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James Alan Tunaley Structure and Activity Investigations of the Cell Fate Determinant, SpoIIE, from Bacillus subtilis. James Alan Tunaley Thesis Submitted for the Degree of Doctor of Philosophy University of York Department of Chemistry September 2013 Abstract For many years the Gram positive bacterium Bacillus subtilis has been a model organism for prokaryotic cell and molecular biology. The asymmetric cell division which B. subtilis undergoes during sporulation is a simple system by which to study the process of cell differentiation. Sporulation is governed by a series of genetic temporal and spatial controls. Gene regulation brought about by a series of σ factors and transcriptional regulators is coupled to key morphological stages or checkpoints. σF initiates the first step in a cascade of complex genetic control which eventually produces a resilient endospore. The activation of σF, the first compartment-specific sigma factor, in the forespore and its regulation through interaction between three proteins; SpoIIAA, SpoIIAB and SpoIIE, is of particular interest. SpoIIE, a protein phosphatase which binds to the asymmetric division septum, is a crucial factor in the selective activation of σF in the forespore. Of three putative domains in SpoIIE only the C-terminal PP2C phosphatase domain has been structurally characterised. The central domain, domain II, of SpoIIE has been assigned a role in interaction with the cell division machinery; however mutational studies have shown that, in addition, this domain is also responsible for the regulation of phosphatase activity. This work describes the isolation and characterisation of three new fragments of SpoIIE containing elements of the central cytoplasmic domain of SpoIIE. These include a fragment found to accurately represent the N-terminal solubility limit of domain II which shows a high degree of oligomeric character. The fragments isolated show specific phosphatase activity against SpoIIAA~P, albeit at reduced rates compared to the free phosphatase domain, which indicates an inhibitory role for SpoIIE domain II against the PP2C domain. Three ultimately unsuccessful approaches were attempted to isolate a co-complex of SpoIIE and SpoIIAA~P for structural characterisation. A tendency for domain II- containing SpoIIE fragments to precipitate in the presence of Mn2+ is also identified. An in vivo investigation into the sporulation efficiencies of amino acid substitutions in a potential regulatory interface between domains II and III of SpoIIE indicated no strong sporulation defects. 1 List of Contents Abstract .............................................................................................................................................................. 1 List of Contents ................................................................................................................................................ 2 List of Tables .................................................................................................................................................... 7 List of Figures ................................................................................................................................................... 8 Acknowledgements .................................................................................................................................... 12 Author’s Declaration .................................................................................................................................. 13 Chapter 1: Introduction ............................................................................................................................ 14 1.1 Spore formation in Prokaryotes................................................................................................ 14 1.2 Bacillus in industry and medicine. ........................................................................................... 15 1.2.1 Risks posed by endospore-forming bacteria. .............................................................. 17 1.3 Bacillus subtilis ................................................................................................................................. 18 1.4 Sporulation ........................................................................................................................................ 21 1.4.1 Entry into the spore formation pathway ...................................................................... 21 1.4.1.1 AbrB ..................................................................................................................................... 22 1.4.1.2 SigH ...................................................................................................................................... 24 1.4.1.3 CodY ..................................................................................................................................... 24 1.4.2 Spo0A ........................................................................................................................................... 26 1.4.3 The sporulation phosphorelay .......................................................................................... 29 1.5 Sigma factors ..................................................................................................................................... 32 1.6 Morphological stages of sporulation ....................................................................................... 35 1.6.1 Coupling cell cycle to sporulation .................................................................................... 36 1.6.2 Sporulation stage I ................................................................................................................. 37 1.6.3 Sporulation stage II ................................................................................................................ 38 1.6.4 Sporulation stage III .............................................................................................................. 40 1.6.5 Sporulation stage IV............................................................................................................... 41 1.6.6 Sporulation stage V ................................................................................................................ 42 1.6.7 Sporulation stage VI............................................................................................................... 43 1.6.8 Sporulation stage VII ............................................................................................................. 43 1.7 The Compartment Specific σ Factor Cascade ...................................................................... 44 1.8 Activation of the First Compartment Specific σ Factor, σF ............................................. 48 1.8.1 Effectors of σF regulation.......................................................................................................... 48 1.8.1.1 SpoIIAB ............................................................................................................................... 49 1.8.1.2 SpoIIAA ............................................................................................................................... 51 2 1.8.1.3 SpoIIE .................................................................................................................................. 53 1.9 The Z-ring and Asymmetric Cell Division ............................................................................. 54 1.9.1 FtsZ ............................................................................................................................................... 54 1.9.2 The B. subtilis divisome ........................................................................................................ 57 1.9.3 Regulating Z-ring assembly during growth ................................................................. 58 1.9.4 Relocation of the division site during sporulation .................................................... 59 1.10 SpoIIE, a Key Cell Fate Determinant ..................................................................................... 61 1.10.1 Developmental behaviour of SpoIIE ............................................................................ 61 1.10.2 Three Domain structure .................................................................................................... 61 1.10.3 How compartment specific activation of σF is achieved ...................................... 63 1.11 Aims ................................................................................................................................................... 66 Chapter 2: Materials and Methods ....................................................................................................... 68 2.1 Agarose gel electrophoresis........................................................................................................ 68 2.2 Small Scale DNA Preparation ..................................................................................................... 69 2.3 Construct preparation ................................................................................................................... 70 2.3.1 Ligation Independent Cloning ........................................................................................... 70 2.3.2 Polymerase chain reaction .................................................................................................. 71 2.3.3
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