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In vitro characterization of the ParA family protein Soj from bacillus subtilis McLeod, Brett 2008

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IN VITRO CHARACTERIZATION OF THE ParA FAMILY PROTEiN SOJ FROM BACILLUS SUBTILIS  by  Brett McLeod  B.Sc., The University of Western Ontario, 1999  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY  in  The Faculty of Graduate Studies  (Microbiology and Immunology)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  July 2008  © Brett McLeod, 2008  ABSTRACT  In a response to deteriorating environmental conditions and high population density the common soil bacterium Bacillus subtilis undergoes a developmental process characterized by an unusual asymmetric division which results in the formation of two different cell types (the forespore and the mother cell) and ultimately the release of a dormant endospore. This developmental process is under multiple levels of control. Mutation in stage zero genes (spoO) block all morphological changes. One stage zero gene spoOJ and a companion gene in the same operon (so]), have been implicated in the negative regulation of sporulation. Soj and SpoOJ are members of ParA and ParB families respectively, implicated in the stable inheritance of low copy number plasmids between daughter cells in bacteria. The role that Soj and SpoOJ play in the negative regulation of sporulation is not fully understood. This thesis characterizes the interaction of Soj with nucleotides, DNA, and SpoOJ in vitro to better understand the activities of Soj in the cell. I characterized the dimerization and DNA binding of wild type Soj and two Soj point mutants (S0jG12V and S0jD4OA). Wild type Soj was shown to bind adenosine nucleotides, form dimers, and hydrolyze ATP. Multiple lines of evidence demonstrated the nucleotide bound state does not alter the dimerization of Soj but does alter its ability to bind DNA. Multiple Soj-ATP proteins bound DNA to form Soj-DNA complexes as determined by DNaseI protection, electrophoretic mobility shift assays, and electron microscopy. Fixed angle light scattering was used to monitor the binding of Soj to DNA. Time resolved light scattering allowed characterization of multiple stages of Soj DNA binding. Soj was also shown to interact with SpoOJ, which resulted in a stimulation of Soj ATPase activity providing evidence that SpoOJ can alter Soj activity. The interaction of Soj and SpoOJ resulted in the formation of large protein complexes detected by light scattering and by electron microscopy. This data indicated that SpoOJ is able to form multimeric structures in response to a Soj interaction. The data were interpreted in a model for changes in the state of Soj and SpoOJ.  11  TABLE OF CONTENTS  ABSTRACT  ii  TABLE OF CONTENTS  iii  LIST OF TABLES  vii  LIST OF FIGURES  viii  LIST OF ABBREVIATIONS AND SYMBOLS ACKNOWLEDGEMENTS  x xii  INTRODUCTION  1  1  Sporulation in B. subtilis  1  2  ParAISoj Belongs to the Mrp/MinD Protein Family  4  2.1  E. coil Mm Proteins  5  3  ParA and ParB Proteins  7  4  Plasmid Par Loci  7  4.1  ParA and ParB Proteins from P1 and F Plasmids  12  4.2  Type II Par System of Plasmid Ri  13  5  Chromosomal ParA and ParB Proteins  14  5.1  Par Proteins from M smegmatis, P. aeruginosa, P. putida, V. cholerae, andHpylori  14  5.2  Streptomyces coelicolorr  15  5.3  Caulobacter crescentus  16  6  Bacillus subtilis  17  6.1  The Role of Soj (ParA) and SpoOJ (ParB) Proteins in Vegetative Growth  17  6.2  The Role of Soj (ParA) and SpoOJ (ParB) Proteins in B. subtilis Sporulation  20  7  Structure of ParAISoj Proteins  22  7.1  T thermophilus  22  7.2  Plasmid pSM19035  8  Experimental Aim  Protein  22 25  111  MATERIALS AND METHODS  .  26  1  Standard Techniques  26  2  Manipulation of DNA and Oligonucleotides  26  2.1  Site Directed Mutagenesis  26  2.2  Oligonucleotide Labeling  30  2.3  Preparation of Labeled DNA Fragments for EMSAs  30  2.4  Preparation of Internally Labeled DNA Fragments for DNase Protection Assays  30  2.5  Preparation of DNA Fragments for Light Scattering Assays  31  2.6  Construction of pET2OspoOJ  31  2.7  Generation of pUCIIGJ8  32  3  Protein Purification  32  3.1  Soj  32  3.2  Soj Mutants  34  3.3  SpoOJ  34  3.4  Protein Quantification  35  4  Protein-Nucleotide UV Crosslinking  35  5  ATPase Assays  36  6  Soj Size Exclusion Chromatography Assays  36  7  Blue Native PAGE Assays  37  8  Electrophoretic Mobility Shift Assays (EMSA5)  37  9  Sedimentation Assays  38  10  DNase Protection Assays  38  11  In Vitro Transcription Assays  39  12  Light Scattering Assays  39  13  Transmission Electron Microscopy  40  13.1  Resolution of Soj DNA Structures  40  13.2  Resolution of Soj-SpoOJ structures  40  13.3  Resolution ofpUCl9 DNA  41  iv  RESULTS  .  42  1  Phylogenetic Analysis of ParA!Soj and MinD Proteins  42  2  Experimental Rationale  42  3  Soj Mutant Overexpression Plasmids  42  4  Protein Purification  44  4.1  Soj Purification  44  4.2  SpoOJ Purification  48  5  ATP Binding and Hydrolysis by Soj  48  6  Characterization of Soj Multimerization  56  6.1  Size Exciution Chromatography of Soj  56  6.2  Blue Native PAGE Analysis of Soj  60  7  Soj DNA Binding  66  7.1  Soj DNA Complexes Resolved by EMSAs  66  7.2  Soj-DNA Sedimentation  71  7.3  Soj Protection of DNA from DNase I Digestion  74  7.4  Soj Inhibition of Transcription in Vitro  78  8  Detection of Soj-DNA Complexes by Light Scattering  82  9  Soj-DNA Complexes Resolved by Transmission Electron Microscopy  96  10  Soj Interaction with SpoOJ  98  10.1  Soj ATP hydrolysis is stimulated by SpoOJ  99  10.2  Soj and SpoOJ interact to Form High Molecular Weight Protein Complexes  103  DISCUSSION  110  1  Technical Modifications  110  1.1  Soj purification  110  1.2  Light Scattering Assays  111  2  Soj ATP Binding and Hydrolysis  111  3  Soj Dimerization and Multimerization by Soj  113  4  DNABindingbySoj  115  5  A Model of Soj DNA Binding Leading to Light Scattering  117  5.1  Insights from SojG12V and SojD4OA for the Soj DNA Binding Model  119 V  IA  OEI  8J  !JO33H  ‘-‘I  8U!a01d  d  fl1m0S0m0q3J0  rncj roods ptw fog oa.z uj o swnsa’Njo ZZT  533M1M313}1  aJ°i II  aoUPAaIa’a  roods puc ‘VNU ‘ppopnj qp suotlolualul fog  oi  foods  B!M  uoanajuj fog  6 8 L 9  LIST OF TABLES.  Table 1.  Plasmids Used in this Thesis  Table 2.  Primers and Templates Used to Generate Site-Directed Mutant Plasmids by PCRs  Table 3.  27  28  Primers and Templates to Generate PCR DNA Fragments Used in this Thesis  29  Table 4.  Bacterial Strains Used in this Thesis  33  Table 5.  Molecular Weight Standards Elution Volumes Used for Size Estimation of Soj Multimers  58  Table 6.  FPLC Absorption Peak Integration  61  Table 7.  Soj Complex Size Estimation from BN-PAGE  65  Table 8.  Initial Rates of Light Scattering for Different Soj Concentrations  88  vii  LIST OF FIGURES.  Fig. 1.  Morphology of sporulation in B. subtilis  Fig. 2.  Sequence alignment of B. subtilis Soj and plasmid encoded ParA Proteins  2  8  Fig. 3.  Sequence alignment of chromosomal ParA proteins  10  Fig. 4.  Motifs of B. subtilis and T. thermophilus Soj  23  Fig. 5.  Phylogenetic analysis of ParA/Soj, and MinD proteins  43  Fig. 6.  Induction of Soj and SpoOJ overexpression  45  Fig. 7.  Analysis of purified hexahistidine tagged Soj  47  Fig. 8.  Nucleotide binding by Soj assayed by UV cross linking  49  Fig. 9.  Specific ATPase activities of Soj, SojG12V, and SojD4OA  52  Fig. 10.  ATP hydrolysis by Soj eluted in different ADP concentration Conditions  54  Fig. 11.  Soj ATPase kinetics  55  Fig. 12.  Elution of molecular weight standards used to estimate size of Soj dimers  Fig. 13.  Analysis of purified Soj and Soj mutants by FPLC size exclusion chromatography  Fig. 14.  57  59  Blue native PAGE of Soj, and Soj point mutants in the presence of ATP, ADP, or absence of nucleotide  63  Fig. 15.  Nucleotide dependence of Soj binding toDNA  67  Fig. 16.  Sedimentation of Soj-DNA complexes in the presence of ATP  72  Fig 17.  DNase protection of DNA by Soj-ATP  75  Fig. 18.  Estimation of DNA sizes protected from DNase I digestion by Soj  77  Fig. 19.  Inhibition of transcription initiation in vitro by Soj  80  Fig. 20.  Light scattering by DNA and Soj-ATP  83  Fig. 21.  Light Scattering by SojG12V  85  Fig. 22.  Light scattering as a function of Soj concentrations  87  Fig. 23.  ATP dependence of Soj light scattering  90  Fig. 24.  DNA concentration dependence of Soj-ATP DNA light scattering  91  Fig. 25.  Effect of DNA size on light scattering by Soj  93  Vu  Fig. 26.  A 24 bp DNA does not stimulate formation of light scattering complexes  Fig. 27.  95  Transmission electron micrographs of negatively stained sedimented Soj-ATP DNA structures  97  Fig. 28.  Stimulation of Soj ATPase by SpoOJ  100  Fig. 29.  Stimulation of Soj ATPase by SpoOJ  101  Fig. 30.  Stimulation of SojG12V ATPase by SpoOJ  102  Fig. 31.  Sedimentation assays of Soj and SpoOJ  104  Fig. 32.  Light scattering by Soj and SpoOJ  106  Fig. 33.  Light scattering Soj or SojG12V, and SpoOJ  107  Fig. 34.  Transmission electron micrographs of negatively stained  Fig. 35.  Soj-SpoOJ structures  108  Interaction of Soj with nucleotides, DNA, and SpoOJ  123  ix  LIST OF ABBREVIATIONS AND SYMBOLS  ATP  Adenosine 5’-triphosphate  BN  Blue native  bp  Base pair  DAPI  4’,6-diamidino-2-phenylindole  dATP  Deoxy adenosine 5’-triphosphate  DNase  deoxyribonuclease  EDTA  Ethylene diamine tetraacetec acid  EMSA  Electrophoretic mobility shift assay  FPLC  Fast Protein Liquid Chromatography  GFP  Green fluorescent protein  GTP  Guano sine 5’ -triphosphate  HTH  Helix turn helix  IPTG  Isopropyl-13-D-thiogalactopyranoside  MW  Molecular weight  NTA  Nitrilotriacetic acid  NTP  Nucleotide 5’-triphosphate  OD  Optical density  oriC  Chromosomal origin of replication  ORF  Open reading frame  P-loop  Structural term used to describe Walker A motif  PAGE  Polyacrylamide gel elecrophoresis  PCR  Polymerase chain reaction  PET  polyethyleneimine  PMT  Photomultiplier tube  PVDF  polyvinylidene fluoride  RNAP  RNA polymerase  spo  Sporulation positive  spo  Sporulation negative  SpoOAP  activated phosphorylated form of SpoOAP  SDS  Sodium dodecyl sulfate  TCA  Trichloroacetic acid x  TE  Tris-EDTA  UV  Ultraviolet  YFP  Yellow fluorescent protein  Abbreviations of measurements conform to the International System of Units (SI).  xi  ACKNOWLEDGEMENTS  I would like to thank past members of the Spiegelman Lab for their help and friendship (Martin Richer, lab tech and all around good guy; Steve Seredick, grad student and intellectual mogul; Barb Turner, grad student and great lab manager). I am also grateful to the directed studies who worked on various aspects of Soj with me (Laura Willihnganz, who taught me Quikchange PCR; David Baker, who characterized Soj in vivo). I would like to acknowledge people close to me who have supported me throughout the course of my degree, including (but not limited to) my family and my partner Laura. I would like to thank the members of my committee; Lindsay Eltis, George Mackie, and Michael Murphy. I would also like to thank my supervisor George Spiegelman for always being available for discussions regarding anything to do with science and sometimes life in general, and also for being a great editor, and an amazingly supportive teacher/instructor/supervisor.  xii  INTRODUCTION Bacillus subtilis has been intensively studied in part due to the genetic tools such as transformation that are available and in part due to interest in developmental process of endospore formation. Genetic screens have identified a small set of genes (‘-4 0) which control entry into sporulation. Surprisingly, two of these genes (spoOJ and sof) encode members of protein families usually associated with plasmid partition and stability in Bacteria, parA and parB. This thesis investigates the in vitro characteristics of the protein Soj and SpoOJ to better understand how they affect sporulation.  1 Sporulation in B. subtilis.  When cells of Bacillus subtilis enter stationary phase they express genes to adopt alternative survival strategies, such as the use of alternative metabolic pathways and the scavenging of nutrients from their surroundings (bacterial motility, and competence), to name a few (Claverys eta?., 2006; Hamoen eta?., 2003; Phillips and Strauch, 2002). When B. subtilis cells sense that survival is unlikely they initiate sporulation that results in the formation of a resistant endospore (Errington, 2003; Piggot and Hilbert, 2004). B. subtilis endospores have been estimated to be able to remain viable for up to —2 million years (Nicholson, 2003). The endospore is capable of germination to resume vegetative growth (Moir, 2006; Setlow, 2003). Spore formation in B. subtilis is characterized by a series of morphological changes that have been used to divide the sporulation event into eight stages (Fig. 1). Stage I of sporulation is characterized by the compaction of the bacterial chromosome throughout the length of the cell to form what is called the axial filament. Stage II is characterized by an asymmetric septation of the cytoplasmic membrane but not the cell wall which creates two unequal compartments (Kroos et a?., 1999); the smaller is called the forespore compartment, and the larger is termed the mother cell compartment. At each stage, compartment-specific transcription of genes is required to complete development of that stage and to progress to the next stage (Hubert and Piggot, 2004). In stage III, one chromosome is actively transported through the asymmetric septum into the forespore (Burton et a?., 2007; Ptacin et a?., 2008). The forespore is then engulfed by the mother cell (Broder and Pogliano, 2006). The fusion of the mother cell membrane around the prespore forms the forespore protoplast which exists as a cell within a cell. In subsequent stages the cortex, a modified form 1  Vegetative Growth  Cortex Formation  Engulfment Asymmetric Septation  0 spoO  Forespore Protoplast  spoil  Coat Formation  IV  112  “1  Maturation  .  spoil  spoill  V spoIV  Release  VII  VI spoV  spoVI  Fig. 1. Morphology of Sporulation in B. subtilis. The main sequence of morphological events begins with the vegetative cell and ends with the release of a mature spore. Roman numerals designate the stages of sporulation characterized by electron microscopy. The italicized spo designation identifies genes whose mutation stalls sporulation at the stage immediately preceding it. For example, mutation of spoOA prevents asymmetric septum formation, while mutation of spoliG prevents engulfment. Stage I represents cells that have replicated their genome and formed axial DNA filaments, but since this is also observed in vegetative cells and no stage I specific mutants have been characterized, the designation has been dropped. Adapted from Errington, 1993, and Seredick, 2006.  2  of cell wall, is synthesized between the forespore and mother cell membranes (Takamatsu and Watabe, 2002), while the mother cell assembles a proteinacieous coat on the outside surface of the spore. During the deposition of these layers, small acid-soluble proteins are synthesized in large amounts to coat the forespore chromosome conferring resistance to UV radiation (Setlow, 2+• 2007), while dipicolinic acid and Ca ions are absorbed resulting in dehydration and  mineralization of the forespore (Henriques and Moran, 2007). After 6 to 8 hours of development, spore maturation is complete, and the mother cell lyses to release a dormant endospore (Lewis, 2000). The signals which trigger the initiation of sporulation are not entirely defined. Some of the signals required for the initiation of sporulation are sensed by at least five histidine kinases (KinA to KinE), which transfer a phosphoryl moiety to SpoOF (Jiang et aL, 2000). SpoOF transfers the phosphoryl group to SpoOB, which in turn transfers it to SpoOA (referred to as the phosphorelay (Varughese, 2002)). Phosphorylated SpoOA (Spo0A’P) both represses transcription (Greene and Spiegelman, 1996), and activates transcription (Satola et a?., 1992) of specific genes, ultimately affecting the expression of over 400 genes (Molle et a!., 2003a). The initiation of sporulation is not favoured until alternate strategies for cell survival have been pursued so B. subtilis tightly regulates the initiation of sporulation. Many regulatory and environmental signals directly impact the flow of phosphate to SpoOA. The autophosphorylation of the histidine kinases can be blocked (Wang et a?., 1997), as is the case for KinA where a protein, Sda, which responds to DNA damage and replication status, binds to KinA preventing its autophosphorylation causing inhibition of sporulation (Burkholder et a!., 2001; Rowland et a!., 2004; Ruvolo et a?., 2006; Whitten et a!., 2007). KinB expression is repressed by CodY whose activity is linked to internal concentrations of GTP and branched chain amino acids (Handke et a!., 2008; Molle et a?., 2003b; Shivers and Sonenshein, 2004). SpoOF and SpoOA are also the targets of multiple phosphatases (Ohisen et a?., 1994; Perego et a?., 1994; Perego, 2001). The Rap phosphatases which reverse phosphorylation of SpoOF are themselves negatively regulated by small pentapeptides that are exported and then re-imported into the cell (Ishikawa eta!., 2002; Perego, 1997). The synthesis, import, and export of these pentapeptides are negatively regulated by CodY (Molle et a?., 2003b). Genes that when mutated block progression to stage II have been termed stage zero genes. Most stage zero genes encode proteins that are part of the phosphorelay or affect the flow of phosphate to SpoOA. The mutation of one gene, spoOJ, has not been shown to affect the phosphorylation level of SpoOA; nonetheless the loss of spoOJ still causes a 1 0 fold decrease in 3  sporulation frequency (Ireton et a!., 1994). Cells with a AspoOJ mutation do not activate stage II gene expression (Ireton et al., 1994). SpoOJ is the second gene in a dicistronic operon and encodes a ParB family protein. The first gene in the operon encodes a ParA family protein (Ireton et aL, 1994). The disruption of this gene in a AspoOJ background relieves the effect of a AspoOJ mutation, resulting in near wild type sporulation frequencies. Therefore, this gene was named soj for suppressor f spo0J (Ireton et a!., 1994). ParA and ParB family proteins were first recognized as proteins that stabilize the vertical transmission of low copy number plasmids (Austin and Abeles, 1983; Austin and Wierzbicki, 1983), so it was striking that members of the ParA family (Soj) and the ParB family (SpoOJ) affected the initiation of sporulation in B. subtilis.  2 ParAISoj Belongs to the MrpfMinD Protein Family.  ParA/Soj proteins are members of the P-loop NTPase fold supergroup (Leipe et a?., 2002). P-loop NTPases are one of the most common families of proteins and comprise 10-18% of all gene products (Koonin et a?., 2000). The P-loop NTPase fold supergroup is divided into 7 lineages (Leipe et al., 2002). ParA/Soj proteins are grouped with the GTPase group based on sequence and structural data. ParA/Soj proteins are a subfamily within the Mrp/MinD family and MinD/Mrp-Etk superfamily (Leipe et a!., 2002). The Mrp/MinD family contains ATPases with various functions, and is divided into subfamilies: Mrp, MinD, Nifl-I, ArsA and ParAJSoj. In some cases, these proteins have been shown to form homodimers and dimerization is believed to be associated with function. In some cases such as those for MinD from E. coli, and T. thermophilus Soj the proteins have been reported to be dimeric when bound to ATP, and monomeric when bound to ADP. The Mrp/MinD family has a conserved overall globular structure of 7 central 13 strands surrounded by 5 a helices. The Mrp/MinD family proteins have what has been referred to as a ‘deviant’ Walker A motif with a conserved sequence of KGGXGK[S/T] [S/TI in the ATP binding site (Koonin, 1993; Lutkenhaus and Sundaramoorthy, 2003). The second lysine is invariant across all Walker motif proteins and is responsible for coordinating ATP binding (Milner-White eta!., 1991; Saraste eta?., 1990; Walker eta?., 1982). This family also has a Walker A’ motif, that contains a conserved aspartic acid residue at the C-terminus of the second 2 13 strand that has been shown to make a direct or water-mediated hydrogen bond to a Mg cofactor in the nucleotide binding pocket (Hayashi et a?., 2001; Schindelin et a?., 1997; Zhou et 4  al., 2000). The structures of monomeric MinD from Pyrococcusfuriosus (Hayashi et al., 2001), P. horikoshii (Sakai et a!., 2001), and Archaeogiobusfulgidus (Cordell and Lowe, 2001) are known. Several of Azotobacter viniandii dimeric NifH (Chiu et al., 2001; Jang et al., 2000; Jang et a!., 2004; Sarma et al., 2007; Schmid et al., 2002; Sen et al., 2004; Sen et al., 2006; Strop et al., 2001), and ArsA from Escherichia coil have also been solved (Zhou et al., 2000, 2001). The structures of monomeric and dimeric Soj from Thermus thermophilus are also known (Leonard et ai., 2005), as is the structure of the 6 protein (ParA) from the Firmicutes plasmid pSM19035 (Pratto eta!., 2008). Mrp/MinD proteins have diverse roles in different bacterial species. Nifil is the obligate electron donor to the MoFe protein and has multiple roles in the catalytic process of biological nitrogen fixation (Burgess and Lowe, 1996; Howard et al., 1986), as well as in the maturation and assembly of nitrogenase (Allen et a!., 1993; Rangaraj et al., 1997). ArsA is the catalytic component of an oxyanion pump that is responsible for resistance to arsenite (As(III)) and antimonite (Sb(III)) (Rosen, 1990). The 6 protein has been implicated in plasmid stabilization (Dmowski et a!., 2006; Pratto et a!., 2008).  2.1 E. coil Mm proteins.  The Mrp/MinD family protein that has been most characterized is MinD from E. coil. MinD together with MinC and MinE is required for accurate placement of the division septum in dividing cells (de Boer et a!., 1989; de Boer et a!., 1991; Lutkenhaus, 2007). Disruption of any of the 3 mm protein genes results in the production of  icells that result from formation  of a septum near the end or pole of the cell rather than the middle (de Boer et a!., 1989). MinD ATP forms dimers in vitro, while MinD-ADP does not readily dimerize (Ru and Lutkenhaus, 2003). MinD-ATP binds liposomes and forms multimeric tube-like structures (5 0-100 nm diameter) in vitro (Hu et a!., 2002; Lackner et a!., 2003). Fluorescence microscopy of cells expressing MinD-GFP shows that MinD binds to the cytoplasmic side of the inner membrane  and forms multimeric structures (Raskin and de Boer, 1999a). Membrane-bound MinD structures bind MinC (Raskin and de Boer, I 999b), and MinC antagonizes the polymerization of FtsZ (Pichoff and Lutkenhaus, 2001). FtsZ is a conserved protein whose polymerization is required for cytokinesis (Margolin, 2005). Destabilization of FtsZ polymers by MinC inhibits cell division. The current model for the interaction of MinCDE is as follows. MinE interacts with MinD, and causes dissociation of MinC from MinD. MinE also stimulates MinD ATPase 5  activity, and the ATPase stimulation causes MinD to dissociate from the inner membrane (Lutkenhaus, 2007). MinD can exchange ADP for ATP then insert into the membrane. MinC subsequently rebinds MinD (Lutkenhaus, 2007). MinE forms a ring-like structure that is observed to oscillate back and forth through the mid-cell region. This MinE oscillation drives the oscillation of MinD structures from one pole, to the other with a periodicity of under a minute (Raskin and de Boer, 1999a). The cause of MinE oscillation in the cell is not well understood, but a model which explains these oscillations is based on the attraction of MinE to MinD bound to the membrane (Lutkenhaus, 2007). MinE binds MinD bound to the membrane and promotes MinD membrane dissociation by stimulating its ATPase activity. The dissociated MinD exchanges ADP for ATP and rebinds the membrane at the opposite cell poles. The newly formed MinD polymers attract MinE and cause MinE progression towards new MinD structures. Pole to pole MinD oscillation provides a system in which MinC concentration is highest at cell poles and lowest at midcell. This oscillation of Mm E inhibits FtsZ polymerization near cell poles, ensuring that cell division occurs at the midpoint in the cell.. The Mm system of B. subtilis is similar to that of E. coil, except for the absence of a MinE homologue in B. subtiiis and the presence of a protein termed DivIVA (absent from E. coil) (Edwards and Errington, 1997; Lee and Price, 1993). There is a low level of sequence identity between MinC proteins of the two species, and a relatively high level of sequence identity between MinD proteins (40%, Marston et al., 1998). In B. subtilis, deletion of either minC or minD causes a minicell phenotype. In B. subtilis MinD-GFP is seen at the cell poles when observed by fluorescence microscopy (Marston and Errington, 1999b). MinC-GFP has a similar polar distribution pattern that is disrupted in minD mutants (Marston and Errington, 1 999b). MinD-GFP does not concentrate at the poles in the absence of DivIVA (Marston et ai., 1998). This indicates that DivIVA is necessary to localize MinD at the poles. A dlviVA deletion is lethal producing large aseptate filaments; however, this phenotype is suppressed by deletion of minC, and the resulting minC dlviVA double deletion mutant strain has a ‘minicell’ phenotype (Marston and Errington, 1999a). The Mm system in B. subtilis is different from E. coil in that the proteins do not oscillate, but are similar in that they function to inhibit FtsZ polymerization in polar regions.  6  3 ParA and ParB Proteins. ParA and ParB proteins are widely dispersed throughout the Bacteria (Leipe et a!., 2002). ParA proteins are ATPases, and ParB proteins bind to specific parS DNA sequences (Bignell and Thomas, 2001; Surtees and Funnell, 2003). These genes were first identified on low copy number plasmids (Austin and Abeles, 1983; Austin and Wierzbicki, 1983; Ogura and Hiraga, 1983). Plasmid-encoded ParA and ParB proteins bind to newly replicated plasmids, and actively segregate them to opposite cell quarters in growing cells (Li et a!., 2004). The ensuing medial division of the growing cell promotes inheritance of the plasmid by both progeny cells. Disruption of any component of the Par system results in unstable plasmid maintenance and loss of the plasmid from a growing population over several generations (Surtees and Funnell, 2003). Studies of plasmid and chromosomal ParA and ParB proteins allow several broad generalizations to be made (Ebersbach and Gerdes, 2005). First, the two proteins interact, and the interaction results in a stimulation of ParA ATPase activity. Second, ParB is a site-specific DNA binding protein. Third, ParA and ParB likely act together on DNA that has a parS sequence to segregate the DNA after it has been replicated. Fig. 2 shows a Clustal W sequence alignment of ParA proteins from plasmid P1 (ParA, type Ia), F plasmid (SopA, type Ia), plasmid TP228 (ParF, type Ib), pSM 19035 (ö, type Ib), and Soj from B. subtilis. These proteins are ones where biochemical and/or structural studies have been carried out. This alignment illustrates that the similarity between these proteins, even between type Ia ParA proteins (P1 and F plasmid), and between type lb ParA proteins (plasmid TP228 and pSM19035) is primarily found in the ATP binding domains (Walker A, A’, and B motifs). This suggests that other than sharing ATP binding and hydrolysis activities plasmid ParA proteins share few properties with chromosomal Soj/ParA proteins. Clustal W sequence alignment of selected chromosomal ParAJSoj proteins is shown in Fig. 3. High sequence identity is observed both throughout the motifs required for ATP binding and hydrolysis and the length of their sequences.  4 Plasmid Par Loci. PlasmidparA and parB genes are usually organized as a single operon (Moller-Jensen et a!., 2000). Plasmid par loci are grouped based on properties of both ParA and ParB proteins (Ebersbach and Gerdes, 2005; Moller-Jensen et a!., 2000). The largest group (type I) has 7  Fig. 2. Sequence alignment of B. subtilis Soj and plasmid encoded ParA proteins. Sequence alignment was generated by Clustal W software v. 2.0.8. Shading indicates protein motifs; Red shading, Walker A motifs; purple shading, Walker A’ motifs; green shading, Walker B motifs.  8  B. subtilis P1 Fplasmid pTP228 pSM19O3S  B. subtilis P1 Fp 1 a smi d pTP228 pSM19O3S  -MSDSSQLHKVAQRANRNLNVLTEQVQLQKDELIThNEFYQVYAKAALAKLPLLTRANVD 58 MFRMKLMETLNQCINAGHEMTKAIAIAQFNDDSPEARKITRRWRIGEAADLVGVSSQAIR 60 -  MGKIIAITNQ 12 YAVSEMEEKGYVFDKRPAGSSMKYANSIQNIIDIYEHRGVPKYRDRYSEAYVIFISNL 118 DAEKAGRLPHPDMEIRGR VEQRVGYT I EQINHMRDVFGTRLRRAEDVFPPVI GvAAH:: 119 MKvIsFLNp: 11 MEKEELKILEELRRILSNKNEAIVILNNYFKO 32 -  **  B. subtilis P1 Fplasmid pTP2 28 pSM19035  PQGNATSGLGIEKADVEQCVY PQSSATMFLSHKHS IGIVNATSAQA PQGTASMYHGWVPDLHIHAEDTLLP PQMSLTNWSK AGKAAFD LQATLTKDLA-ICTFKVELPRVN  RtTTSVNLGACLAYI GKRV VVSTVSLAHAMRAHPHLLMEDLRI GLRV P*1’SVSVHLAQDLALK GYNI EJKTrAVINIATALSRS NLKV GGKWCLSTMFAYLTDKL *  *:  -  -  -  -  -  61 177 173 56 81  *  *  B. subtilis P1 Fp 1 a smi d pTP228 pSM19O3S  DILVDDAIJ--VIDIIKATTVENLDVIPATIQLAGAEIELVPTISR EVRLKR-A MLQNVSREELLEEFIVPSVVPGVDVMPASIDDAFIASDWRELCNEHLPGQNIHAVLKENV FYLGEKDD- -VTYAIKPTCWPGLDIIPSCLALHRIETELMGKFDEGICLPTDPHLMLRL-A VFTAASEK--DVYGIRKDLAD FYEGLKNG- -NLASSIVHLTDNLDLIPGTFDLM LLPKLTRSWTFENESRLLAT-L  111 237 230 75 133  B. subtilis P1 Fplasmid pTP228 pSM19O3S  LEAVKQNY y 9 ifltCPPSLGLLTINALTASDSWIPVQCEYYALEGLSQLLNTVRLVQKH I DKLKSDYflFILVtSGPHLDAFLKNALASANI LFTPLPPATVDFHSSLKYVARLPELVKL I ETVAHDZ&IIVZtSAPNLGI GT INVVCAADVL IVPTPAELFDYTSALQFFDMLRDLLKN YDFAIVDGAGSLSVITSAAVMVSDLVIIPVTPSPLDFSAAGSVVTVLEAQAYS LAPLKSDYDtI I IDTVPTPSVYTNNAIVASDYVMI PLQAEEESTNNIQNYI SYLIDLQEQ  171 297 290 128 193  **  ::*  .  .::::.  *  .  B. subtilis P1 Fplasmid pTP228 pSM19O3S  LNTDLMIEGVLLTMLDARTNLGIQ- -VIEEVKKYFR-DKVYKTVIPRNVRLSEAPSHGK I SDEGCECQLATNIGFMSKLSNKADHKYCHSLAKEVFGGDMLDVFLPRLDGFERCGESFD VDLKGFEPDVRILLTKYSNSNGSQSPWMEEQIRDAWG- SMVLKNVVRETDEVGKGQIRMR RKVEARFLITRKIEMATMLN VLKESIKDTG-VKAFRTAITQRQVYVKSILDGD FNPGLDMIGFVPYLVDTDSATIKSN- -LEELYKQHKEDNLVFQNI IKRSNKVSTWSKNG  B. subtilis P1 Fplasmid pTP228 pSM19035  PIILYDPRSR GAEVYLDLAKEVAANG TVISANPATYVGSADALKNARIAAEDFAKAVFDRIEFIRSNTVFEQAIDQRSSTGAWRNALSIWEPVCNEIFDRLIKPRWEIR SVFESSDGAAKG EIEILTKEIVRIFE -ITEHKGYDK KVLSMYKNVFFEMLERIIQLENEKE  -  227 357 349 180 250  253 398 391 206 284  9  Fig. 3. Sequence alignment of chromosomal ParA proteins. Sequence alignment was generated by Clustal W software v. 2.0.8. Shading indicates protein motifs; Red shading, Walker A motifs; purple shading, Walker A’ motifs; green shading, Walker B motifs; blue shading, residues implicated in Soj DNA binding activity by Hester and Lutkenhaus (2007).  10  B. subtilis T. thermophi lus S. coelicolor C. crescentus P. aeruginosa H.pylori M. smegmat is P.putida V. cholerae B. subtilis T. thermophilus S. coelicolor C. crescentus P. aeruginosa H.pylori M. smegmat is P.putida V. cholerae  ML 2 MS 2  MGSGQNKGQGVVQKPNVSRETWDSATSTWVTDAAMDTPIAAEAEQATRVLHSSARRQLPR 60  TSVNLGACLAYIGICRVZ4WD1tPQGNATSGLGIEKADVEQ 58 TAINLAYLRLGKRV*flpLPPQGNATSGLGVR- -AER 59 TTVNLAAsLALHGARvt0vrn,DpQGNAsTALGI DHHADVP 54 TAINLGTALAACGERVi4D.tPQGNCSTGLGIGRTQRRT 62 !TCINLAASLVATKRRV.I4PLDPQGNATTGSGIDKHNLEH 58 TAvNLAAsLAAnEKKIzz;DFnpQANATssLGFRRoKIoY 59 AVNVAAALALQGLRTWflIJDPQGNASTALS I EHRPGTP 120 TC INLAASLAATKRRVI t’IDLPPQGNATMGSGVDKHELEH 58 3 TCINLAASMAATKRKVLflDLDPQGNATMASGVDKYQVDS 58  -MGKI IAITNQ RAKVRRIALi MVVAN( ANPLRVLAIAN -MAKVFAIANQ -MMNEI IAVANQ PERQRVFT] -MAKVFAIAN -MGKIVAIAN( -  -  -  -  -  • *fl k*******  B. subtilis T. thermophilu S .coelicolor C. crescentus P. aeruginosa H.pylori M. smegmat is P.putida V. cholerae  -  .:  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  **.*  **.*.  .  *  *  .*.*  *. .****  LNTDLMIEGVLLTMLDATNLGIQVIEEVK1CYFRDKVYKT LNPRLRLLGILVTMYDGaTLLAQQVEAQLRAHFGEKVFWT LNPTLHVSTILLTMYDG:TRLASQVADEVRSHFGEEVLRT  :  .:  **  *  SIVRLSEAPS SJVRLAEAPS 3VRI SEAPS SWRVSEAPS SIVRLAEAPS 3VKLAESPS 3VKVSEAPG 41 RLAEAPS 4VRLAEAPS  224 217 221 230 224 236 288 224 224  *  HGKPIILYDPRSRGAEVYLDLAKEVAANG FGKTIAQHAPTSPGAHAYRRLAEEVMARVQEA YGQTVLTYDPGSSGALSYLEA.kREIALKGVGVTYDATHAHLGAQNDPSMVEGTQ FGKPVLLYDLKCAGSQAYLKLAREVISRERDRQAKAA FGMPALVYDKQSRGAIAYLALAGELVRRQRAKGRAàTA FGKPILLYDIKSNGSIAYQKLAQSILQG YGMTILNYDPGSRGALSYLDASREIAERGAPPRQQ FGMPALAYDKQSRGALAYLALAGELVRRQRRPSRTAQTT HGKPANYYDKQSAGAKAYLALAGEMLRREEIPA *:  171 164 168 177 171 176 235 171 171  *.  VIP VIP SIP LNPRLEIQGVVLTMYDRNSLSEQVAKDVRAHFGDKVYDA VIP LNPTLKIEGLLRTMYDP:ISLTNDVSAQLQEEFGDTLYST VIP TNPIKLKIRGFLPTMHVPLNLTKGVLAELFICYFDSEFFRDSATGEYIMIP VIP LNPELSVSTVILTMYDGPTKLADQVAEDVREHFGDKVLRT LNPELKIEGLLRTMYDPgLSLNNDVSAQLKEHFGPQLYDT VIP VNDNLKIEGILRTMYDPNRLANEVSDQLKKHFGSKVYRT VIP *  113 105 110 117 113 118 176 113 113  *:  .  AVKQN- 1CPPSLGLLTINALTASDSVVI PVQCEYYALEGLSQLLNTVRLVQKH ALRDEG- 4JAPPSLSPLTLNALAAAEGVVVPVQAEYYALEGVAGLLATLEEVRAG AYEQP ICPPSLGLLTVNALVAGQEVLI PIQCEYYALEGLGQLLRNVDLVRGH Al RANGP4JCPPSLNVLTVNAMTAADAVFVPLQCEFFALEGLTQLMRTI ERVRGS PIREN- -4jCPPSLSMLTVNALTAADGVIIPMQCEYYALEGLTDLMNSIQRIGQL SVVKL- -SPPALGPLTINSLSAAHSVIIPIQCEFFALEGTKLLLNTIRMLQKS ELKNHN- PJCPPSLGLLTINALVAAPEVLIPIQCEYYALEGVGQLLRNIEMVKAH PIRDN- -4CPPSLSMLTLNALVASDGVI IPMQCEYYALEGLSDLVDNIKRIAAR SVRDN- JCPPSLNLLTINANAAADSVLVPMQCEYFALEGLTALMDTISKLAAV  *  B. subtilis T. thermophilus S. coelicolor C. crescentus P. aeruginosa H.pylori M. smegmat is P.putida V. cholerae  -  •  .*  B. subtilis T . the rmophilus S. coelicolor C. crescentus P. aeruginosa H.pylori M. smegmat is P.putida V. cholerae  ****:  CVYDILVDDADVIDIIKAT-TVENL]JVIPATIQLAGAEIELVPTISREVR-LKRALE -LR- -E GVYHLLQGE- PLEGLVHPV- -DGFHLLPATPDLVGATVELAGAPT- -A-LQRAIT SIYDVLVESRPLSEVVQPVPDVEGLFCAPATIDLAGAEIELVSLVARESRTLYDVLMGEAPVVDAAVKT -ELPGLDVI PADADLSGVEIELGQTARRSYRLRDALE LRNALA S IYDVLTGECNLAEANQFS -EHGGYQLLPANRDLTAAEVVLLEMDMKENRDIYHVLIGRKQISQVILKT-QMPFLDLVPSNLGLAGFEKTFYDSQDENKRGELMLKNALG -LRTALA SSYEVLIGEIPVEEALQQSPHNERLYCIPATIDLAGAEIELVSMVAREGRSVYDLLIGECDLAQAMHYS -EHGGFQLLPANRDLTAAEVVLLEMQVKESR-LRNALA -LKNALA TAYELLVEDAPFDQVVCRK-TTGHYDLIAANGDVTAAEIKLMEVFAREVR*:*  B. subtilis T. thermophilus S. coelicolor C. crescentus P. aeruginosa H.pylori M. smegmat is P.putida V. cholerae  *::*  :*: .  253 249 275 267 262 264 323 263 257  *  11  Walker A/B (P-loop) ATP binding motifs in the ParA proteins (Koonin, 1993). An ActinlHsp7o ATP binding motif in ParA characterizes type II par loci (Bork et al., 1992). Type I can be further divided based on the type of protein encoded byparB. Type Ia loci encode large ParB (>310 amino acid) with helix-turn-helix (HTH) DNA binding motifs, and type lb loci encode smaller ParB proteins (<140 amino acid) with ribbon helix DNA binding motifs. The general function of most plasmid encoded Par proteins is thought to be promoting equal distribution of plasmids to daughter cells (Ebersbach and Gerdes, 2005; Schumacher, 2008).  4.1 ParA and ParB Proteins from P1 and F Plasmids.  The best studied ParAB system is that from the temperate P1 prophage (type Ia) which can exist as a low copy number plasmid in E. coli (Prentki et al., 1977). P1 ParA-ATP dimers interact with ParB multimers bound to parS sequences and form the ‘partition complex’ (Bouet and Funnell, 1999). The partition complex is observed in vivo by fluorescence microscopy to localize ParB, ParA and plasmid DNA to specific sites in the cell (Erdmann et a!., 1999). ParA ADP binds specifically to the par operon operator (parOP) to negatively regulate the expression of the par operon by acting directly as a repressor of transcription (Davey and Funnell, 1994; Davis et al., 1992). ParA-ADP autoregulation plays an important role in the proper expression of ParA and ParB proteins and the maintenance of plasmid P1. Overexpression of either, or both, proteins causes a loss of plasmid stability (Funnell, 1988). ParA contributes to the formation of partition complexes in vivo (Erdmann et a!., 1999; Li et a!., 2004). In the absence of ParA, plasmids bound by ParB multimers mislocalize, often failing to form plasmid complexes at the cell centre. Furthermore without ParA, plasmid DNA bound by ParB does not segregate to opposite cell quarters and is inherited by only one progeny cell. This results in plasmid loss (Li et a!., 2004). At the partition complex the ATPase activity of ParA-ATP is thought to be needed to eject paired ParB-parS complexes away from each other towards opposite cell poles, although the mechanisms of how ParA and ParB move paired plasmids to opposite poles is not well understood (Edgar et a!., 2006). In vitro ParA-ATP interacts with ParB-parS DNA complexes (Bouet and Funnell, 1999). In vitro ParA ATPase activity is stimulated in the presence of DNA and further increased in the presence of ParE and parS DNA (Davis et al., 1992). The Par system of the F plasmid of E. coli (S0pAB, type Ia) functions similarly to the P1 Par system (Surtees and Funnell, 2003). Disruption of SopA, SopB or the parS equivalent sopC 12  leads to a random distribution of F-plasmids in E. coli (Hirano et al., 1998). SopA-ADP binds to the sop promoter and the Sop proteins have been demonstrated to participate in autoregulation of their genes (Hirano et a?., 1998). The largest difference between SopA and ParA is that SopA-ATP forms protein filaments in the absence of DNA that vary in size (200-500 nm long, 10-40 nm wide) (Bouet et al., 2007). SopA filamentous structures have been observed in vivo but the role they play to support plasmid stabilization is unclear (Hatano et al., 2007). The autoregulation activities of type Ia ParA proteins such as P1 ParA and SopA is due to a domain (‘-100 residues) N-terminal to the Walker A motif (Ebersbach and Gerdes, 2005; Moller-Jensen et a?., 2000; Surtees and Funnell, 2003). This N-terminal DNA binding motif is absent from type Ib, and all chromosomal ParA proteins, consistent with reports that only type Ia ParA proteins are able to regulate the expression of their own operon (Ebersbach and Gerdes, 2005; Moller-Jensen eta?., 2000; Surtees and Funnell, 2003).  4.2 Type II Par System of Plasmid Ri.  The Par proteins of the E. coli Ri plasmid, ParM (ParA) and ParR (ParB), constitute a type II Par system (Ebersbach and Gerdes, 2005; Moller-Jensen et a?., 2000). As mentioned there is little similarity between type II ParA proteins (actin/hsp7o motif) and type I and chromosomal ParA proteins (WalkerAB motif) except that they are all ATPases. However ParR is similar to the ribbon helix type lb ParB proteins. Functionally there are major differences between the par loci of type II and type Ia plasmids (Ebersbach and Gerdes, 2005). ParR (ParB), and not ParM (ParA) is responsible for efficient autoregulation of the Ri par genes. ParR is responsible for plasmid pairing in vitro and in vivo. ParM has been shown to form filaments in vitro (Moller-Jensen et a?., 2002; van den Ent et a?., 2002), and similar filamentous structures have been observed in vivo (Moller-Jensen et a?., 2003). In vitro filamentation of ParM is stimulated by interaction with ATP or non hydrolysable ATP analogues. The long straight filaments can reach lengths up to 1 jim and are structurally similar to filaments formed by F-actin (Orlova et a?., 2007). It has been proposed that the insertional polymerization of ParM-ATP monomers at the ParM-ParR interface causes the ParM filaments to grow bidirectionally, thereby pushing the two plasmids apart toward opposite ends of the cell (Garner et a?., 2007; Moller-Jensen et a?., 2003; Popp et a?., 2008).  13  5 Chromosomal ParA and ParB Proteins.  Chromosomally encoded ParA/Soj and ParB/SpoOJ do not fit well in the plasmid-based nomenclature system which is based on the characteristics of plasmid ParA and ParB proteins. Chromosomal ParAJSoj proteins lack the N-terminal HTH DNA binding domain, and therefore are similar to ParA proteins from type lb Par systems with respect to domain organization (Ebersbach and Gerdes, 2005; Gerdes eta!., 2000; Moller-Jensen eta!., 2000). Chromosomal ParB/SpoOJ more closely resemble ParB proteins from type Ta (HTH DNA binding motif), and are very different from the small ribbon helix domain proteins in type lb loci (Ebersbach and Gerdes, 2005; Gerdes et al., 2000; Moller-Jensen et a?., 2000). Of the major prokaryotic phyla only Thermatoga are completely without any species that encode chromosomal ParA and ParB proteins (Leipe et a?., 2002). However within certain phyla chromosomal ParA and ParB proteins are absent from some species, most notably from E. coil and its close relatives. Recent studies on the chromosomally encoded Par proteins from species other than B. subtilis will be briefly reviewed.  5.1 Par Proteins from Mycobacterium smegmatis, Pseudomonas aeruginosa, Pseudomonasputida, Vibrio cholerae, and Helicobacter pylon.  Consistent with a role in DNA segregation, P. putida or V. cholerae chromosomal Par systems can each stabilize a plasmid in the absence of selection in E. coii (Godfrmn-Estevenon et a?., 2002; Yamaichi et a?., 2007). In each case plasmid stabilization is dependent on both Par proteins, and having a parS sequence on the plasmid. This indicates that ParA and ParB interact with a parS plasmid and act independently or with cellular factors present in both E. coil to increase plasmid stability. However, in most of these experiments complete plasmid stabilization is not observed. This contrasts with plasmid ParAB systems which maintain plasmids in growing cultures over large numbers of generations. This indicates that chromosomal ParAB proteins are not as effective at plasmid stabilization as their plasmid encoded counterparts. V cho?erae is unusual in that it possesses two chromosomes that encode different Par systems, and different parS sequences which are responsible for segregation of the chromosome on which they are encoded (Saint-Dic et a?., 2006; Yamaichi et a!., 2007). The generation of a V. cholerae AparBl, or zparAB1 deletion on the large chromosome has not been successful 14  despite extensive efforts (Saint-Dic et al., 2006). This indicates that the pan locus of the large chromosome might be essential. An increased frequency of anucleate cells has been reported in parA and/or parB deletions or overexpression strains in M smegmatis, P. putida, and P. aeruginosa (Godfrmn Estevenon et a!., 2002; Jakimowicz et a!., 2007a; Lewis et a!., 2002). In these vegetatively growing par mutant cultures, anucleate cells constitute a minority (usually <10%) of the population, and for each species neither protein is essential. Therefore, ParA and ParB proteins are not the primary chromosome segregation machinery, and play a dispensable role in maintaining proper chromosome segregation in growing cells. M smegmatis and P. aeruginosa are reported to grow slower upon deletion or overexpression of Par proteins (Bartosik et a!., 2004; Jakimowicz et a!., 2007a; Lasocki et a!., 2007). In V cholerae the large chromosome is mislocalized in AparA and AparS mutants as observed by fluorescence microscopy (Saint-Dic et a!., 2006; Yamaichi et a!., 2007). For all the organisms in this section, ParB has been shown to bind parS (Bartosik et a!., 2004; Jakimowicz et a!., 2007a; Lee et a!., 2006; Yamaichi et a!., 2007). In vitro the presence of ParA from M smegmatis increases the binding of ParB to parS indicating a ParA-ParB interaction (Jakimowicz et a!., 2007a). Purified H. pylon Soj and SpoOJ co-immunoprecipitate in vitro. Moreover, the inclusion of H pylon SpoOJ in reactions with Soj and ATP leads to Soj ATPase stimulation (Lee et a!., 2006). An interaction between ParA and ParB from P. aeruginosa has been inferred from yeast two hybrid assays (Bartosik et a!., 2004). While ParA and ParB from these diverse organisms have similar activities, disruption of their par genes results in different DNA segregation and growth phenotypes. Thus the chromosomal Par systems appear to play diverse roles in vivo, or have similar roles with diverse effects on each species physiology.  5.2 Streptomyces coelicolor.  S. coe!ico!or sporulates by forming aerial hyphae. In the aerial hyphae multiple chromosomal replications creates a multigenomic compartment which undergoes synchronous septation at evenly spaced sites to form unigenomic prespore compartments. These compartments then develop into mature spores (Chater, 2001; Claessen eta!., 2006). Transcription of the panAB operon is dramatically upregulated upon sporulation (Jakimowicz et a!., 2006; Kim et a!., 2000). In par gene mutants, hyphal prespore compartment sizes are 15  disturbed, and a high frequency of anucleate spores are observed (26%) (Jakimowicz et a?., 2007b). ParB binds to 20 parS sites near oriC (Jakimowicz et a!., 2002), and ParB-GFP has been observed by fluorescence microscopy to form DNA-associated foci in vivo (Jakimowicz et a?., 2005). In the absence of ParA, the ParB-GFP foci are less condensed and mislocalized; in addition FtsZ polymerization (Z-rings) is also mislocalized (Jakimowicz et al., 2007b). In vivo, ParA-GFP forms helical structures in hyphal tips with a pitch that corresponds to the length of a prespore compartment (Jakimowicz et a?., 2007b). ParAK44A-GFP, (ATPase deficient) still forms helical structures but behaves as a null mutant (anucleate prespore compartments observed). These results indicate that ATPase activity is not important to form ParA structures but it is crucial for proper chromosome segregation during sporulation (Jakimowicz et a?., 2007b). S. coelicolor ParA ATPase is stimulated in vitro by ParB, and the presence of ParA increases in vitro DNA binding efficiency of ParB to parS sequences (Jakimowicz et a?., 2007b). ParA but not ParAK44A interacts with ParB as indicated by E. coli two hybrid assays (Jakimowicz et a?., 2007b). An interesting model for the role of ParA and ParB from S. coelicolor has been presented (Jakimowicz et a?., 2007b). Upon entry into sporulation the par operon expression is upregulated. ParB binds parS, and ParA forms helical structures in the hyphal tips. The ParA condenses ParB-parS structures and act as a ‘ruler’, ensuring appropriate DNA localization which produces a high frequency of cells with a single chromosome upon septation. This model postulates that ParA positions both ParB-DNA complexes and FtsZ polymerization within hyphal tips.  5.3 Caulobacter crescentus.  Caulobacter crescentus is the only reported bacterial species in which both parA and parB genes are essential (Mohl and Gober, 1997; MohI eta?., 2001). C. crescentus is unlike most bacteria in that every cell division is a developmental process which produces two morphologically different cells (Laub et a?., 2007). To create mutants, second copies ofparA and/or parB were inserted in the genome under control of an inducible promoter to allow the deletion of the genes (Easter and Gober, 2002; Mohl and Gober, 1997; MohI eta?., 2001). When ParB is depleted cells fail to divide, and instead grow into long filaments. If these cells divide, the placement of the division septa is irregular and results in anucleate minicells (Easter and Gober, 2002; Mohl eta?., 2001). Overexpression of ParA results in a phenotype similar to 16  ParB depletion (Easter and Gober, 2002). The overproduction of both ParA and ParB results in viable cells, but cultures of these cells have a high frequency of anucleate cells (Easter and Gober, 2002). These results indicate ParA and ParB have an essential role and function prior to cell division. In vitro ParB binds specific sequences found near the origin of replication (Mohl and Gober, 1997). In vitro ParA-ATP, but not ParA-ADP interacts with ParB-DNA complexes and ejects ParB from DNA (Easter and Gober, 2002). ParA-ATP can also interact with ParB and prevent ParB-DNA interactions (Easter and Gober, 2002). The ATPase of ParA is stimulated in vitro by ParB by acting as a nucleotide exchange factor (Easter and Gober, 2002). Cells depleted for ParB grow as filaments which have high concentrations of ParA-ADP, so it is thought that in the absence of ParB the increased level of ParA-ADP inhibits cell cycle progression (Easter and Gober, 2002; Figge et a!., 2003).  6 Bacillus subtilis.  The soj/spoOJ locus of B. subtilis was one of the first chromosomal Par systems to be identified (Ireton et a!., 1994). SpoOJ was recovered in screens to identify genes which blocked sporulation initiation (Mysliwiec eta!., 1991). A number of studies have investigated Soj and SpoOJ function during vegetative growth as well as during sporulation.  6.1 Role of Soj and SpoOJ in Vegetative Growth  The deletion of spoOJ results in a low levels of anucleate cells during vegetative growth (0.1 % anucleate cells; Ireton et a!., 1994; Lee and Grossman, 2006; Ogura et a!., 2003). The same low level of anucleate cell phenotype is also present when the whole operon is deleted, but not in cultures of tssoj cells (Ireton et a!., 1994; Lee and Grossman, 2006; Ogura et a!., 2003). These results are partially consistent with a DNA segregation role for Soj and SpoOJ such as that established for plasmid ParA and ParB proteins, but the Soj and SpoOJ cannot be the primary determinant of chromosome segregation in vegetative cells. In contrast, consistent with a role in chromosome segregation, Soj and SpoOJ significantly stabilize a plasmid with a parS sequence in growing B. subtilis and E. coli cultures in the absence of selection (Hester and Lutkenhaus, 2007; Lin and Grossman, 1998; Yamaichi and Niki, 2000). In vitro SpoOJ binds to DNA with a preference for binding to parS sequences (measured to be approximately 40:1, parS DNA:non-specific DNA, Lin and Grossman, 1998). SpoOJ 17  spreads on DNA to form SpoOJ multimers on DNA in vivo and in vivo, this spreading was shown to be dependent onparS in vivo (Murray et al., 2006). Fluorescence microscopy observation of B. subtilis cells that express SpoOJ-GFP shows foci distributed near oriC (Lin et al., 1997) and SpoOJ-GFP foci correspond to the number of replicated chromosomal origins (1:1 ratio of SpoOJ-GFP foci to oriC; Glaser et al., 1997). Shortly after cell division SpoOJ-GFP foci associated with oriC are found at one quarter and three quarter positions relative to cell poles. However, SpoOJ does not direct chromosomal segregation because the oriC region still localizes correctly at cell quarters in the absence of SpoOJ (Webb et a!., 1998). In addition, placement of parS sequences at sites away from oriC is not sufficient to localize that portion of the chromosome to cell quarters (Lee et a!., 2003). In iXsof mutants, numerous smaller less intense foci of SpoOJ-GFP are observed throughout the cell but still associated with DNA (Marston and Errington, 1 999a) but the foci associated with oriC are absent. These data indicate that Soj affects an aspect of SpoOJ that is important for its distribution in vivo, although it is not clear what role this localization plays. In B. subtilis cells that express Soj-GFP it appears to be located primarily at cell poles or newly formed septa, with some foci within the cytoplasm (Murray and Errington, 2008; Quisel eta!., 1999). Disruption of spoOJ leads to Soj-GFP association along all chromosomal DNA (Autret et a!., 2001; Autret and Errington, 2003; Marston and Errington, 1 999a; Quisel et a!., 1999), a pattern that is strikingly different from the polar localizations observed in wild type cells. Soj-GFP is distributed in the cytosol away from the chromosome in a AminD strain, but is associated with the nucleoid in a spoOJ/minD double deletion mutant (Murray and Errington, 2008). These data were interpreted to mean that Soj is directed to cell poles and division septa by MinD, and SpoOJ. In the absence of SpoOJ, Soj associates with chromosomal DNA. Effects of mutations in the Soj Walker motif on Soj-GFP distribution have been reported (Autret and Errington, 2003; Murray and Errington, 2008; Quisel eta!., 1999). In cells with iXsoj and A(soj-spoOJ), ectopically expressed SojKl6A and SojDl25A GFP fusion proteins are found uniformly throughout the cytosol rather than at the poles, in foci, or distributed on DNA (Murray and Errington, 2008; Quisel et a!., 1999). These point mutants affect invariant residues in the Walker A (K16) and Walker B (D125) motifs and would be expected to confer a loss of ATP binding. In Asof and i(soj-spoOJ) cells SojG12V-GFP is observed at the cell poles and as cytosolic foci (Autret and Errington, 2003; Murray and Errington, 2008; Quisel eta!., 1999). SojG12V-GFP is not observed to associate with chromosomal DNA under any condition. In a Asof background SojD4OA-GFP is detected exclusively as bright foci that completely colocalize 18  with SpoOJ-YFP foci on the chromosome, but not at poles or division septa (Murray and Errington, 2008). In I(soj-spoOM cells SojD4OA-GFP is distributed along all chromosomal DNA in a pattern similar to wild type Soj-GFP in the same background. The expression of SojD4OA in a wild type or AspoOJ background results in a low sporulation frequency, but the sporulation frequency is 100 fold higher than the sporulation frequency when wild type Soj is expressed in a zspoOJ mutant (0.03% vs. 2 x 10%) (Murray and Errington, 2008). This indicates that SojD4OA is less effective at inhibiting sporulation than is wild type Soj in a AspoOJbackground. For the Soj Walker motif mutants (SojK16A, SojK16Q, SojD125A, S0jG12V) loss of sporulation inhibition is associated with loss of the ability to form bright foci, or associate with DNA (in AspoOJ strains). This indicates a role for nucleotide binding or hydrolysis in sporulation inhibition by Soj, and supports the idea that when Soj is bound to the chromosome, sporulation in blocked. In vegetative growth, wild type B. subtilis stained with DAPI show a compact chromosomal DNA staining pattern when observed by fluorescence microscopy (Autret et a!., 2001; Ogura et al., 2003). Vegetatively growing AspoOJor cells that overproduce Soj show chromosomes that appear abnormally elongated and diffuse (Autret et a!., 2001; Ogura et a!., 2003). These observations have been used to suggest an overall decrease in chromosomal DNA structure in mutants lacking Soj or SpoOJ that correlates with increased frequencies of anucleate cell in AspoOJ, and t(soj-spOJ) mutants (Lee and Grossman, 2006), and minicell production in A(soj-spoOJ) mutants (Ogura et a!., 2003). Chromosomal DNA structure in B. subtilis is affected by multiple proteins. Smc is proposed to compact chromosomal DNA (Britton et al., 1998). Asmc cells have slightly elevated rates of anucleate cells and of cells that place division septa through chromosomal DNA (—5% total) instead of between chromosomes (Lee and Grossman, 2006). In a Asmc background, deletion of either soj, spoOJ, or both results in 27-30% of the cells being anucleate, and division septa are observed over chromosomes rather than between them (Lee and Grossman, 2006). In a i’\smc background the effect of a so] or a spoOJ deletion on the phenotype is equal (Lee and Grossman, 2006). This differs from a wild type background where i\spoOJ, but not /Xsoj, contributes to anucleate cell production (Ireton et a!., 1994; Lee and Grossman, 2006; Ogura et a!., 2003). Two studies have reported that disruption of one or both genes of the soj/spoOJoperon alters the DNA:protein ratio in vegetative B. subtilis cultures. One study directly analyzed the protein and DNA content from cultures (Lee and Grossman, 2006), and the other study analyzed 19  the number of chromosomal origins per cell by flow cytometry (Ogura et a!., 2003). Both groups reported that there is overinitiation of chromosomal DNA replication in a AspoOJ mutant. However, Lee et a!. detected similarily high DNA:protein ratios in Asoj and A(soj spoOJ) mutants, whereas Ogura et al. detected a wild type DNA:protein ratio only in a A(soj spoOJ) strain. Elevated DNA:protein ratios similar to AspoOJ strains were seen by Ogura eta!. when Soj was overproduced but when both Soj and SpoOJ were overproduced the DNA:protein ratios returned to levels observed in wild type strains. The discrepancy between the two groups provides two different possibilities for the role of Soj in relation to replication. On the one hand, the absence of Soj or SpoOJ may lead to increased replication initiation, whereas on the other hand, Soj (in the absence of SpoOJ) may function to stimulate replication initiation and this activity is antagonized by SpoOJ. While both results indicate a role of the soj/spoOJoperon in properly timed DNA replication, the contradictory results make the role unclear.  6.2 The Role of Soj (ParA) and SpoOJ (ParB) Proteins in B. subtilis Sporulation.  As its name suggests disruption of spoOJ blocks sporulation initiation prior to asymmetric septation (Ireton et a!., 1994). This block in sporulation is also characterized by low transcription of spollA, spollE, and spoliG operons (Ireton eta!., 1994; Quisel and Grossman, 2000). SpollA and spoJIG operons encode forespore and mother cell compartment specific sigma factors, respectively (Clarke and Mandelstam, 1987; Trempy eta!., 1985). SpollE encodes a protein required for properly timed activation of the forespore-specific  cYF  encoded by  the spollA operon (Arigoni eta!., 1996). Deletion of both soj and spoOJ results in wild type sporulation frequencies, and the restoration of spollA spollE, and spoliG transcription (Ireton et ,  al., 1994; Quisel and Grossman, 2000). Disruption of soj alone does not produce an effect on sporulation efficiency (Ireton et a!., 1994). The available evidence indicates that SpoOA phosphorylation (Spo0A’P) is not affected by deletion of spoOl Repression of the abrB gene in stationary phase and during sporulation requires Spo0AP, and increased transcription of the spoOA gene upon entry into sporulation is also dependent on Spo0A—P levels. In /spoOJ strains (sp&), both abrB repression and spoOA transcription are similar to wild type B. subtilis (Cervin et al., 1998). Immunoprecipitation of DNA chemically crosslinked to Soj in vivo found that Soj was associated with spollA spollE, and spoliG (Quisel et a!., 1999). The amount of specific stage II ,  DNA crosslinked to Soj increased in the absence of SpoOJ. This result supports the idea that Soj 20  bound to the chromosome represses transcription which blocks sporulation, and SpoOJ modulates Soj chromosome association. The correlation between a chromosomal DNA distribution of Soj and sporulation inhibition is supported by analysis of divlB mutants (Real et al., 2005). DiviB (a homologue of FtsQ in Gram-negative bacteria) is a bitopic membrane protein which interacts with multiple components of the division apparatus, and promotes its stability (Daniel eta!., 2006). When DivIB levels are reduced by -30% (divIB8O mutation) sporulation is inhibited, SpoOJ-GFP is found in scattered small foci (very similar to Asof mutants), and Soj-GFP is chromosomally distributed (as described for AspoOJ mutants). The sporulation inhibition in cells with reduced DivIB levels is rescued by deletion of the soj/spoOJoperon and more specifically by deletion of soj (Real et al., 2005). Thus like deletion of spoOJ, lowering DivIB concentrations in the cell results in the distribution of Soj on DNA and sporulation inhibition. Murray and Errington recently found (personal communication; in press, 2008) that in a AspoOJ background, DnaA-dependent replication initiation increased. The same trend was observed for SojD4OA in a wild type or \spoOJ background. In a IX(soj-spoOJ) background replication initiation was also slightly elevated. When mutant Soj proteins (S0jG12V, SojKl 6A) that do not localize to the chromosome were expressed replication initiation was less stimulated than when wild type Soj was expressed. These data indicate that Soj might either inhibit DnaA-dependent replication initiation, or stimulate it depending on Soj localization in the cell. Thus when Soj is bound to the chromosome it is not able to inhibit the activity of DnaA and the chromosome is replicated, but when Soj is antagonized by SpoOJ or mutated so that it no longer binds DNA, it inhibits replication initiation by DnaA. In support of this model, Soj-DnaA interactions were detected in vivo. The study suggests that Soj localization to the chromosome results in over initiation which in turn activates a DNA replication checkpoint (Sda), Sda then inhibits sporulation possibly by inhibiting KinA. In support of this model the spo phenotype of a AspoOJmutant was rescued by an additional sda mutation (a spoOJsda double deletion mutant was spo), indicating that Sda might be responsible for the sporulation inhibition observed in /xspoOJ strains). This model indicates that a key question regarding Soj and SpoOJ is what affects the DNA binding properties of Soj.  21  7 Structure of ParAISoj Proteins.  7.1 T. thermophilus  While Soj (ParA) and SpoOJ (ParB) from Thermus thermophilus have been studied in vitro, nothing is known about how Soj and SpoOJ affect T thermophilus physiology. SpoOJ from this organism is a site-specific DNA binding protein, which constitutively forms dimers (Leonard et a!., 2004). The two HTH DNA binding motifs of the SpoOJ dimer align in a manner that suggests it binds to successive major grooves in the DNA helix (Leonard et a?., 2004). The crystal structure of T thermophilus Soj-ADP is monomeric (Leonard et a?., 2005, Fig. 5A) and Soj dimers are formed only in the presence of ATP (Leonard et a?., 2005). A point mutant of Soj (S0jD44A) was reported to be required for the determination of a Soj ATP dimer structure (Leonard eta!., 2005, Fig. 4A). Soj-ATP binds DNA with little sequence specificity, and can form multimeric Soj structures on DNA. The ATPase activity of Soj is stimulated by SpoOJ in vitro (Leonard et a?., 2005). Soj from B. subti!is is similar to Soj from T. thermophi!us at the level of amino acid sequence and motif organization (Figs. 3, 4B). BlastP alignment of B. subtilis vs. T thermophi!us sequences, and the inverse alignment indicate 50% identity and 65% similarity between the two. For this reason the structure of B. subtilis and T thermophilus Soj are likely very similar. The sequence identity is the strongest close to the ATP binding pocket. Strikingly Soj from either T thermophi?us or B. subti!is does not have a classic DNA binding motif. Recently Hester and Lutkenhaus (2007) used the structure of T. thermophi!us Soj to identify residues that when mutated in B. subtilis Soj resulted in loss of DNA binding  in vivo and in  vitro, indicating that these residues are in a motif which is responsible for DNA binding .While Soj from T thermophilus has not been well characterized in vivo, I used the structure of this protein as a model for studies of B. subtilis Soj due to their sequence similarity.  7.2 Plasmid pSM19O35 ö Protein.  Recently a structural and functional characterization of the dimeric 6 protein (ParA) from the Streptococcus pyogenes plasmid p5Ml9035 was published (Pratto eta?., 2008). The structure of the 6 protein differs from that of T thermophi?us Soj. The 6 protein made a ‘U like’ dimer where the nucleotide binding pockets were solvent-exposed.  In vitro the 6 protein 22  Fig. 4. Motifs of B. subtilis and T. thermophilus Soj. (A) The structure of Soj from T thermophilus. The Soj dimer structure was solved by x-ray crystallography of SojD44A bound to ATP (Leonard et al., 2005). The Soj monomer structure was solved by x-ray crystallography of wild type Soj bound to ADP. For each structure nucleotides and Mg 2 are coloured orange, x-helices are coloured yellow, 13-sheets are coloured turquoise, and motifs are coloured as follows; Walker A motifs (red), Walker A’ motifs (puruple), Walker B motifs (green), and residues involved in DNA binding by B. subtilis Soj (blue). The residues equivalent to positions G12 and D40 in B. subtilis Soj are indicated as space filled residues in the T thermophilus Soj structures. Structure images were generated using Rasmol. (B) Linear representation of Soj motifs. The sequences, and positions of the Walker A motifs (red), Walker A’ motifs (purple), Walker B motifs (green), and residues involved in DNA binding by B. subtilis Soj (blue) are indicated for Soj from both B. subtilis and T thermophilus.  23  A  Dimer  Monomer  90  900  B B. subtilis 1 11-18  35-40  120-125  189  218  N-I• KGGVGKTT LLVDID WalkerA A’  DYIIID Walker B  253  1- C  DNA binding motif  T. thermophilus 1 15-22 NH  3  >)c  KGGVGKTT LLVDLD Walker A A’  118  DLVIID Walker B  182  211  249  1- C  R DNA binding motif  24  is primarily dimeric in the presence of either ATP or ADP, and also in the absence of nucleotide. When the invariant lysine of the Walker A motif was mutated so the protein no longer bound or hydrolyzed ATP, the protein was still observed to dimerize in vitro. The formation of the dimer in all conditions was stabilized by intermonomer interaction of a large hydrophobic patch, and by several intermonomer salt bridges. The ö protein along with co protein (ParB) were required for stabilization of the plasmid pSM 19035. In B. subtilis cells harbouring pSM19035 observed by fluorescence microscopy the ö protein fused to GFP was observed to make dynamic structures that made spirals and associated with cellular DNA. BlastP alignment of B. subtilis Soj vs. ö protein sequences, as well as the inverse alignment, indicated 28% identity and 49% similarity between the two. The sequence identity was the strongest for the Walker motif sequences, and residues which are close to the ATP binding pocket in the 6 protein structure (Fig. 3). While the structure of the 6 protein is interesting, it does not share high sequence similarity with Soj from B. subtilis, and this structural information was not used the work described below.  8 Experimental Aim.  Previously the functions of Soj and SpoOJ in B. subtilis have been inferred through the behavior of Soj and SpoOJ in vivo, and by their contribution to sporulation initiation. These experiments have indicated that Soj prevents sporulation initiation, and is antagonized by SpoOJ. The role of ATP binding and hydrolysis has been suggested to be important to Soj function on the basis of mutants, but biochemical characterization of these activities had not been carried out. In addition, the association of Soj with DNA, observed by microscopy, correlates with inhibition of sporulation, and with a decrease in sporulation-specific gene transcription, but there had been no investigation of this property in vitro, To study the role of ATP and ATP hydrolysis, I started this work by examining the ATPase activity of B. subtilis Soj. The in vitro characterization of Soj and two Soj point mutants predicted to affect the ATPase activity (S0jG12V and S0jD4OA) was included to link their in vitro activities to the effect on sporulation. Experiments were carried out to investigate how adenine nucleotides affect association with DNA and SpoOJ in vitro. The experiments in the thesis indicated that Soj forms multiple complexes with DNA and that this property is dependent on ATP binding. The experiments contribute to a more complete characterization of the role of Soj in B. subtilis physiology. 25  MATERIALS AND METHODS  1 Standard Techniques.  Standard molecular biology techniques (precipitation of DNA, extraction of protein from nucleic acids, digestion of DNA with restriction endonucleases, ligation of DNA restriction fragments, SDS-PAGE of proteins, polyacrylamide and agarose electrophoresis of DNA) were performed as described (Sambrook et a!., 1989). The sources of enzymes were as follows; Restriction endonucleases, DNA ligase, and DNase I (Invitrogen), Pfu turbo DNA polymerase (Stratagene), Pfu DNA polymerase (Fermentas). All chemicals were reagent grade. Oligonucleotide primers were purchased from Alpha DNA (Montreal, QC).  2 Manipulation of DNA and Oligonucleotides.  Plasmid DNA and other DNAs used in this thesis are listed in Table 1. Primers and plasmid templates used to generate site-directed mutant plasmids are listed in Table 2. All DNA fragments used in this thesis and the primers and templates used to prepare them are listed in Table 3. For all molar DNA concentrations throughout this thesis, the values are given per base pair of DNA (assuming 650 Da!bp DNA) unless stated otherwise.  2.1 Site-Directed Mutagenesis.  Plasmids generated by QuikChange PCR reactions in this thesis are listed in Table 2. PCRs were composed of the indicated primer pairs (125 ng, -0.28 jiM final each), 250 jiM dNTPs, 1 unit Pfu Turbo DNA polymerase (Stratagene) in lx Pfu Turbo reaction buffer in a 50 jil reaction. The reactions were subject to 18 cycles of 30 s at 95°C, 1 mm at 55°C, and 4 mm at 68°C. The reaction products were treated with 10 units of DpnI for 1 hour. The DNA was ethanol precipitated, resuspended in 20 j.tl of water, and used to transform E. coli DH5cL. Mutations in plasmids recovered from transformants were verified by DNA sequencing (Nucleic acids and protein services (NAPS), UBC).  26  Table 1. Plasmids Used in this Thesis  Plasmida  Description  Reference or Source  pET2OSoj  pET2Ob-based Soj over-expression plasmid. pET2Ob-based SojGl2V over-expression plasmid generated by site directed mutagenesis PCR. pET2Ob-based SojD4OA over-expression plasmid generated by site directed mutagenesis PCR. pETl6b-based SpoOJ over-expression plasmid. Used to generate pETsp0OJQC. pETl6b-based SpoOJ over-expression plasmid generated by site directed mutagenesis to remove a NdeI site. pET2Ob-based SpoOJ overexpression plasmid.  (Cervin, et a!., 1998)  pET2OSojG12V  pET2OSojD4OA  pETl6spoOJ pET165p0OJQC  pET2OspoOJ pUC 19 pUCIIGtrpA pUCIIGBglIItrpA pUCIIGJ8 pUCA2trpA pJM5 134 P29  240 bp spoliG promoter fragment in pUCl9trpA. pUCIIGtrpA with g—*t at position -22 of the spoliG promoter. pUCIIGtrpA with 18 bp spacer, generated from pUCIIGBglIItrpA 200 bp A2 promoter fragment in pUCl9trpA. abrB promoterfragment containing plasmid. Bacteriophage ç 29 genomic DNA  This work  This work  (Glaser, et a!., 1997) This work  This work Invitrogen (Satola, et al., 1991) Gift, V. Mendoza This work (Dobinson, et al., 1985) (Greene, et al., 1996) Lab stock  a. all plasmids contained ampicillin resistance markers (amp’).  27  Table 2. Primers and Templates Used to Generate Site-Directed Mutant Plasmids by PCRs.  Plasmid  Primer  Primer Sequence  Template  pET2Osoj G 1 2V  soj Gi 2V-F soj G 1 2V-R soj D4OA-F sojD4OA-R OJQC-F OJQC-R  gaaccaaaaagtcggggtcggcaaaaca gccgaccccgactttttggttcgtaat gtagatattgctccgcagggaaatgcg tccctgcggagcaatatctaccagcag gggagtcatacgcctgagcctcttcaag gaggctcaggcgtatgactcccttttg  pET2Osoj  pET2OsojD4OA pET 1 6spoOJQC  pET2Osoj pET 1 6spoOJ  28  Table 3. Primers and Templates to Generate PCR DNA Fragments Used in this Thesis.  DNA  Primer Primer Sequence  Template  2 kb  2k-R gacctcgagttagctagtgcgttcatga 2k-F ctgaagcttgttaggcacacaagacaacg 1k-F gtcgaattcgctagaattgtttgcggctg 1 k-R cagctcgagttatagagccgctaccgct 500-F gcgtcacatatgccattaacgccaaat 500-R gctgcactcgagttccttttcctcaaa 300-F cgagtctagcggcactg 3 00-R gataggatagttaaagtc 100-F catcgtggaacaaatgag 1 00-R cttaactgcattgtgtaactactc 24-F gagtagttacacaatgcagttaag 1 00-R cttaactgcattgtgtaactactc 0J20-F gcgtcacatatggctaaaggccttgga 0J20-R gctgcactcgagtgattctcgttcaga  p29 genomic DNA  1 kb 0.5 kb 0.3 kb 0.1 kb 24 bp SpoOJ insert  29 genomic DNA p B. subtilis JH642 genomic DNA  29 genomic DNA c p29 genomic DNA no PCR (annealed) pET 1 6spoOJQC  29  2.2 Oligonucleotide Labeling.  32 (8.3 .tM, --6000 Ci 7 [ Primer l00-R (100 pmol) was incubated with 3.5 jil P]-ATP mmol’, GE Healthcare) and 5 units of polynucleotide kinase (Roche) in 10 il of lx Kinase P]-ATP 32 Buffer (Roche) at 37°C for 3 hrs. The primer was separated from unincorporated [‘y and ADP by use of a nucleotide removal kit (Qiagen). The primers eluted from nucleotide removal columns were ethanol precipitated on crushed dry ice and resuspended in 60 tl of Tris HC1-EDTA (TE) pH 8.  2.3 Preparation of Labeled DNA Fragments for EMSAs.  Radiolabeled primer 1 00-R was used to create 100 bp PCR products for use in electrophorectic mobility shift assays (EMSA5). Reactions contained labeled primer 100-R and primer 100-F (0.15 M each), 1 ng j.tl’ bacteriophage (p29 genomic DNA, 250 iM dNTPs, and 1 unit Pfu DNA polymerase in 50 gi of 1 x Pfu reaction buffer. The reactions were subjected to 30 cycles of 95°C for 30s, 55°C for 1 mm, and 68°C for 1 mm in a Biometra T Gradient thermocycler (Montreal Biotech). Labeled PCR products were purified using a PCR cleanup kit (Qiagen). Labeled 24 bp DNA was generated by annealing 5 tl of radiolabeled primer 100-R (8.3 pmol) and 5 il of unlabeled primer 24-F (9 pmol) in 30 p1 of 2 x SSC buffer. The reaction was heated to 95°C for 2 mm in a Biometra T Gradient thermocycler (Montreal Biotech), and the temperature was decreased by 5°C every minute until the temperature reached 50°C.  2.4 Preparation of Internally Labeled DNA Fragments for DNase Protection Assays.  Internally [a P]-dATP labeled 1 kb DNA fragments were generated by PCR. Reactions 32 were cycled for 16 cycles of 95°C for 30s, 55°C for 1 mm, and 68°C for 1 mm. Reactions contained primer 1k-F and primer lk-R (0.15 jiM each), 1 ng ji1’ bacteriophage (p29 genomic DNA, and 1 unit Pfu DNA polymerase in 50 jil of 1 x Pfu reaction buffer. Concentration of each dNTP was 200 jiM, except for dATP, which was [a P]dATP (50i.tM, —‘20 Ci mmo[’, GE 32 Healthcare). Unlabelled dATP was added to PCR tubes at a final concentration of 200 jiM for 14 additional cycles. The 1 kb PCR products were subjected to 4.5% PAGE through a 4.5% native gel in lx Tris-acetate (Sambrook eta?., 1989), the position of the 1 kb PCR product in the gel was resolved by autoradiography and the band was excised from the gel. The 1 kb products 30  were electroluted from the gel slice into dialysis tubing (12-14 kDa MW cutoff, SpectraPor). The DNA was collected, ethanol precipitated, and resuspended in 50 j.il of TE pH 8.  2.5 Preparation of DNA Fragments for Light Scattering Assays.  The different length DNA fragments used in light scattering experiments were generated by PCR using primers and templates listed in Table 1. PCRs were composed of the indicated primers (0.15 iM each), 1 ng j.tl’ DNA template, 250 jiM dNTPs and 1 unit Pfu DNA polymerase in 50 jil of 1 x Pfu reaction buffer. The reactions were subjected to 30 cycles of 95°C for 30s, 55°C for 1 mm, and 68°C for 1 mm. The products were purified using a PCR purification kit (Qiagen). PCR products were quantified by calculating the concentration from the 0D 260 values after being diluted 50 fold. All PCR products used in light scattering assays were also quantified by densitometry to compare the intensity of bands of interest to those of serially diluted DNA ladders of known concentrations following agarose gel electrophoresis and staining of the gel with ethidium bromide.  2.6 Construction of pET2OspoOJ.  pETI 6spoOJ (a gift from M. Perego) was subject to QuikChange site-directed mutagenesis to generate pET16sp0OJQC, containing a silent mutation that removed an internal NdeI site from the SpoOJ coding region (see section 1.2). To redone the mutated spoOJ, the spoOJ coding region, flanked by NdeI and XhoI restriction endonuclease sites at the 5’ and 3’ end respectively, was generated by PCR amplification. The PCR was composed of primer OJ-F and OJ-R (7.5 pmol each), 1 ng  .r’ pET16spoOJQC, 250 p.M dNTPs and 1 unit Pfu DNA 1  polymerase in 50 p.1 of 1 x Pfu reaction buffer. The reactions were subjected to 30 cycles of 95°C for 30s, 55°C for 1 mm, and 68°C for 1 mm in a Biometra T Gradient thermocycler (Montreal Biotech). The PCR products were purified using a PCR cleanup kit (Qiagen), and were treated with NdeI and XhoI. The NdeIJXhoI treated PCR fragments were gel purified (Qiagen) and ligated into gel purified pET2Ob treated with NdeI and XhoI. The ligation products were used to transform E. coil DH5ct. Plasmids purified from tranformants were screened for the appropriate size insert by agarose elecrophoresis of NdeI and XhoI treated plasmids. Candidate plasmid preparations with the appropriate sized insertion were sequenced (NAPS, UBC). 31  2.7 Generation of pUCIIG18.  The spoIIG]8 promoter was created from plasmid pUCIIGBglIItrpA. pUCIIGBglIItrpA is a derivative of pUCIIGtrpA, in which the G at position —22 of the promoter was changed to T, introducing a unique BglII site. pUCIIGBglIItrpA was treated with BglII, ethanol precipitated and resuspended in TE, pH 8. Mung bean nuclease (3.3 units, Invitrogen) was added to 8 jig of the BglII treated DNA in mung bean nuclease buffer in a final volume of 30 p1, incubated at 50°C for 1 hour and stopped by the addition of 1p1 of 10% (w/v) sodium dodecyl sulfate (SDS). The reaction was phenol extracted, and the DNA was precipitated by the addition of ethanol. The DNA precipitate was collected, dried and redissolved in 10 mM Tris-HC1, pH 8 and re circularized by ligation. The ligation reaction was extracted with phenol, and the DNA was precipitated by addition of ethanol, collected by centrifugation, and resuspended in React 2 buffer (Invitrogen) and treated with BglII prior to transformation of E. coli DH5ct. To identify clones with shortened promoters, transformants were selected by slot lysis (Sambrook et al., 1989) and sequenced to confirm deletions (NAPS, UBC).  3  Protein Purification.  3.1 Soj.  Plasmid pET2OSoj, containing the Soj coding sequence ligated into the NdeI and XhoI sites of pET2Ob (C-terminal hexahistidine tagged overexpression plasmid) was a gift from J. Hoch. A fresh E. coli BL2 1 (pLysS) pET2OSoj transformant colony was used to inoculate 1 litre of Luria-Bertani broth supplemented with 100 jig mr’ ampicillin and 35 jig ml’ 600 of 0.8 was chloramphenicol. Cultures were grown with vigorous shaking at 37°C until an 0D reached. Isopropyl-J3-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.5 mM. Cultures were grown at 37°C for 3 hours. The cells were harvested by centrifugation (12k x g, 15 mm) and cell pellets from 1 litre of culture were stored at -70°C. All purification steps were carried out at 0-4°C. The cell pellet from 1 litre was resuspended in 10 ml of lysis buffer (20mM HEPES (pH 8), 10mM magnesium acetate (Mg acetate), 80 mM potassium acetate (K acetate), 1.9 M NaC1, 1 mM ADP, complete protease cocktail tablet (Roche), 1 mM f3-mercaptoethanol) with 20 mM imidazole. Cells were lysed by addition of lysozyme to a final concentration of 1 mg m1’ and sonication on ice (Ultrasonic 32  Table 4. Bacterial Strains Used in this Thesis  E. coli Strain  Genotype  Source  DH5a  [hsdRl 7 (rj mK) supE44 thi-] rexA] gyrA (Naf) relAl A(lacZYA-argF U169] F ompT hsdSB (rBmB) gal dcm (DE3) pLysS (CamR) F ompT hsdSB (rBmB) gal dcm rne]3 1 (DE3)  Invitrogen  BL2 1 (DE3) pLysS BL2 1 Star (DE3)  Invitrogen Invitrogen  33  processor XL2020, 15 x 20 s at power level 3.5, Misonix Inc.). The lysate was cleared by centrifugation (25k x g, 20 mm) and the supernatant was stirred on ice with Ni-NTA agarose (2.5 ml, Qiagen) pre equilibrated with lysis buffer for 45 minutes and loaded into a 10 ml column. The column was washed with 30 ml of lysis buffer with 20 mM imidazole, 30 ml of lysis buffer with 60 mM imidazole, and then was equilibrated with 30 ml elution buffer (20 mM HEPES, 10 mM Mg acetate, 80 mM K acetate, 300 mM NaCl, 100 !.tM ADP, 1 mM f3mercaptoethanol, 0.5 mM phenylmethylsulfonyl fluoride) with 20 mM imidazole. Protein was eluted with elution buffer, with 300 mM imidazole. Fractions (-0.3 ml) were immediately aliquoted, snap frozen on crushed dry ice and stored at -70°C.  3.2 Soj Mutants.  Over expression vectors were made by site directed mutagenesis of pET2OSoj (section 2.1). Induction of mutant Soj overexpression and purification of Soj mutants was like wild type except cell pellets from 0.5 litre of induced cells were used in the lysis reaction and the final wash buffer and elution buffer contained 1 M NaC1.  3.3 SpoOJ.  E. coli BL2 1 Star was transformed with pET2OspoOJ. A single transformant colony was used to inoculate 1 L of Luria-Bertani broth supplemented with 100 ig  mr’ ampicillin.  600 of 0.6 was reached, IPTG was added to a final Cultures were grown at 37°C until an 0D concentration of 1 mM. Cultures were grown at 37°C for 3 h, and the cells were harvested by centrifugation (12k x g, 15 mm) and pellets from 0.5 L of culture were stored at -70°C. Cell pellets from 0.5 L were resuspended in 10 ml of SpoOJ lysis buffer (20 mlvi HEPES, 10 mM MgAc, 80 mlvi KAc, 1.9 M NaC1, Complete protease cocktail tablet (Roche), 1 mM [3mercaptoethanol) with 20 mM imidazole. Cells were lysed by addition of lysozyme to a final concentration of 1 mg m1’ and sonication on ice (Ultrasonic processor XL2020, 15 x 20 s at power level 3.5, Misonix Inc.). The lysate was cleared by centrifugation (25k x g, 20 mm) and the supematant was stirred on ice with Ni-NTA agarose (2.5m1, Qiagen) pre equilibrated with lysis buffer for 45 minutes and loaded into a 10 ml column. The column was washed with 30 ml of SpoOJ lysis buffer with 20mM imidazole, followed by a wash of 30 ml of SpoOJ lysis buffer with 65mM imidazole, then equilibrated with 30 ml SpoOJ elution buffer (20mM HEPES, 34  10mM MgAc, 80 KAc, 300 mM NaC1,) and 20mM imidazole. Protein was eluted with SpoOJ elution buffer with 250mM imidazole. Fractions (0.3 ml) were immediately aliquoted and snap frozen on crushed dry ice and stored at -70°C. Because the imidazole and NaC1 were not dialyzed from Soj and SpoOJ protein preparations, in assays that compared the effect of protein concentration input of these salts was kept constant. This was achieved by diluting the protein in elution buffer and/or adding additional dilution buffer to the reactions.  3.4 Protein Quantification.  Protein concentrations were determined by the Bradford assay kit (Bio-Rad) standardized to BSA. Purified proteins used in this thesis were judged to be over 95 % pure by Coomassie brilliant blue stained 12% SDS-PAGE gels of recombinant purified protein samples.  4  Protein-Nucleotide UV Crosslinking.  The protein was incubated with P-labeled 32 nucleotide at 37°C in 20 jil of transcription x buffer (10 mM HEPES pH 8, 10 mM Mg acetate, 80 mM K acetate) with 100 mM NaCl (protein, nucleotide concentrations, and specific activities are indicated in figure legends). The samples were transferred to a 96 well plate on ice and irradiated for the indicated time in a GS Gene Linker UV chamber (Bio-Rad). The reaction was transferred to a microfuge tube, and 20 jil of 1 mM unlabeled nucleotide was added. The well was washed with 100 jil of water which was added to the microfuge tube. Proteins were precipitated by addition of trichloroacetic acid (TCA) to 10% and incubation on ice for 40 minutes. The precipitate was collected by centrifugation for 40 minutes at 4°C. The pellet was washed with 10% TCA, followed by an acetone wash and the sample was then dissolved in 40 jil of SDS-PAGE loading buffer and separated by eleetrophoresis through a 12% SDS polyacrylamide gel. The gel was stained with Coomassie Brilliant Blue, dried, and exposed to a phosphorimager intensifying screen.  35  5  ATPase Assays.  ATPase assays were performed in 30 tl of ATPase buffer (20 mM HEPES pH 8, 10 mM Mg acetate, 80 mM K acetate) with 100 mM NaC1. The indicated concentrations of Soj were mixed with cL P-ATP (12.4-0.62 Ci mmoP’) and incubated at 37°C. At the indicated time 32 points 2 p1 of the reaction were removed and stopped by addition of EDTA to 20mM, and SDS to 0.05%. Samples were spotted on dry PEI cellulose thin layer chromatography (TLC) plates (Sigma-Aldrich) which were developed in 1 M formic acid and 0.5 M LiC1. TLC plates were dried, exposed to a phosphorimager intensifying screen, scanned, and exposed pixels were quantified using Imagequant software v.5.2 (GE Healthcare). ADP production was calculated by dividing the volume of exposed pixels in the phosphorimage spot corresponding to ADP by the sum of the volume of exposed pixels in the spots corresponding to ATP and ADP. All values were corrected by subtracting background P-ADP 32 levels of control reactions with no x protein addition, SpoOJ alone, and DNA alone for each time point where appropriate to reflect Soj ATPase activity. GTPase activity was assayed as described for ATPase activity assays except ct P-GTP was added to the reactions to the indicated final concentrations (specific 32 activities are indicated in figure legends).  6  Soj Size Exclusion Chromatography Assays.  The indicated protein  (- 60 tg, 80 jiM) was incubated in the presence of ATP or ADP  (1 mM) in a 25 p1 reaction at room temperature for 5 mm in ATPase Buffer supplemented with 150 mM NaC1. The reaction was applied to a Superdex200 3.2/30 column on an ATKA purifier FPLC system (GE Healthcare) equilibrated with ATPase buffer supplemented with 150 mM NaCl, 10 % glycerol, and 100 jiM of the indicated nucleotide at a flow rate of 70 jil min’. Protein elution from the column was detected by monitoring the absorption of the eluate at 230 and 280 nm and the absorption was plotted as a function of elution volume by Unicorn 5.11 FPLC system software. Absorption peaks at 230 nm corresponding to protein elution were analyzed with Unicorn software.  36  7  Blue Native PAGE Assays.  Blue native PAGE (BN PAGE) of purified proteins was performed essentially as described (Wittig et a!., 2006) except the level of Coomassie dye was greatly reduced. The indicated protein (2 1 iM) was incubated with the indicated nucleotide (1 mM) in ATPase Buffer with 100 mM NaC1 in a 20 iil reaction for 5 mm at 37°C. Two i1 of 10 x BN PAGE loading buffer (10 x ATPase buffer with 2 x 10% (wlv) Coomassie blue G-250) were added to the reactions and the reactions were applied to a 6-20% acrylamide gel, made in a 1 x gel buffer composed of 75 mM imidazole, and 1.5 M 6-aminohexanoic acid-HC1, pH 7. The cathode buffer (50 mM tricine, 7.5 mM imidazole, 2 x i0 4 % Coomassie blue G-250, pH 7), anode buffer (25 mM imidazole-HC1, pH 7), and gel were supplemented with the indicated nucleotide and Mg acetate (100 1 iM, and 4 mM respectively). For no nucleotide BN PAGE assays, the conditions were the same as above, except nucleotides and Mg acetate were omitted, and EDTA (1 mM) was added to reaction buffers, gel buffers, cathode, and anode buffers. Following electrophoresis (50 mm, 130 V), 12 cm gels were silver stained with a BioRad silver stain kit (BioRad), and digital images of gels were generated by scanning the stained gels using an Epson 2450 Photo scaimer. Digital gel images were analyzed with Imagequant software v5.2.  8  Electrophoretic Mobility Shift Assays (EMSAs).  For EMSAs using polyacrylamide gels, the indicated concentrations of Soj were P end labeled incubated for 1 mill with the indicated nucleotide (1mM) prior to the addition of 32 DNA (40k Cerenkov counts per reaction) in 10 tl of ATPase buffer supplemented with 100 mM NaC1, 0.1 mg m1 BSA, and 0.5% glycerol. Reactions were incubated for 5 minutes at 37°C and 5 p.1 aliquots were loaded onto running 7.5 or 5.5 % acrylamide gels (23:1, acrylamide: bisacrylamide), made with 0.5x tris borate buffer (Sambrook et a!., 1989). Both gel and reservoir buffer were supplemented with 5 mM Mg acetate, and 100 p.M of the indicated nucleotide. Samples were separated on a 12 cm gel at 120 V for 45 mm at room temperature, the gels were dried, and phosphorimages of gels were generated using a Phosphorimager SI (GE Healthcare). For EMSAs using agarose gels, the indicated concentrations of Soj were added to reactions with the indicated nucleotide (1 mM, same conditions as EMSAs using acrylamide gels) for 1 mm prior to the addition ofpUCl9 DNA (1.5 ng p.1’ final concentration). The 40 p.1 37  reactions were incubated for 5 minutes prior to electrophoresis through 12 cm, 0.5% agarose gels for 50 mm at 90V, where both gel and buffer (0.5X Tris borate) were supplemented with 5 mM Mg acetate, and 100 jiM of the indicated nucleotide. Gels were stained with ethidium bromide, rinsed briefly, and images were captured using an Alphalmager Gel documentation system (Alphalnnotech).  9  Sedimentation Assays.  To investigate Soj-DNA interactions, Soj was incubated in 40 p1 of ATPase buffer with 100 mM NaC1, at 37°C, with 1mM of the indicated adenosine nucleotide for 1 mm prior to the addition of linearized pUC19 DNA (4 ng  r’ final concentration) where indicated. 1  Reactions  were further incubated for 5 minutes before centrifugation in an Eppendorf 5415C microfuge (16k x g) for 20 mm. Supernatants were removed and pellet fractions were resuspended in 40 jtl of ATPase buffer. Where protein sedimentation was investigated, 1X SDS-PAGE loading buffer was added to the fractions, and they were heated in a boiling water bath and subjected to 12% SDS-PAGE. The gels were stained with Coomassie Brilliant Blue. Where DNA sedimentation was investigated fractions were treated with Pronase (0.4 mg ml’ final concentration, Roche) prior to electrophoresis through 0.8% agarose in 1X Tris Acetate (Sambrook et a!., 1989). Gels were stained with ethidium bromide and analyzed using a gel documentation system (Alphaimager EC, Alpha Innotech Inc.). To investigate Soj-SpoOJ interactions, Soj was incubated in 40 jil of ATPase Buffer with 100 mM NaCl, at 37°C, with 1mM of the indicated adenosine nucleotide for 1 mm prior to the addition of SpoOJ. Reactions were incubated for 5 mm and treated as described for Soj-DNA sedimentation reactions to resolve protein sedimentation. Protein and DNA band density was determined from digital gel images using ImageJ (Abramoff et a!., 2004) or Imagequant v5 .2 software.  10 DNase Protection Assays.  DNase I protection assay reactions were performed in 20 p1 of ATPase buffer supplemented with 100 mM NaC1, and 100 jtg m1’ BSA (New England Biolabs) at 37°C. Soj was preincubated with either 1mM ATP or ADP for 1 mm prior to the addition of internally labeled 1 kb DNA and incubation for 5 mm. DNase I (4.8 units, Invitrogen) was added to the 38  reaction and after 1 mm the reaction was terminated by addition of SDS and Pronase (0.1%, and 400 ug m1’ final concentrations, respectively) and vigorous vortexing. Ten jil of DNase reactions were electrophoresed through 20 cm, 6% polyacrylamide (23:1, acrylamide: bisacrylamide) gels in lx Tris acetate (Sambrook et al., 1989). The gels were dried and a phosphorimage of the gel was generated using a Phosphorimager SI (GE Healthcare). Phosphorimage analysis used Imagequant v5.2 (GE Healthcare). Electrophoretic mobilities of P end-labeled primers, and undigested 1kb 0.1 kb and 0.3 kb standards generated by PCR with 32 DNA used for DNase assay were used to interpolate the size of partial DNase I protected species.  11 In Vitro Transcription Assays.  Transcription assays were carried out in a 20 jil final volume of Transcription buffer (10 mM HEPES pH 8, 10 mM Mg acetate, 80 mM K acetate) with 0.1 mg mV’ BSA (Sigma, fraction V). Linearized plasmids containing promoter template DNA (6 nM DNA molecules) were incubated with the C-terminal domain of SpoOA which constitutively activates RNAP transcription from the spoliG promoter (SpoOAC, 1 jiM, gift of Steve Seredick), when P]-GTP (7.7 jiM, 23 32 indicated, RNAP (25 nM, gift of Steve Seredick), ATP (0.6 mM), and [aCi mmol’) and the indicated concentrations of Soj for two minutes at 37°C. Complexes were then challenged with heparin plus UTP and CTP and allowed to elongate for 5 minutes at 37°C. Final concentrations were: heparin, 15 jig  mr’; UTP, 0.6 mM; CTP, 0.6 mM. After elongation,  transcription reactions were stopped by the addition of 10 jil of 8 M urea, 0.1% xylene cyanol, 0.1% bromophenol blue in 0.5x TBE. Transcripts were separated on an 8% polyacrylamide gel containing 7 M urea in 0.5x TBE and exposed to a phosphorimager intensifying screen (Molecular Dynamics, GE Healthcare). Phosphorimages of the gels were generated using a Phosphorimager SI (GE Healthcare). Phosphorimage analysis used Imagequant v5.2 (GE Healthcare).  12 Light Scattering Assays.  Light scattering assays were performed in a Cary Eclipse fluorescence spectrophotometer (Varian Inc., excitation and emission wavelengths 350 nm, slit widths 5 nm) 39  in the Laboratory of Molecular Biophysics Shared Spectroscopy and Kinetics Hub at UBC. Reactions were performed in ATPase buffer supplemented with indicated concentrations of nucleotides at 30°C and a final reaction volume of 1.5 ml in a stirred cuvette (4.5 ml, UV/vis plastic, 10 mm light path). Soj or mutant Soj protein was added at 1 mm, the indicated concentrations of DNA were added at 2 mm, and reactions were monitored for up to 10 mm. In light scattering assays to detect Soj-SpoOJ interactions SpoOJ was added at 2 mm in lieu of DNA. Data points were collected at 0.2 s intervals. The photomultiplier tube (PMT) voltage varied among experiments and is noted in figure legends. Changes in the PMT voltage result in scale differences for light scattering intensities (in arbitrary units) but light scattering intensity was reproducible for many different experiments and Soj preparations.  13 Transmission Electron Microscopy.  13.1 Resolution of Soj DNA Structures.  Soj sedimentation reactions in the presence of 4 tM Soj, 1 mM ATP and 2 ng p1’ linearized pUC19 were performed as described for Soj DNA sedimentation assays. Following centrifügation supernatant was removed and the pellet fraction was resuspended in 10 p1 of ATPase buffer without nucleotide, and 5 iil was placed on a glow discharged (Balzers carbon evaporator and glow discharge apparatus, Bal-Tec) carbon coated 400 mesh copper grid (Structure Probe incorporated). After 1 mm 5 p1 of 0.5% uranyl acetate was placed on grid for a further minute before wicking the majority of the reaction off the grid with filter paper, and the remaining material on grid was allowed to dry. Grids were examined with a Hitachi H7600 transmission electron microscope (80 kV filament voltage) at the UBC Biolmaging Facility. 150 000 times direct magnification micrographs were captured using a side mount AMT Advantage CCD camera. Micrographs were analyzed using ImageJ software (Abramoff et a!., 2004).  13.2  Resolution of Soj-SpoOJ structures.  Soj and SpoOJ (1 tM each) were incubated for 5 mm at 37°C in ATPase buffer with 100 mM NaC1 and 100 tM ATP. Five p1 of the reaction was placed on a glow discharged carbon coated 400 mesh copper grid (Structure Probe incorporated) for one minute. The majority of the 40  sample was removed by wicking with filter paper briefly; the grid was washed with 5 .tl of distilled water before wicking dry. Samples on the grid were stained, the grids were examined and electron micrographs were captured as described for the resolution of Soj DNA structures.  13.3  Resolution of pUC19 DNA.  pCU19 DNA linearized by incubation with Hindill was ethanol precipitated and resuspended in sterile distilled water to a final concentration of 1 ng pY’. Five tl of the DNA solution was placed on a glow discharged carbon coated grid (same specifications as above), and was stained and washed as described for the resolution of Soj-SpoOJ structures. Grids were examined and electron micrographs were captured as described for the resolution of Soj DNA structures.  41  RESULTS  1 Phylogenetic Analysis of ParAISoj and MinD Proteins.  Phylogenetic analysis of plasmid and chromosomal ParA protein, and MinD protein sequence alignments was carried out using PAUP phylogenetic software v.4.0. An unrooted phylogram tree and an unrooted radial tree are shown in Fig. 5. No extensive analysis of these data was carried out but in general the patterns indicate that chromosomal ParAJSoj proteins group together, and are distinct from plasmid ParA proteins and MinD proteins). The separate grouping of plasmid and chromosomal ParA/Soj proteins suggests these proteins have functions dissimilar to those of plasmid ParA proteins. As a result the paradigms of plasmid encoded ParA family proteins might not be good guides for Soj activity and function.  2 Experimental Rationale.  To better understand the activities of Soj two mutants were also selected for characterization in addition to examining wild-type protein. The properties of the mutants are described in the Introduction. Briefly, SojG12V cannot repress sporulation in a A(soj-spoOJ) mutant background, and a SojG12V-GFP fusion does not have the same chromosomal distribution as wild type Soj-GFP in A(soj-spoOJ) cells (Murray and Errington, personal communication; Quisel et al., 1999). SojD4OA was examined because it was predicted to increase the stability of dimer formation and to be ATPase-deficient but still inhibit sporulation initiation.  3 Soj Mutant Overexpression Plasmids.  Both SojG12V and SojD4OA expression plasmids were created by site-specific mutagenesis using pET2obsoj as a template. This plasmid encodes wild type Soj with an C terminal hexahistidine tag fusion. The primers used are listed in Table 1, and the conditions used to generate the mutant protein overexpression plasmids are described in the Materials and Methods.  42  Bacterial Divsion  A  ParF-pTP228  Soj-H.pyl  e-proteobacteria cz-proteobacteria firmicutes  ParA-C.cre Soj-B.sub ParA-S.coe ParA-M.sme ParA-V.cho ParA-P.aer  }  deinococi  }  Mm D-B.sub y-proteobacteria  10 B  pA-Fplasmid Mm D-B.sui  ParF-pTP228  Soj-H pyl .  Soj-B.sub  ParA-S.coe 10  irA- M .sme  Fig. 5. Phylogenetic analysis of ParAISoj, and MinD proteins. Protein sequences were analysed by PAUP phylogenetic software package v. 4.0 to generate an unrooted phylogram tree (A), and an unrooted radial tree (B) based on the neighbour-joining method. Bootstrap values are shown for each branch in panel A and are based on 100 replicates. The proteins and species or plasmids are indicated. Species abbreviations are as follows; B .sub, Bacillus subtilis; E.col, Escherichia coli; H.pyl, Helicobacter pylon; C .cre, Caulobacter crescentus; P.put, Pseudomonasputida; P.aer, Pseudomonas aeruginosa; V.cho, Vibrio cholerae; T.the, Thermus thermophilus, M. sme, Mycobactenium smegmatis; S .coe, Streptomyces coelicolor. Species phylogentic divisions are indicated. Scale bar indicates 10 units of phylogenetic distance as calculated by PAUP phylogenetic software. 43  4 Protein Purification.  Purified Recombinant Soj and SpoOJ used in these experiments were both expressed and purified as C-terminal hexahistidine fusion proteins.  4.1 Soj Purification.  The goal of my thesis was to investigate Soj activities in vitro to better understand interactions of Soj with nucleotide cofactors, DNA, and SpoOJ. To achieve this goal a Soj purification protocol which maximized yield while ensuring high purity and conservation of Soj activities was required. The hexahistidine extension of purified recombinant Soj was assumed to not affect Soj activities in vitro because expression of C-terminal hexahistidine Soj protein is capable of blocking sporulation in a AspoOJB. subtilis strain, which indicates that the protein is active in vivo (Cervin et al., 1998). Also in B. subtilis strains Soj-GFP hybrid proteins with GFP translationally fused to the C-terminus of Soj are capable of negative regulation of sporulation in a z\spoOJ strain (Quisel et a!., 1999). Early experiments using an overnight culture to inoculate flasks for Soj production with gave inconsistent yields. After multiple trials I found that when Soj was over expressed by induction of a 1 litre culture inoculated with cells from a single freshly transformed E. coli BL2 1 (pLysS) pET2Obsoj colony, the protein yield was far more consistent. The cultures were 600 O.8 by addition of IPTG and shaken for 3 h. Growth was observed to halt induced at an 0D shortly after IPTG addition, and microscopic examination revealed filamentous E. coli cells. The induced cells overproduced Soj despite the arrested growth, as observed by SDS-PAGE analysis of cell lysates (Fig. 6). Initial attempts of Soj purification followed a standard protocol for purification of a hexa-histidine tagged proteins using Ni-NTA agarose (Cervin et a!., 1998). ATPase assays (described in Introduction section 5) indicated that Soj purified by this protocol had almost no activity which demonstrated that a significant fraction of purified Soj was not active (not shown). During my studies a report was published that indicated that isolation of active recombinant C. crescentus ParA purified from E. coli required the presence of ATP or ADP throughout the purification protocol. In light of those results, purification protocols were  44  IPTG  -+-  +  SpoOJ Soj  pETSoj  pETOJ  Fig. 6. Induction of Soj and SpoOJ overexpression. One litre of LB containing ampicillin (100 ig m1 ) and chioramphenicol (35 g m1’) was seeded with a fresh E. coil 1 BL21(pLysS) pET2Osoj transformant. One litre of LB with ampicillin (100 pg m[’) was seeded with a fresh E. coil BL2 1 Star pET2OspoOJ transformant. Cultures were grown at 37°C with vigorous shaking. Protein expression was induced by addition of IPTG to 0.5 600 of 0.8 for 3 h. One mM (BL21 pET2Osoj) or 1 mM (BL21 Star pET2OspoOJ) at an 0D ml samples of cultures before IPTG addition (-), or after 3 h of IPTG addition (+) were removed, centrifuged, resuspended in 50 p1 SDS loading buffer, and boiled for 5 mm. Five jil of the indicated lysate was subjected to SDS PAGE and staining with Coomassie blue. Protein bands corresponding to overexpressed recombinant hexahistidine Soj and SpoOJ are indicated. The molecular weight of low-range standards (Bio-Rad) are indicated in kDa.  45  modified to include ATP or ADP in all purification buffers. This modification resulted in Soj which was 95% pure as estimated by Imagequant densitometry of images of Soj samples subjected to SDS-PAGE and stained with Coomassie blue (Fig. 7A) and had significant ATPase activity (see Section 3). Control experiments done while examining DNA binding with early preparations of Soj, showed the presence of DNA in protein purified using buffers that had NaC1 concentrations below 1 M in both the cell lysis and Ni-NTA column steps. This is shown in Fig. 7. Agarose gel electrophoresis of 3 jig of purified Soj and ethidium bromide staining of the gel detected fluorescing material (Fig. 7B). The incubation of Soj with DNase I, but not RNase A or Pronase prior to electrophoresis removed the fluorescing material, indicating that the contamination was DNA of varied length with an average size of approximately 500 bp (Fig. 7B). I estimated approximately 120 ng of DNA was co-purified per jig Soj, by comparing the staining intensity of the DNA in the pronase condition to the 0.5 kb MW ladder in Fig. 7B. To eliminate the DNA contamination, I tried increasing the ionic strength in the lysis buffer, Ni-NTA agarose binding buffer and column wash buffers. As seen in Fig. 7C when Soj was purified using a high ionic strength buffer (1.9 M NaCl) for the lysis, Ni-NTA binding, and wash steps, the DNA contamination, assessed by agarose gel electrophoresis and staining with ethidium bromide of 9 jig of purified Soj, was greatly reduced (Fig. 7C). Presumably, this DNA contamination represented DNA bound to Soj. Since high ionic strength in the Soj preparations might affect subsequent assays, after loading Soj onto the Ni-NTA agarose, packing the loaded resin into a column and washing the column with buffer containing 1.9 M NaC1, the column was washed with buffer containing 300 mM NaC1. Soj was then eluted using a buffer containing 300 mM NaC1, 100 jiM ADP, and 300 mM imidazole. In numerous trials of different conditions I found that Soj precipitated and ATPase activity decreased more than two-fold when the purified Soj protein was subject to dialysis to remove either imidazole or nucleotide. As a result Soj eluted from the Ni-NTA agarose column in 300 mM NaC1 and 100 jiM ADP was immediately aliquoted and snap frozen. Long term storage (more than —30 days) at either -20 or-70 °C also led to a 60-70 % reduction of ATPase activity, even though Soj degradation as assayed by SDS-PAGE was not observed. This loss of ATPase activity during storage occurred regardless of storage conditions. Approximately 2 mg of recombinant Soj were purified per litre of induced E. coil culture.  46  A  C H *  IL  Fig. 7. Analysis of purified hexahistidine tagged Soj. (A) SDS-PAGE (12% polyacrylamide) of Soj (4.6 tg) purified under high ionic strength conditions stained with Coomassie brilliant blue, Soj was judged to be over 95% pure by Imagequant v5.2 software densitometry. Low-range molecular weight standards are shown on the left (from bottom; 21, 28, 35, 50, 93, 113 kDa, Bio-Rad Laboratories). (B) Agarose gel electrophoresis of Soj purified using low ionic strength lysis and wash buffers. After elution from a Ni-NTA column, Soj was pretreated with: buffer (b), DNase I (d), RNase A (r), or Pronase (p) prior to electrophoresis through 0.8% agarose along with a sample of 1 kb DNA ladder (kb, New England Biolabs). The gel was stained with ethidium bromide. (C) Effect of ionic strength on DNA in Soj preparations. Soj was purified by Ni-NTA chromatography using high (H; 1.9 M NaC1), or low (L; 0.3 M NaC1) salt in lysis and column wash buffers. After elution a sample was separated by electrophoresis through agarose. P indicates pronase treatment prior to electrophoresis. 1 kb (kb) and 100 bp (bp) ladders were included (New England Biolabs). Arrow heads on the right indicate DNA molecular weight standards (from bottom, 1 00-700bp, increasing by 100 bp).  47  4.2 SpoOJ Purification.  SpoOJ was over expressed by IPTG induction of a culture of E. coli BL2 1 star transformed with pET2O-OJ (Fig. 6). The SpoOJ purification protocol followed a published purification protocol (Cervin et a!., 1998), with a modification in which the cell lysate was bound to Ni-NTA agarose, and washed with 1.5 M NaC1 to remove a significant ATPase activity. SpoOJ was eluted at lower ionic strength in order to not affect subsequent applications. SpoOJ overproduction did not halt cell growth as did Soj overproduction, and there was no evidence of DNA contamination. Approximately 5 mg of SpoOJ was purified per litre of induced E. coli culture, and could be stored at -70 °C for prolonged periods of time (6 months).  5 ATP Binding and Hydrolysis by Soj.  Phylogenetically Soj is a member of a large Walker AB motif containing NTPase superfamily (Leipe et al., 2002). Therefore it was expected that purified Soj would bind, exchange, and hydrolyze ATP. The importance of Soj ATP binding and hydrolysis activities has been suggested by the altered localization and loss of sporulation inhibition observed for B. subtilis strains expressing mutant Soj proteins predicted to have altered ATP binding and hydrolysis compared to wild type Soj. The in vitro characterization of the ATP binding and hydrolysis activities of Soj was central to my thesis for two reasons. First, it would establish that purified Soj protein preparations contained active Soj protein. Second, it was possible that the nucleotide bound state of Soj would alter its properties. The ability to monitor ATP binding and hydrolysis could be a tool to better understand the function of Soj. Binding of nucleotide triphosphates by Soj was investigated by UV cross linking assays. 32 labeled NTPs and the reactions were incubated prior Soj was added to reactions containing P 32 to Soj. Following UV x to irradiation with UV light which covalently links bound P-NTP irradiation the protein in the sample was precipitated by addition of TCA. The precipitate was collected by centrifugation, washed with acetone, resuspended in SDS-loading buffer and the P nucleotide by UV protein was subjected to SDS-PAGE. Proteins covalently linked to the 32 irradiation were detected on a phosphorimage of the gel. As seen in Fig. 8A, ATP was readily linked to Soj. To investigate the NTP binding specificity of Soj, the purified protein was incubated with P 32 labeled ATP, dATP, or GTP (Fig. 8A). A lower intensity band was x observed when Soj was incubated with labeled dATP instead of ATP prior to crosslinking. 48  A  ÷  Soj  +  +  BSA  -  ———+  Nudeotide Al?  +  ——  +  B Incubation (mm) 0  .25 .5  1  2  3  4  5  ATP GTPdATPATPGT No  uv  C  -.  D Incubation time (mm)  >.  b  0.4  1 2.5 5 10 15 20 SojGl 2V  Soj D4OA  .1  a,  ITh1  0  0  50 100 150 200 250 300 350 time (s)  Fig. 8. Nucleotide binding by Soj assayed by UV cross linking. (A) -P-labeled nucleotides (50 .tM, 24.8 Ci 32 Nucleotide binding specificity of Soj. ci mmol’) were incubated with 12.5 jiM Soj or 0.1 mg m[’ of bovine serum albumin (BSA) for 5 mm in 20 jil of ATPase buffer with 100 mM NaC1. Samples were irradiated with UV for 5 mm on ice, followed by trichioroacetic acid precipitation and solubilization in SDS-PAGE loading buffer. Samples were subjected to 12% SDS-PAGE. The gel was dried and a phosphorimage was generated as described in Materials and Methods (section 4). (B) Time course of Soj ATP binding detected by UV cross linking assays. Reactions of Soj (5 jiM), and & P-ATP (200 jiM, 6.2 Ci mmol’) were incubated at 37°C for 2 the indicated time, and then irradiated with UV for 2 mm on ice. Samples were treated and a phosphorimage was generated as described for panel A. (C) Phosphorimage densitometry of the major cross-linked bands from panel B were determined using Imagequant v5.2. Data were normalized to the density of the band in 5 mm incubation and plotted as a function of incubation time. (D) SojG12V and SojD4OA ATP binding assayed by UV cross-linking. The 32 (500 jiM, 2.5 Ci a indicated Soj mutant (5 jiM) was incubated with P-ATP 20 jil reactions, time at 37°C in mmo[’) for the indicated and irradiated with UV for 3 mm on ice. Reactions were then treated as described in panel A. 49  When Soj was incubated with labeled GTP prior to crosslinking no band was observed. To test 32 but was not UV irradiated, and BSA, a the validity of this assay Soj was incubated with P-ATP 32 prior to UV a a protein with no nucleotide binding activity, was incubated with P-ATP irradiation. In the latter two cases, no signals corresponding to protein covalently linked to 32 were observed, which indicated that the UV cross-linking assay was an indication of a P-ATP nucleotide binding, and that Soj specifically bound ATP (and dATP to a lesser extent), but not GTP. ATP binding by Soj as a function of both time and temperature was assayed by UV cross-linking. To test the rate of ATP binding, Soj was added to reactions which contained PATP and the reactions were allowed to incubate at 37 °C for various times before UV 32 cL irradiation on ice (Fig. 8B). Soj binding of ATP increased rapidly reaching 90% of maximum at approximately 90 s (Fig. 8C). When either SojG12V or SojD4OA were included in cross linking reactions with 32 P-ATP a maximum crosslinking signal was reached by 1 mm (Fig. 8D) indicating both mutants could bind ATP. While these assays show that the mutant Soj proteins bind ATP, is it possible that the kinetics of binding were different in the first minute of the assay. This point was not considered of great importance, since it was clear that a 1 mm incubation with ATP would give maximum binding. A more detailed examination of SojG12V and SojD4OA ATP binding activities at earlier time points such as those in Figs. 8B and 8C would be required to determine the ATP binding constants and ATP binding rates for these proteins. The amount of ATP crosslinked to Soj was calculated by comparing the densitometries 32 The amount of ATP crosslinked to Soj a of Soj bands to those of known amounts of P-ATP. was determined using the specific activity of P-ATP 32 in the crosslinking reactions. At a maximum binding of Soj to ATP, 0.05 % of Soj was crosslinked to ATP. The low level may reflect the fact that UV irradiation is an inefficient method of crosslinking. On the other hand possibly the Soj that was purified was primarily inactive. I was not able devise a second ATP binding assay which could determine a specific Soj activity. It seems unlikely that the Soj purified was only 0.05% active since the ATPase level (see below) was within the range seen for other MinD family members. An independent ATP binding assay would have greatly improved my analysis of Soj by indicating how much Soj was active from a specific Soj purification. UV-crosslinking efficiencies for other Walker A motif ATPases have not been reported, to my knowledge, so it is possible that the values observed in Fig. 8 are reasonable.  50  Because of the low crosslinking efficiency, I considered the possibility that a contaminant which binds ATP and migrates in an SDS-PAGE gel is responsible for the crosslinking observed in Fig. 8. Crosslinking assays with a Soj mutant which is predicted to not bind ATP (such as SojKl 6A, a Walker A motif invariant lysine mutant) was not done. If such an experiment resulted in greatly reduce UV-crosslinking it would confirm the UV-crosslinking of ATP to wild type Soj and not a contaminant in Fig. 8. Minor bands were observed in the UV cross-linking assays that migrated differently than Soj, and were not present in conditions without UV cros slinking. I investigated the identity of the anomalous protein bands. Soj was subjected to SDS-PAGE. The area of an SDS-PAGE gel stained with Coomassie brilliant blue which contained the larger species was excised from the gel, subject to in gel proteolysis, and analyzed by mass spectroscopy at the University of Victoria. The only detectable peptides were derived from Soj. A directed studies student separated samples of purified Soj by SDS-PAGE, transferred the proteins to a PVDF membrane, and probed the membrane with x-Soj antibodies (a gift from M. Perego, data not shown). Both the upper and lower bands cross reacted with the a-Soj antibodies indicating that the bands contained Soj or Soj breakdown products. The lower band may have been a degradation product of Soj, and the higher band appeared to be Soj with anomalous electrophoretic mobility. The higher band constituted only a very small fraction of purified Soj and the cause of this anomaly is unknown. Later Soj preparations used buffers supplemented with dissolved protease inhibitor cocktail tablets (Roche), and fewer Soj degradation products were observed. Soj hydrolysis of ATP to ADP was investigated by following the production of ADP. The purified protein (3 jiM) was incubated with ct P-ATP. Aliquots of the reactions were 32 removed and the ATPase was stopped by the addition of SDS (0.2 % final concentration) and EDTA (50 mM final concentration). Four jil of the quenched aliquot were spotted on a PEI cellulose plate which was then developed to separate cL P-ATP and P-ADP. 32 32 Phosphorimage a intensities of the spots corresponding to both 32 P- ATP and 32 P-ADP were used to calculate the percentage of ADP in the reaction or total moles of ADP produced. The production of ADP by Soj was linear for the first 60 mm of the assay (Fig. 9). Incubation of Soj G 1 2V with P-ATP 32 also resulted in the production of P-ADP L 32 and at a x similar rate as seen with wild type Soj. SojD4OA showed a reduced ADP production compared to both Soj and SojG12V. The reduced ATP hydrolysis by SojD4OA was a catalytic defect of the protein, since the mutation did not affect its ATP binding as shown in Fig. 8D. This was the expected result as the mutated residue has been shown in the T thermophilus structure of Soj to 51  12  10  8 >.  >0  4-’  oE ca.  C.) V  6  0 C)  I-— <0  ‘-.3  a.  a.  E 4  0  2  0 0  10  20  30  40  50  60  time (mm)  Fig. 9. ATPase activities of Soj, SojG12V, and SojD4OA. The indicated P]ATP (1.33 Ci mmol’) in 30 32 protein (4 pM) was incubated with 250 jiM [ajil of ATPase Buffer containing 100 mM NaC1. Aliquots of reactions were removed at the indicated times, stopped as described in Materials and Methods (section 5), and subjected to thin layer chromatography. The amount of ATP hydrolyzed was calculated from the densitometry of ATP and ADP spots as described in Materials and Methods (section 5). The data points labeled Soj * correspond to GDP production from GTPase reactions (same P-ATP and the 32 conditions as ATPase reactions except for the absence of cL presence of P-GTP 32 (250 jiM, 1.33 Ci mmo[’)). Each time point is the a average value of 3 independent reactions; error bars represent standard deviations for each time point. Linear trend lines were fitted by Microsoft Excel. Values were corrected for trace ADP or GDP presence detected in reactions containing no protein for each time point.  52  2 ion cofactor required for ATP hydrolysis but does not affect coordinate the binding of a Mg ATP binding by the Walker A motif of Soj. Since SojD4OA was purified the same way as Soj and SojG12V, it was expected that any contaminants would be present equally in all of the recombinant proteins. Therefore, this result also provided evidence that ADP production was mediated by Soj and not by a contaminating ATPase. The result that SojG12V was a functional ATPase and produced similar levels of ADP as wild type was surprising as the equivalent mutation in related ATPases has been shown to severely reduce ATPase activity (addressed in the Introduction and Discussion). As an additional control, the ability of Soj to hydrolyze GTP was assayed (indicated as Soj* in Fig. 9). No significant GDP production was observed, consistent with nucleotide cross linking experiments that detected no GTP binding. The latter result supported the idea that there was not a contaminating purine nucleotidase activity in purified Soj samples. I also noted that the presence of ADP rather than ATP during purification was more effective at stabilizing the solubility of Soj after elution, resulting in higher yields of soluble Soj (data not shown). Therefore, Soj was purified in the presence of 100 jiM ADP. The inclusion of ADP in Soj purification protocols resulted in the presence of small concentrations of ADP  (  10 jiM) in all experiments. The presence of ADP in Soj ATPase assays had a negative effect on Soj ATPase activity, especially at lower ATP concentrations (data not shown). These results suggested that ADP was a ligand for Soj and that the presence of ADP in kinetic assays could cause product inhibition giving inaccurate data. To address this problem the Soj purification protocol was changed for protein to be used in kinetic analyses. Prior to Soj elution the Ni-NTA agarose column was equilibrated with ten column volumes of purification buffer with reduced ADP concentration, or without nucleotide. This modification did have an impact on ATPase (Fig. 10), but the effect was observed to be small but reproducible. Despite the small decrease in ATPase activity, Soj used in the initial velocity kinetic assays was prepared with a ‘no nucleotide’ wash to eliminate the worry about product inhibition. To determine the initial velocity of Soj ATPase, purified Soj was incubated with the 32 and the production of ADP was measured over time. An a indicated concentration of P-ATP example of ADP production plotted versus time at 10 jiM ATP is shown in Fig. hA. The slope of the best fit line was used to calculate the initial velocity (v, pmol/nmol Soj/min) of Soj ATPase. The initial velocity of Soj ATPase was calculated for a range of ATP concentrations  53  240  200 E I4 V.  0  120 0  E  0.  80  40 <  20 0 0  30  100  ADP (pM)  Fig. 10. ATP hydrolysis by Soj eluted in different ADP concentration conditions. Soj was purified as described in Materials and Methods (section 3.1) except the Ni-NTA column was equilibrated with buffer containing the indicated concentration of ADP, and the protein was eluted from the column in elution iM) was incubated with 50 buffer with the indicated ADP concentration. Soj (3 1 with in ATPase buffer pM P-ATP 32 for 15 mm at 37°C a 100 mM NaC1; each reaction was repeated in triplicate. Reactions were stopped and ADP was detected and quantified as described in Materials and Methods (section 5). Values were corrected for trace radioactive ADP present in reactions containing no protein.  54  A 70  y=3.2x+5.9  60 50 0  20  15  10  5  time (mm)  0 300  -  E 250 o200  E 150 • 100  E  0.  50 ‘I  U.  I  0  200  400  600  800  1000  ATP (pM) Fig. 11. Soj ATPase kinetics. (A) An example of the determination of the initial rate of ATP hydrolysis for kinetic analysis. Soj (3 i.tM) was incubated with ct P-ATP (10 32 .iM, 24.8 Ci mmo[’) in a 40 l of ATPase buffer containing 100 mM NaC1, at 37°C. At 1 the indicated times aliquots were removed and stopped. ADP was detected and quantified as described in Materials and Methods (section 5). The ADP production was plotted as a function of time and the rate was calculated from a trend line slope fitted by Microsoft Excel assuming linearity. (B) The rate of ATP hydrolysis was determined from curves such as shown in panel A. The rate was divided by the quantity of Soj to give the initial velocity (v, pmol ADP/ nmol Soj / mm) The initial ATPase velocity of Soj was determined at the indicated ATP concentrations. The initial velocity was plotted as a function of ATP concentration. Each point represents the average initial velocity calculated from at least 3 independent reactions; error bars representing standard deviations are indicated.  55  and the initial velocity of Soj ATPase activity as function of ATP concentration is plotted in Fig. 11B. Visual examination of Fig. 11B. (initial velocity versus ATP) indicated a Vm of 175180 pmol ATP consumed / nmol of Soj I mm and an apparent Km of 175±62 jiM ATP. At Vmax, . Due to the large standard 1 the turnover number (koat) was calculated to be 3±1 xl 0 s deviations of Km and kcat values the calculation of kcat/Km ranged between 8.4-35.4 M  The  range of kcat/Km values indicated that Soj is not an efficient ATPase. These values are similar to other ParA/MinD proteins that also have low ATPase activity (Davis et a?., 1992; de Boer et a?., 1989).  6 Characterization of Soj Multimerization.  6.1 Size Exclusion Chromatography of Soj.  As discussed in the Introduction, the ParAIMinD protein family members undergo nucleotide-dependent dimerization and in some instances can form higher order complexes. Size exclusion chromatography was used to monitor the formation of Soj multimers in the presence of either ATP or ADP. For these experiments Soj  (‘--  60 pjg, -80 jiM) was incubated  with 1 mM of the indicated nucleotide for 5 mm at room temperature in a 25 111 volume and loaded onto an analytical Superdex200 column equilibrated with buffer containing 0.1 mM of the same nucleotide at room temperature. The elution profile was monitored by absorption at 230 run and was plotted versus the elution volume. The elution volumes for a set of molecular weight standards (Fig. 12, Table 5), was also determined. The elution profiles of wild type and mutant Soj had higher peak intensities when detected by absorbance at 230 nm compared to 280 rim. Peptide bonds, tryptophan residues and tyrosine residues all absorb at 230 mm Both absorption spectra at 230 rim and 280 nm were monitored simultaneously and showed the same elution patterns. Only Soj elution profiles monitored by absorption at 230 rim are shown. Soj showed absorption peaks at 1 .78-1.81 ml and 2.15-2.17 ml corresponding to the molecular weights of 58 kDa (possible Soj dimers) and 29 kDa (possible Soj monomers) respectively (Fig. 13). In the presence of ATP, the peak centred at 1.78 ml showed a shoulder that was not seen in the presence of ADP suggesting the presence of a Soj species larger than that of a dimer (possibly a tetramer, 1 05 kDa), or a fraction of the protein with an altered shape.  56  3 2.5  0 LU  1 0.5 0  0  50  100  150  200  MW (kDa)  Fig. 12. Elution of molecular weight standards used to estimate size of Soj dimers. The elution volume of molecular weight standards (listed in Table 5) from a Superdex 200 3.2/30 size exclusion chromatography column attached to an ATKA purifier FPLC system was plotted as a function of molecular weight.  57  Table 5. Molecular Weight Standards Elution Volumes Used for Size Estimation of Soj Multimers.  Standard  MW (kDa)  Creatine Phosphokinase dimer Alcohol Dehydrogenase BSA SpoOJ Lysozyme ATP  162 141 66 32 14 0.551  Elution (ml) 1.25 1.30 1.80 2.10 2.40 2.60  58  D 1400 —  M  NT  Soj-ATP  1200  D  A  I 600  800 600  1’  400 <  NT  800  I  < 1000  M  4 44. iooo Soj-ADP  /  200  / \/ \ I.  0 1400  S0jGI 2V-ATP  SojGl 2V-ADP f  1200  2000  I  j \ /\ /  200  o 2500  /“ “  /  400  1000 1500 a C.,  H  H  U’.,’.,  Ii  .1000 o  R /1  500  \\J\  __1’  o  Ii  400  J.  200  So] D4OA-ADP  800  H  o  400  /  H  E 1000  •  Ii  /  1200  Soj D4OA-ATP  1200  600  1  0  1400  •  11.  600  H  600  /( \  /  400  J  200  \_._  [\  200  1  /  —  U  1  1.5  2  2.5  Elution (ml)  3  1  1.5  2  2.5  3  Elution (ml)  Fig. 13. Analysis of purified Soj and Soj mutants by FPLC size exclusion  chromatography. The indicated protein (—60 jig, 80 was incubated in the presence of ATP or ADP (1 mM) in 25 jil of Soj elution buffer at room temperature for 5 mm as described in the Materials and Methods (section 6). The mixture was applied to a Superdex 200 3.2/30 column attached to an ATKA purifier FPLC system equilibrated in ATPase Buffer with 100 mM NaC1, 10 % glycerol, and 100 .tM of the indicated nucleotide. The A 230 of the elution was monitored and plotted as a function of the elution volume. Vertical arrows indicate the elution position of peaks that correspond to Soj dimers (D), Soj monomers (M), and nucleotides (NT). 59  The elution of Soj G 1 2V in the presence of either ATP or ADP showed absorption peaks centred at 2.15-2.17 ml and 1.68-1.75 ml which correspond to the elution volume expected from proteins with masses equal to Soj monomers and dimers respectively. The elution of SojD4OA in the presence of ATP or ADP showed an absorption peak centred at 2.15-2.17 ml corresponding to the elution volume expected for a protein equal in size to a Soj monomer. However, SojD4OA eluted differently in the presence of ATP or ADP. In the presence of ATP, the first absorbance peak was centred at 1.57 ml, whereas in the presence of ADP the first absorbance peak detected was centred at 1.74 ml. The SojD4OA peak at 1.57 ml corresponded to the elution volume of the shoulder of the peak in the elution profile of wild type Soj in the presence of ATP, suggesting the presence of Soj protein complexes with higher molecular weight in the presence of ATP (possibly a tetramer, -405 kDa), or a significant change in shape. For both Soj and SojD4OA in the presence of ATP, the presence of a peak eluting at a lower elution volume indicating a shape change in Soj dimers or the elution of a tetramer was not due to loading error as the elution of monomeric Soj and ATP remained constant throughout these assays. FPLC system software was used to determine the area of the peaks corresponding to Soj monomers, dimers and ATP or ADP. Peak elution volumes, areas, and areas relative to the area of the peak corresponding to Soj dimers are shown in Table 6. No clear trend in the ratio of the peaks between ATP and ADP conditions was observed for Soj or SojG12V which indicated that ATP and ADP did not differentially affect the dimerization of Soj from B. subtilis. For all three Soj proteins regardless of which nucleotide was present no absorbance peaks were observed in the void volume of the column eluate (0.6 ml). This indicates that under the conditions of this chromatography, larger multimers of Soj were not detected. However I routinely found that the back pressure in the FLPC colunm increased in successive runs, implying that Soj might be forming large complexes that come out of solution (see below). In agreement with that possibility, I noted incomplete recovery of Soj A 230 units in the eluates. 6.2 Blue Native PAGE Analysis of Soj.  It was reasoned that observed Soj dimerization from size exclusion chromatography could be influenced by the high concentrations of Soj  ( 60 .tg, -80 M) loaded on the column.  Therefore, blue native PAGE (BN-PAGE) was used as an independent technique to assay for Soj multimerization with the advantage of small sample volumes (20 d) with low Soj 60  Table 6. FPLC Absorption Peak Integrationa.  Nucleotide ATP  ADP  Protein  Peak” Elution (ml)  Area (mAU*ml)  Relative Area°  D M NT  1.78 2.15 2.59  262 67 115  1 0.26  SojG12V  D M NT  1.66 2.15 2.60  222 118 196  1 0.53  SojD4OA  D M NT  1.57 2.15 2.59  234 71 103  1 0.30  D M NT  1.73 2.15 2.60  159 50 89  1 0.31  SojG12V  D M NT  1.75 2.17 2.60  208 126 137  1 0.60  SojD4OA  D M NT  1.68 2.17 2.60  136 82 122  1 0.60  Soj  Soj  a. Integration of the peaks from elution profiles in Fig. 13 was performed with Unicorn FPLC software. b. Peaks identified corresponding to Soj dimer (D), Soj monomer (M), or nucleotide (NT). c. The relative area of indicated peak relative to the area of the dimer peak in the same chromatograph.  61  concentrations (2 tM). BN-PAGE includes Coomassie brilliant blue in the loading and anode (top) buffers which confers negative charge to proteins in their native state which mediates protein mobility through a gradient gel. This technique has been reported to allow resolution of high molecular weight complexes (Switzer et a!., 1979). To assay for the effect of ATP or ADP on higher order Soj interactions, reactions of Soj were incubated with ATP, ADP, or buffer without nucleotide at 3 7°C, and were loaded onto blue native gels. When Soj was subjected to BN-PAGE with nucleotides the gels and buffers were supplemented with the indicated nucleotide and Mg acetate. When Soj was subjected to BN-PAGE without nucleotides, nucleotides, and Mg acetate were omitted from reactions, gels, and running buffers, and EDTA was added to each at final concentration of 1 mM. When Soj was subject to BN-PAGE multiple bands were resolved. The apparent molecular weights of these bands corresponded to multiple Soj complexes (Fig. 14A). The electrophoretic mobilities of molecular weight standards subject to BN-PAGE were calculated by Imagequant software analysis and were plotted as a function of their molecular weight (Fig. 14B). The curves from Fig. 14B were used to estimate the molecular weight of Soj bands which ranged from 30-900 kDa (summarized in Table 7). The purity of Soj analyzed by SDS-PAGE ensured that resolved bands were only composed of Soj protein and were not contaminants. The high molecular weight Soj species (>175 kDa) were not seen when Soj was incubated in the absence of nucleotide. Instead smaller molecular weight Soj species (30- 110 kDa) were resolved. Similar banding patterns observed for wild type Soj, SojG12V, and SojD4OA in BN PAGE assays in the different nucleotide conditions. One notable exception was the different banding pattern observed with SojD4OA in the absence of nucleotide; a single sharp band of higher molecular weight was observed but the pattern was not similar to SojD4OA in the presence of either ATP or ADP. These results indicate that Soj may form multiple high molecular weight complexes some of which require ATP or ADP binding. It is unclear how to interpret the BN-PAGE pattern. The key question is whether the Soj multimers were artifacts caused by the electrophoresis conditions (e.g. non-specific aggregation). One piece of evidence against non-specific aggregation is that differences in apparent molecular mass between the complexes detected is not random. For example, aside from the monomer and dimer bands, adjacent band differed by more than the mass of a single Soj monomer. In fact, adjacent bands above l7OkDa (the size of Soj hexamers) increased by a mass that suggested addition of hexamers. A second piece of evidence suggesting the higher  62  Fig. 14. Blue native PAGE of Soj and Soj point mutants in the presence of ATP, ADP, or in the absence of nucleotide. (A) Soj (wt), SojG12V (G12V), or SojD4OA (D4OA) were incubated at a final concentration of 2 jiM with ATP or ADP (1 mM), or EDTA (1 mM) at 37°C in 20 jil of ATPase buffer with 100 mM NaCl for 5 mm. Incubations containing ATP or ADP were subjected to BN PAGE on a 6-20% gel with the gel, anode, and cathode buffers supplemented with the indicated nucleotide (100 jiM), and Mg acetate (4 mM). “No nucleotide” conditions were the same except that nucleotide and Mg acetate were omitted from, and EDTA was included in the gel, anode, and cathode buffers. After electrophoresis as described in Materials and Methods (section 7) the acrylamide gels were stained using the BioRad silver stain kit and a digital image of the gel was generated with an Epson 2450 Photo scanner. A Native protein molecular weight ladder (L, Native Mark, Invitrogen) was included on each BN PAGE. Arrows indicate molecular weight ladder bands (from bottom, 20, 66, 146, 242, 480, 720, 1048, 1236 kDa). (B) Estimation of the size of Soj complexes from BN PAGE. The mobility of the native protein molecular weight standards, determined by Imagequant analysis of the digital images of the gels from panel A, is plotted as a function of protein size. The mobility of wild type Soj complexes in each condition (determined by Imagequant) is shown by vertical arrows on the molecular weight axis.  63  A ATP  ADP  NoNT  So] wt GI2VD4OA L —  wt GI2VD4OA L  —  J4 —4 4  —4  B  800  ATP 600 0  400 200 44.4,4,  4,  4,  4,  4,  4,4,  800  ADP 600  400 200 4.4,4, 4,  0 800  \ .  4,  4,  4,  4,  4,4,  NoNT  600 400 200 44 4, 4, ‘1  0  200  400  600  800  1000  1200  1400  MW (kDa)  64  Table 7. Soj Complex Size Estimation from BN-PAGE Nucleotide Condition Molecular Weight Estimate (kDa)a Oligomeric State Estimateb ATP  38 59 105 175 360 530 685 787 885  1 2 4 6 12 18 24 27 30  (29) (58) (116) (174) (348) (522) (696) (783) (870)  ADP  39 60 105 167 352 510 660 771 865  1 2 4 6 12 18 23 27 30  (29) (58) (116) (174) (348) (522) (667) (783) (870)  No Nucleotide  25 50 105  1 2 4  (29) (58) (116)  a. Molecular weight estimate values are the average of two molecular weight estimates from independent BN-PAGE assays. b. Molecular weight of the Soj complex resolved by BN-PAGE was divided by the molecular weight of recombinant his-tagged Soj (29 kDa), and rounded to the closest whole number. The expected molecular weight (in kDa) for the indicated oligomeric Soj species is shown in parentheses.  65  molecular weight species are specific is that Soj multimers larger than a tetramer were not observed in the absence of nucleotide. These BN-PAGE results are difficult to interpret without further experimental investigation. The relevance of higher order multimers resolved by BN PAGE that were not also observed by size exclusion will not be addressed in the thesis Discussion or in the general model given for Soj interaction with nucleotides, DNA, and SpoOJ. These results have been included because they were reproducible and striking and to indicate that Soj multimerization is an aspect of this research that warrants more investigation.  7 Soj DNA Binding.  When this work was started, the possibility that Soj would bind to DNA was predicted from multiple lines of evidence from genetic and microscopic techniques. In vivo, inhibition of sporulation by Soj is characterized by low transcription of stage II sporulation genes suggesting transcriptional repression. In AspoOJ B. subtilis strains that express a Soj-GFP fusion protein, the GFP signal is observed to localize evenly with chromosomal DNA. As mentioned in Section 2.1 Soj purified under conditions of low ionic strength contained large amounts of contaminating DNA. It seemed likely that a Soj DNA binding activity would be detectable in vitro. Recently a group investigating the DNA binding activity of B. subtilis Soj identified surface-exposed residues that, when mutated, abrogated DNA binding by Soj (Hester and Lutkenhaus, 2007). A structural model of Soj bound to DNA based on the structure of T. thermophilus Soj was presented. This model suggests a DNA binding surface on the Soj dimer where the residues implicated in the DNA binding activity of Soj are situated. Although the authors did not report experimental evidence, approximately 20 bp of DNA are closely associated with the proteins, predicting that Soj would have a 20 bp DNase I footprint.  7.1 Soj DNA Complexes Resolved by EMSAs.  To characterize the putative DNA binding activity of Soj gel electrophoretic mobility shift assays (EMSAs) were performed using 32 P-labeled 24 bp or 100 bp DNA. Initial experiments indicated that Soj exhibited no sequence preference for DNA binding. Therefore, DNAs were amplified from the genome of p29, a B. subtilis bacteriophage. These DNA sequences were previously used to assay for SpoOJ DNA binding and could be easily generated in large quantities by PCR as described in the Materials and Methods. For the EMSA reactions, 66  Fig. 15. Nucleotide dependence of Soj binding to DNA. (A) Soj-ATP dependent shift of DNA elecrophoretic mobility. Soj, SojG12V, or SojD4OA at the indicated concentration was preincubated with ATP (1 mM) in ATPase buffer with 100 mM iM final bp NaCl, prior to the addition of either 100 bp (‘--0.2 ng ji[’, ‘--0.3 1 P-labeled DNA concentration) or 24 bp (‘—10 pg rd’, ‘—8.4 nM final bp concentration) 32 fragments in a 10 jil final reaction volume. After 5 mill at 37°C, half the reaction was loaded onto a running acrylamide gel. Both the gel and running buffers were supplemented with 5 mM Mg acetate and 100 iM ATP. Reactions containing 100 bp or 24 bp fragments of DNA were separated by electrophoresis through 5.5% and 7.5% , 45 mm). Gels were dried and bands were 1 acrylamide gels respectively (10 V cm detected using a Phosphorimager SI as described in Materials and Methods (section 8). Horizontal arrows indicate wells of the gel. (B) Electrophoretic mobility shift of plasmid DNA in the presence of Soj and ATP. The indicated concentrations of Soj were preincubated with ATP or ADP (1 mM) where indicated prior to the addition of linear pUC19 DNA (1.5 ng t[’, 2.3 iM bp final concentration). The reactions were . 1 loaded onto running 0.5% agarose gels and electrophoresed for 50 mm at 7.5 V cm gel stained with ethidium bromide and a digital Following electrophoresis the was image was generated as described in the Materials and Methods. (C) Hill plot of Soj binding the 100 bp DNA fragment. The fraction of 100 bp DNA bound by Soj in EMSAs in panel A at each Soj concentration relative to binding observed with 8 jiM Soj and 100 bp DNA was determined by Imagequant densitometry. Relative binding values were used to construct a Hill plot. The Hill coefficient (slope of the best fit curve at the x-axis, H) calculated from the linear best fit curve using Microsoft Excel is shown.  67  A  SoJGI 2V (pM)  SoJD4OA (pM)  00.5124800.51248  00.51248  SoJ (pM)  lOObp  -p  “a  ATP  ADP  24bp  .. p  ATP -p  ADP  *fl4w1flSS  B Soj  C  2 y4.2x-1.1  1.5 V  = 0  .0 Cu  V  0  -0.5 0  -J  -Il -1.5  * -  0  0.1  0:2  0:3 0:4 Log [Soj]  0:5  0:6  0.7  68  Soj was incubated with ATP or ADP (1 mM) for 1 mm at 37°C before addition of DNA (24 bp, 8.4 nM final bp concentration; 100 bp, 0.3 jiM final bp concentration) in a final volume of 20 jil. After an additional 5 mm, 10 jil of the reactions were loaded onto a running gel where the gel buffer and running buffers were supplemented with the indicated nucleotide, and Mg acetate (100 jiM and 4 mM respectively). At a concentration of2 jiM, in the presence of ATP, Soj was able to bind and alter the mobility of both DNA species tested (Fig. 1 5A). When higher concentrations of Soj were incubated with labeled DNA prior to electrophoresis further changes in DNA mobility wereobserved. At 4 jiM Soj the mobility of all 100 bp DNA was altered, and at 8 jiM all 24 bp DNA had altered mobility. The observed patterns of DNA mobility shifts mediated by Soj-ATP for both 24 bp and 100 bp DNA were unusual. A single altered DNA mobility complex, which would indicate the presence of only a single protein-DNA species was not observed. Instead, EMSAs using wild type Soj and the 24 bp DNA produced two different Soj-DNA species with distinct electrophoretic mobilities. Since Hester and Lutkenhaus (2007) suggest that the DNA binding site involves both subunits of the Soj dimer, it is possible that these two species with lower mobility represent DNA binding by a Soj dimer, and Soj tetramer. The binding of a Soj dimer to the 24 bp DNA would be consistent with the 20 bp footprint in the structural model of Soj DNA binding. The structure of a Soj tetramer bound to a 24 bp DNA fragment is not easily predicted from the structure of the dimer, but as shown below there is evidence that Soj dimers might interact. For EMSAs using the 100 bp DNA fragment, three discrete complexes of Soj -DNA complexes with different electrophoretic mobility, and Soj-DNA trapped in the well were observed (Fig. 1 5A, bands labeled i, ii, iii, and the origin are indicated). The relative mobilities (mobr) of observed bands were calculated by dividing the mobility of the species by the mobility iM Soj lane), and iii of unbound DNA. The bands i (mob 0.87, 1 jiM Soj lane), ii (mobr 0.75, 2 1 (mobr 0.43, 8 jiM Soj lane) observed over increasing Soj concentrations suggest the binding of multiple Soj proteins (presumably mostly dimers) to a single molecule of DNA, respectively. Soj-DNA trapped in the well could represent higher order Soj-DNA multimers of sufficient size to prevent electrophoresis into the gel. The observation of a single Soj-DNA electrophoretic species at a particular Soj concentration instead of a mixture of two or three Soj-DNA electrophoretic species was unexpected. Possible explanations for the appearance and disappearance of single electrophoretic species between Soj inputs are given in the Discussion. Soj induced DNA mobility shifts were not observed in the presence of ADP. SojG12V was not able to alter the mobility of either DNA species under any condition indicating that 69  SojG12V is deficient in binding DNA. SojD4OA was also observed to alter DNA electrophoresis, but only in the presence of ATP, indicating that ATP binding but not ATPase activity is required for DNA binding. In the case of SojD4OA EMSAs with 100 bp fragments of DNA, bands with similar iM mobilities to bands i and ii seen with wild type Soj were observed (band iv (mob 0.92, 4 1 SojD4OA lane), band v (mobr 0.77, 8 pM SojD4OA lane)). However, a band with a similar relative mobility to band iii was not observed. Instead SojD4OA-DNA complexes were detected in the wells. This finding might indicate that SojD4OA could more readily form Soj-DNA complexes that cannot enter the gel than is wild type Soj. I tested Soj binding to linearized pUC19 DNA, using agarose gels to separate bound and free plasmid (Fig. 1 5B). Agarose EMSA reactions were performed as described for the 24 bp, and 100 bp EMSA reactions, except pUC 19 DNA linearized by Hindill treatment was added to the reaction (1.5 ng jt1’, 2.3 jiM final bp concentration). The reactions were loaded on a 0.5% agarose gel where the gel and running buffers were supplemented with the indicated nucleotide and Mg acetate (100 pM, and 4 mM respectively). At 2 jiM Soj, the intensity of the band corresponding to unbound plasmid DNA decreased however discrete bands of plasmid DNA with altered mobilities were not observed; rather, the plasmid DNA became trapped in the wells of the agarose gels. In the presence of ADP plasmid became trapped in the wells only when 8 jiM Soj was added. Even with the lower molecular weight DNAs, addition of 8 jiM Soj led to some trapping of the DNA in the wells, so possible this is due to a non-specific effect. The presence of ATP and magnesium in the reaction, gels, and running buffers were required to support Soj-dependent mobility shift of DNA probes (data not shown), further supporting the importance of ATP in Soj DNA binding activity. The disappearance of free 100 bp DNA or linearized plasmid DNA in the EMSAs seemed to occur over a very narrow Soj concentration range. As a simple measure for DNA binding by Soj, I measured the amount of unbound 100 bp DNA at each Soj input using band densitometry using Imagequant v5.2 software. I constructed a Hill plot using the data for the binding to the 100 bp DNA, using the intensity value of unbound DNA from the highest Soj concentration to represent maximum binding. While based on only three data points and the assumption of a linear relationship at the x-axis intercept the apparent Hill coefficient for Soj binding to 100 bp DNA was approximately 4 (Fig. 1 5C) indicating that Soj binding to DNA was cooperative.  70  7.2 Soj-DNA Sedimentation.  The possibility that Soj binding to DNA resulted in the formation of large Soj -DNA complexes was investigated by sedimentation assays using low speed centrifugation. These experiments used a plasmid DNA to provide many Soj binding sites which could allow oligomeric protein structures to form. To carry out the sedimentation assay, Soj was preincubated with either ATP iM bp concentration final) and or ADP for 1 mm. pUC19 DNA was then added (4 ng p1 , 6.2 1 1 after an additional 5 mm, the reaction was centrifuged at 16k x g for 20 mm in an Eppendorf 5414C microfuge. Aliquots from the supematant and resuspended pellet were subjected to SDS-polyacrylamide gel electrophoresis (PAGE, Fig. 16A). The digital image was analyzed by band densitometry using Imagequant v5.2 software. A small amount (12%) of Soj was found in the pellet fraction when the reaction contained only Soj and ATP. In contrast, inclusion of DNA with Soj and ATP resulted in a 4-fold increase in sedimented Soj compared to reactions of Soj incubated with ATP alone (Fig. 16A, compare lanes 2 and 4). When the reaction buffer did not contain magnesium, even in the presence of ATP and DNA, Soj sedimentation was reduced to background level (Fig. 16A-B). Since magnesium is reported to be required as a cofactor for nucleotide binding by Walker box motifs such as found on Soj, this result indicated that a bound nucleotide is required to form a structure that could be pelleted. When the sedimentation assays contained ADP instead of ATP the fraction of Soj in the pellet was lower, although still appreciable. In retrospect I noticed that it looks like the total amount of Soj in the plus ADP condition in Fig. 1 6A is lower that in the other lanes, although all had the same protein input. One possibility is that there was a loading error of the Soj+ADP+DNA supematant (but not the pellet of the same condition) onto the SDS-PAGE gel, which would have artificially increased the fraction of total protein observed in the pellet. I also investigated the fate of the DNA in the sedimentation assays, by analyzing the pellet and supematant fractions by agarose gel electrophoresis. Using a digital image of the gel, the amount of DNA in each fraction was analyzed by Imagequant software (Fig. 16B). DNA sedimentation was observed only in the presence of Soj, ATP, and Mg acetate. Fig. 16C shows that as the concentration of Soj in the sedimentation assays increased, the amount of DNA sedimented also increased, with a concomitant loss of DNA from supernatant fractions (Fig. iM Soj, and at 6 1 iM essentially all DNA 16C and D). DNA sedimentation was detected at —2 1 was found in the pellet fraction. DNA sedimentation was not a linear function of Soj input since at low concentrations of Soj (<1 jiM) only traces of DNA were detected in the pellet fraction. 71  Fig. 16. Sedimentation of Soj-DNA complexes in the presence of ATP. (A) Soj sedimentation in the presence of adenosine nucleotide and DNA. Soj (4 tM) was incubated for 1 mm with indicated nucleotide (NT) prior to the addition of linearized pUC19 DNA (4 ng j.i1’, 6.2 jiM bp final concentration). Supematant (s) and pellet (p) fractions were separated by centrifugation for 20 mm at 16k x g at room temperature. Fractions were treated as described in the Materials and Methods (section 9), separated by SDS-PAGE and stained with Coomassie Brilliant Blue. Numbers in parentheses indicate the band density of the pellet fraction divided by the sum of densities of supernatant and pellet fraction bands as determined by ImageJ software. (B) DNA sedimentation in the presence of Soj and ATP. Sedimentation assay conditions were identical to those in part A, except Soj was included at 5 jiM, nucleotides were included at 1 mM (T indicates ATP; D, indicates ADP), and DNA was included at 4 ng ji[’ where indicated. Supematant and pellet fractions were treated as described in the Materials and Methods (section 9). Fractions were separated by agarose gel electrophoresis and the gel was stained with ethidium bromide. Negative image shown. (C) DNA sedimentation as a function of Soj concentration. Sedimentation assays were identical to those in panel A except they contained the indicated concentration of Soj and 1 mM ATP. Supernatant and pellet fractions were treated as described in the Materials and Methods (section 9). Fractions were separated by agarose gel electrophoresis and the gel was stained with ethidium bromide. Negative image shown. Numbers in parentheses indicate the band density, determined by Image J, of the pellet fraction divided by the sum of supernatant and pellet fraction band densities. (D) The supernatant (+) and pellet (.) band density values from C were plotted as a function of Soj input. Densities were normalized relative to the value of the pellet fraction with 6 jiM Soj. (E) Hill plot of Soj-ATP mediated DNA sedimentation values from panel D. Relative sedimentation values observed for each Soj concentration from panel D were used to construct a Hill plot. The slope of the linear best fit curve at the x-axis (Hill coefficient, nH) calculated from the indicated equation using Microsoft Excel is shown.  72  A Soj pUC19 NT Fraction  +  +  +  —  +  +  +  +  +  —  ATP s p  ATP s p  ATP ADP ADP s p s s p p (.1 (.24) (.07)  ‘  Sojø  —  No  B + Soj T NT Fraction s  +  +  D s  T s p no  Mg  C 0  Soj (pM)  p  S  0.5 sp  1 sp  1.5 S  3 6 2 2.5 4 psps PSPS PS P  II  pUC19I (15)  (0)  (0)  (18)  (5)  (37)  (24)  (85) (100)  E  D  4 0 C  y=7.2x-3.2  C  a 0.8  0  2  0.6  CU  0  w 0.4  C  -2  N  0  Cu  -4  0.2 0  0  -J  z  0  1  2  4 3 Soj(pM)  5  6  -0.2  Ô  0.2 0.4 0.6 0.8 Log [Soil  73  The fraction of pelleted DNA as a function of Soj concentration was used to construct a Hill plot, using sedimentation observed at 6 jiM Soj as a maximum. Assuming the Hill plot is linear, the best fit line of the data had a slope at the x-axis approximately 7 (Fig. 1 6E). This would indicate a very high degree of cooperativity in forming these sedimentable Soj-DNA complexes.  7.3 Soj Protection of DNA from DNase I Digestion.  It was reasoned that the cooperative binding observed in EMSAs and DNA sedimentation reflected protein-protein interactions between adjacent Soj dimers as they bind to DNA. The general model for the binding would be represented by a ‘spreading’ process on DNA. To investigate this, I employed a DNase footprint assay. Since I expected that the initial Soj binding event on a DNA would be random, instead of using an end-labeled DNA, I used an internally labeled 1 kb PCR product to test DNase protection. Primers lkb(+)-F and lkb(+)-R were used with bacteriophage p29 genomic DNA as a template, and P-dATP 32 was included to a internally label the PCR products. The accurate resolution of DNase products in the assays required that the labeled 1 kb DNA did not contain smaller molecular weight DNA species that would interfere with experimental analysis, so the products of the PCR reaction were subjected to PAGE through a 4.5% non-denaturing gel. The position of the 1 kb PCR product in the gel was determined by autoradiography and the band was excised from the gel. The 1 kb product was electroluted from the gel slice into dialysis tubing, and the electroluted DNA was collected and ethanol precipitated as described in the Materials and Methods. To test DNase I protection, labeled DNA (O.38 jiM final bp concentration) was added to reactions with Soj and nucleotide. After 5 mm, DNase I was added for a minute before the reaction was stopped. The products were separated by electrophoresis and phosphorimages were generated from dried gels to determine the size of the protected DNA fragments. I first tested dilutions of DNase to establish the amount I needed to add so that there would be no full length product in the absence of Soj (Fig. 1 7A). In the absence of Soj, DNase I treatment (1/50) reduced the 1 kb internally labeled DNA fragment to a distribution of small DNA fragments with a median size less than 0.1 kb (Fig. 17 A and B). Preincubation of the DNA with 4 jiM Soj in the presence of ATP partially protected the DNA fragments from digestion by DNase (Fig. 17A, 1/50 DNase dilution). When ADP was substituted for ATP, DNase I protection was lost  74  Fig. 17. DNase protection of DNA by Soj-ATP. (A) Calibration of DNaseI. Soj (4 jiM) was incubated with 1mM ATP (T) or ADP (D) for 1 mm prior to the P-dATP labelled 1 kb PCR product (0.25 ng addition of an internally 32 —M.38 jiM bp concentration final). The reactions were incubated for 5 mm. DNase I was added to the reaction for 1 mm at the indicated dilution, where indicated, and the reaction was stopped. Reaction products were separated by electrophoresis and a phosphorimage of the dried gel was generated. Arrows indicate the mobility and size of molecular weight standards from lane 1 (0.3 kb, and 0.1 kb) and 1 kb labeled PCR product. (B) Protection at varying Soj concentrations. Soj was preincubated with 1 mM ATP for 1 mm prior to the addition of an internally 32 P-dATP labelled 1 kb PCR product. Reactions were treated with a 1/50 dilution of DNase I for 1 mm before the reaction was stopped. Reaction products were separated by electrophoresis and a phosphorimage of the dried gel was generated. Lane 1, 0.3 kb and 0.1 kb standards. Lanes 2-10, reactions contain indicated Soj concentrations. Arrows indicate the mobility of molecular weight standards and 1 kb labeled PCR product. (C) Densitometry profiles of lanes 2-10 from panel B. Densitometry profiles were generated by Imagequant software v5.2 and lanes 2, 7-10 are indicated. The hash mark on the horizontal axis at the top indicates a density of 1500 arbitrary units. The densitometry scan of lane 2 reached a maximum at 10 000 a.u. (D) Protection of 1 kb DNA from DNaseI digestion as a function of Soj concentration. The fraction of 1 kb completely protected DNA from DNaseI digestion (determined by densitometry using Imagequant v5.2) relative to the densitometry of 1kb DNA without DNaseI treatment (panel A, lane 2) was plotted as a function of Soj concentration. (E) Hill plot of appearance of fully protected 1 kb DNA. Relative protection values (P) observed for each Soj concentration from panel D were used to construct a Hill plot. The slope of the best fit curve at the x-axis (Hill coefficient, flH) calculated from the indicated equation, using Microsoft Excel is shown.  75  A  1234 1/50  DNase Soj (pM) NT  -  T  +  +  T T  -  D T T  —  I kb  1/300  1/100 -  ++-  -  1/500 +  +  T D T T T  -  D T  -  ++  T T D  114  $  0.3 kb  0.1 kb%  B 1 2 3 4 Soj(pM) DNaseI  -  I  -  -  5 6 7 8 9 10  010.5 1  2  4  6  C  8  ÷1+1+1+1+1+1+1+1  Density (au.)  lkb 2  V’\c’?  0.3 kbb  %.10 8  7 0 lkbk  [9  D  E  1  1  0.8  z  a 0 * (U S -0.5 a a —1 0) -1.5 0  DC  .2 0.6 o.$ c9 .2 a 0.4 13 0.2  •4.4-  .-‘  ‘I  I Soj (pM)  /4.1  -2  /.,  0 0.1  y=4.lx-3.2  0.5  7  10  -2.5  -  I)  0.2  0.4 0.6 log [Soj]  0.8  76  A  ci)  E  0 (0 C  *  ci) ci)  0 0. U)  0  pixels  B 900  \1c%\  800  700 U)  600  y= 3.23 &0028x 81  500 400  z U  300 200 100 0 0  0.2  0.4  0.6  0.8  1  1.2  DNA length (kb)  Fig. 18. Estimation of DNA sizes protected from DNaseI digestion by Soj. (A)  Densitometry profiles of lanes 2-10 from Fig. 13B. Discreet length DNA species appeared over a range of Soj inputs as a series of small peaks (*) on the densitometry profiles. (B) Size estimation of the DNAs. The electrophoretic mobility of 1 kb, 0.3 kb, and 0.1 kb molecular weight standards from Fig. 1 5B were used to estimate a mobility curve. A best fit curve equation was calculated x) and was used to estimate the sizes of DNA bands detected as 28 (y=8l3 .23e peaks in densitometry profiles in panel A (perpendicular jointed arrows).  77  and the pattern was similar to that generated by DNase I treatment without Soj (Fig. 1 7A, compare lanes 3 and 4). I then tested the protection of DNA from DNase I digestion (at 1/50 dilution) using a range of Soj concentrations (Fig. 17B). There was a dramatic shift in the pattern of protected DNA fragments between Soj concentrations of 1 iM and 4 pM. The densitometry profiles of protected fragments at greater than 2 tM Soj showed multiple small peaks most obvious in the 4, 6 and 8 jiM Soj conditions. These peaks corresponded to visually observable bands on the phosphorimage (Fig. 17 B and C, Fig. 1 8A). The size of DNA fragments being protected changed over a small range of Soj concentrations, which indicated cooperativity of Soj DNAbinding. The appearance of 1 kb DNA, which represented full DNase protection, was observed at 4 jiM Soj where 15% of DNA was fully protected. The fraction of full length DNA protected from DNase by Soj binding increased sharply over a narrow range of Soj concentration (Fig. 17D), and appeared to be cooperative. Imagequant was used to calculate the amount of full length DNA protected at the various Soj concentrations, and those data were used to produce a Hill plot (Fig. 17E). Assuming that the Hill plot is linear at the x-axis, it had an intercept slope of approximately 4 which supported cooperative binding Soj along the length of DNA. The DNase protection profiles were analyzed assuming that the small peaks observed represented specific DNA species. The sizes of these protected DNA species were estimated by comparing their electrophoretic mobilities to the electrophoretic mobilities of 1 kb, 0.3 kb, and 0.1 kb DNA (Fig. 1 8B). The mean differences in size between adjacent DNA species detected in multiple Soj input conditions and replicate experiments were -47 or 90 bp. The approximate 20 bp DNA footprint of a Soj dimer on DNA proposed by Hester and Lutkenhaus suggest the --45 bp gradations between protected DNA species could reflect the expected size of DNA occupied by the binding of a Soj tetramer. However, the a 47 bp difference in the length of protected DNA could also result from the simultaneous binding of two Soj dimers to both ends of a cluster of Soj proteins.  7.4 Soj Inhibition of Transcription in Vitro.  The sporulation negative phenotype observed in z\spoOJ cells is characterized by low transcription of essential stage II sporulation genes (spollA, spollE, spoliG, (Ireton et al., 1994; Quisel and Grossman, 2000)). The deletion of so] in a AspoOJ background rescues the sporulation negative phenotype of a AspoOJ strain, and could indicate that Soj could directly 78  inhibit sporulation by acting as a repressor of transcription (Cervin et a!., 1998; Ireton et a!., 1994; Quisel et a!., 1999; Quisel and Grossman, 2000). It has been noted that transcription of the spollA , spollE, and spoliG operons is activated by the phosphorylated form of SpoOA (Spo0AP) (Ireton et a!., 1994; Quisel and Grossman, 2000). Non-specific binding of Soj to DNA to act as a direct repressor of all transcription is not compatible with the operon-specific transcription inhibition observed in vivo, but Soj could inhibit sporulation by selectively repressing transcription activated by Spo0AP. To investigate this possibility, transcription P32 repression by Soj was examined by single round transcription assays in vitro. Soj, ATP+x GTP, and a DNA fragment with the indicated promoter (P) were incubated for two minutes prior to the addition of purified B. subtilis RNAP. SpoOAC (the constitutively active C-terminal DNA binding domain of SpoOA) was added when transcription from PspoIIG was assayed. The reactions were incubated for another two minutes to allow transcription initiation. Transcription was limited to a single round by adding heparin, which binds and inactivates free but not initiated RNAP, along with the remaining nucleotides. Reactions were incubated further and stopped by addition of urea. The products of transcription were analyzed by denaturing PAGE. A phosphorimage of the gel was used to quantify the major 32 P-labelled transcript by densitometry using Imagequant. The inhibition of transcription from four different promoters by Soj was investigated. PspoIIG was selected because it is known to be repressed in vivo in a AspoOJ mutant and therefore is a presumed target for Soj transcription inhibition, and transcription initiation at this promoter in vitro has been well studied. I constructed a mutant of the PspoIIG (PspoIIGl8) by removing 4 bp between the -10 and -35 consensus sequences (Fig. 19A). This change was predicted, and I showed experimentally (Fig. 1 9B) to change the promoter from being dependent on Spo0A’—P for activity to being Spo0A—P independent. The basis of the Spo0A—P dependence is that the separation between the -10 and -35 sequences is 5 bp greater than the optimal distance. Thus this promoter is identical in sequence to PspoIIG, except for the internal deletion. Two other promoters PabrB, and PA2 were used as controls because transcription of these genes has not been shown to be negatively regulated by Soj in vivo; moreover, they are amenable for use in the single round transcription assay. The product of abrB is a transition state regulator which controls the expression of multiple genes in late log phase and early stationary phase. The A2 promoter from bacteriophage ç29 drives expression of several genes during the early stages of the lytic cycle in B. subtilis.  79  Fig. 19. Inhibition of transcription initiation in vitro by Soj. (A) Sequence of PspoJIG and shortened spoliG promoters. The region between +1 and —60 is indicated. The —10 and —35 sequences are in boldface letters. Underlined bases indicate OA boxes. Dashes indicate bases deleted from BglII-treated pUCIIGBglIItrpA by incubation with mung bean nuclease followed by ligation (described in Materials and Methods (section 2.7)). Italicized letters in PspoIIGBglII indicate a BglII site that was introduced into pUCIIGtrpA. (B) SpoOA dependence of transcription from PspoIIG, and independence of transcription from PspoIIGl8. Transcription from PspoIIG and PspoIIGl8 in the presence and absence of SpoOAC was assayed. Transcription assays were composed and performed as indicated in the Materials and Methods (section 11). Products from transcription assays were separated by electrophoresis through an 8% denaturing polyacrylamide gel. The major transcript was detected and quantified by exposure of the gel to a phosphor screen followed by analysis with Imagequant v5.2. Error bars represent the standard deviation from the average of four independent assays. (C) Soj-mediated transcription inhibition from different promoter templates. Four promoter templates were used: PspoIIG (i); PA2 (ii); PabrB (iii); PspoIIG]8 (iv). The indicated concentrations of Soj were incubated with PspoIIG (the insert indicates transcription at lower Soj concentrations), or PA2 and ATP plus GTP (and SpoOAC at 1 jiM when PspoIIG was used) for 2 mm in transcription buffer with BSA (0.1 mg ml-1) at 37°C before the addition of RNAP. After another 2 mm, heparin plus GTP and UTP were added, and the reaction was stopped after 5 mm of elongation. Relative transcription was measured as the percentage of transcription in the absence of Soj. To maintain a consistent ionic strength among samples, Soj was diluted into elution buffer and a constant volume was added to each reaction. The same volume of storage buffer alone was added to the control reactions. Assays of Soj-mediated transcription inhibition at PabrB were performed as described for PA2, except the initiation nucleotides used were ATP plus GTP plus UTP, and the transcripts were separated by electrophoresis on a 12% denaturing polyacrylamide gel. Soj-mediated transcription inhibition at PspoIIGl8 in the presence (.) or absence (+) of 1 jiM SpoOAC was assayed and analyzed as described for PspoIIG. Error bars represent the standard deviation from the average percentage of at least three independent transcription assays.  80  A PspoIIG PspoIIGl 8 PspoIIGBgl II  ‘  5’ TTTTCCTCTCAAQATTAATTGACAGACTTTCCCACAGAGCTTGCTTTATAcTTATGAAGCA- 3’ 5’ TTTTCCTCTCAACATTAATTGACAGACTTTCCCACA- -TTGCTTTATAcTTATGAAGCA- 3’ -  - -  -  5’ TTTTCCTCTCAACATTAATTGACAGACTTTCCCACAGATCTGCTTTATATTATGAAGCA -3’ -  I  I -60  -40  -50  I  -30  -20  -10  +1  B  150000  (0 C  ,1O0000 E  5000  0  —  —  —  \‘  C  120  i. PspoIIG  100  120  PabrB  ii. PA2  N iv. PspoIIGl8  I Soj (pM)  12 0246810 Soj (pM)  12  81  As seen in Fig. 1 9C the addition of increasing concentrations of Soj resulted in a decrease in the amount of transcript from all promoters used in the transcription assays. In all cases the inclusion of 10 jiM Soj resulted in >85% reduction of the primary transcript compared to conditions where Soj elution buffer alone was added. The effect of increasing Soj concentrations differed when PspoIIG was used as the template compared to other promoter templates. A 50% reduction in relative transcription from PspoIIG was observed when Soj was included at 1 jiM. Approximately 5-fold higher Soj concentrations were required for the same reduction in relative transcription from the other 3 promoters. The higher concentrations of Soj required for a 50% reduction in relative transcription from PspoIIGl8 compared to PspoIIG indicated that Soj -mediated transcription inhibition at PspoIIG was likely not due to Soj binding to a specific sequence because only 4 bp differed between the two. The data support a model where Soj preferentially antagonizes SpoOA-dependent transcription activation.  8 Detection of Soj-DNA Complexes by Light Scattering.  Size exclusion chromatography indicated that the majority of Soj is dimeric (Fig. 13). Soj binding to DNA characterized by EMSAs and DNase protection assays, indicated that multiple Soj dimers bound to DNA cooperatively as indicated by Hill plots of the data. Data from DNase protection assays indicated that the binding of Soj dimers along the length of the DNA could completely cover the fragment. Sedimentation assays in Fig. 16 indicated that Soj DNA structures were of sufficient size to be cleared from solution by low speed centrifugation, and EMSAs indicated that they were too large to enter 0.5% agarose or 4.5% acrylamide gels (Fig. 15). Furthermore, I noticed that reactions containing Soj, ATP and DNA in conditions which resulted in DNA sedimentation, were visibly turbid. I was interested in characterizing the formation of the complexes with a method that would allow a more continuous assay rather than the discontinuous assays of EMSA and sedimentation. I chose to use 90° light scattering for this assay because of the availability of suitable instruments. To monitor light scattering Soj-DNA structures I used 90° light scattering using a Cary Eclipse fluorescence spectrophotometer. The instrument has a lamp which emits 350 nm wavelength light, a detector at 90° from the incident light, and a photomultiplier tube with adjustable voltage to change the detector sensitivity. The instrument also has the capability to record multiple data points over time. To measure the light scattering, Soj was added to reactions with ATP or ADP in a cuvette (1 cm light path) with moderate stirring, at 30°C. After 82  Cu 0 0 C  1  C, C I  0 Cu C) 0  .C C,  0  2  4 time (mm)  6  8  Fig. 20. Light scattering by DNA and Soj-ATP. Soj (1 jiM) was incubated with 1 mM of the indicated adenosine nucleotide in a UV/vis plastic cuvette (10 mm light path) in 1.5 ml of ATPase buffer with 100 mM NaC1. After 1 mm, pUC19 DNA (0.5 ng ji1’, 0.78 jiM bp concentration final) was added where indicated and light scattering in arbitrary units (a.u.) was monitored. Fluorescence spectrophotometer PMT voltage in this assay was 600 V.  83  1 minute, DNA was added and the extent of light scattering (350 nm) was followed over approximately 8 mm. Some differences among the light scattering profiles from different light scattering experiments with similar conditions were noted, however the overall trends in the extent, rates and requirements for light scattering were observed in a large number of experiments, and typical examples are shown. Fig. 20 shows results from experiments where pUC19, Soj and either ATP or ADP were mixed and light scattering was monitored over 6-8 mm. When 1 jiM Soj was added to a reaction with ATP, a small immediate increase in light scattering was observed (Fig. 20) and light scattering slowly increased over the following 10 mm. The same trend was observed when Soj was added to a reaction with ADP (data not shown). When pUC19 DNA (0.5 ng j.tr’,0.78 jiM final bp concentration) was added to Soj plus ATP, a second large increase in light scattering was observed. This DNA-dependent increase was not observed when ADP was substituted for ATP. The DNA-dependent light scattering was unaffected by whether the DNA was circular, or linearized by restriction endonuclease treatment (data not shown). Whatever the molecular mechanism that is responsible for the increase in light scattering in the presence of DNA, it was dependent on the presence of ATP. The similarity between the charge, counter ions, et cetera associated with adding the ATP versus ADP makes it unlikely the trinucelotide dependence is due to some ionic interaction. Thus it seemed likely that the observed changes in light scattering when DNA was added to Soj and ATP depended on an effect of ATP on Soj. Mixtures of Soj, ADP, and DNA consistently scattered less light than Soj incubated with ATP in the absence of DNA. In fact Soj, ADP, and DNA scattered less light than any other condition including Soj and ADP without DNA (not shown). At present it is not known why this result was observed, but it could indicate that Soj-ADP might transiently interact with DNA to stabilize a Soj species which scatter less light than Soj bound to either ATP or ADP in the absence of DNA. SojGl2V did not alter the electrophoretic mobility of DNA (Fig. 15A), although it was capable of ATP hydrolysis (Fig. 9). SojG12V was added to light scattering assays to investigate whether SojG12V differed from wild type Soj with respect to light scattering. Mixtures containing SojG12V, DNA, and ATP were monitored over time only small changes in light scattering were observed when SojGl2V (Fig. 21). The presence of ADP instead of ATP did not change the light scattering profile for SojG12V plus DNA (data not shown). Thus conditions required for the light scattering by Soj and SojG12V paralleled the conditions required for DNA binding as observed in other assays (Fig. 15), which indicated that light 84  1  = I.  0 .  0  1  2  3  4  5  6  time (mm)  Fig. 21. Light Scattering by SojG12V. Soj, or SojG12V (1 .iM), was added to a light scattering assay, as described in Materials and Methods (section 12) containing the indicated nucleotide (1 mM). Following 1 mm of incubation pUC19 DNA (0.5 ng j.tl’, 0.78 1 .iM bp concentration final) was added to each mixture. PMT voltage in these assays was 600 V.  85  scattering monitored the formation of Soj-DNA structures. In addition the data suggested that ATP but not ADP was a co-factor for forming these structures. Light scattering by wild type Soj  +  ATP  +  DNA in Fig. 21 had a faster rate of formation compared to the same conditions in  Fig. 20, and reached a plateau within two minutes after DNA addition whereas a plateau was not observed in Fig 20. I considered these minor variations in protein activity between different preparations of purified Soj. For example, the wild type Soj used in Fig. 20 could have been less active than that used in Fig. 21. The difference could also result from differences in the purity of the ATP used in each assay. Older solutions of ATP may have contained more ADP from spontaneous breakdown of ATP duringstorage (and therefore a lower concentration of ATP) resulting in lower rates of light scattering observed in Fig. 20. As a result of these minor variations I did not attempt to measure “specific activities” of complex formation and I used comparative experiments done on a single day to examine properties such as the effect of DNA length (see below). Monitoring formation of SojD4OA-DNA complexes as measured by light scattering presented a technical hurdle that turned out to insurmountable. SojD4OA alone resulted in high levels of light scattering independent of nucleotide and/or DNA (data not shown) and no additional effects of adding DNA could be determined. EMSA results which indicated that the DNA binding activity of SojD4OA was ATP-dependent (Fig. 1 5A), suggested that SojD4OA light scattering in the absence of DNA binding (no DNA, or DNA in the presence of ADP) was due to some characteristic of SojD4OA that promotes aggregation of the protein itself. This effect could be related to a larger fraction of —1 05 kDa species observed in size exclusion chromatography of SojD4OA-ATP compared to wild type Soj-ATP or a high MW band observed in BN-PAGE of SojD4OA in the absence of nucleotides (Figs. 13, 1 4A). However, the basis of SojD4OA light scattering effect is not known at this time, and the discrepancies between the light scattering of SojD4OA and size exclusion chromatography and BN-PAGE assays remain unresolved. I next investigated the effect of Soj concentration on rates and levels of light scattering changes. Varying concentrations of Soj were pre-incubated with ATP for 1 mm and then pUC19 DNA was added (0.5 ng jff’, 0.78 jiM final bp concentration) and the light scattering monitored. The light scattering intensities, from the time of DNA addition, for 1, 2, and 4 jiM Soj are shown in Fig. 22A. Because of the high scattering intensity observed with 4 jiM Soj, it was necessary to decrease the PMT voltage in this experiment compared to the setting used in Figs. 20-21. The low PMT voltage resulted in a small increase in light scattering at 1 jiM Soj 86  A 160  pM  R 140 120  I  40  .2  20  A  1pM  0  2  4 time (mm)  3  5  6  B —14O  ...  34  Soj(pM)  Fig. 22. Light scattering as a function of Soj concentrations. (A) Light scattering at 3 Soj concentrations. DNA (pUC19, 0.5 ng pJ’, 0.78 jiM bp concentration final) was added at 2 mm to mixture containing the indicated concentration of Soj preincubated with 1 mM ATP as described in Materials and Methods (section 12). Fluorescence spectrophotometer PMT voltage was 500 V in this assay. (B) Maximum net light scattering as a function of Soj concentration. The net change in light scattering between 2 mm (DNA addition) and 4.5 mm time points in panel A is plotted as a function of the Soj concentration in the reaction. 87  Table 8. Initial Rates of Increase of Light Scattering for Different Soj Concentrations.  Soj fliM)  Rate of Light Scattering Increase  (a.u. min  25 432 858  1 2 4  a. Light scattering rates for each Soj concentration are the average of two replicate light scattering experiments.  88  compared to experiments in Fig. 20-21. Increased Soj concentrations increased both the levels and rates of light scattering upon DNA addition. The initial rate of increase in light scattering, calculated from the slope of the linear portion of light scattering curves from two replicate experiments, for each Soj concentration is indicated in Table 8. The rate increased almost 20fold between 1 jiM and 2 jiM Soj, but increased only -2 fold between 2 jiM and 4 jiM Soj. These rates indicated that Soj-ATP DNA structure formation might be cooperative but saturates between 2 jiM and 4 jiM Soj. The net increase in light scattering was calculated by subtracting the light scattering value at the time of DNA addition from the light scattering value at 6 mm. While the initial rate of increase in light scattering was not a linear function of Soj, the overall amount of light scattering was roughly a linear function of Soj concentration (Fig. 22B). The effect of Soj concentration in EMSAs, and DNase protection assays showed similar trends, where small increases in Soj concentration led to a large increase in Soj interaction with DNA. In EMSAs no DNA binding was observed at 1 jiM Soj, while at 2 jiM Soj —50% of the DNA was bound by Soj (Fig. 15). In DNase I protection assays no full length DNA was observed at 2 jiM Soj, whereas 4 jiM Soj led to appearance of full length DNA (Fig. 17B). One significant difference between light scattering assays and other assays described above was the detection of light scattering at 1 jiM Soj (in the presence of ATP and DNA). Other assays did not detect Soj interaction with DNA at 1 jiM final Soj concentration so possibly the light scattering assay is one of the most sensitive measures of Soj -DNA interaction. The effect of ATP concentration on the formation of Soj-DNA structures was investigated. DNA was added to reactions of Soj preincubated with various ATP concentrations and light scattering was observed (Fig. 23A). A change in light scattering in reactions containing Soj and DNA was observed in the presence of ATP compared to light scattering by Soj and DNA without ATP, as previously observed. The change in light scattering level 6 mm after DNA addition was calculated for each ATP concentration, and then the observed change in an identical reaction without ATP was subtracted. The rate of light scattering over the first 6 mm increased as the ATP concentration was increased (Fig. 23B). Comparing the effect of ATP concentrations on light scattering, with the effect on the initial velocity of Soj ATPase (Fig. 11 B), indicated that the concentration of ATP required for 50% activity for each assay differed by a factor of two. The Km for ATP in enzymatic hydrolysis of ATP was determined to be 180 jiM, while the concentration of ATP which gave 50% of the light scattering rate observed at 1 mM ATP occurred at approximately 360 jiM, indicating similar levels of ATP binding are required for both ATPase activity and formation of light scattering complexes. 89  A ATP (mM) 300  JU  I  LILi’” 1  I  0.75  0.5  250  0.25  ‘  0.1  200  0  Cu Ci Cu ‘I  -lul) 0  I  I  1  2  I  I  I  I  I  3  4  5  6  7  8  time (mm)  B 100  *_________________________________  C  E  80  Cu  40  20  0  I  0  0.2  I  0.6 0.4 ATP (mM)  0.8  1  Fig. 23. ATP dependence of Soj light scattering. (A) Effect of ATP concentration on Soj-DNA light scattering. Soj (1 M) was incubated with the indicated concentration of ATP for 1 mm as described in Materials and Methods (section 12). After 1 mm pUC19 DNA was added (0.5 ng pf’, 0.78 M bp concentration) and light scattering was monitored for another 6 mm. PMT voltage was 600V in this assay. (B) The net change in light scattering of Soj and DNA reactions between 2 mm (DNA addition) and 8 mm for each ATP concentration condition was corrected by subtraction of the net light scattering amplitude from light scattering reactions of Soj and DNA without ATP in the same time interval from panel A. The net change in light scattering was plotted as a function of ATP concentration. 90  200 180 160  140 120 100  8O 0  60 0  .40 20  0 0.01  I  0.1  ii  III  1  10  DNA input (nglpl)  Fig. 24. DNA concentration dependence of Soj-ATP DNA light scattering.  The net change in light scattering was determined 2.5 mm (0), and 5 mm (+) after addition ofpUCl9 DNA to I 1 iM Soj preincubated with 1 mM ATP as described in Materials and Methods (Section 12). PMT voltage in these assays was 600 V.  91  The characterization of light scattering dependence on DNA was monitored by addition of pUC19 DNA at various concentrations to reactions containing 1 jiM Soj preincubated with 1 mM ATP. The observed net increase in light scattering at 2.5 and 5 mm after DNA addition is plotted versus DNA concentration in Fig. 24. At both time points the maximum increase in light scattering occurred at —O.5 ng jil’ (0.78 jiM final bp concentration) DNA. Lower DNA concentration (0.01  -  0.1 ng jil’) resulted in lower levels of light scattering. A ten-fold increase  in DNA concentration input (to 5 ng  jir’, 7.8 jiM final bp concentration) also led to a decrease  in light scattering. At low concentration DNA may be limiting in the formation of Soj-DNA light scattering structures. Since high DNA concentration resulted in levels of light scattering below the maximum, possibly at high DNA input Soj dimers are too dispersed on the DNAs to form structures that scatter light. If this were the case it would have implications for the structure of the light scattering complex. The formation of Soj DNA structures that scattered light was also investigated by varying the length of DNA added to reactions of Soj-ATP. DNA molecules ranging from 0.1 to 2.6 kb were created using primers and templates indicated in Table 3. Primers, templates, and unincorporated dNTPs were separated from PCR products using a Qiagen PCR cleanup kit. Plasmid DNA (2.6 kb) was linearized by digestion of CsCI gradient-purified pUC19 with Hindill with no further cleanup. The concentration of the DNAs were calculated from the 260 of a small PCR sample, and checked by comparing band intensities of small samples with 0D the band intensities of known DNA concentrations from a DNA ladder following agarose gel electrophoresis. The determination of DNA concentrations by two independent methods was used to increase the possibility that any differences in light scattering profiles were due to changes in DNA length, and concentration. The effect of DNA length on the formation of light scattering structures is shown by the data in Fig. 25A. In this experiment all DNA concentrations were 0.5 ng  jir’ (0.78 jiM bp  concentration); however, the molar concentration of the DNA fragments will vary. All of the DNAs supported light scattering with Soj-ATP. The increase in light scattering was characterized by a lag before the curves increased linearly for a few minutes and then approached an apparent saturation. There was a longer lag when smaller DNA lengths were added to the reactions (see enlarged scale in Fig. 25B). The lag time is plotted versus DNA length (Fig. 25C). The lag time decreased as the DNA length increased, and was essentially absent with a 2.6 kb DNA. The slope of the linear portion of the light scattering time course 92  Fig. 25. Effect of DNA size on light scattering by Soj. (A) Formation of light scattering complexes over time using DNA of different length. Soj (1 iiM) was incubated with ATP (1mM) as described in Materials and Methods (section 12). DNA fragments of indicated lengths were added at 2 mm to a final concentration of 0.5 ng tl’ (0.78 jiM bp concentration) and light scattering was monitored. PMT voltage in this assay was 600V. (B) The portions of light scattering profiles from panel A between 2 mm and 4.5 mm are shown. (C) Changes in apparent lag time for formation of light scattering complexes after addition of DNAs of different lengths. The lag time, measured as the time between DNA addition and when the rate of light scattering increase became linear as a function of time, was plotted as a function of the DNA length added to light scattering reactions in panel A. (D) Changes in the rate of increase in light scattering for different DNA lengths. The rate of increase in light scattering was calculated from the average slope of the linear portion of the light scattering curves from panel A. These values were plotted as a function of the DNA length added to light scattering reactions. (E) The net change in light scattering observed after addition of different DNA lengths. The net change in light scattering from 2 mm (DNA addition) to 8 mm was plotted as a function of the DNA length added to light scattering reactions.  93  A 0.5 kb 0.3 kb >  I kb  .4-  U,  2 kb 2.6 kb (un pUCI9)  .4-  I  0.1 kb  w U U, .4-  .=  0  2  4 time (mm)  6  B  8  C 1-ti  U,  ::  0 .4-  120 100  0  ‘S  1  0  44-  40 20 0  U  (I) 4-  2  3 time (mm)  a)  1  2  3  DNA length (kb)  4  E  D 4-  0  180 160 140 120 100 80 60 40 20 0  450 C,  ‘ 400 oR 350 ‘  300 —  S 0  2 1 DNA length (kb)  3  o  i DNA length (Rb)  3  94  A ,  pUCI9 29005 ng!pI 270  U,  a, 250 .9 230  24 bp  4-  .  a,  4-  190  ‘  170  4-  .  1 nglpl  210  0 5 ng4iI 5ngIpI  150 01234567 time (mm)  B  24 bp (ng/pI)  325 300  410  80 .  70 60  275  pUCI9,.’  .9 250 .  C  50  24 bp  ;40  225  30  a, i 200 4-  4-  I  4  20 1o 0  150 0  1  3 4 2 time (mm)  5  6  —  0  3 6 24 bp DNA (nglpl)  9  Fig. 26. A 24 bp DNA does not stimulate formation of light scattering complexes. (A) Soj (1 jiM) was incubated with 1 mM ATP for 1 mm as described in Materials and Methods (section 12), before either a 24 bp duplex DNA (final concentration indicated) or pUC19 DNA (0.5 ng ji1 ) was added and light scattering was monitored. (B) The final 1 concentrations of 24 bp DNA indicated on the right were added to Soj+ATP as described in Materials and Methods (section 12). After a 1 mm incubation, pUC19 DNA was added 1 final) and light scattering was monitored. (C) The net change in light (3 mm, 0.5 ng jiF scattering determined from the curves in panel B 3 mill after addition ofpUCl9 DNA is plotted as a function of 24 bp DNA concentration added (at 2 mm).  95  also differed with DNA length. The slope was greatest with the 500 bp DNA addition and was lower with both shorter and longer molecules (Fig. 25D). The net changes in light scattering levels, measured at 6 minutes after DNA addition, also differed with DNA length (Fig. 25E). The light scattering levels were observed to peak at about 500 bp in three independent light scattering assays with different Soj protein preparations. In control experiments with the shorter DNA molecules the formation of Soj and DNA light scattering complexes did not occur in solution containing ADP or no nucleotide (data not shown), and so appeared to require ATP. As detected by EMSA (Fig. 15), Soj-ATP was capable of binding to a 24 bp duplex oligonucleotide. However, addition of a 24 bp double stranded DNA to Soj and ATP did notcause an increase in light scattering over a range of DNA concentrations (Fig. 26A). Furthermore, preincubation of Soj-ATP with the 24 bp double stranded DNA prior to addition of pUC19 DNA inhibited light scattering observed when plasmid DNA alone was added to Soj ATP (Fig. 26B). The inhibition of plasmid DNA-mediated light scattering by the 24 bp DNA increased with the concentration of the 24 bp double stranded DNA (Fig. 26C). These data indicated that 24 bp DNA did not allow formation of structures that scatter light. Since the 100 bp DNA did support formation of complexes that scattered light, the minimum length of DNA required to form light scattering structures was apparently between 24 bp and 100 bp. The effect of DNA lengths on Soj-ATP light scattering lag times was unexpected. Identical masses of DNA should represent the addition of identical amounts of Soj binding sites. A simple model where Soj bound DNA cooperatively due to a preference for binding sites adjacent to a Soj dimer already bound to DNA does not predict the different lags observed upon addition of different length DNA. The data could be explained by a model where multiple Soj dimers bound to adjacent DNA sites form ‘tracts’ of Soj multimers on DNA which are able to interact with other Soj ‘tracts’ either on the same or different DNA molecules, and this possibility will be further addressed in the Discussion.  9 Soj-DNA Complexes Resolved by Transmission Electron Microscopy.  Direct visualization of Soj bound to DNA by negative stain transmission electron microscopy was attempted to examine the ultrastructure of a Soj-DNA complex. Soj-ATP was incubated with pUC 19 DNA and the complexes were collected by centrifugation. The pellet fraction was resuspended in buffer without ATP, and quickly applied to a carbon coated grid The grids were stained with uranyl acetate (Fig. 27). Micrographs of the linearized pUC 19 96  B  Fig. 27. Transmission electron micrographs of negatively stained sedimented Soj ATP DNA structures. Soj (4 jiM) was incubated with nucleotide (1 mM) and DNA (2 ng ji1’) in ATPase buffer with 100 mM NaC1 and treated as sedimentation assays described in Materials and Methods (section 9). Pellet fractions were resuspended in buffer and placed on the glow discharged carbon coated TEM grids and stained with uranyl acetate as described in the Materials and Methods. (A) Micrograph of linear plasmid DNA used in assays without Soj. (B-D) Micrographs of resuspended sedimented complexes from reactions containing Soj, ATP, and plasmid DNA. (E) Digitally magnified inset of boxed area from D. Bars in panels A-E are 100 nm. (F) Boxed area of panel E where the greyscale is inverted and the brightness and contrast have been adjusted by Photoshop v6.0 to highlight points of electron densities (white circles). (G) Representative field of view from a grid prepared without Soj or DNA (an embedded carbon coat impurity shows a focal reference, scale bar 500 nm). Roman numerals i-ui in panels A-E indicate structures of varying diameter described in the text. 97  DNA template alone used in sedimentation assays (Fig. 27A) showed linear molecules of uniform diameter. When Soj-ATP and DNA were incubated, sedimented and applied to the grids, a variety of structures were observed. After many attempts no higher quality pictures were obtained. The widths of these structure, where it was possible to determine them, were approximately 6-12 nm or 17-22 nm (labeled i and ii, respectively, in Fig. 27B-D). I selected one area that is boxed in Fig.27D and used digital magnification to look for areas of greater electron density that might represent the position of the Soj proteins.(Fig. 27E). The points of electron density were further highlighted by inverting the greyscale and adjusting the brightness and contrast of Fig. 27E. Points of electron density with dimensions approximately 5-6 nm in width appear to be visible (Fig. 27F). The dimension of these dots would be consistent with the previously published dimensions of a Soj dimer from T. thermophilus (Leonard et a!., 2005). The 5-6 nm wide electron dense structures are potentially in a higher order repeating structure. All of the micrographs also showed DNA not bound by Soj-ATP (labeled iii in Fig. 27). This was expected given that at 4 pM Soj DNA sedimentation was incomplete in sedimentation assays and at 4 jtM Soj, protection of DNA from DNase I digestion was incomplete (Figs. 16 and 1 7B). DNA unbound by Soj could also represent decay of complexes during grid preparation as the dehydration requires wicking away the buffer that contains the ATP. Structures seen in Fig. 27B-E were not observed in conditions without ATP or DNA  (  Fig. 27F)  The structures with widths equal to or larger than 17 nm in Fig. 27B might represent Soj-Soj interactions between complexes bound to different DNA molecules, indicating the possibility that Soj can form higher order structures and bridge DNA molecules (addressed in the Discussion).  10 Soj Interaction with SpoOJ  SpoOJ is required in vivo to antagonize the negative regulation of sporulation by Soj. A simple model by which SpoOJ affects Soj is by a direct interaction. Several lines of experimental evidence support such a direct interaction between Soj and SpoOJ. Plasmids with a parS sequence are stabilized in B. subtilis and E. coli in the absence of antibiotic selection only when both Soj and SpoOJ are expressed (Hester and Lutkenhaus, 2007; Lin and Grossman, 1998; Yamaichi and Niki, 2000). Chemical cross-linking treatment of B. subtilis followed by resolution of Soj-SpoOJ conjugates by Western blots using aSoj and cLSpoOJ antibodies, also support a direct Soj interaction with SpoOJ (Ogura et a?., 2003). The interaction of Soj and 98  SpoOJ in vitro was assayed by multiple techniques with the expectation that these interactions would affect the in vitro activities and characteristics of Soj) or SpoOJ.  10.1 Soj ATP hydrolysis is stimulated by SpoOJ.  The ATPase activity of some members of the ParA/MinD family of proteins has been shown to be stimulated in vitro by the cognate ParB (or the protein encoded by the second ORF of the operon). The MinE stimulation of the ATPase of MinD is required for the proper function of MinD in vivo. A point mutation in MinE that cannot stimulate MinD ATPase in vitro leads to the production of minicells when expressed in E. coli (Hu and Lutkenhaus, 2001). If Soj and SpoOJ followed the MinD/MinE pattern, SpoOJ would stimulate Soj ATPase activity, and this stimulation could be detected in ATPase assays. Purified recombinant SpoOJ was added to Soj and P-ATP, 32 in the presence or absence of DNA. The production of ADP a was assayed as described in the Results Section 3, and the Materials and Methods. ATPase values were corrected for the presence of radioactive ADP in the ATP preparations. Soj ATPase values were also corrected by subtracting the values of ADP produced by the indicated concentration SpoOJ or DNA alone for each time point. This ensured that the calculated stimulation were observations of Soj ATPase activity stimulation by SpoOJ. In a typical experiment the background without Soj was 5% of total ADP produced when Soj was added, and SpoOJ reactions without Soj were 15 % of total ADP produced when Soj was added. Fig. 28 shows the data from a representative experiment to monitor the effect of SpoOJ on ATPase activity, with or without DNA. In the absence of DNA, SpoOJ stimulated a 1.7 +10.4 fold increase in ADP production by Soj compared to ADP production by Soj alone (measured as nmol ADP produced / nmol Soj, n14 independent ATPase assay reactions, P value -0.0l9 as calculated by a two tailed Student’s T test). The inclusion of plasmid DNA along with SpoOJ and Soj led to a further increase in the ATP hydrolysis activity of Soj. In the presence of SpoOJ and DNA, ATP hydrolysis by Soj (measured as nmol ADP/nmol Soj) increased by a factor of 2.5 +1-0.7 compared to the levels of ATP hydrolysis by Soj alone (n17 independent ATPase assay reactions, P value —1.3 x l0 as calculated by a two tailed Student’s T test). No stimulation of Soj ATPase activity was observed by the addition of DNA alone in any condition. The stimulation of Soj-dependent ATPase activity by SpoOJ was further characterized by monitoring ATP hydrolysis by Soj over a range of SpoOJ concentrations in the presence of DNA (Fig. 29). As the concentration of SpoOJ increased, so too did the production 99  1200  Soj+SpoOJ+DI ._  1000  0  E  800  Soj+SpoO N -  0 I V .=  600  400  Soj  200  Soj+  0 I  0  I  0  10  20  30  40  50  time (mm)  Fig. 28. Stimulation of Soj ATPase by SpoOJ. Soj (3 jiM) and ct P-ATP (250 32 jiM, 1.33 Ci mmol’), were incubated with SpoOJ (3 jiM) where indicated, with or without pUC19 DNA (6 ng ji[’), in 30 jil of ATPase buffer with 100 mM NaC1 at 37°C. At the indicated time aliquots were removed, ATPase stopped, and ADP was detected and quantified as described in Materials and Methods (section 5). The amount of ADP produced was plotted as a function of time. ADP produced from parallel reactions containing no protein, SpoOJ, and/or DNA was assayed at the indicated time points. These values were subtracted from ATPase reactions containing Soj, SpoOJ, and/or DNA where appropriate.  100  I U.J  600 0  500  .  400  300 200  100 0 0  2  1  3  SpoOJ (pM)  Fig. 29. Stimulation of Soj ATPase by SpoOJ. Soj (3 jiM), P-ATP 32 (250 jiM, x 1.33 Ci mmo[’), and pUC19 DNA (6 ng i’’) were incubated with the indicated concentration of SpoOJ in 20 jil of ATPase buffer with 100 mM NaC1 at 3 7°C. After 15 mm reactions were stopped and ADP was detected and quantified as described in Materials and Methods (section 5). The amount of ATP hydrolyzed was plotted as a function of SpoOJ concentration. For each point, ATPase reactions containing the indicated concentration of SpoOJ without Soj were assayed. These values were subtracted from the ATPase values from reactions of Soj with the appropriate SpoOJ concentration to give corrected Soj ATPase values.  101  1200 SojG12V+Spo0J+DNA\  1000 800  SojG12V+Spo0J ,•_  /7  & 600  /  400  -  200 0 0  10  20  30  40  50  time (mm)  32 a Fig. 30. Stimulation of SojG12V ATPase by SpoOJ. SojG12V (3 jiM) and PATP (250 jiM, 1.33 Ci mmo[’), were incubated with SpoOJ (3 jiM) where indicated, with or without pUC19 DNA (6 ng ji[’), in 30 jil of ATPase buffer with 100 mM NaC1, at 37°C. At the indicated time aliquots were removed, ATPase stopped, and ADP was detected and quantified as described in Materials and Methods (section 5). The amount of ADP produced was plotted as a function of time. ADP produced from parallel ATPase reactions containing no protein, SpoOJ, and/or DNA was assayed at the indicated time points. These values were subtracted from SojG12V ATPase reactions containing SpoOJ and/or DNA where appropriate to give Soj ATPase values.  102  of ADP. The increase in ATP hydrolysis by Soj as a function of SpoOJ concentration was roughly linear over the range tested. The stimulation of SojG12V ATPase by SpoOJ was also investigated. The ATPase activity of SojG12V was stimulated by SpoOJ in the presence, but not the absence, of DNA. Results from a representative experiment are shown in Fig. 30. Inclusion of both DNA and SpoOJ in SojG12V ATPase reactions resulted in a 1.7 +7- 0.1 fold stimulation (n=6 independent ATPase assay reactions, P value 2.3 x 1 06 as calculated by a two tailed Student’s T test). Unlike wild type Soj, no stimulation of SojG12V ATPase by SpoOJ alone was observed. As with wild type Soj, SojG12V ATPase activity was not stimulated by DNA alone (not shown). The stimulation of Soj ATPase activity by SpoOJ could be caused in two ways. First, the interaction of SpoOJ with Soj could stimulate an increase in the catalysis of ATP hydrolysis by Soj. Alternatively, SpoOJ could act as a Soj nucleotide exchange factor. Stimulation of ATP hydrolysis, and stimulation of ATP exchange which leads to increased ATPase levels have both been demonstrated in different ParA/MinD ATPases by the cognate partner protein. The possibility that SpoOJ is a nucleotide exchange factor was investigated by monitoring ATP binding by Soj in the presence of SpoOJ by UV crosslinking assays. Results from these experiments were inconclusive, so this question remains unanswered.  10.2 Soj and SpoOJ interact to Form High Molecular Weight Protein Complexes.  Other members of the ParA and ParB family of proteins have been demonstrated to form large protein complexes or ‘fibres’ that consist of ordered protein multimers. The possibility that the interaction of Soj and SpoOJ could result in the formation of larger protein complexes was assessed. It was reasoned that if Soj and SpoOJ interacted to form large protein complexes these complexes could be sufficiently large to be pelleted by low speed centrifugation. Sedimentation assays were performed by mixing Soj (3 tM) and SpoOJ (3 jiM) in the presence or absence of bacteriophage p 29 genomic DNA, with either ATP or ADP (described in the Materials and Methods). Bacteriophage p 29 genomic DNA has several SpoOJ binding sites (parS). Following centrifugation of the reactions in a microfuge (“46k x g, 20 mm), the proteins were identified by SDS-PAGE of aliquots of the supernatants and resuspended pellets from the centrifuged mixtures. The amount of protein corresponding to Soj and SpoOJ was estimated by densitometry of digital images of Coomassie brilliant blue stained gels using Imagequant, and 103  SpoOJ Soj DNA ATP ADP  1  2  3  4  5  6  + +  + +  + + +  + + +  +  +  -  -  +  +  -  PS  +  -  PS  S  PS  +  -  +  +  -  P  -  + + +  -  +  -  -  P  -  7  -  S  PS  -  P  S 4—SpoOJ 4—Soj  SpoOJ (%) (72) Soj (%) (16)  (55) (8)  (78*) (27*)  (58*) (11*)  (27)  (27*) (44*)  Fig. 31. Sedimentation assays of Soj and SpoOJ. The indicated components (Soj and Spo0J, 3 riM; nucleotide, 1 mM; bacteriophage 029 genomic DNA, 6 ng jit’ (0.5 nM)) were incubated in 40 jil of ATPase buffer with 100 mM NaC1, for 5 mill at 37°C. Supernatant (S) and pellet (P) fractions were separated by centrifugation for 20 mm at 16k x g at room temperature. Fractions were treated as described in Materials and Methods (section 9), separated by SDS-PAGE and stained with Coomassie Brilliant Blue. An image of the gel was generated using an Aiphalmager Gel documentation system. Numbers in parentheses indicate the band density of the pellet fraction over the sum of supematant and pellet fraction band densities (given as percent values) for the indicated protein as determined by Imagequant v5 .2. Values in parentheses with asterisks represent the average of two independent sedimentation reactions.  104  these values were used to calculate the fraction of each protein recovered in the pellet and supernatant fractions from the sedimentation assay. As seen in Fig. 31 when SpoOJ was incubated with Soj in the presence or absence of DNA or either nucleotide, then subjected to centrifugation, a large fraction of SpoOJ was recovered in the pellet (Fig. 31, lanes 1-4). The pellets contained only a small fraction of Soj. Relatively low levels of SpoOJ were recovered in the pellets following centrifugation of reactions containing only SpoOJ, with or without DNA in the presence of ATP (Fig. 31, lanes 5 and 6). Comparing the effect of ATP versus ADP (Fig. 31, compare lanes 1 and 2, and lanes 3 and 4) indicated the presence of ATP resulted in a small increase in the fraction of sedimented SpoOJ compared to the same reaction conditions where ADP was present. In reactions where Soj was incubated with SpoOJ, ATP, and DNA the fraction of Soj that sedimented was reduced compared to the fraction seen with ATP and DNA without SpoOJ (Fig. 31, compare lanes 3 and 7). This suggested that Spo0J may reduce the formation of sedimentable Soj DNA complexes. The sedimentation of a large fraction of total SpoOJ but a relatively low fraction of Soj from mixtures containing SpoOJ incubated with Soj, indicated that the protein complexes were composed mostly of SpoOJ. Formation of complexes between SpoOJ and Soj was monitored by light scattering. Light scattering was assessed as described in Materials and Methods Section 12. Soj (1 tiM) was incubated in the presence of either ATP or ADP at 3 0°C. After one mm, an equimolar amount of SpoOJ was added and light scattering was monitored for another 3 mm. Upon addition of SpoOJ to Soj incubated with either ATP or ADP, a large increase in light scattering was consistently observed (Fig. 32; duplicate experiments for each nucleotide condition are indicated). This large increase in light scattering was not observed when Soj or SpoOJ were incubated alone in the same reaction conditions (Figs. 20-21, and data not shown, respectively), although Soj did form much less intense light scattering signals. The slopes of the linear portion of the light scattering curves were steep and slightly increased when ATP was included compared to ADP. A slightly higher level of light scattering was observed in the presence of ATP compared to ADP which was compatible with the slightly increased sedimentation of SpoOJ from mixtures of Soj and SpoOJ in the presence of ATP compared to ADP from Fig. 31. The ability of SojG12V to interact with SpoOJ in the presence of ATP was investigated by light scattering assays in a similar manner as wild type Soj. SojG12V (0.5 jiM) was incubated with ATP for one mm, an equimolar amount of SpoOJ was added and light scattering was monitored for 3 minutes (Fig. 33). A sharp increase in light scattering upon addition of SpoOJ (at 2 mm) 105  900-j--  —_______  ATP  8001 .  I  7001  Ei  Soj \%ADH  Fig. 32. Light scattering by Soj and SpoOJ. Soj (1 1 iM) was incubated with 1 mM of the indicated adenosine nucleotide ml .5 ml of ATPase buffer with 100 mM NaC1 as described in the Materials and Methods (sectionl2). After 1 mm, SpoOJ (1 pM) was added and light scattering was monitored. Fluorescence spectrophotometer PMT voltage in this assay was 600 V.  106  Soj+SpoOJ\  —  300 >‘  250 a  Soj a  200  SpoOJ auue S0jGI2V+SpoOJ  150 100 50  I  0  1  I  2  3  4  5  time (mm)  Fig. 33. Light scattering by Soj or SojG12V, and SpoOJ. Where indicated Soj, or SojG12V (0.5 jiM) was incubated with 1 mM of the indicated adenosine nucleotide in 1.5 ml of ATPase buffer with 100 mlvi NaCI as described in Materials and Methods (section 12). After 1 mm, Spo0J (0.5 jiM) was added and light scattering was monitored. Fluorescence spectrophotometer PMT voltage in this assay was 600 V.  107  D  4  Fig. 34. Transmission electron micrographs of negatively stained Soj-SpoOJ structures. Soj and SpoOJ (1 1 iM) were incubated with ATP (1 mM) in 20 jil of ATPase buffer with 100 mM NaC1. A small sample of the reaction was applied to a carbon coated grid and stained with 1 % uranyl acetate as described in the Materials and Methods (section 13.2). Grids were examined by transmission electron microscopy and micrograph images were captured as described in Materials and Methods (section 13.2). (A) 30 000 times magnification from a grid prepared with Soj and SpoOJ. Scale bar 500 mm (B-C) 200 000 times magnification from a grid prepared with Soj and SpoOJ. Scale bars 100 nm. (D) 30 000 times magnification of a grid prepared with SpoOJ and nucleotide. Scale bar 100 nm.  108  was observed with both Soj G 1 2V and wild type Soj. The rates of increase of light scattering during the linear portions of the curves were similar for Soj and SojG12V. These results indicated that like wild type Soj, SojG12V was able to stimulate formation of light scattering protein complexes in the presence of SpoOJ. The protein complexes which resulted from incubation of Soj and SpoOJ were examined by staining with uranyl acetate and transmission electron microscopy using the same methodology as used for Soj. Electron micrographs of structures resulting from incubating Soj and SpoOJ are presented in Fig. 34A-C. The overall dimensions of these structures were approximately 400-700 nm in length and approximately 25-45 nm in width. These filamentous structures appeared to be composed of stacked striations that are approximately 4 nm to 7 nm across. These ends of the filamentous structures appeared to be frayed and in several examples filaments might be joined to other stacked striated structures. Filaments were observed only in conditions where Soj and SpoOJ were incubated together prior to application to the EM grid. The filaments were absent if either Soj or SpoOJ were omitted from the incubation prior to application to the grid. A representative field of view of SpoOJ alone is shown in Fig. 34D where a carbon coat imperfection is used as a focal reference. The crystal structure of the N-terminal domain of SpoOJ from T thermophilus has been solved (Leonard et al., 2004). BlastP protein sequence alignment of the T. thermophilus protein fragment used to solve the crystal structure showed it to be 47% identical to the SpoOJ protein from B. subtilis. Using the analysis of the truncated T. thermophilus SpoOJ crystal structure with DeepView! Swiss-pdb viewer (Guex and Peitsch, 1997), I estimated that an approximate dimensions of 62A x 47A x 42A for the truncated SpoOJ dimer. Since the sedimentation assays of Soj and SpoOJ (Fig. 31) indicated that the structures from Fig. 34 should consist primarily of SpoOJ. The width of the striations in the structures in Fig. 34, would be similar to the dimensions of a SpoOJ dimer.  109  DISCUSSION  Experiments in this thesis addressed the properties of Soj, a protein that negatively regulates sporulation. The characterization of Soj addressed how it interacts with adenosine nucleotides, and how interaction with nucleotides affects the DNA binding activity of Soj in vitro. A characterization of the interaction between Soj and SpoOJ was also presented. A better understanding of these interactions in vitro is important to understand how these ParA and ParB family proteins affect sporulation. In the Discussion I will first outline the technical advances that were important for these experiments. Second, the in vitro properties of Soj will be related to its function in vivo, and compared to the properties of Par proteins in other organisms. A model of how Soj interacts with DNA in an ATP-dependent manner will be presented, as well as an outline of how Soj interacts with SpoOJ and DNA. Models for the role of Soj and SpoOJ in B. subtilis and the impacts of these models on sporulation will be presented.  1 Technical Modifications.  1.1 Soj purification.  Previous to this work, the recombinant hexahistidine tagged Soj purified from E. coil could not hydrolyze ATP, or bind double-stranded DNA (Cervin et ai., 1998). I used information from studies of the C. crescentus ParA protein to modify the purification scheme incorporating adenosine nucleotides in the purification buffers (Easter and Gober, 2002). The inclusion of ATP or ADP in the purification protocol resulted in Soj preparations that consistently bound and hydrolyzed ATP. I also included very high salt washes of the protein while it was on the Ni-NTA agarose resin (Fig. 7). The inclusion of high salt in the buffers for purification of Soj prevented DNA contamination which interfered with the characterization of Soj DNA binding. The exclusion of a dialysis or buffer exchange step to remove either imidazole or nucleotides improved the yield of active, soluble Soj, as did the immediate freezing of Soj on dry ice following elution from the Ni-NTA agarose column. Even with the purification modifications, Soj tends to form precipitates in liquid solution in 1-2 hours. This was the rationale behind eliminating the step which removed imidazole or nucleotide by dialysis. This lack of solubility was likely not a result of a lack of charge as at pH 8 (the pH of buffers used for purification and all the activity assays). Soj had a net negative 110  charge and therefore was predicted to remain in solution. Other researchers have noted poor solubility of ParA proteins (Barilla et al., 2005; Leonard et a?., 2005). It is possible that in general ParA proteins tend to multimerize which results in reduced solubility in vitro.  1.2 Light Scattering Assays.  The possibility that Soj could form multimeric structures on DNA was indicated by work that characterized Soj from T thermophilus (Leonard et a?., 2005). I reasoned that these structures would have distinctive optical properties. Light scattering was found to be a sensitive assay which could detect Soj DNA interactions at low concentrations of Soj and DNA. The effectiveness of this assay allowed me to generate a refined model for the interaction of Soj on DNA that is discussed further in this section.  2 Soj ATP Binding and Hydrolysis.  The specificity of Soj binding to ATP was demonstrated by UV-crosslinking assays. Soj was shown to possess some dATP binding activity although the yield was low compared to the binding of ATP (Fig. 4A). Soj binding to ATP reached a maximum quickly (l mm). Both Soj mutants (SojG12V and SojD4OA) also bound ATP (Fig. 8D). Using thin layer chromatography, Soj was demonstrated to possess a low level ATPase activity. The initial rate of ATPase as a function of ATP concentration indicated Michaelis 1 corresponded to a turnover number Menton kinetics. The Vmax of 1 80 pmol ATP nmol’ min 3 s’ at saturating ATP concentrations, and a Km of 175 jiM (Fig 7B). The low (kcat) of 3 x10 kcat/Km values for Soj indicated that it was not an efficient ATP hydrolyzing enzyme. The low ATPase is consistent with the idea that Soj is not likely to provide energy to drive biological processes such as chromosome segregation. Soj ATP binding and hydrolysis activities were similar to those of MinD from E. coli, which is the most extensively characterized MinD/Mrp family ATPase (Lutkenhaus, 2007). MinD can bind ATP, ADP and dATP, and it hydrolyzes ATP with a Vmax of 62 pmol ATP nmoF 1  3 s and a Km of 40 jiM (de Boer eta?., 1 corresponding to a turnover number of 1 x10 min  1991). Although their ATPase activities are 3-fold different, Soj and MinD are similar and distinct from other WalkerAB ATPases that exhibit ATPase specific activities several orders of magnitude higher (Leipe et a?., 2002). To date no other ParA!Soj protein has been subject to a 111  detailed enzymatic analysis of its ATPase activity that would allow a comparison to the parameters calculated here. The ATPase activity of SojG12V is similar to wild type Soj (Fig. 9). This result was unexpected and not predicted by the characterization of an equivalent mutant MinD/Mrp protein (Barilla et a?., 2005). This indicated that the loss of function phenotype observed for B. subtilis strains expressing SojG12V instead of wild type Soj was not due to loss of the ATPase activity of Soj. Protein sequence data indicate that the G12 residue of Soj is conserved in the MinD/Mrp family (Leipe et a?., 2002; Lutkenhaus and Sundaramoorthy, 2003). Structural data indicate that MinD/Mrp family proteins form sandwich dimers, where the ATP molecules bound by the Walker A motifs (P-loops) are in close proximity. The structures of dimers of Nifli (Chiu et a?., 2001; Jang eta?., 2000; Jang eta?., 2004; Sarma eta?., 2007; Schmid eta?., 2002; Sen eta?., 2004; Sen eta?., 2006; Strop eta?., 2001), ArsA (Zhou eta?., 2000), ö protein (Pratto eta?., 2008), and T thermophi?us Soj (Leonard et a?., 2005) have been solved. In all these structures the residues equivalent to G 12 of B. subtilis Soj are buried in the dimer interface and are at the closest points of contact between the two ATP binding motifs of the monomers. The SojG12V mutant was constructed by another research group in the hope that it would be an equivalent mutation to a Ha-ras p21 mutant, because the valine residue is conserved in the Walker A motif in that protein (Quisel et a?., 1999). However Ha-ras p21 is a monomeric GTPase protein with a Walker A motif that is different from the motif in the Mrp/MinD family (Bhattacharya eta?., 2004; Leipe eta?., 2002). The equivalent G12V mutation in Ha-ras p21 does not abrogate GTP hydrolysis; rather, it renders the mutant protein insensitive to activation by GTPase activating proteins (Maegley et a?., 1996; Vogel et a?., 1988). However, it was assumed that the mutant was not able to hydrolyze ATP. The altered localization of SojG12V-GFP in B. subti?is was attributed to loss of this function (Quisel eta?., 1999). The equivalent T. thermophi?us Soj mutant (SojGl6V) has been briefly described, but no data were presented regarding its ATPase activity (Leonard et a?., 2005). The ATPase activity of ParF (a type Tb plasmid ParA) and a mutant of ParF (G11A, equivalent to G12V) indicated that the G11A mutation reduced ATPase activity (Barilla eta?., 2005). It is unclear why a decrease in ATPase activity was reported for this mutant, but the report also indicates that a mutation at an invariant lysine (K15Q) was ATPase proficient (Barilla eta?., 2005). The invariant lysine is required for proper binding of ATP to coordinate ATP hydrolysis in numerous Walker AB ATPases, so the ATPase activity of ParF Ki SQ contradicts a large body of data in the literature regarding WalkerAB ATPase proteins (Leipe et a?., 2002; Saraste et a?., 112  1990). This discrepancy has not been resolved. Data presented here indicate that Soj G 1 2V hydrolyzes ATP, so its altered characteristics in vivo are due to some other property of this mutant. That SojD4OA could bind, but not hydrolyze, ATP was expected (Fig. 8D, 9) (Leonard et al., 2005). Protein sequence and structural data indicate that the aspartic acid residue in the equivalent position in the larger superfamily of GTPases is situated at the catalytic core of the protein and coordinates the Mg 2 cofactor required for hydrolysis (Leipe et a!., 2002). The presence of ADP in Soj ATPase reactions had a small, negative impact on Soj ATPase, suggesting that Soj could bind ADP but that it had a higher affinity for ATP than ADP. A striking increase in ATPase activity resulting from the addition of either ATP or ADP during the purification of Soj was noted, although why ATP or ADP inclusion during the purification protocol impacted its ATPase activity is not well understood. This requirement has been noted in the purification of at least one other ParAISoj protein (Easter and Gober, 2002).  3 Soj Dimerization and Multimerization.  Members of the MinD/Mrp superfamily of proteins, including Soj are usually reported to form nucleotide-dependent dimers (Leipe et al., 2002). In my work size exclusion chromatography indicated that Soj forms dimers in the presence of either ATP or ADP (Fig. 13). There was no indication that either ADP or ATP differentially affected dimerization, and the majority of the protein in preparations used in this thesis was in the dimeric form (75-80%). The monomer-dimer profiles obtained by size exclusion chromatography in the presence of ADP for SojG12V, and SojD4OA were similar to those of wild type Soj (Fig. 13). Nucleotide exchange by Soj likely requires that the protein dissociate into monomers, as the nucleotide binding pocket of other Mrp/MinD family members is buried in the dimer interface. The structure of ö, a ParA protein from pSM19035, bound to Mg-ATPyS indicates that this protein could exchange nucleotide while the protein is a dimer as the nucleotide binding pockets are solvent exposed (Pratto et a!., 2008). Soj from B. subtilis has 25% sequence identity and 49% sequence similarity to the  protein (compared to 50% sequence identity and 65% sequence  similarity to Soj from T. thermophilus). More data are required to ascertain whether the nucleotide binding pockets of B. subtilis Soj are solvent exposed or not, and therefore if it can exchange bound nucleotide as a dimer or a monomer.  113  In the presence of ATP, the size exclusion chromatograms of wild type Soj, and SojD4OA showed a peak eluting at lower volumes than the dimer form. For wild type Soj this larger species was observed as a shoulder on the dimer (Fig. 13). For SojD4OA this larger species was the majority of the protein (76%). Estimation of the molecular weight from the elution of molecular weight standards indicated an approximate size of 100 kDa for the larger Soj species. In both cases, these data suggest formation of species approximately the size of a Soj tetramer. The apparent quaternary structure of Soj was not different in the presence of ATP versus ADP. This result is similar to those found with P1 ParA and ö from pSM19035 where the formation of dimers is equal in the presence of either ATP or ADP binding. However, this result contrasts to those found with Soj from T thermophilus, and MinD from E. coil, where dimerization was observed to be ATP-dependent (Hu and Lutkenhaus, 2003; Leonard et al., 2005). The recent publication by Hester and Lutkenhaus (2007) indicated that B. subtilis Soj dimerization was also ATP-dependent. Multiple experimental approaches throughout the course of my research (size exclusion chromatography shown here, and additional data not shown) showed that Soj preparations were a mixture of monomers and dimers. It is unclear why Hester and Lutkenhaus (2007) observed Soj dimers only in the presence of ATP. A possible explanation is that the ATP-dependent dimerization reported for Soj from T thermophiius and B. subtilis was carried out with proteins that were not purified in the presence of nucleotides and this may have affected the behaviour of Soj in both cases. Stabilization of a Soj dimers by ADP would be compatible with structural data from MinD/Mrp superfamily proteins. An important feature of the Mrp/MinD family is a universally conserved lysine (Kl 1 in B. subtilis Soj), 5 residues N-terminal to the universal lysine responsible for ATP binding by all P-loop NTPases (Leipe et al., 2002; Lutkenhaus and Sundaramoorthy, 2003). In the crystal structure of a NifH dimer (one of the family members) bound to ADP, the equivalent lysine makes intermonomer contacts to the  13 phosphate of ADP  bound by the other monomer (Chiu et al., 2001; Jang et al., 2000; Jang et al., 2004; Sarma et al., 2007; Schmid et al., 2002; Sen et al., 2004; Sen et al., 2006; Strop et al., 2001). So it is possible that the Ki 1 in Soj can stabilize a dimeric conformation while bound to ADP, as the f3 phosphate is still present as a ligand. The dimerization of NifH bound to ADP could also be a result of other determinants elsewhere on the dimer interface, which may also have parallels in B. subtilis Soj. What these determinants might be is difficult to identify as sequence alignment does not indicate any known dimerization motifs. The dimer of pSM19035 ö protein is 114  stabilized by interaction of a hydrophobic surface at the dimer interface that would be otherwise solvent exposed, and reciprocal inter-monomer salt bridges (Pratto et al., 2008). The distribution of hydrophobic residues in the dimer interface of the T thermophilus structure is similar to that of 6 protein structure, and charged residues are absent throughout most of this interface. The sequence similarity between Soj from T. thermophilus and B. subtilis is high, especially for residues situated at the dimer interface. The dimerization of B. subtilis Soj-ADP could be stabilized by either hydrophobic interactions or inter monomer salt bridges.  4 DNA Binding by Soj.  DNA binding by Soj was demonstrated by EMSAs, sedimentation assays, and DNase protection assays. In all of the assays a strict ATP dependence was observed for the interaction of Soj with DNA. In EMSAs Soj was able to bind 24 bp, 100 bp, and 2.6 kb DNA (Fig. 15). The binding of Soj to DNA equal to or larger than 100 bp was cooperative. A model based on the structure of Soj from T thermophilus, and protein residues involved in the DNA binding activity of B. subtilis Soj, predicts an approximate Soj DNA footprint of 20 bp based on the length of DNA that would be in close contact with the protein (Hester and Lutkenhaus, 2007). In this model the DNA-binding surface is formed only in the context of a Soj dimer, as the Soj monomer is not predicted to have structural motifs compatible with DNA binding. Binding of Soj to 24 bp DNA was tested by EMSAs and gave rise to multiple species with different electrophoretic mobilities. The simplest explanation of the patterns is that one species corresponds to one Soj dimer bound to a 24 bp DNA, and the second species corresponds to two Soj dimers binding to each other, with either one or both dimers binding a separate DNA molecule. EMSAs with 100 bp DNA fragments showed an unusual pattern; for each Soj input a single species of a specific electrophoretic mobility was resolved. The electrophoretic mobility of the single observed species changed as Soj input increased. If Soj dimers bound independently to DNA and multiple Soj dimers could bind to a single 100 bp DNA, the expected EMSA would show multiple electrophoretic species corresponding to the binding of 15 Soj dimers seen gradually shifting over increasing Soj inputs (100 bp DNA is expected to bind 3-5 Soj dimers given a 20-25 bp footprint). One explanation of these results is that in this assay, the different Soj-DNA species do not resolve due to similar electrophoretic mobilities (for example DNAs with 3, 4 and 5 Soj might have the same mobility for some reason). Another 115  explanation is that binding of Soj dimers may not be independent. Soj binding could be highly cooperative, so that when a single Soj dimer binds to DNA another binds almost instantaneously so that Soj-DNA intermediates expected at a given Soj concentration are not observed. Another possibility is that Soj forms multiple higher order complexes in solution in a concentration dependent manner which can then bind DNA. At 2 1 iM Soj dimers might predominate in solution, therefore the DNA would be bound by Soj dimers. The doubling of Soj concentration could favour the formation of the next highest order Soj multimer. If that Soj multimer binds DNA with higher affinity than a Soj dimer, it out compete the dimer for DNA binding even though there are still dimers in solution. Eight tM Soj could favour the formation of the next higher order form, such as two interacting tetramers or a hexamer which could bind to DNA, which could out compete lower order Soj multimers by binding with a higher affinity resulting in the detection of only one electrophoretic species. Further tests of this hypothesis could be done by examining the DNA binding patterns at varying protein to DNA ratios. Evidence supporting the binding of multiple Soj dimers (up to several hundred) to a single DNA molecule was obtained by low speed sedimentation of both Soj and DNA in the presence of ATP (Fig. 16). It is not likely that the binding of a single Soj dimer to DNA would result in plasmid DNA sedimentation at low centrifugal force. The finding that Soj and DNA could be sedimented from mixtures of Soj and DNA supported the binding of multiple Soj dimers to a single DNA molecule. DNA sedimentation was not a linear function of Soj input. Calculation of the amount of DNA bound (done by measuring loss of free DNA) versus Soj concentration indicated cooperativity in Soj binding by a Hill plot, DNA binding by Soj was also followed using a DNase I protection assay (Fig. 17), and evidence for stepwise additions of Soj was obtained from DNase I cutting pattern. The size differences between adjacent DNA sizes indicated an approximate “footprint” of 45 bp (Fig. 18). Given the structural prediction that 20 bp of DNA is closely associated with a Soj dimer (Hester and Lutkenhaus, 2007) this footprint could result from the binding of two Soj dimers to the DNA, either both at one end or one to each end of a cluster of Soj dimers. Evidence for tetramers was found in size exclusion chromatography (Fig. 13). Thus the possibly that Soj tetramers form in solution and bind to DNA directly should be further investigated. Solutions containing Soj-ATP and DNA were found to scatter light. This technique appeared to be the most sensitive assay to detect Soj-DNA interactions. As light scattering was observed with a concentration of Soj as low as 1 jIM; a DNA binding by Soj was not detected by other assays at this level of Soj input. SojG12V was not able to form light scattering 116  complexes with (Fig. 21). The other biochemical analyses indicated that SojG12V was specifically deficient in DNA binding, supporting the notion that the observed light scattering required ATP-dependent DNA binding. The Soj-DNA complexes were examined by transmission electron microscopy. The resulting structures supported data which indicated Soj spreading on DNA. The varying diameters of Soj DNA structures suggested that areas covered by Soj might interact to form a “bridge”. This bridging would form larger complexes, possibly those which scatter light as well as the complexes that can be pelleted at moderate g force.  5 A Model of Soj DNA Binding Leading to Light Scattering.  Binding of Soj to 24 bp DNA was observed in EMSAs (Fig. 15), but did not lead to light scattering in any condition assayed (Fig. 26). A 24 bp duplex DNA could accommodate the binding of one Soj dimer according to a structural model based on the structure of a T thermophilus Soj dimer (Hester and Lutkenhaus, 2007). These data indicate that a population of Soj dimers bound to 24 bp DNA complexes cannot adopt a conformation required for light scattering. The approximate 45 bp Soj DNA “footprint” detected by DNase I assays (Figs. 1718) would be consistent with two Soj dimers binding to a DNA either independently or as a tetramer that forms before DNA binding. Soj association with DNA, assayed by DNase I protection was interpreted to reflect Soj spreading (an initial random binding with additional Soj dimers bound to adjacent sites on the same DNA molecule). Since Soj bound to a 24 bp DNA did not scatter light, while Soj bound to the 100 bp DNA (that can bind multiple Soj dimers) did scatter light, it appears that multiple Soj dimers bound to the same DNA molecule are required for the formation of light scattering complexes. Logically, the formation of the complexes that scatter light might involve multiple sequential steps beginning with binding of a single Soj dimer to make an initial complex I (ci). In subsequent steps, additional Soj dimers bind the same DNA molecule cooperatively, resulting a higher order complex II (cii) on the same DNA molecule which would scatter light. The formation of ci and cli from Soj-ATP dimers and DNA equal to or larger than 100 bp is represented by reaction 1 and 2:  117  (Reaction 1)  Soj  +  (Reaction 2)  ci  (Soj)  +  DNA  .  ci  cii  The comparison between the light scattering patterns seen with the 24 bp and 100 bp DNA leads to the interpretation that for light scattering to occur Soj multimers must be bound to the same DNA molecule. A useful future experiment would be to assay DNA fragments between 24 bp and 100 bp in length for their ability to generate light scattering complexes when combined with Soj-ATP. The idea that cii, which contains multiple Soj dimers is required for light scattering is consistent with results from experiments reported here. For example light scattering dependence on DNA concentration showed that both high and low concentrations of DNA decreased light scattering compared to the levels of light scattering at moderate DNA concentrations of 0.5 ng p1’ (Fig. 24). As well, the presence of 24 bp DNA inhibited formation of light scattering structures on plasmid DNA. This suggests that at high concentrations, 24 bp DNA could compete for Soj dimers with complexes formed on larger molecules reducing formation of cii. A model where cli itself caused light scattering may not entirely explain the results of the light scattering experiments where DNA of different lengths were tested (Fig. 25). The key observation was that the lag time (defined as the time before the light scattering increase began to be linear) decreased as the length of DNA added increased. There is no obvious reason for the lag if light scattering were measuring only the binding of Soj dimers onto individual DNAs to create a population of independent cli complexes. The lag in light scattering could be explained by a model where ci and cii form as described by reaction 1 and 2 (see above), but an additional step, the interactions of cils is responsible for light scattering. These cli interactions could take place between cii formed on different DNA molecules (in the case of smaller DNA) or between cIIs formed on the same DNA molecule (in the case of longer DNAs). The absence of the lag when 2.6 kb DNA was used could result if cils formed on the same DNA molecule can fold back to interact. This 118  would be predicted to be a faster reaction than when the clis are on different DNAs since cli interactions are uni-molecular (therefore zero order and basically at infinite concentration) In essence the diffusion contribution to the rate of cli interaction has been eliminated. These cli interactions are consistent with electron micrograph images of Soj and DNA where there appear to be structures of varying diameters. The cli interactions may bridge multiple Soj-DNA nucleoprotein resulting in larger diameter structures. Despite extensive searches of the literature, and requests for technical specifications from the manufacturer of the spectrophotometer used in these studies, I was unable to find the necessary specifications to provide an estimate for the minimum size of particle that would scatter light in my experiments. Such information would greatly improve our ability to interpret the light scattering results. At present the light scattering experiments presented here cannot separate changes in the structure of Soj bound to DNA in solution from an increase in the total number of Soj-DNA particles in solution. In spite of this deficiency, the technique was useful to probe the nucleotide requirements of Soj DNA interaction. More detailed electron micrographs and/or a more detailed optical characterization (i.e. dynamic light scattering, particle sizing) of Soj-DNA nucleoprotein will be needed to give a clearer indication of the interactions of Soj spread on DNA.  5.1 Insights from SojG12V and SojD4OA for the Soj DNA Binding Model.  SojD4O and SojGl2V both behave as partial loss of function mutants in vivo; however, in vitro each mutant exhibits a different defect compared to wild type Soj. SojD4OA bound ATP, but was ATPase negative. Like wild type Soj, SojD4OA formed dimers and multimers, and had ATP-dependent DNA binding activity. SojG12V ATPase was catalytically similar to wild type Soj, and was able to dimerize. However SojG12V, unlike Soj and SojD4OA, did not show evidence of a tetrameric species in size exclusion chromatography, and was not able to bind DNA under any condition. Since SojG12V and SojD4OA mutants both bound ATP and formed dimers it appeared that dimerization was not sufficient for DNA binding. SojD4OA, an ATPase deficient mutant, bound DNA; therefore, ATP hydrolysis was not required for the DNA binding activity of Soj. However, the effect of ATP on DNA binding showed that it was required for association with DNA. SojG12V was an active ATPase, was able to dimerize, but like Soj-ADP, SojGl2V-ATP could not bind DNA. One way to interpret these results is that like a Soj-ADP dimer, a 119  SojG12V-ATP dimer was not able to adopt a conformation required for DNA binding. In this model ATP is an allosteric effector that modifies the shape of the Soj dimer to drive DNA binding. The hypothesis of ATP as an allosteric effector of Soj is supported by structural evidence of such a shift in NifH, another member of the MinD/Mrp superfamily. When NiflI is crystallized bound to ADP-tetrafluoroaluminate 4 (A1F ) , which mimics the ATP bound state or the transition state of ATP hydrolysis, the Walker A motif (P-loop) of each subunit is —4 apart. In contrast a —10  A  A distance separates the P-loops observed in the structure of the ADP  bound NifH dimer (Schindelin et aL, 1997). It is possible that such a conformational change could occur in Soj. The B. subtilis Soj G12 equivalent residue in T thermophilus Soj and other Mrp/MinD structures is situated at the middle of the P-loop (Leipe et al., 2002). It is possible that a valine at this position modifies the ATP-dependent conformation by causing a steric clash, so that SojG12V-ATP cannot bind DNA. Recent experiments that described ParA-ATP binding non-specifically to DNA were presented during the proceedings of the Canadian Society for Microbiology annual general meeting (B. Funnell et al.; Calgary, 2008). Data presented suggested that ParA-ATP adopts a conformation required for non-specific DNA binding not achieved by a ParA-ADP dimer. ParA-ADP binds specifically to the parOP site as a dimer through its N-terminal DNA binding motif (absent from Soj) and regulates par operon expression (Davey and Funnell, 1994).  6 Soj Interaction with SpoOJ.  The ATPase activity of Soj was stimulated 1.7 fold by SpoOJ, and 2.5 fold by SpoOJ in combination with DNA. The ATPase stimulation in both conditions was determined to be statistically significant. Stimulation of Soj ATPase by SpoOJ was increased by the presence of DNA. This result could be interpreted to indicate that either Soj bound to DNA is more sensitive to stimulation by SpoOJ, or that SpoOJ which is itself a DNA binding protein is a better ATPase stimulator of Soj when it is bound to DNA. Investigation of SojG12V ATPase stimulation by SpoOJ indicated that SpoOJ alone was not able to stimulate SojG12V ATPase activity. The addition of DNA to reactions containing SojG12V and SpoOJ resulted in a 1.7 fold increase in Soj ATPase, which was also determined to be statistically significant. Since SojG12V cannot bind DNA but is otherwise unaffected in ATPase activity compared to Soj, it is  120  more likely that the effect of DNA is on the ability of SpoOJ to stimulate the ATPase activity of Soj, rather than Soj bound to DNA being more sensitive to ATPase stimulation. A feature of many MinD/Mrp family proteins is the modulation of their ATPase activity. Several cases have been studied where ATPase activity is stimulated by their cognate partner protein. For example, ATPase stimulation of plasmid encoded ParAs vary: plasmid P1 ParA ATPase is stimulated 9-10-fold by interaction with ParB-parS (Davis et al., 1992); plasmid TP228 ParF ATPase is stimulated 13-fold by ParG (Barilla et al., 2005); and plasmid pSM19035 6 protein ATPase is stimulated 3.4-fold by interaction with o protein bound to parS (Pratto et a!., 2008). E. coil MinD bound to phospholipids vesicles ATPase is stimulated 9-fold upon interaction with MinE (Hu and Lutkenhaus, 2001; Lackner et al., 2003). H pylon Soj ATPase is stimulated 2-fold by interaction with SpoOJ-parS (Lee et al., 2006). C. crescentus ParA ATPase is stimulated 5-fold by ParB (Easter and Gober, 2002). However C. crescentus ParA and ParB are unusual in that stimulation of ParA is due to nucleotide exchange catalyzed by ParB (Easter and Gober, 2002). The stimulation of the ATPase activity of plasmid ParA proteins is thought to be required for the segregation of replicated sister plasmids; however, the mechanism by which ParA ATPase promotes separation of plasmid DNA is unclear, and may differ among plasmids. In E. coil the ATPase stimulation of MinD by MinE is required to release MinD from the membrane so that it can oscillate between poles (Lutkenhaus, 2007). Stimulation of ParA in C. crescentus (which is by nucleotide exchange) is required to balance the intracellular levels of ParA-ATP and ParA-ADP, which are important for cell cycle progression, as high levels of ParA-ADP in cells depleted of ParB correlate with decreased growth and cell division (Easter and Gober, 2002; Figge et ai., 2003). Mixing Soj and SpoOJ led to formation of protein complexes that could be sedimented by low speed centrifugation. SDS-PAGE analysis of the supernatant and pellet fractions indicated that the sedimented protein was mostly SpoOJ. The presence of ATP instead of ADP slightly increased the sedimentation of SpoOJ, indicating that Soj-ATP interacted better with SpoOJ to stimulate formation of SpoOJ complexes. The presence of DNA did not affect the sedimentation of SpoOJ complexes. A striking feature of the Soj-SpoOJ interaction was the relative absence of Soj in the sedimented protein. The level of Soj in these pellet fractions was not above background observed with Soj alone or with Soj plus DNA and ADP (when Soj does not bind DNA). This indicates that Soj acted either as a nucleus for SpoOJ complexes or that Soj was catalytic in formation of sedimentable SpoOJ complexes. 121  The sedimentation of Soj-ATP after incubation with SpoOJ and DNA was reduced by almost two fold compared to its sedimentation when incubated with DNA alone. One explanation of these results is that the interaction of Soj with SpoOJ inhibits the binding of Soj to DNA, and the formation of Soj-DNA. SpoOJ is also a DNA binding protein so it could compete with Soj for DNA binding. This possibility was not investigated as the sedimentation of DNA in these assays was not measured. The formation of large structures containing SpoOJ was also measured using light scattering mixtures containing Soj and SpoOJ with either ATP or ADP. The formation of complexes that scattered light was immediate upon addition of SpoOJ to Soj, and did not depend on whether ATP or ADP was present. However, the level of the increase in light scattering was higher in the presence of ATP compared to ADP. This agreed with the increased sedimentation of SpoOJ in Soj-SpoOJ experiments with ATP versus ADP, and indicates the stimulation of SpoOJ multimer formation by Soj was favoured by the presence of ATP. SojG12V was able to interact with SpoOJ and to stimulate the formation of light scattering structures to a similar extent as Soj. This result indicates that the block to DNA binding was not a block to interaction with SpoOJ, and that the proposed ATP-induced conformational shift in Soj was not required for interaction of Soj and SpoOJ. Structures resulting from the interaction of Soj and SpoOJ were examined by transmission electron microscopy. These structures were long and filamentous, composed of stacked layers or striations. These striations were similar in width to the estimated dimensions of a Spo0J dimer (Leonard et a!., 2004). Stacked layers were observed to branch from some of the larger filaments and rejoin other larger filamentous structures making bridges between the filaments. These filamentous structures were unique to reactions where Soj and SpoOJ were incubated together before application to the EM grid, and were qualitatively different from Soj DNA structures. The filamentous structures resulting from Soj-SpoOJ interactions likely contained only small amounts of Soj, because Soj sedimentation was minimal after incubation with SpoOJ.  7 Soj Interactions with Nucleotide, DNA, and SpoOJ.  A diagram of Soj interactions with adenosine nucleotides, DNA and SpoOJ shown in this thesis is presented in Fig. 35. Size exclusion chromatography indicates that Soj exists as both  122  2 SpoOJ-(Soj-ATP)  -(Soj-ADP) SpoOJ 2  2 SpoOJ pi 4  Soj-ATP  ‘(Soj-ATP)  ;“‘  ATP  2 (Soj-ADP)  SojADP  ADP  (Soj-ATP)-DNA  Fig. 35. Interaction of Soj with nucleotides, DNA, and SpoOJ. Soj bound to either ATP or ADP exists as monomers (e.g. Soj-ATP) and ). Soj can exchange bound ADP for ATP. Multiple 2 dimers (e.g. (Soj-ATP) Soj-ATP dimers can bind to DNA to create Soj-DNA nucleoprotein complexes. Soj bound has a weak ATPase activity that can be stimulated by interaction with SpoOJ (hatched line). Interaction of Soj with SpoOJ results in the formation of multimeric SpoOJ structures that likely contain -(Soj-ATP) where n are whole SpoOJ , small amounts of Soj (e.g. 2 numbers to indicate SpoOJ dimer multimers).  123  monomers and dimers bound to either ATP or ADP, but the majority of Soj are dimers. Soj has a weak ATPase activity similar to other MinD and ParA proteins. Soj-ATP dimers bind DNA and form multimeric structures on DNA. SpoOJ stimulates the ATPase activity of Soj-ATP dimers (Fig. 35, dotted arrows). The interaction of Soj and SpoOJ resulted in the formation of SpoOJ multimers, but it is not known if the multimers of SpoOJ stimulated by the presence of Soj were able to bind DNA because DNA sedimentation in those assays was not measured. The interaction of Soj-ADP with SpoOJ also resulted in the formation of multimeric SpoOJ filaments.  8 Relevance of Results to In Vivo Soj and SpoOJ Data.  In wild type cells, Soj-GFP fusions are observed distributed at cell poles, division septa, or as foci of Soj in the cytosol (Murray and Errington, 2008; Quisel et a?., 1999). Only a small fraction of Soj-GFP in the wild type cell is associated with the chromosome. In AspoOJ cells (spo), Soj is distributed on the chromosome indicating that when Soj is bound to the chromosome sporulation is inhibited. These results also indicate that the role of SpoOJ in the antagonism of Soj could be to prevent association of Soj with chromosomal DNA either directly or indirectly. SpoOJ could antagonize such association by stimulation of the ATPase activity of Soj (results presented here). Soj-ADP that would be released from the chromosome could exchange nucleotide to rebind ATP and either rebind the chromosome (only to be dissociated from DNA again by SpoOJ) or associate with the cell poles. Association with the poles requires the cell contain the MinD protein but the mechanism for this requirement is not known. A polar distribution is observed for SojG12V but not SojK16A (cytosolic distribution) (Murray and Errington, 2008; Quisel eta?., 1999). Data in my thesis indicate that SojG12V is capable of binding ATP, and data in the literature indicate that SojK16A is likely unable to bind ATP. Therefore, it seems likely that polar distributed Soj is bound to ATP. Interactions of Soj-ATP with proteins (MinD) at the pole likely keep Soj-ATP from interacting with SpoOJ or from binding the chromosome. Expression of SojG12V in a AspoOJ background does not inhibit sporulation, which correlates to its failure to associate with the chromosome (Murray and Errington, 2008; Quisel eta?., 1999). Data presented here demonstrate that the failure of SojG12V to associate with the chromosome as observed in vivo could be due to a loss of DNA binding activity that I propose requires a conformational change in Soj. Presumably localization of Soj at the poles of the cells in vivo does not require the conformational change induced by ATP. 124  Examination of cells which express SojD4OA-GFP reveals fluorescent foci which co localize with SpoOJ when it is present, but are distributed on chromosomal DNA in a iXspoOJ background (Murray and Errington, 2008). These results are consistent with SojD4OA being able to interact with both SpoOJ and DNA as seen in the work reported here. Given the highly cooperative nature of Soj-ATP binding to DNA, the inability of SojD4OA to hydrolyze ATP (demonstrated here) might result in SojD4OA-ATP being trapped on the chromosome in vivo. This view is consistent with observations that SojD4OA inhibits sporulation both in the presence and absence of SpoOJ, and is distributed on the chromosome in a AspoOJ background or colocalized with SpoOJ-parS foci when SpoOJ is present. Finally I present a general model for Soj and SpoOJ function in B. subtilis. This is based on the assumption that the primary role of Soj/SpoOJ in B. subtilis is to negatively regulate initiation of sporulation rather than to carry out specific functions associated with vegetative DNA replication, segregation or cell division. One key finding from analysis of Soj-GFP distribution in various genetic backgrounds is that the inhibition of sporulation correlates with the distribution of Soj more or less evenly along the chromosome (Marston and Errington, 1 999a; Quisel et a!., 1999). I have demonstrated in this work that Soj DNA-binding activity is modulated by its nucleotide-bound state. In order for Soj to bind the chromosome it must be in an ATP-bound state. The stimulation of Soj ATPase activity by SpoOJ would shift the Soj nucleotide-bound state to be ADP-bound and loss of SpoOJ in vivo would promote Soj association with DNA because the ATPase activity of Soj would be lower. The association of Soj with DNA in a AspoOJ background is associated with a decrease in the expression of stage II sporulation genes (Ireton et a!., 1994; Quisel et a!., 1999; Quisel and Grossman, 2000). Two possibilities exist for how this could happen. The first is that Soj could bind DNA at the stage II genes and directly repress transcription. Data which support this idea include the finding that Soj was found specifically associated with stage II sporulation gene promoters by chromosomal Soj immunoprecipitation experiments, and that this promoterspecific association was increased in a zspoOJbackground (Quisel et a!., 1999; Quisel and Grossman, 2000). Data that support Soj acting directly as a repressor was presented here. I found that in vitro, SpoOA dependent transcription was more sensitive to Soj than SpoOA independent transcription (Fig. 19). In particular PspoIIGl8, (a mutant of PspoIIG) was less sensitive to inhibition of transcription initiation compared to PspoIIG (Fig. 15). Sensitivity of PspoIIG to Soj was not likely due to Soj binding a specific PspoIIG sequence because PspoIIGl8 was so 125  similar to PspoIIG. The increased sensitivity of PspoIIG could be explained if Soj DNA binding interfered with Spo0AP DNA binding. Soj could also interact with Spo0A—P on or off DNA to inhibit Spo0AP- RNAP. Sequence-specific binding of Soj to DNA was not observed in any of my experiments so that although Soj inhibited PspoIIG transcription at a lower concentration than from the control promoters, a more detailed analysis of why this was observed is still required. The second possibility for how Soj inhibits sporulation is that Soj acts indirectly to affect Spo0AP levels and therefore sporulation-specific transcription. Support for this possibility comes from recent observations which indicate that in I\spoOJ strains, an additional mutation in sda rescues sporulation (spo+) (Murray and Errington, 2008). This finding implies Soj and Sda are part of the same regulatory pathway. Sda has been shown to inhibit KinA and likely KinB autophosphorylation directly (Rowland et al., 2004; Whitten et a!., 2007). Sda therefore would reduce the concentrations of Spo0AP in the cell and decrease expression of stage II sporulation genes that depend on SpoOAP. Sporulation is transiently blocked during artificial replicative stress (UV irradiation, elongation inhibition, mutations in replication initiation proteins) and this blockage requires Sda (Burkholder et a?., 2001; Ruvolo et a?., 2006). In wild type cells under these stress conditions, sda transcription increases and Sda protein levels in the cell increase transiently, later decreasing due to proteolysis. Sda gene expression is positively regulated by DnaA (Ishikawa et a?,, 2007). DnaA-ATP binds to specific DNA sequences termed DnaA boxes and promotes initiation of replication (Moriya et a?., 1988). A DnaA box is found in the promoter region of sda and co immunoprecipitation experiments have detected DnaA-ATP binding to the sda promoter. Several studies have suggested that transcription from sda changes when DnaA levels in the cell are altered. In the report describing that a sda deletion rescues the spo phenotype of a AspoOJ strain, the authors also presented data which suggested a direct interaction between DnaA and Soj (Murray and Errington, 2008). The authors also suggested that this interaction could alter DnaA-dependent replication initiation at oriC. These interesting results leave unanswered how Soj could impact DnaA function at oriC and how this interaction influences expression of sda. In a AspoOJ strain (spo  ), Soj-GFP is found spread along the chromosome and  replication increases as measured by an increase in the ratio of origins to termini (Lee and Grossman, 2006; Murray and Errington, 2008; Ogura et a?., 2003). In vivo SojD4OA-GFP is primarily associated with SpoOJ (which is itself associated with the chromosome), and to a lesser extent with the chromosome. In a AspoOJ background SojD4OA-GFP associates evenly 126  with the chromosome, sporulation is inhibited, and the cell contains an increase in the number of origins relative to the wild type. These data would be consistent with Soj acting as a positive regulator of replication initiation when bound to the chromosome. However increased replication initiation is also observed in /ssof and A(soj-spoOJ) mutants, arguing against Soj acting as a positive regulator of replication initiation. One way to explain these conflicting data is that Soj negatively regulates DnaA, but when Soj is spread along the chromosome, the negative effects are prevented. Obviously in a Asoj strain negative regulation of DnaA by Soj would be lost. Both IXsoj and A(soj-spoOJ) lead to increased initiation of DNA replication (Lee and Grossman, 2006; Murray and Errington, 2008; Ogura et a!., 2003). The increased replication initiation in a AspoOJ background, and in cells expressing SojD4OA is accompanied by inhibition of sporulation that depends on Sda. In contrast, in Asoj or A(soj-spoOf) strains the increase in replication initiation happens without inhibiting sporulation, indicating that Sda is not activated. This could indicate that Soj is required for expression of Sda, possibly in cooperation with DnaA. Despite the logic of the argument above, there are data in the literature that are not consistent with Soj acting to alter sporulation gene expression through Sda. One study indicated that the deletion of spoOJ does not affect the phosphorylation of SpoOA based on analysis of abrB repression and spoOA induction during stationary phase and sporulation (Cervin et a!., 1998). Both abrB repression and spoOA activation require Spo0AP; however, there was little difference in abrB repression or spoOA induction during stationary phase between AspoOJ and wild type cultures. If Sda were activated, then the phosphate flow to SpoOA should be reduced which should be reflected in abrB and spoOA expression. Further investigation of this discrepancy is required. The two possibilities for how Soj inhibits sporulation in the absence of SpoOJ need not be exclusive. Soj could both affect Spo0AP levels through the phosphorelay by activation of Sda, and independently repress the transcription of stage II genes. If the role of Soj is to inhibit sporulation quickly, this could happen more efficiently if Soj both repressed gene transcription and prevented SpoOA phosphorylation. Both possibilities for how Soj inhibits expression of stage II genes and inhibit sporulation merit further investigation to uncover specific mechanisms. The number of Soj molecules per cell has not been experimentally determined. It is not clear how many Soj dimers bound to chromosomal DNA in vivo would create nucleoprotein structures that could affect the initiation of sporulation. The cooperative nature of DNA binding 127  and formation of Soj nucleoprotein presented herein indicate that even several hundred copies of Soj bound to the chromosome might impact chromosome structure by the formation of Soj nucleoprotein. DNA replication in a Asoj or A(soj-spoOJ) mutants is not altered to the point where sporulation cannot proceed, which indicates that control over replication initiation is not the primary role of Soj and SpoOJ. When B. subtilis exits vegetative growth it must ensure that replication is coordinated so that both the forespore and mother cell receive a full chromosome upon sporulation. More than 35 years ago it was proposed that the replication status of the chromosome affects sporulation (Mandelstam et a!., 1971; Mandelstam and Higgs, 1974). Researchers postulated a window of time following replication initiation in which initiation of sporulation is negatively regulated so that B. subtilis does not sporulate with the improper genetic content. After that window of time, the status of chromosome replication is compatible with sporulation until a new round of DNA replication is initiated and sporulation is again inhibited. Upon replication initiation (controlled by DnaA) the signal that transmits inhibition of sporulation must be transient so that the cell can resume its capacity to sporulate as its genetic content reaches the requirement of two full chromosomes. Sda could provide such a signal. The negative regulation of sporulation by Soj requires Sda, and current data suggest that Soj could affect Sda through its interaction with DnaA. It is not clear what state of Soj associates with DnaA but it likely depends on the nucleotide bound to Soj. Given the possibility that Soj-ATP binds the chromosome and does not inhibit DnaA activity, it is tempting to propose that Soj-ADP interacts with DnaA to inhibit replication. The level of Soj-ADP in the cell is critically regulated by the activity of SpoOJ which can stimulate the ATPase of Soj. Signals which control SpoOJ activity are unclear, but identification of such a signal would be an important contribution to understanding the complex mechanism of how chromosome replication and sporulation initiation are properly timed.  9 The Role of Chromosomal Par Proteins in Bacteria.  A striking feature of ParA and ParB systems is the diversity in function and contribution to cell physiology among bacterial species. The Par system of C. crescentus is unique among bacteria because both Par proteins are essential (Easter and Gober, 2002; Figge et al., 2003; Mohl and Gober, 1997; Mohl et al., 2001). Depletion of either causes a division defect which results in filamentation. This indicates that in C. crescentus both Par proteins are required for 128  productive cell division. It is worth noting that cell division is a developmental process in this organism and like sporulation in B. subtilis results in the production of two cell types (Laub et al., 2007). In S. coelicolor ParA and ParB are not essential genes, but do affect an aspect of coordinating spore compartment septation and chromosome segregation (Jakimowicz et a!., 2007b). Unlike B. subtilis and C. crescentus, development of S. coelicolor can proceed when the Par system is mutated. The only other bacterial species aside from C. crescentus where a Par protein has been suggested to be essential is V. cholerae. The parB gene encoded on the large chromosome has not been successfully deleted despite reported attempts (Saint-Dic et al., 2006). This indicates that the role of ParB in this organism is indispensable for vegetative growth. V cholerae reproduces by binary fission and does not engage in a developmental process, so Par function in this species is unlike that of C. crescentus and B. subtilis. The literature indicates that chromosomal Par protein systems have been adapted to perform different functions associated with the chromosome in different cells. 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