (Ben-Yehuda et al.,  2  Vegetative ° Growth  _ .„ Engulfment  + u  Asymmetric Septation  Cortex Formation Forespore Protoplast  o  II, spoO  II spoil  III  2  spoil  Coat Formation  o  o  O  K A  Maturation  iv spoIII  o  o  v spoIV  Release  VI spoV  VII spoVI  Figure 1. Sporulation morphology. The development o f 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 I V 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 twoequally sized daughter cells by forming a structure called a ' Z - r i n g ' 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 o f new cell wall material in the space between the membranes (Errington, 2003). During sporulation, the formation o f a single medial Z-ring is inhibited and two Z-rings form, one at each pole (Ben-Yehuda and Losick, 2002). Constriction o f one o f the two Z-rings and disassembly o f the other leads to formation o f a polar septum at one end o f the cell, creating the mother cell and forespore (Errington, 2003).  Formation o f the polar septum captures approximately one-third o f the chromosome in the forespore; the remainder o f 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 o f 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 o f 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 o f endospore development.  The spore cortex and "primordial cell wall", composed o f peptidoglycan, are produced and deposited between the two membranes in stage I V (Losick et al, 1986). Endospore development continues with production o f the proteinaceous spore coat from deposition o f 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 o f large amounts o f dipicolinic acid and calcium ions from the mother cell (Errington, 2003). In the last stage o f development the  4  fully developed spore is released into the environment by lysis o f the mother cell (Losick et al, 1986). When the spore is again located i n a nutrient-rich environment it w i l l germinate and outgrow into a vegetative cell (Paidhungat and Setlow, 2002).  1.1.3. The sigma factor cascade Expression o f genes required for sporulation is regulated by the sequential production o f five sporulation-specific sigma factors (Errington, 1993; Piggot and Losick, 2002). The activated, phosphorylated form o f SpoOA, SpoOA~P, initiates this "sigma factor cascade" through indirect regulation o f transcription o f the first "alternate" sigma factor, a . During H  vegetative growth, transcription o f spoOH, the gene encoding G , is inhibited by the h  transition state regulator, A b r B (Weir et al, 1991). A t the onset o f sporulation, transcription o f abrB is repressed by SpoOA, facilitating expression o f o and expression o f over 87 genes H  within the o regulon (Britton et al, 2002; Hahn et al., 1995). The next sigma factors produced, a and o , are responsible for initiating a specific program o f gene expression in E  F  the mother cell and forespore, respectively (Dworkin, 2003).  Transcription o f the spolIG and spoIIA operons, encoding a and a , is activated b y SpoOA E  F  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 o f a and c activation are regulated. Activation o f rj is E  F  F  regulated through the activity o f an inhibitory protein that binds to a whereas activation o f F  c  E  is regulated by post-translation processing o f a p r o - a form (Errington, 2003). Activation E  o f each sigma factor takes place only in the appropriate compartment.  Engulfment o f the forespore b y the mother cell at the end o f stage II leads to activation o f a third sporulation-specific sigma factor, a (Sun et al, 1989). Expression o f a is limited to the forespore as transcription o f the gene encoding o , spoIIIG, is dependent upon R N A G  polymerase containing r j ( R N A P - o ) (Partridge and Errington, 1993). Activity o f a is F  F  G  regulated post-translationally, possibly by an anti-sigma factor, and inhibition is not relieved until engulfment is completed (Partridge and Errington, 1993). Engulfment o f the forespore also triggers activation o f the last sporulation-specific sigma factor in the cascade, o . This K  5  sigma factor is produced i n the mother cell and its activity is regulated at multiple levels. Transcription o f sigK occurs specifically in the mother cell under direction o f R N A P - o (Kunkel et al, 1988) after excision o f a prophage that is integrated at the sigK locus and interrupts the coding region o f the sigK gene (Kunkel et al, 1990). a activity is also K  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 i n the surrounding environment is often controlled by the activity o f "two-component signal transduction systems". Hundreds o f two-component systems have been identified i n 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 o f degradative enzymes, the development o f competence and endospore formation (Msadek, 1999).  The simplest two-component system is composed o f a sensor kinase, responsible for detection o f 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 o f a conserved histidine residue within the sensor kinase core. The response regulator catalyzes transfer o f 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 o f the phosphorelay signal transduction system that governs the commitment o f 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 o f the diversity o f 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 Sensing domain  N  -(  Kinase core  M ^ 1  )-'  PO3 ADP ATP aut.ophosphoryla.tion  phosphorylation  Response Regulator Receiver domain  N-  Output domain  Asp  PO  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 H i g h cell density, nutrient deprivation, cell cycle progression  ATP  ADP  KinA KinB, KinC, KinD. KinE  KinA-P  SpoOF  SpoOF~P |-  SpoOB  SpoOB~P  V  Kipl V  KipA  RapA  SpoOA  RapB RapE  phr pentapeptides  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). U p o n 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 activation/transcription stimulation are indicated by > .  1 and  8 and E) capable o f 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 o f 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 o f the expression o f 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 o f the response regulator family o f proteins. Response regulators generally contain two domains joined by a linker o f 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. W h i l e the receiver domains o f response regulators share a common fold (Stock et al, 1989a), the structures o f the output domains vary. W i t h the exception o f several members involved in chemotaxis, most response regulators have D N A - b i n d i n g 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 o f the receiver domain o f 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). L i k e 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 o f phosphorylation is located within a pocket located at the C terminal end o f strand p3 (Lewis et al, 1999; Muchova et al, 1998). Phosphorylation o f the receiver domain o f SpoOA results in a concerted and conserved rearrangement o f 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 d o m a i n 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 o f phosphorylation (D56) is found at the C-terminal end o f (33 (shown with arrow). The structure shown is from Bacillus stearothermophilus, differing from the receiver domain o f 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; D a Re et al, 1999; Fiedler and Weiss, 1995; McCleary, 1996). Unlike the response regulators PhoB, N t r C , and FixJ (Birck et al, 2003; D a Re et al, 1999; Fiedler and Weiss, 1995; McCleary, 1996), the structure o f the dimer form o f SpoOA indicates that a dimer can be formed by unphosphorylated receiver domains through an exchange o f helix oc5 (Lewis et al, 2000b). However, it is suspected that this unusual dimer form may be a consequence o f 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 o f 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 D N A - b i n d i n g domain is conserved only among SpoOA homologues from endospore-forming bacteria (Brown et al, 1994). The crystal structure indicates the D N A - b i n d i n g domain is composed o f six a-helices joined by short segments o f polypeptide and contains a helix-turn-helix ( H T H ) 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 o f 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 o f a -dependent promoters (Buckner et al, 1998; Hatt and A  Youngman, 1998). The crystal structure o f the D N A - b i n d i n g domain in complex with a consensus SpoOA-binding site (from the site o f repression o f the abrB gene) indicates that SpoOA D N A - b i n d i n g 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% o f the surface area o f each monomer in the dimer interface (Zhao et al, 2002). The dimer interface is formed by helix aF o f the upstream SpoOA monomer and helix a B o f the downstream SpoOA monomer (Zhao et al, 2002). The recognition helix, a D , fits perpendicularly into the major groove o f 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 ( a A - a F ) . 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 D N A - b i n d i n g 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 9 0 ° 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 a E ) o f 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; a E , 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 o f sporulation involves integration o f signals from both external and internal environments concerning the nutrient status o f the environment, cell density, and progression o f the cell cycle (Burkholder and Grossman, 2000; Perego and Hoch, 2002; Trach and Hoch, 1993).  1.2.2.1. Nutrient deprivation One o f the main signals for sporulation initiation is nutrient deprivation (Burkholder and Grossman, 2000). The nutrient status o f the environment is detected by the transition state regulator C o d Y (Serror and Sonenshein, 1996). C o d Y senses intracellular levels o f guanine nucleotides and branched chain amino acids; when bound to G T P or when stimulated b y 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 o f alternate energy sources (Ratnayake-Lecamwasam et al, 2001; Serror and Sonenshein, 1996). When G T P levels decrease at the onset o f stationary phase, repression o f 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 o f phosphoryl groups to SpoOA, thus facilitating sporulation initiation.  1.2.2.2. High cell density A second requirement for the initiation o f sporulation is high-cell density (Perego and Hoch, 2002). The cell-density o f the environment is detected in a mechanism reminiscent o f quorum-sensing and involves the secretion and import o f short peptides. The peptides are produced from proteolytic processing o f the products o f the phrA and phrE genes, transcribed with the gene encoding the phosphatases which they inhibit, R a p A and RapE (Perego and Hoch, 1996). A t low cell density, the R a p A 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 o f the Rap  14  phosphatases (Bongiorni et al, 2005; Perego et al, 1994), thus allowing phosphoryl transfer to SpoOA and facilitating the onset o f 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 w i l l not commence due to inhibition o f K i n A autophosphorylation by the Sda protein (Ireton and Grossman, 1992, 1994; Lemon et al, 2000; Rowland et al, 2004). Normally, transcription o f the sda gene is repressed by the D n a A 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 o f the phosphorelay facilitates many points for regulation o f SpoOA activation (Figure 3). The existence o f different sensor kinases permits transfer o f varying levels o f phosphate through the system and permit response to a variety o f inputs. Although the activity o f K i n C and K i n D produce a level o f Spo0A~P sufficient for regulating abrB transcription, the activity o f K i n A has proved most critical during sporulation (Antoniewski et al, 1990). Autophosphorylation o f 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 o f SpoOF is negatively regulated by three phosphatases, R a p A , B , and E (Jiang et al, 2000a) and the activity o f each o f these is subject to negative regulation by small peptides (Jiang et al, 2000a; Perego, 1999). SpoOA is dephosphorylated by the SpoOE, Y i s I and Y n z D phosphatases (Ohlsen et al, 1994; Perego, 2001). Transcription o f the kinA, spoOF, spoOA, and rapA genes is activated by Spo0A~P (Errington, 2003; Hilbert and Piggot, 2004; M o l 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 encoding a  H  H  (McQuade et al, 2001), and transcription o f the gene  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 o f expression o f a complex genetic network as shown i n Figure 7. During vegetative growth, intracellular levels o f SpoOA~P are low due to constitutive expression o f spoOA from the a -dependent spoOApv promoter, SpoOA-mediated A  repression o f the higher-activity spoOAps promoter (Strauch et al, 1992), and reduced phosphorylation o f SpoOA by regulation o f the phosphorelay as described previously. Upon detection o f nutrient deprivation, high cell density, and normal cell cycle progression, there is an increase in transfer o f phosphate through the phosphorelay to activate SpoOA. Activated SpoOA initiates two programs o f 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 o f SpoOA activation is repression o f the global regulatory protein, A b r B (Strauch et al, 1990). During vegetative growth, A b r B regulates the expression o f 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 o f enzymes needed to search for alternate carbon sources (Strauch and Hoch, 1993), thus A b r B is usually thought o f as a transition state regulator. Repression o f abrB also permits expression o f 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 o f abrB inhibits the activity o f ScoC, itself a global regulator. ScoC represses transcription o f sinl (Kallio et al, 1991) and the opp operon (Koide et al, 1999; Perego and Hoch, 1988). Continued repression o f abrB indirectly leads to increased levels o f Spo0A~P and thus entry into stationary phase.  1.2.4.2. Alleviation of SinR repression A n additional effect o f abrB repression is expression o f Sinl, an antagonist o f a negative regulator o f sporulation, SinR (Bai et al, 1993). The two proteins are encoded i n 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 o f the diagram and those expressed during later stages o f sporulation are indicated progressively from the top. The regulatory inputs affecting transcription o f 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  ±±  -L-tr—  tr  spoOH  sigA  I  T  spoOE  ktnE  J J Z  v  soj  ±  sinIR  T 44 spoQApg spoOApy  spoOF  OPP  ^JL-Lj—>  - - Y t r L  scoC  L  ii  spoIIA  spoUG  rig*:  spoWD  ix Mother Cell  spoIUG  spoVT  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 o f sporulation, SpoOA~P repression o f abrB allows synthesis o f o and the subsequent H  transcription o f sinl (Shafikhani et al, 2002). This permits expression o f SinI which interacts with SinR to relieve SinR inhibition o f key sporulation gene promoters (Bai et al, 1993).  1.2.4.3. Antagonism by a negative regulator of sporulation, Soj Another key regulator o f 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 ( L i n and Grossman, 1998). However, in the absence o f 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 b y SpoOJ, which may antagonize Soj by retaining Soj at the poles o f cell (Marston and Errington, 1999; Quisel et al, 1999).  1.2.4.4. Induction of a expression O f particular importance for the initiation o f sporulation is de-repression o f the spoOH gene which encodes the sigma factor, o ( W e i r et al, 1991). Expression o f spoOH and postH  transcriptional stimulation o f c -directed transcription by the C l p X protease ( L i u et al, H  1999; L i u and Zuber, 2000) permits expression o f sporulation genes such as spoVG, required for spore coat synthesis (Rosenbluh et al, 1981), transcription o f 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 o f the Rap phosphatases (Britton et al, 2002). In addition, transcription o f the spoOF, and ftsAZ genes is increased due to promoter switching to the o -dependent promoters o f these genes (Britton et al, 2002; Predich et al, H  1992).  19  The most significant effect o f a  expression is induction o f a positive-feedback loop that  leads to increased transcription o f spoOA from the a -dependent, SpoOA-dependent promoter of spoOA (spoOAps). Together, the accumulative effects o f abrB repression, alleviation o f SinR and soj repression, and o -dependent transcription are to increase intracellular levels o f H  phosphorylated SpoOA. This increase in SpoOA~P is required for regulation o f "highthreshold SpoOA" genes (Fujita et al, 2005), such as those encoding other sporulationspecific 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 o f the F  E  spoIIA and spoIIG operons and subsequent activation o f 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 o f o~ permits transcription o f sigK and spoIIID, which in turn activate transcription o f the final mother cell-specific transcription factor, gerE. Similarly, transcription o f the final forespore-specific transcription factor, spoVT, is activated following o -directed transcription o f spoIIIG, encoding the a F  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 o f gene regulation, in addition to the evolution o f new genes, may have played a significant role in generating much o f the biological diversity observed today (Ptashne and Gann, 2002). In principle, regulation o f gene expression may affect any step leading to a functional gene product. There are four levels o f 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 o f 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 o f functional gene product ultimately produced, to initiate new metabolic or developmental activities requires induction o f  20  transcription o f new genes. Thus much o f the control over regulation, from bacteria to higher eukaryotes, involves the control o f initiation o f transcription b y 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 o f R N A P is a stable non-covalent assembly o f four subunits: two a subunits, one P subunit and one P' subunit (Darst, 2001). In vitro, the core w i 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 o f 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 o f 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 o f entire batteries o f genes (Gross et al., 1998; Gruber and Gross, 2003; Helmann and Moran, 2002; Paget and Helmann, 2003). In E.coli, a whereas alternate a-factors such as a  3 8  7 0  is produced during exponential growth,  and o~ are activated during stationary phase and i n 54  response to nitrogen limitation, respectively. Although sigma factors confer the ability o f R N A P to recognize different classes o f promoters, they can be categorized into one o f 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 o f initiation has been extensively studied. The basic elements o f the transcription cycle have been elucidated through study of E.coli and involve R N A P complexed with the major vegetative sigma factor, a  70  , leading to the mechanism described below. The transcription cycle begins with  21 location o f the promoter. The sigma subunit bound to R N A P recognizes two conserved 6base 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 o f the -35 hexamer also contribute to promoter binding. A T - r i c h sequences ("UP elements"), found 40 to 60 base pairs upstream o f the transcription start site, stabilize R N A P at the promoter by binding the C-terminal domain o f at least one o f the a subunits o f 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 o f core promoter sequence and spacing relative to an idealized consensus promoter. Deviations from this ideal reduce the amount o f 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 o f 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 <-> RP 2 *-> RPoi <-> RP 2 <"> RPinit C  0  In the case of E.coli the initial complex formed by R N A P at a strong promoter such as P| uv5 ac  is referred to as an initial "closed" complex (RPci). In this complex, E a  7 0  is bound to only  one face o f the double helix and the D N A near the start site o f transcription is doublestranded 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 o f the transcription start site. Downstream o f the  22  -10 element polymerase contacts both strands indicating that the D N A is enveloped by RNAP.  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 i n 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 o f 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 o f ternary initiation complexes (RPjnit). The ternary initiation complex advances by binding subsequent N T P s , the P subunit catalyzing covalent bond formation. R N A P characteristically undergoes a process o f "abortive initiation" in which short transcripts are continually synthesized and released to reform RPc2The transcription cleavage factors G r e A and GreB (Borukhov et al, 1993; Sparkowski and Das, 1990) are involved i n increasing the efficiency o f 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 o f the a-subunit from the core and synthesis o f 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 o f 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 o f initiation (Record et al, 1996). These key regulatory proteins, "activators" and "repressors", underlie the ability o f 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 o f E G  7 0  to  specific promoters. In this scenario the holoenzyme is i n a 'constitutively active' state and cooperative binding o f 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 , are not constitutively active. In this case the activator, such as N t r C , activates a 54  holoenzyme that is pre-bound to the D N A by inducing a conformational change i n E r j  54  such  that it becomes capable o f initiating transcription (Ptashne and Gann, 2002; Zhang et al, 2002). A fourth proposed mechanism o f transcriptional activation involves binding o f an activator, such as M e r R , to induce a conformational change in the conformation o f promoter D N A , thus leading to initiation o f transcription (Heldwein and Brennan, 2001). In this mechanism it is unclear whether M e r R 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 i n activation, there are multiple mechanisms whereby transcriptional repressors can reduce the frequency o f 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 D N A - b i n d i n g sites are adjacent to promoter D N A , cooperative repressor binding causes intervening regions o f 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 C y t R 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 G a l repressor and A b r B , inhibit D N A - b o u n d 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 o f activation o f transcription initiation has been extensively studied in E.coli using the transcriptional activator, C A P . C A P is responsible for activating transcription o f over one-hundred genes in the presence o f 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 o f 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 o f R N A P to promoter D N A . In this mechanism, the binding site for the C A P dimer is located upstream o f the conserved promoter elements (typically located between -62 and -93), such as at the lac promoter (Busby and Ebright, 1999). Interactions between the D N A - b o u n d C A P dimer and the C-terminal domain ( C T D ) o f one o f the a subunits o f 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 o f an initial closed promoter complex (RPci) and stimulation o f 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 o f R N A P leading to formation o f 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) o f 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 o f 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 o f SpoOA to OA boxes 5' o f the transcription start site increases the rate o f transcription initiation (Bird et al, 1996; Rowe-Magnus and Spiegelman, 1998). Promoters activated by SpoOA include the spoIIA, and spoIIG operons, which encode the foresporespecific sigma factor a , and the mother cell specific transcription factor a F  E  (Hoch and  Silhavy, 1995). In contrast, binding o f SpoOA to specific D N A sequences 3' o f the transcription start site causes repression o f 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. A t the spoIIA promoter, SpoOA interacts with R N A P H  containing a  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 ( M c L e o d 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 o f SpoOA in transcription activation o f a -dependent promoters such as spoIIA is not understood. H  1.6. Experimental rationale A large number o f mutations within SpoOA have been identified and tested. One SpoOA mutant which contains a substitution o f 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 o f 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 o f the crystal structure o f the isolated C T D (Lewis et al, 2000a) has determined that this mutation lies i n helix aF. A more recent structure o f 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 o f two suppressor mutations o f SpoOA(A257V) within the dimer structure support this hypothesis. The suv4 (H162R on helix a A ) and suv3 (L174F on helix a B ) (Perego et al, 1991b) mutations have been proposed to suppress the A 2 5 7 V mutation by strengthening intermolecular interactions between two molecules o f 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 o f formation o f several orientations upon D N A binding. If SpoOA does activate transcription as a dimer, a recent model o f SpoOA activation suggests that dimerization is mediated by interactions between the receiver domains and not by interactions between D N A - b i n d i n g domains (Lewis et al, 2002), possibly minimizing the role o f the A 2 5 7 V mutation in disruption o f crucial interactions within a dimer.  Another possible explanation for the effect o f 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 o f 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 . Since interaction with o H  H  is needed to increase levels o f  Spo0A~P which is required for stimulation o f 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 o f a  A  dependent genes. To determine i f the A 2 5 7 V mutation  specifically effected c -dependent transcription initiation, I have conducted an in vitro H  characterization o f 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 S p o 0 A ( A 2 5 7 V ) expression was due to a defective interaction between SpoOA and a  H  or between SpoOA dimers I analyzed activation o f a o - and a a -dependent A  H  promoter in vivo when w i l d 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 o f SpoOA to levels sufficient to activate transcription o f 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 ( L B ) 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 o f 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 o f 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  p E T 1 6 b A 9 V . p E T 1 6 b A 9 V was transformed into E.coli B L 2 1 ( l D E 3 ) p L y s S , creating strain BTA9V.  29  Table 1. Bacterial strains used in this study Strain E. coli DH5a  BL21(A,DE3)pLysS  Genotype"  Source or Reference  [hsdR\l(x\c,m^+)supE44 Thi-1 recAl gyrA (NaT) relAl A(lacZYA-argF)U169 (cp80lacZAM15) F" ompT hsdS (r "m ") gal dcm (DE3) p L y s S ( C m ) BL21(?J)E3)pLysS p E T 1 6 b A 9 V B L 2 1 ( ^ D E 3 ) p L y s S pET16bOA  Invitrogen  B  B  Invitrogen  B  R  BTA9V MCOA B. subtilis JH642  This study Lab Stock  b  trpC2 phe-1  JH695 JH16124  J. Hoch, Scripps Research Institute L a Jolla California U S A Ferrari et al, 1985 M . Perego, Scripps Research Institute L a Jolla California U S A M . Perego, Scripps Research Institute L a Jolla California U S A This study  trpC2 phe-1 spoOA(A257V) trpC2 phe-1 amyE:: (spoil'AlacZ), C m trpC2 phe-1 amyE::(spoIIGlacZ), K a n JH642 s/w&4::(pMNSpoOAN), Erm JH695 ^ o 6 » ^ . : ( p M N S p o O A N ; , Erm BT2001 amyE::(spoIIA-lacZ), Cm BT2001 amyE::(spoIIG-lacZ), Kan BT2002 amyE:.(spoil'A-lacZ), Cm BT2002 amyE::(spoIIG-lacZ), Kan R  JH16304  R  BT2001  R  BT2002  This study  R  BT2003  This study  R  BT2004  This study  R  BT2005  This study  R  BT2006  This study  R  spoIIA-lacZ  a  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 , chloramphenicol resistance; K a n , kanamycin resistance; E r m , R  R  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  pGEM-T pGEMA9V  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 P I 4 6 . A m p , K a n 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 , E r m integration vector used for systematic inactivation o f coding sequences in B. subtilis. Features a spoVG-lacZ translational fusion, the LacIrepressed/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-lacZ ) and a 874 bp insert bearing the spoOA ribosome binding site and coding sequence up to E263. p M N S p o O A with a 855 bp deletion (AspoOA) and a 481 bp insert bearing the spoOA ribosome binding site and coding sequence up to P I 4 6 . p U C 1 9 with a 240 bp Hindlll to BamUl D N A fragment bearing the spolIG promoter and 100 bp downstream o f the transcription start site encoding the trpA terminator Cloning vector with an 814 bp D N A fragment bearing the -703 to +37 region o f abrB, relative to the P2 promoter start site. Includes two abrB transcription initiation sites ( P I , P2) and two downstream and one upstream OA box.  Promega This study  pGEMSpoOA pGEMOAN pET16b pET16bOA  pET16bA9V  pMUTIN-4  pMNSpoOA  R  R  This study This study Novagen Lab stock  This study  Vagner et al., 1998  This study  umA  pMNOAN  pVCIIGtrpA  pJM5134  pUC//G27  down p\JCIIG2.2 down  pXJCIIGtrpA with mutation o f the site 2.1 OA box pVCJIGtrpA  with mutation o f the site 2.2 OA box  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 OA-4 abrB-F abrB-R IIG 2 X M13R BTOARBS OAEco BTOAlinker  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 ' 5' - C G G G A T C C A A A G A C G T T T G A T - 3 ' 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 ' 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 ' 5'-GGGGATCCCTCGAGGTCA-3' 5' - C A G G A A A C A G C T A T G A C C - 3 ' 5'-AAGCTTGGTGAATCCTGTTA-3' 5'-TCTAACCTCAGCTTATCCGC-3' 5'-GTCGACAGGCTGGCTGCTGCGTATAAT-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 o f protein following heparin-agarose affinity purification. Concentrated protein was dialyzed overnight at 4°C against 2 L o f buffer C ( 2 0 m M sodium phosphate (pH 8.0), 1 m M ethylenediaminetetraacetic acid ( E D T A ) , and 1 m M phenylmethylsulfonyl fluoride (PMSF)) + 5 0 m M N a C l . Following dialysis the protein was loaded directly onto a 30 m l D N A cellulose column equilibrated with buffer C + 50 m M N a C l and eluted with a 150 m l linear gradient o f 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 S p o 0 A ( A 2 5 7 V ) 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 o f buffer C containing 150 m M N a C l , 0.1 m M dithiothreitol ( D T T ) 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 o f 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 S p o 0 A ( A 2 5 7 V ) 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 o f w i l d type and mutant SpoOA proteins were isolated as described previously (Grimshaw et al, 1998; Z a p f et al, 1996; Zhou et al, 1997).  In vitro phosphorylation o f SpoOA and SpoOA(A257V) was compared by incubating 0.5, 1.0, or 2.0 u M SpoOA or S p o 0 A ( A 2 5 7 V ) 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 u C i o f [ y - P ] A T P (6000Ci/mM; Amersham Biosciences) in l x 32  transcription buffer in a final volume o f 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 o f w i l d 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 u C i o f [ y - P ] A T P ( 6 0 0 0 C i / m M ; Amersham 32  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 o f 0.5 u M SpoOA or S p o 0 A ( A 2 5 7 V ) and were added directly to 5 ul o f 2x S D S - P A G E buffer (100 m M T r i s - C l , p H 6.8, 4 % S D S , 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 o f 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 o f either p\JCIIG2.1 down, or p\JCIIG2.2down  pUCIIGtrpA,  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 G e l 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 o f pJM5134 (Greene and Spiegelman, 1996; Perego et al, 1988). This  fragment includes both abrB transcription initiation sites ( P I , P2) i n addition to a pair o f 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 o f different forms o f SpoOA were completed as described previously (Bird et al, 1993; Greene and Spiegelman, 1996). For spoIIG transcription, template D N A (4 n M ) and 0-1200 n M unphosphorylated or phosphorylated SpoOA and SpoOA(A257V) were incubated with the initiating nucleotides, A T P (0.4 n M ) and G T P (5 u M ) , 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 o f initiated complexes. For abrB transcription, template D N A (4 n M ) 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 o f C T P and heparin. The reactions were terminated after five minutes by the addition o f 5 p i o f 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 o f 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 o f pUCIIGtrpA  plasmid D N A was incubated with 30 U BamHl to  linearize the vector 135 bp downstream o f 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 o f calf intestinal alkaline phosphatase ( C I A P ) . The dephosphorylation reaction was terminated with the addition o f 0.1% S D S and 20 m M ethyleneglycol-bis(Paminoethyl)-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 o f the precipitated D N A in l x forward kinase buffer (0.5 M T r i s - H C l , p H 7.6; 0.1 M M g C l ; 50 m M D T T ; 1 m M 2  spermidine; 1 m M E D T A , p H 8.0) and incubation with 1333 u C i [ y P ] A T P (6000 32  Ci/mmol) ( I C N Biomedicals) and 12 U T 4 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 o f 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 o f 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 o f passive elution buffer (500 m M N H 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 , 4  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 redissolved 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 S p o 0 A ( A 2 5 7 V ) ~ P binding were carried out as described previously (Bird et al., 1996). Briefly, 2.0 x 10 C P M 5  [ y P ] A T P end-labeled spoIIG promoter D N A was incubated with 0-600 n M Spo0A~P or 32  SpoOA(A257V)~P in l x T2 transcription buffer (40 m M Hepes, p H 8.0; 10 m M M g A c ; 1 m M E D T A , p H 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 o f 75 pi o f DNase stop buffer (0.1 % S D S ; 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 , p H 8.0; 1 mg/ml xylene  35  cyanol; 1 mg/ml bromophenol blue) and the activity o f labeled D N A in each reaction was estimated from Cerenkov radiation in the sample. Approximately 1.13 x 10 C P M o f labeled 5  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 D N a s e l protection patterns o f 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 o f 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 filling i n the two base overhangs by incubation with 0.016 U Klenow Fragment (Invitrogen), 16.6 u M d G T P , 16.6 u M d A T P , 20 u M dTTP (Amersham Biosciences), and 30 u C i [ a - P ] d T T P 32  (3000Ci/mmol) in l x React 2 buffer (Invitrogen) in a final volume o f 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 o f 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). T o 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 C P M (8 n M ) o f labeled duplex D N A in l x transcription buffer at 37°C. After two minutes, 3.3 ul o f 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  4  36 duplex D N A by electrophoresis at 12 V c m " for 1.5 hr and detected using a Molecular 1  Dynamics Phosphorlmager SI system. The fractional saturation o f 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 + x) using the program SigmaPlot version 8.0 (SPSS). This calculation assumes d  that SpoOA binds as a dimer to the two OA boxes which act as a single site on the duplex.  2.8. C l o n i n g of Yspac-spoOA(l-146) Plasmid p M N O A N is the integrative vector used to create strains o f B. subtilis which encode a copy ofspoOA or spoOA(A257V) under control o f the LacI/IPTG-inducible Pspac promoter. This plasmid was created in a two-step process which involved removing most o f the spoVGlacZ translational fusion from the vector p M U T I N 4 in the first step and sub-cloning the N terminal sequence o f 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 o f the Pspac promoter, an origin o f replication for growth and maintenance in E.coli  {pri .coii), E  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 . To remove the spoVG ribosome 2  binding site and create a truncated, non-functional copy o f 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, p G E M S p o O A , to create the integrative vector p M N S p o O A (Figure 8 B , C ) . The plasmid p G E M S p o O A had been created by ligation o f 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). p G E M S p o O A encodes the first 263 amino acids o f the spoOA coding sequence and 37 bp of sequence upstream o f the G T G start codon which encodes the spoOA ribosome binding site.  37  F i g u r e 8. Construction o f V -spoOA(l-l46). spaQ  (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 o f the LacI-repressed/IPTG-inducible P p S  promoter, an origin o f replication {ori . oii) E C  a(  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 . The Hindlll-Sacl 2  inserted into Hind\\l-Sac\ spoVG-lacZ  region o f p G E M S p o O A (B) was  digested p M U T I N 4 to create p M N S p o O A (C), replacing the  translational fusion in the parent vector with sequences encoding a truncated  spoOA gene and the spoOA ribosome binding site i n p M N S p o O A . T o construct the P c spa  spoOA strains, the HinAWl-Sall region o f p M N S p o O A was replaced with the  Hindlll-Sall  region o f 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 b y sequences  encoding the N-terminal domain o f 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 o f spoOA under the control o f Pspac, JH642 chromosomal D N A was used to amplify the first 146 amino acids o f 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 o f p G E M O A N (Figure 8D) generated a D N A fragment encoding the N-terminal domain o f spoOA. This fragment was ligated with the 6604 bp Hindlll-Sall  fragment from p M N S p o O A .  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 o f spoOA (amino acids 1- 146). The sequence o f the spoOA gene was confirmed to be free o f errors by sequencing completed by the Nucleic A c i d and Protein Service Unit, University o f British Columbia.  2.9. Construction of Yspac-spoOA and T*spac-spoOA(A257V) B. subtilis strains To create strains o f B. subtilis which encoded a copy o f spoOA or spoOA(A257V) under control o f 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 o f 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 I P T G induction using 0, 1, and 4 m M I P T G .  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 I P T G when required. After 28 hours growth the cultures were sampled and the total number o f viable cells per m l o f culture was determined by serial dilution and plating on L B agar supplemented with antibiotic ( i f required). The number o f spores was determined by treating each dilution with 0.1 volume of chloroform and plating onto L B agar containing the appropriate antibiotic ( i f required).  40 After 24 hours incubation, the number o f colonies on plates containing between 30 and 300 colonies were counted and the sporulation frequency determined. Sporulation frequency is defined as the number o f 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 m l samples were collected at selected times and harvested by centrifugation. The cell pellets were rinsed with wash buffer (50 m M T r i s - H C l , p H 8.0; 10 m M E D T A , p H 8.0; 10% glycerol; 1 M K C 1 ; 1.7 m M P M S F ) and resuspended in 1 m l o f lysis buffer (20 m M T r i s - H C l , p H 8.0; 1 m M E D T A , p H 8.0; 300 m M KC1; 100 m M M g C l ; 1.7 m M P M S F ; 0.1 m M D T T ) prior to breaking open the cells by 2  sonication (3 x 30 sec, level 4, Sonicator® Ultrasonic Liquid Processor M o d e l X L 2 0 2 0 , Misonix Inc). The protein concentration o f 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 o f 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 o f rabbit-antiSpoOA 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 o f 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 o f 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 ( G E 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 o f SpoOA and SpoOA(A257V) activity i n strains encoding mutant or w i l d type proteins under transcriptional control o f 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 spoIIAlacZ 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 I P T G induction.  2.12.2. P-Galactosidase activity To assay for P-galactosidase activity, 50 m l o f S S M supplemented with the appropriate antibiotic was inoculated with a 1/100 dilution o f an overnight culture o f the appropriate strain. Four millimolar I P T G was either added to the cultures at the time o f inoculation or was added to the cultures at various time points during growth, as indicated. The growth o f each culture at 37°C was followed by measurement o f optical density at 525 nm. A t various times during logarithmic growth and stationary phase, 1 m l samples o f each culture were collected in triplicate and harvested by centrifugation. The cell pellets were frozen at -20°C until assayed for P-galactosidase activity. C e l l pellets were thawed by resuspension in 730 pi o f Z-buffer (60 m M N a H P 0 ; 40 m M N a H P 0 ; 10 m M KC1; 1 m M M g S 0 ; 50 m M p2  4  2  4  4  Mercaptoethanol). Cells were permeabilized by addition o f 0.1 % Triton X following 5 minutes incubation with 0.1 mg/ml lysozyme. O-nitrophenol-P-D-galactoside ( O N P G ) , a chromogenic p-galactosidase substrate, was added to a final concentration o f 0.45 mg/ml and incubated with the permeabilized cells at 28°C. After 15 minutes incubation the reactions were terminated with the addition o f N a C 0 3 tol 80 m M . The absorbance at 420 nm was 2  measured for each reaction and the enzyme specific activity was determined using the calculation: specific activity (Miller Units) = ( A o nm x 6 6 . 7 ) / O D 5 n m O f the culture. 42  52  42  3. RESULTS In this study I have investigated the in vitro and in vivo characteristics o f a substitution mutant o f the response regulator SpoOA. I was interested in how changes in the D N A binding domain affect the ability o f SpoOA to activate transcription initiation. It was previously reported that a single amino acid substitution made within the D N A binding domain o f SpoOA, A 2 5 7 V , resulted in a sporulation-deficient phenotype (Ferrari et al., 1985). Although the substitution was associated with loss o f activation o f transcription initiation in vivo from both a -and c -dependent promoters by SpoOA (Perego et al., 1991b; A  H  Rowe-Magnus et al., 2000), it did not affect inhibition o f 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 o f SpoOA. In this thesis I have investigated the uncoupling o f the ability o f 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 o f 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 o -dependent promoter or bind D N A from that promoter? A  Finally, did the A 2 5 7 V mutation interfere with a positive feedback mechanism in vivo which leads to upregulation o f spoOA transcription?  3.1. Examination of the effect of the A257V mutation on SpoOA phosphorylation in vitro The ability o f SpoOA to stimulate transcription is dramatically increased by transfer o f a phosphoryl group through a phosphorelay signal transduction system to D56 located within the N-terminal domain o f SpoOA (Bird et al., 1993). Although the A 2 5 7 residue is distant from the site o f phosphorylation and is located in the C T D o f SpoOA, it was possible that the valine substitution could alter the structure o f the protein such that it became a poor substrate for the phosphotransferase protein SpoOB, and that decreased phosphorylation could explain the lack o f 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 o f protein phosphorylation using an in vitro phosphorylation assay composed o f reconstituted phosphorelay components. The rate o f phosphorylation was  43  determined by incubation o f SpoOA and S p o 0 A ( A 2 5 7 V ) with phosphorelay components K i n A , SpoOF, SpoOB and [ y P ] A T P for various times prior to separation by S D S - P A G E and 32  quantification o f phosphorylation by Phosphorlmager analysis (Figure 9 A ) . The initial rates o f phosphorylation o f w i l d 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 o f w i l d 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 o f 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 o f 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 o f 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 o f both phosphorylated and unphosphorylated w i l d type and mutant proteins to repress abrB transcription in an in vitro transcription assay.  Unphosphorylated and phosphorylated w i l d type and mutant proteins were incubated with a linear D N A fragment encoding both the P I and P2 transcription initiation sites o f the abrB promoter (abrBp), initiating nucleotides ( A T P , U T P and G T P ) , and R N A P - a . After two A  minutes incubation, transcripts were permitted to elongate with the addition o f C T P and heparin. Transcripts resulting from a single round o f 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 K i n A , 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) SpoOA  500  1000 2000 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 S p o O A ( A 2 5 7 V ) 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, S p o 0 A ( A 2 5 7 V ) . A representative Phosphorlmage is shown. The values reflect the average o f three independent experiments and their standard deviations.  46  A SpoOA ' SpoOA~P  SpoOA(A257V)fc 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. U T P , and G T P and a linear D N A fragment encoding both the P I 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 ) 2 p _ i J  a D e  j j abrB e t  transcripts were detected by autoradiography and (B) the level o f transcripts produced from the abrB promoter was determined by Phosphorlmager analysis. S y m b o l s : open circles, SpoOA; filled circles, S p o 0 A ~ 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 o f three  independent experiments and their standard deviations.  47 Consistent with earlier results, increasing amounts o f SpoOA reduced the amount o f run-off transcript produced from the abrB promoter, and SpoOA(A257V) was capable o f repressing transcription from abrB (Figure 1 I B ) . 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 o f SpoOA to repress abrB transcription by 3.3-fold, whereas phosphorylation o f SpoOA(A257V) only enhanced repression by 1.3-fold. This may be a consequence o f the unexpected effectiveness o f unphosphorylated SpoOA(A257V) in repressing transcription. The significance o f the apparently effective repression by SpoOA(A257V) is uncertain as in vivo experiments have indicated that the temporal pattern o f repression o f the abrB gene in a strain with SpoOA(A257V) mimics that in the w i l d type strain (Perego et al, 1991b). Critically, these data confirmed that the A 2 5 7 V substitution did not substantially affect the ability o f 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. A m o n g the first sigma factors produced are the E  a  F  and a sigma factors which direct gene expression in the mother cell and forespore,  respectively (Dworkin, 2003). The o sigma factor is transcribed from the o -dependent E  A  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 , but is not transcribed in a spoOH mutant. For A  both the spolIG and spoIIA operons, transcription requires that S p o 0 A ~ P reaches a threshold sufficient for activation (Fujita et al, 2005). This increase in concentration o f phosphorylated SpoOA appears to require increased transcription o f spoOA and the phosphorelay components kinA and spoOF.  48 Previous studies have indicated that the A 2 5 7 V eliminates activation o f transcription initiation from the spolIG promoter in vivo. However, because activation o f transcription initiation at this promoter in vivo is not only dependent upon SpoOA~P, but also upon a a H  dependent increase in concentration o f SpoOA~P, it was possible that the A 2 5 7 V mutation did not change the ability o f the protein to stimulate a -dependent transcription initiation. I A  tested whether the A 2 5 7 V mutation affected the ability o f SpoOA to activate transcription initiation in vitro testing both the initial rate o f transcription and the effect o f 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 was added to the A  reactions and the proteins were allowed to form initiated complexes for 0-120 seconds before elongation was permitted with the addition o f heparin and the remaining nucleotides. Phosphorylated SpoOA was able to stimulate the rate o f spolIG transcription initiation to a greater extent than the other proteins tested (Figure 12A, B ) . The number o f transcripts increased rapidly in the presence o f 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 o f stimulation o f spolIG transcription initiation, although the unphosphorylated protein stimulates the rate at a much reduced level. Similarly, in the presence o f phosphorylated SpoOA(A257V) there was an increase in the number o f 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 o f transcription initiation was approximately ten times slower than stimulation by SpoOA~P.  The effect o f varying the concentration o f unphosphorylated and phosphorylated w i l d 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 for two minutes to allow formation o f initiated complexes. Transcription was A  limited to a single-round from initiated complexes by the addition o f 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 w i t h a linear D N A fragment encoding the spolIG operon promoter (spolIGp) and initiating nucleotides A T P and G T P .  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 b y the addition o f transcription stop buffer at the times indicated. Elongated 32p-]abelled spolIG transcripts were separated b y 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, S p o O A ~ P ; open squares, S p o O A ( A 2 5 7 V ) ; filled squares, S p o O A ( A 2 5 7 V ) ~ 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. F o l l o w i n g a live-minute incubation, elongated transcripts were separated by electrophoresis through an 8% denaturing polyacrylamide gel. ( A ) 3 2 p _ i b e l e d spoIIG a  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. S p o 0 A ( A 2 5 7 V ) ~ P . Representative films are shown. Values reflect the average o f three independent experiments and their standard deviations.  51  Increasing amounts o f SpoOA~P led to increasing amounts o f transcription over the range o f 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 o f transcripts produced in the absence o f SpoOA. Both phosphorylated and unphosphorylated SpoOA(A257V) were capable o f stimulating transcription initiation, but only to levels approximately 50% o f those stimulated by w i l d type SpoOA. Over the range o f concentration at which S p o 0 A ( A 2 5 7 V ) was tested, transcription increased linearly and at 1200 n M the amount o f 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 o f both proteins was four-fold more effective i n stimulating transcription than was the unphosphorylated protein. These data demonstrate that S p o 0 A ( A 2 5 7 V ) was capable o f stimulating transcription from a a -dependent promoter A  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 b y SpoOA. A t these sites SpoOA makes both base-specific contacts and contacts with the D N A backbone (Zhao et al, 2002). A t 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 o f 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 o f 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 o f 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 P ] A T P end-labeled 32  fragment o f 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 o f 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 o f 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 o f 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 o f 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 o f unphosphorylated and phosphorylated wild type and mutant proteins were incubated with [ a P ] N T P end-labeled duplex D N A encoding the two OA 32  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 o f 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" M (standard error = 7  0.22) compared to an apparent dissociation constant o f 3 x 10" M (standard error = 0.44) for 7  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 S p o O A ( A 2 5 7 V ) (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 w i t h [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 S p o O A ( A 2 5 7 V ) 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, S p o O A ~ P ; open squares, S p o O A ( A 2 5 7 V ) ; filled squares, S p o O A ( A 2 5 7 V ) ~ 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 o f 1 x 10" M . The binding o f SpoOA(A257V) to the duplex D N A was negligible, 6  precluding an accurate assessment o f 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 o f 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 o f 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 o f 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 o f this model is that the active form o f SpoOA is a dimer. The crystal structures o f 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).  A t 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 o f the spolIG promoter. When the site 2 OA boxes are occupied by a dimer o f Spo0A~P, only the downstream monomer is expected to be close to the sigma subunit o f 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 o f 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 o f 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 o f interaction between each monomer and one OA box and the strength o f interaction between the two monomers. I f dimer-binding conditions were compromised, so that simultaneous contact o f the monomers and OA boxes was altered, then potentially one o f 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 o f SpoOA~P with the sigma subunit is critical to transcription activation at the spoIIG promoter, it should be more affected by binding o f the downstream monomer o f the SpoOA dimer than binding o f the upstream monomer. To test this idea I altered the spoIIG promoter through mutation o f each o f the site 2 OA boxes separately and tested whether the effects o f these mutations would be exaggerated for SpoOA(A257V). In the case o f w i l d type SpoOA, where the protein-protein interactions are most favorable, mutation o f either OA box should have roughly the same effect, reducing the sum o f contacts stabilizing dimer binding to the D N A . On the other hand, in the case o f SpoOA(A257V), potentially only one o f 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 o f the downstream 2.2 OA box might have a more deleterious effect on transcription than mutation o f the upstream 2.1 OA box. Thus I measured the effects o f mutations in each o f 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 o f 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). T o mutate the 2.2 OA box (creating the "2.2down" 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  c  A spoIIG  -50 -40 CCTCTCAACATTAA7TGACAGAC G G A G A G T T G T A ATTA 4CYGICJG  6000000,  CCTCagAAgATTAA7TGACAGAC 2.1 down GGAGtcTTcTAATTA/f(7r(rTCTG CCTCTCAACATTAAcTGAgAGAC 2.2 down G G AG AGTTGTA KTTeA CTcTCTG  B  SpoOA~P  SpoOA(A257V)~P  spoIIG  200  400  600  800  1000 1200  [Protein] (nM)  2.1 down 2.2 down  Figure 16. W i l d type and mutant SpoOA stimulation of transcription from mutant spoIIG promoters in vitro. Phosphorylated or unphosphorylated SpoOA or S p o O A ( A 2 5 7 V ) were incubated w i t h a linear D N A fragment encoding either w i l d 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. F o l l o w i n g a five-minute incubation, elongated transcripts were separated b y 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 b y SpoOA are shown i n bold and those expected to be contacted b y the a A subunit o f R N A polymerase are italicized. Mutated bases are shown i n lower case. ( B ) 32p-labeled transcripts were detected by autoradiography and (C) the level o f transcripts produced were determined b y Phosphorlmager analysis. Symbols: black circles, S p o 0 A ~ P (spoIIGp): black squares, S p o O A ( A 2 5 7 V ) ~ P ) (spoIIGp); grey circles, S p o 0 A ~ P (promoter templates: 2.1 down, —  ; 2.2 down,  (promoter templates: 2.1 down,  ); grey squares, S p o 0 A ( A 2 5 7 V ) ~ P  ; 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 o f R N A P and eliminating one c-specific contact, as predicted from the crystal structure (Campbell et al, 2002). The ability o f SpoOA~P and SpoOA(A257V)~P to stimulate transcription initiation from w i l d type and mutant promoter templates was determined using an in vitro transcription assay (Figure 16B). Mutation o f either the 2.1 or 2.2 OA boxes decreased, but did not abolish, the ability o f phosphorylated SpoOA or phosphorylated SpoOA(A257V) to stimulate transcription (Figure 16C). Overall the pattern o f transcription stimulation as a function o f activator input was similar to that seen with the w i l d type spolIG promoter; transcription in the presence o f SpoOA~P reached a maximum at 800 n M whereas stimulation by SpoOA(A257V)~P increased i n 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 o f transcription stimulation by both phosphorylated SpoOA and S p o 0 A ( A 2 5 7 V ) as would be expected i f the binding affinity o f 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 o f 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. W h i l e the difference between the effects o f the OA box mutations on stimulation by the two types o f activator proteins was not dramatic, the combination o f the A 2 5 7 V mutation and loss o f one o f the OA boxes lead to very low levels o f 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 o f SpoOA and that sporulation requires a high level o f SpoOA (Chung et  59  al, 1994; Fujita et al, 2005). Genes which require low-levels o f 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 o f SpoOA for activation and are referred to as 'highthreshold activated genes' o f the SpoOA regulon (Chung et al, 1994; Fujita et al, 2005). One possible explanation for the uncoupling o f transcription stimulation from transcription repression in B. subtilis JH695 is that S p o 0 A ( A 2 5 7 V ) is expressed to lower levels than SpoOA. Reduced expression o f the mutant protein would be sufficient to repress lowthreshold genes such as abrB, but would not be o f sufficient threshold to activate highthreshold genes such as the spolIG and spoIIA operons nor be o f sufficient concentration to permit sporulation.  To test this idea, we observed expression o f w i l d type and mutant SpoOA proteins over time in an immunoblot analysis. Lysates o f whole cell-extracts were collected from w i l d 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 o f both wild type and mutant SpoOA proteins increased over time and reached a maximum level approximately two hours after the onset o f stationary phase (T+2). However, whereas previous reports suggested that similar amounts o f 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 o f SpoOA expression (35ng SpoOA(A257V) vs.52 ng SpoOA in samples taken at T ) (Figure 17A). + 2  To correlate the reduction i n 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 o f 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 o f sporulation in spoOA(A257V) B. subtilis strains.  60  A  purified protein JH642 JH695 T ,  T  T  0  +  T+2 T  + 3  T+4 1 l n g 33ng  B Strain  No. C F U (ml )  No. Spores (ml )  6.3 x 10 6.2 x 10  3.3 x 10' 0  1  JH642 JH695  x  8  1  Sporulation Frequency 5.2 x 10"' < 1 . 0 x 10" 8  Figure 17. W i l d type and mutant SpoOA protein expression B. subtilis JH642 and JH695. B. subtilis strains J H 6 4 2 and J H 6 9 5 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 o f total protein from whole cell extracts and samples containing 11 and 33 n g o f purified recombinant SpoOA and S p o O A ( A 2 5 7 V ) were separated b y 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 . The spoOA gene can be transcribed from one o f two H  promoters, spoOApv or spoOAps (Strauch et al, 1992). During vegetative growth, transcription from the c -dependent vegetative promoter (spoOApv) results in low-level A  expression o f SpoOA. Increased expression o f SpoOA required for sporulation occurs during the onset o f sporulation as a consequence o f a positive feedback mechanism i n which SpoOA stimulates transcription o f its own gene from the a -dependent, sporulation specific promoter H  (spoOAps). Similarly, an increase in SpoOA activity could be achieved by increased phosphorylation as a result o f greater synthesis o f the phosphorelay components spoOF and kinA whose transcription also depends on c (Britton et al, 2002; Predich et al., 1992). H  One possible explanation for the reduction in SpoOA protein expression observed in B. subtilis strain JH695 is that A 2 5 7 or the region around it is required for specific interaction with o - R N A polymerase. Strains containing the spoOA(A257V) mutation would not only be H  unable to sporulate because o f lack o f activation o f SpoOA-dependent, o -dependent H  promoters but also because the threshold level o f SpoOA would never increase sufficiently to permit activation o f G -independent, high threshold SpoOA activated promoters, such as the H  a -dependent spolIG promoter. One way to test this hypothesis in vivo was to create B. A  subtilis strains which over-expressed either w i l d type or mutant SpoOA proteins and monitor transcription activation o f both a a -dependent and a rj -dependent promoter in vivo. H  A  To test this hypothesis I created strains o f B. subtilis in which expression o f w i l d type or mutant SpoOA protein was placed under the control o f 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 o f the spoOA gene downstream o f 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 V -spoOA spac  and P -spoOA(A257V). spac  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 o f 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 o f the lacZ gene, and the transcription termination sequences To, T i and T . p M N O A N was transformed into B. 2  subtilis strains JH642 and JH695 and integrated into the spoOA locus o f 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 o f SpoOA transcribed from the native sporulation and vegetative promoters  spoOA  and spoOA , in addition to either the full length spoOA or spoOA(A257V) gene transcribed ps  from the V  spac  promoter.  pv  0\  64  The strains created, BT2001 and BT2002, encoded both a full length copy o f either the w i l d type or mutant spoOA gene under control o f the Pspac promoter in addition to a truncated spoOA gene encoding the first 146 amino acids o f the N-terminal domain downstream o f 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 w i l d type or mutant SpoOA proteins and sporulate in the presence o f inducer. Strains BT2001 and BT2002 were grown in S S M supplemented with 0, 1 or 4 m M I P T G . After 16 hours o f growth a sample o f each culture was removed and tested for expression o f wild type or mutant SpoOA proteins using an immunoblot analysis (Figure 19A). Lysates o f whole-cell extracts collected from cultures o f BT2001 and BT2002 were probed for presence o f mutant or w i l d type SpoOA proteins using anti-SpoOA antibodies. A s expected, the cultures grown in the absence o f inducer did not produce detectable amounts o f w i l d type or mutant SpoOA whereas both BT2001 and BT2002 expressed protein reacting with the aSpoOA antibody in response to I P T G . Under these conditions it was estimated that both samples contained approximately the same amount o f mutant and w i l d 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 o f growth strains BT2001 and BT2002 were tested for the ability to sporulate. Only strain BT2001, which encodes the w i l d type spoOA gene under the control o f Pspac (Vspac-spoOA), was able to sporulate, albeit poorly, with the addition o f I P T G (Figure 19B). Strain BT2002, which encodes Vspac-spoOA (A25 7 V), was unable to sporulate at both I P T G concentrations tested. Strain BT2001 was also able to sporulate at an extremely l o w frequency (3.4 x 10" spores/ml) in the absence o f I P T G . Addition o f 1 m M I P T G increased 6  the ability o f BT2001 to sporulate by a factor o f 100 while addition o f 4 m M I P T G caused a 1000-fold increase in sporulation frequency. However, while higher levels o f I P T G caused an increase in the ability o f strain BT2001 to sporulate, there was no obvious difference in expression o f mutant or wild type proteins from Pspac at 4 m M I P T G (Figure 19A). Moreover, although I P T G induction o f Pspac resulted in an increased ability o f strain  65  A  [IPTG](mM)  1  0 50 ng  4  1  0  BT2001  4  BT2002  purified SpoOA  B Strain  No. C F U (ml )  No. Spores (ml )  Sporulation Frequency  0 1 4  3.5 x 10* 7.9 x 10 7.1 x 10  0 1 4  1.5 x 10* 6.3 x 10 5.7 x 10  1.2 x 10 5.6 x 10 1.5 x 10 0 0 0  3.4 x 10* 7.0 x 10" 2.0 x 10" <1.0x 1 0 < 1 . 0 x 10~ < 1 . 0 x 10"  [IPTG] ( m M )  1  BT2001  BT2002  s  8  s  8  1  3  5  6  4  3  s  8  8  Figure 19. W i l d 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 B T 2 0 0 1 and B T 2 0 0 2 were grown i n 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 w i l d type B. subtilis strains.  3.10. Investigation of the effect of varying IPTG concentration on expression from Pspac-spoOA (A25 7V) The goal o f constructing the inducible B. subtilis strains was to alleviate the requirement o f S p o O A - a interaction for increased expression o f SpoOA. However, it appeared that protein H  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 o f sporulation i n 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 S p o 0 A ( A 2 5 7 V ) expression and the ability to sporulate in response to increasing concentrations o f I P T G . Cultures were grown in S S M supplemented with 0-16mM I P T G for 16 hours prior to harvest for immunoblot analysis or for 28 hours before sporulation assay. Lysates o f whole cell extracts were probed for SpoOA(A257V) using anti-SpoOA antibodies. In the absence o f any I P T G , no protein reacting with the anti-SpoOA antibody was detected (Figure 2 0 A , lane 2). Addition o f 1 m M I P T G induced expression o f SpoOA(A257V) (Figure 2 0 A , 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 o f I P T G lead to induction o f 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 o f the mutant protein to be phosphorylated, the mutation did alter binding to OA boxes, transcription repression and activation o f a -dependent transcription. A n A  unanswered question is the effect o f spoOA(A257V) transcription directed by o . One way to H  test this interaction is to compare the ability o f strains expressing equal levels o f 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] (mM) 0 1 2 4  s 16  No. C F U (ml ) 1  1.1 4.0 3.3 4.2 3.4 3.2  x 10* x 10 x 10 x 10 xlO x 10 s  8  8  8  s  No. Spores (ml )  Sporulation Frequency  0 0 0 0 0 0  <1.0xl(P < 1 . 0 x 10" < 1 . 0 x 10~ < 1 . 0 x 10" < 1 . 0 x 10~ < 1 . 0 x 10"  1  8  8  8  8  8  Figure 20. Spo0A(A257V) expression in BT2002 as a function of I P T G induction. Samples o f B. subtilis strain B T 2 0 0 2 were collected and harvested from cultures grown i n 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 S p o O A ( A 2 5 7 V ) ; lane 2-7: lysates from B T 2 0 0 2 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 c -dependent and o -dependent lacZ reporter A  H  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 o f the chromosome. A double recombination event resulted in integration o f the lacZ translational reporter into the amyE locus o f the chromosome o f strain BT2001 and BT2002, creating strains BT2003, BT2004, BT2005, and BT2006. I first tested the ability o f the strains to sporulate in the absence and presence o f inducer (Figure 21 A ) . Cultures were grown in S S M supplemented with 0 or 4 m M I P T G 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 I P T G and the addition o f 4 m M I P T G increased sporulation frequency by at least 100-fold, although this frequency was still 100-fold lower than w i l d 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  I P T G . Analysis o f protein expression in strains BT2003, BT2004, BT2005 and BT2006 with and without I P T G revealed that all strains expressed either the mutant or wild type proteins to similar levels (Figure 2 I B ) .  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 I P T G at the time o f 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 w i l d 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 o f spoIIA-lacZ transcription in strains BT2003 and BT2005 was negligible, even with the addition o f 4 m M I P T G . In general, there was little or no difference in the (3galactosidase activity measured in strains grown with or I P T G , although the activities  69  A lane  m 1  2  3  4  5  —  6  7  8  9  mm -  B Strain BT2003 BT2004 BT2005 BT2006  [IPTG] (mM) 0 4  No. C F U (ml ) 5.5 x 10' 7.2 x 1()  No. Spores (ml ) 4.0 x 10" 7.1 x I 0  Sporulation Frequency 7.2 x 10" 9.9 x 10"  0 4  2.1 x 10  8  6.8 x 10  s  2.0 x 10" 8.4 x 10  9.5 x 10"' 1.2 x 10~  0 4  4.6 x 10 8.0 x 10  8  0 4  1.3 x 10 1.7 x 10  8  1  8  8  8  1  5  5  0 0 0 0  6  4  3  <1.0x <1.0x <1.0x <1.0x  10" 10" 10" 10"  8  8  8  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 B T 2 0 0 3 and B T 2 0 0 4 encode Fsp&c-spoOA and strains B T 2 0 0 5 and B T 2 0 0 6 encode PspacspoOA(A257V). Samples o f B. subtilis strains B T 2 0 0 3 - B T 2 0 0 6 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: B T 2 0 0 3 ; lane 3: B T 2 0 0 3 + 4 m M I P T G ; lane 4: B T 2 0 0 5 ; lane 5: B T 2 0 0 5 + 4 m M I P T G ; lane 6: B T 2 0 0 4 ; lane 7: B T 2 0 0 4 + 4 m M I P T G ; lane 8: B T 2 0 0 6 ; lane 9: B T 2 0 0 6 + 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, B T 2 0 0 3 and B T 2 0 0 5 were grown i n S S M +/- 4 m M I P T G and samples were collected in half-hour intervals from m i d exponential growth and assayed for (3-galactosidase activity. T i m e '0' indicates the end o f exponential growth. Symbols: closed triangles, JH16124, open circles. B T 2 0 0 3 (no I P T G ) ; filled circles, B T 2 0 0 3 + 4 m M I P T G ; open squares, B T 2 0 0 5 (no I P T G ) ; filled squares, B T 2 0 0 5 + 4 m M I P T G . Values reflect the average o f three independent experiments and their standard deviations.  71  measured i n 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, spoIIAlacZ 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 o f strains BT2003 and BT2005 to activate  spoIIA-lacZ  transcription was due to inappropriate timing o f induction. If spoOA or spoOA(A257V)  were  expressed early i n growth and i f I P T G 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 w i l d type or mutant SpoOA would be below the threshold required for activation o f stage II sporulation genes such as spoIIA and spoIIG. T o address this potential problem I attempted to optimize the timing o f I P T G 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 I P T G at half hour intervals during exponential growth in hopes o f inducing w i l d type and mutant SpoOA protein expression coordinately with an increase in phosphorelay component expression. Samples o f each culture were harvested 3 hours after the onset o f stationary phase and tested for P-galactosidase activity (Table 4).  Altering the time o f I P T G induction did not lead to a significant increase in the pgalactosidase activity measured in induced cultures o f BT2003, BT2004, BT2005 and BT2006. Addition o f I P T G at different time points during exponential growth had little effect upon activation o f 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 o f the uninduced cultures and occurred when I P T G was added 1.5 hours prior to the onset o f stationary phase. Activation o f spoIIA-lacZ transcription was greatest upon I P T G addition during exponential growth approximately 2 to 2.5 hours before the onset o f 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 o f 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. Genotype  Induction Time (hr)  3  JH642 spoOA::(pMNspoOAN ermAM)  JH695 spoOA::(pMNspoOAN ermAM)  uninduced T-2.5 T-2.0 T-1.5 T-1.0 TO uninduced T-2.5 T-2.0 T-1.5 T-1.0 TO  0  0  [IPTG] (mM)  P-galactosidase Activity (Miller Units)  0 4 4 4 4 4 0 4 4 4 4 4  spoIIA-lacZ spoIIG-lacZ 4.05 2.96 20.18 3.63 21.00 4.85 15.47 6.35 4.77 8.76 6.7 4.79 4.13 1.85 7.65 2.09 7.87 3.04 8.01 4.30 7.75 4.07 6.56 3.77  b  B. subtilis strains used in this experiment also encode amyE: :(spoIIA-lacZ)  a  amyE': :(spoIIG-lacZ)  C m or R  Kan . R  B. subtilis strains BT2003-BT2006 were grown in S S M and supplemented with 4 m M  b  I P T G at half hour intervals during exponential growth through the onset o f stationary phase (T-2.5 to TO). Samples o f each culture were harvested 3 hours after the onset o f stationary phase and tested for P-galactosidase activity. Values shown are the average o f three independent experiments. C  A culture o f each B. subtilis strain was grown in S S M without addition o f I P T G . Samples o f  each culture were harvested and tested for P-galactosidase activity similarly to the test cultures. Values shown are the average o f 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 i n 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 o f  phosphorylated w i l d type or mutant protein is not o f 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 o f 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 o f sporulation. Sporulation is a tightly regulated process and initiation o f sporulation is ultimately controlled by the response regulator SpoOA. SpoOA directly controls the expression o f 121 genes by repressing the expression o f 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 o f the abrB gene which encodes a transition state regulator. During vegetative growth, A b r B represses transcription o f many stationary phase genes and some genes required for sporulation (Strauch and Hoch, .1993). Repression o f abrB permits expression of the spoOH gene, encoding o , an alternate o factor that together with SpoOA regulates H  transcription during the onset o f sporulation. Together the two proteins cooperate to increase transcription o f the spoOA gene itself as the result o f a positive feedback mechanism. o  H  is  also responsible for increasing the concentration o f activated, phosphorylated SpoOA by increasing transcription o f the genes o f 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 D N A - b i n d i n g domain o f SpoOA affect the ability o f SpoOA to activate transcription. One substitution mutation in SpoOA, A 2 5 7 V , was found to differentially affect the ability o f SpoOA to activate and repress transcription. The A 2 5 7 V mutation abolishes the ability o f B. subtilis cells to sporulate but does not affect the ability o f SpoOA to repress transcription at the abrB promoter. Instead, the mutation eliminates the ability o f SpoOA to activate both o -dependent transcription o f the spoIIG operon promoter A  and o -dependent transcription o f the spoIIA operon promoter (Perego et al, 1991b). H  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, P I and P2 (Perego et al, 1988), and two consensus OA boxes located downstream o f the second transcription start site (Strauch et al, 1990). SpoOA~P binds to the OA boxes at abrB by interacting with only one side o f 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 o f R N A P with the abrB promoter (Greene and Spiegelman, 1996). It has been presumed, although not directly proven, that the formation o f 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 a -dependent transcription by SpoOA A  H o w SpoOA stimulates transcription is best understood at the a -dependent spoIIG promoter. A  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 o f OA boxes lying immediately upstream o f and overlapping with the - 3 5 element, placing it in a position to interact with the 4.1 region o f o~ . Importantly, the distance separating the conserved -35 A  and -10 elements o f this promoter and another SpoOA-dependent, o -dependent promoter, A  spoIIE, are 22 and 21 base pairs respectively, instead o f 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 o f these promoters (McLeod and Spiegelman, 2005). The requirement for Spo0A~P during transcription initiation at spoIIG can also be bypassed by artificially denaturing the - 1 0 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 - 1 0 element. A recent study has also shown that Spo0A~P bound to the OA box overlapping with the - 3 5 element appears to re-position R N A P to facilitate the interaction o f the a subunit o f R N A P with the A  non-template strand o f the -10 region o f heteroduplex spoIIG promoters (Seredick and Spiegelman, 2004). From these data, one would predict that SpoOA re-positions o  A  for  recognition o f the double-stranded - 1 0 element and for nucleation o f promoter melting.  76  Thus, transcription initiation at spoIIG involves at least three steps: formation o f an inactive KNAP-spoIIG  complex; activation o f the RNAP-spoIIG complex by SpoOA, including open  complex formation; and initiation o f transcription and promoter clearance. However, it is not clear whether the binding o f 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 - 3 5 element (Seredick & Spiegelman, unpublished data).  4.3.3. Activation of o -dependent transcription by SpoOA Little is known o f the role SpoOA plays in transcription initiation at promoters transcribed by R N A P - a . Genetic studies suggest that SpoOA contacts homologous regions in o H  H  and o  A  (Baldus et al, 1995). However, the arrangement and orientation o f OA boxes and the spacing o f the - 3 5 and - 1 0 elements at a -dependent promoters differs from those at a H  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 o f the spoIIA promoter indicate that deletion o f sequences 64 bases 5' to the transcription initiation site completely abolish promoter activity in vivo ( W u et al., 1991), suggesting that the OA box located upstream is critical for activation. W h i l e a cr contact surface on SpoOA A  has been defined (Buckner et al., 1998; Hatt and Youngman, 1998; K u m a r et al., 2004; Schyns et al., 1997), the region responsible for interaction with o has not been identified. H  Genetic screens designed to identify the a  H  contact surface (Hatt and Youngman, 1998) may  have been unsuccessful because o f the complexity o f the genetic regulatory network controlling SpoOA synthesis and activation, or for other technical reasons.  4.4. Objective of thesis The objective o f this study was to resolve how a single amino acid substitution within the D N A binding domain o f SpoOA could prevent sporulation while differentially affecting the  77  -100 SO  -60  -40  -20  +1  +20 +40 +  spoIIGp ' ^ ^  1  spoLJAp  oH  +  spoOApsL^  oH  +  spoOApvl—  aA  -  abrBp L—  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 o f the +1 transcription start site o f 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 o f spoOA and the abrB promoter. The number, location (bp relative to +1 indicated above promoters) and orientation o f OA boxes within target promoters is not conserved. OA boxes are indicated by arrows, and the direction o f the arrow indicates orientation. Promoters recognized by R N A P complexed with and are indicated.  78  ability o f the protein to activate and repress transcription (Perego et al, 1991b; RoweMagnus 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 o f the protein to compromise it as a substrate for the phosphotransferase protein SpoOB. This indicated that insufficient phosphorylation o f SpoOA(A257V) could not account for the absence o f sporulation in vivo. Similarly, S p o 0 A ( A 2 5 7 V ) was able to repress transcription from the abrB promoter in vitro, although to levels approximately half those achieved by the w i l d 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 o f SpoOA to activate c -dependent transcription in vitro. SpoOA(A257V) stimulated spolIG transcription, A  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 o -dependent spolIG promoter A  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 o f SpoOA to stimulate transcription activation but suggested the mutation could possibly represent a defect in activation o f c -dependent H  transcription.  One possible explanation for the reduced ability o f 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 w i l d type protein (Figure 14). This indicated that the A 2 5 7 V mutation o f SpoOA did not alter the specificity o f binding or alter the interaction o f the mutant protein with D N A bases or the D N A backbone.  To quantitatively measure the effect o f the A 2 5 7 V mutation on the binding affinity o f SpoOA for OA boxes I used an E M S A to compare the ability o f w i l d type and mutant proteins to bind consensus OA boxes encoded within duplex D N A . Analysis o f D N A binding revealed that the affinity o f 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 o f both o - and o -dependent A  H  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 o f these residues compromises the ability o f SpoOA to repress abrB in vivo as well, arguing against the possibility that A 2 5 7 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 o f 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 o f the upstream monomer. This indicated that altered packing o f the D N A binding domain, as a consequence of the A 2 5 7 V substitution, reduced the affinity o f the mutant proteins for the OA boxes. However, this effect was relatively modest and seemed unlikely to be able to account for the loss o f sporulation in vivo. In sum, these results indicated that the A 2 5 7 V mutation did not significantly effect on the ability o f SpoOA to stimulate transcription activation. The lack o f  80  a dramatic effect on transcription activation by R N A P - a points to the possibility that the A  mutation could possibly represent a defect in activation o f a -dependent transcription. H  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 o f the SpoOA to activate o -dependent transcription. SpoOA(A257V) A  was able to both activate and repress transcription in vitro, albeit to levels approximately half that achieved by w i l d 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 o f SpoOA for OA boxes and appeared to have a negligible effect on the packing o f SpoOA dimers against R N A P . The data from this in vitro investigation indicated noticeable but slight effects o f 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 o f 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 w i l d type SpoOA (Figure 17). This result was unexpected since previous work had suggested that both w i l d type and mutant proteins were expressed to the same extent (Perego et al, 1991b). This suggested that the lack o f sporulation in B. subtilis strains encoding spoOA(A257V) could be attributed to insufficient mutant SpoOA protein expression.  SpoOA expression increases at the onset o f stationary phase as a consequence o f a positive feedback mechanism in which transcription o f the spoOA gene is increased due to activation o f a second promoter for the spoOA gene transcribed by R N A P - c r . A similar regulatory H  mechanism exists controlling expression o f the master regulator o f competence in B. subtilis, comK. L i k e SpoOA, there are multiple regulatory inputs affecting transcription from the  81  comK promoter that determine the threshold level o f comK expression (Hahn et al, 1996; Smits et al, 2005; van Sinderen and Venema, 1994). A t some point, the concentration o f 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 o f biological systems such as competence and sporulation (Hofer et al, 2002; Smits et al, 2005).  The reduction in expression o f S p o 0 A ( A 2 5 7 V ) observed in this study could be attributed to a deficiency in the positive feedback loop leading to upregulation o f 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 o f 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 o f wild type and mutant proteins was inducible and then tested the ability o f cells over-expressing w i l d type or mutant protein to activate transcription from both a a -dependent and a o -dependent A  H  promoter in vivo. I predicted that i f the A 2 5 7 V mutation caused a defective interaction between SpoOA and a , cells expressing the mutant protein would activate o -dependent H  A  promoters, but not o -dependent promoters whereas cells expressing the wild type protein H  would be able to activate both a -dependent and a -dependent transcription. If the A 2 5 7 V A  H  mutation caused a defective interaction between SpoOA monomers leading to low levels o f SpoOA in vivo, over-expression o f SpoOA(A257V) would overcome the defective interaction and permit activation o f both o - and o -dependent transcription. A  H  Although wild type and mutant SpoOA proteins were expressed in response to I P T G induction, the levels o f expression o f either protein were low and were insufficient to permit wild type levels o f sporulation or drive activation o f either o - or o -dependent transcription. A  H  Increasing the concentration o f I P T G 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 o f SpoOA induction was not  82  synchronized with the expression o f other proteins required for sporulation as induction at various points during growth did not activate either a -dependent or a -dependent A  H  transcription.  There are two possible explanations for the decreased levels o f sporulation and lack o f activation o f 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 o f SpoOA during mid-exponential growth and demonstrated that sudden high level expression o f SpoOA exerts a dominant-negative effect on sporulation. From their study, the authors postulated that a gradual increase in the threshold concentration o f activated SpoOA is required for sporulation. Alternatively, decreased levels o f sporulation and lack o f activation o f stage II promoters in the inducible-SpoOA B. subtilis strains used in this study may have been observed because there were insufficient levels o f phosphorylated wild type or mutant protein available for stimulation o f transcription and subsequent sporulation. The low concentration o f phosphorylated protein available may have been a consequence o f the genotype o f the strains created. For example, the strains created here encode a w i l d type or mutant spoOA gene under transcriptional control o f Pspac and one or more copies o f the N terminal receiver domain o f SpoOA (SpoOA ) transcribed under the control o f the native N  vegetative- and sporulation-specific promoters o f spoOA. The spoOA gene duplication arose as a result o f the recombination event that inserted the Pspac-controlled spoOA allele into the chromosomal spoOA locus. It is conceivable that i f SpoOA was expressed it would compete N  for phosphorylation by the phosphorelay in vivo. Furthermore, i f multiple copies o f p M N S p o O A N were integrated into the spoOA locus, there would be many duplications o f the truncated spoOA gene encoding S p o O A and only a single copy o f the full length wild type or N  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 and the full length wild type or mutant SpoOA proteins. This may be sufficient to N  reduce the concentration o f phosphorylated full length w i l d type or mutant SpoOA below the level required for activation and repression o f high-threshold SpoOA-dependent promoters and prevent sporulation. The hypothesis has some support from previous research that has shown that SpoOA can successfully compete with the full length protein for phosphorylation N  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 o f approximately 14 K D a was observed which could represent SpoOA (data not shown). However, since the N  polyclonal antibody used in the analyses reacted with other proteins in cell lysates, it was unclear i f this band represented SpoOA or another cellular protein. N  There are several methods by which one might increase the low levels o f SpoOA and SpoOA(A257V) expression in the reporter strains. One alternative is to eliminate competition by SpoOA for phosphorylation. This can be accomplished by reconstructing the N  Pspac-spoOA and Pspac-spoOA(A257V)  strains encoding spoIIA-lacZ  and  spoIIG-lacZ  translational fusions i n either a AspoOA background, or by removing the native spoOA promoters from the existing strains. Alternatively, expression o f the full length w i l d type or mutant SpoOA proteins could be increased in vivo without removing the spoOA  N  sequence.  One method to achieve this is to introduce a second copy of Vspac-spoOA or YspacspoOA(A257V) into a site on the chromosome separate from the spoOA locus. Alternatively w i l d 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 o f spoOA from the vegetative promoter (Dixon and Spiegelman, 2002; Yamashita et al, 1989). The latter approaches w i l l increase the concentration o f w i l d type or mutant SpoOA protein but do not eliminate the possibility o f competition for phosphorylation by S p o O A . Repetition o f the sporulation and p-galactosidase assays using true overN  expression strains should provide a more definitive answer to whether the A 2 5 7 V substitution interferes with the interaction o f SpoOA and o or whether the results are more H  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 o f 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 o f the D N A binding domain o f 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 o f salt bridges, hydrogen bonds and hydrophobic patches. The dimer interface in the crystal structure is formed by helix ccF o f the upstream SpoOA monomer and helix a B o f 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 o f the helix within which A257 is located (Zhao et al, 2002). It has been suggested that two suppressor mutations o f spoOA(A257V), H162R and L174F, repress the effects o f 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 A 2 5 7 (Zhao et al, 2002) (Figure 24). This orientation strengthens hydrophobic interactions between monomers and permits formation o f a new hydrogen bond between H 1 6 2 R and F236 (Zhao et al, 2002).  L i k e the A 2 5 7 V mutation, deletion o f the last 10 amino acids o f SpoOA and the D 2 5 8 V and L 2 6 0 V mutations abolish the ability o f SpoOA to activate both c - and a -dependent H  A  transcription but do not effect the ability o f the protein to repress transcription (RoweMagnus et al, 2000). These residues are also located at the dimer interface and as such may also affect SpoOA activity because o f 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 o f the L 2 6 0 V mutation is similar to that o f the A 2 5 7 V mutation in that it likely weakens intermolecular interactions by decreasing the flexibility o f helix aF (Zhao et al, 2002).  Similarly to SpoOA, members o f the OmpR-PhoB family o f response regulators bind D N A in a head-to-tail orientation (Blanco et al, 2002). The C-terminal domains o f 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 a B ) and the A 2 5 7 V mutation (aF) together at the dimer interface, strengthening hydrophobic interactions between monomers and permiting formation of a new hydrogen bond between. H I 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 9 0 ° 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 o f response regulators such as SpoOA and PhoB to D N A . L i k e 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 o f all members o f the O m p R - P h o B family are direct repeats with identical orientation with respect to the direction o f transcription (Blanco et al, 2002). In contrast, OA boxes do not always occur in the same orientation. Dimerization facilitated by interactions between adjacent D N A - b i n d i n g domains is also suggested from the crystal structure o f another response regulator, N a r L , although N a r L dimers are oriented in an antiparallel orientation (Maris et al, 2002). L i k e the spoOAAW allele in which the last 10 amino acids (residues 253 to 263) have been removed (Rowe-Magnus et al, 2000), deletion o f the last 7 amino acids o f the N a r L homolog U h p A abolishes the ability o f the regulator to activate transcription (Webber and Kadner, 1995). Based on the N a r L structure bound to D N A , the last 7 amino acids in U h p A would also be predicted to form part o f the dimer interface. W h i l e the C-terminal domain structures o f SpoOA, P h o B / O m p R and U h p A / N a r L are different, the dimer interface o f 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 o f 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 N t r C , PhoB, F i x J , and PhoP, and speculated in N a r L (Baikalov et al, 1996) in response to phosphorylation (Birck et al, 2003; D a 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; D a Re et al, 1999). While the D N A - b i n d i n g domain interactions revealed from the crystal structures should not be ignored, such interactions would seem to be redundant in light o f the ability o f the receivers to dimerize (Lewis et al, 2002) and the ability o f 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 o f 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 o f SpoOA at 'high-threshold SpoOA-activated' promoters but not at promoters responding to low concentrations o f SpoOA. This prediction is consistent with the lack o f activation o f the 'high-threshold SpoOA-activated' (Fujita et al, 2005) spoIIA and spolIG operon promoters in vivo (Perego et al., 1991b) and repression o f 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 o f induction o f the 'low-threshold SpoOA-activated' (Fujita et al, 2005) a -dependent, H  sporulation-specific promoter o f spoOA (Chibazakura et al, 1995) in vivo and the subsequent decrease in SpoOA(A257V) expression observed i n 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 o f activated protein, SpoOA may dimerize on the D N A instead o f in solution. Under these conditions, monomers o f 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 o f the monomers, interaction o f the phosphorylated receiver domains would serve to stabilize the S p o 0 A ~ P - D N A complex. The stability o f the complex would be a function o f the sum o f interdomain interactions between N-terminal and C-terminal domains, and the interactions o f 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 S p o 0 A ( A 2 5 7 V ) 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 o f cooperativity. None o f the binding experiments carried out so far show any indication o f 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 o f protein. The choice o f 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 o f activator to the reactions to minimize the effects o f ionic and organic solvent (glycerol). Thus, the concentrations o f 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 o -SpoOA interaction surface H  The second possible explanation for the effect o f the A 2 5 7 V mutation on SpoOA function is that residue A 2 5 7 is important for contact with the a subunit o f R N A P and that the A 2 5 7 V H  mutation diminishes this interaction. This hypothesis would explain the lack o f activation o f the o -dependent spoIIA promoter (Perego et al, 1991b) and the lack o f transcript from the o -dependent sporulation-specific promoter o f spoOA (Chibazakura et al, 1995) observed in vivo. Furthermore, this hypothesis accounts for the lack o f activation o f the 'high-threshold SpoOA-activated' a -dependent spoIIG promoter in vivo since increased expression o f A  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 o f transcription activation o f promoters that do not contain tandem OA boxes and presumably bind monomelic SpoOA~P, such as spoOAps (Figure 23). Moreover, because the direction o f the OA boxes at a -dependent promoters (eg. spoIIA, spoOAps) H  differs from the direction o f OA boxes at a -dependent promoters (eg. spoIIG), the face o f A  the D N A - b i n d i n g domain o f 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 A 2 5 7 and/or the region around it represents that a  H  contact  region within SpoOA accounts for the in vivo and in vitro characteristics o f SpoOA(A257V) thus far, the structural data available run counter to this hypothesis. The region within SpoOA that contacts o during transcription activation (the S A A R ) is located within and A  around the a E helix o f the D N A binding domain o f SpoOA (Lewis et al, 2000a) (Figure 5).  89 Head-to-tail binding o f a dimer o f C-terminal domains o f SpoOA to tandem OA boxes places the a E helix o f 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 o f 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 , we would expect that H  suppressor mutations o f A 2 5 7 V would be located near this residue. This is not observed as two o f three A 2 5 7 V suppressor mutations are located far from A 2 5 7 on the opposite face o f the D N A binding domain o f SpoOA. The third A 2 5 7 V suppressor mutation, sofll4  (a D 9 2 Y  substitution), was isolated as a suppressor o f a spoOF deletion mutant (Spiegelman et al, 1990). D92 is located in the receiver domain o f SpoOA and because the structure o f the full length SpoOA protein has yet to be determined, it is possible that the orientation o f the two domains places D 9 2 Y near A 2 5 7 V . However, a more likely explanation for suppression o f 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, A 2 5 7 V ) since previous work demonstrated that the D 9 2 Y mutation enhanced stability o f S p o O A - R N A P complexes (Cervin and Spiegelman, 1999).  Further research could determine i f residue A 2 5 7 and/or the region around it identifies the o  H  interaction region within SpoOA. A direct test o f this hypothesis in vitro would be to include S p o 0 A ( A 2 5 7 V ) ~ P and R N A P - o in an in vitro transcription reaction and assess the H  stimulation o f 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. 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