Unravelling the role of the bacterial cell division protein DivIB

Kimberly Wadsworth (2009). Unravelling the role of the bacterial cell division protein DivIB PhD Thesis, Institute for Molecular Bioscience, The University of Queensland.

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Author Kimberly Wadsworth
Thesis Title Unravelling the role of the bacterial cell division protein DivIB
School, Centre or Institute Institute for Molecular Bioscience
Institution The University of Queensland
Publication date 2009-01-01
Thesis type PhD Thesis
Supervisor Dr Glenn F King
Total pages 153
Total colour pages 18
Total black and white pages 135
Subjects 270000 Biological Sciences
Abstract/Summary The molecular mechanics of bacterial cell division remain one of the most fundamental unsolved problems in bacterial cell biology. During bacterial cytokinesis, bacteria divide symmetrically to give rise to two identical daughter cells. This tightly regulated process is orchestrated by an assembly of essential cell division proteins that form a supramolecular structure known as the divisome. The divisome, which forms at the cell centre, is responsible for the invagination and fusion of the cell’s membrane and peptidoglycan layers. The Escherichia coli divisome is comprised of at least ten essential proteins whose individual functions are mostly unknown. These divisomal proteins are recruited in a semi-hierarchical order, with the early recruits being predominantly cytoplasmic and the later recruits being predominantly extracytoplasmic or multi-pass transmembrane proteins. DivIB and its ortholog FtsQ are essential members of the divisome in Gram-positive and Gram-negative bacteria, respectively. DivIB is a bitopic membrane protein composed of an N-terminal cytoplasmic domain, a single-pass transmembrane domain, and a C-terminal extracytoplasmic region comprised of three separate protein domains. The α domain is located next to the transmembrane segment and is a polypeptide-transport-associated (POTRA) domain. The β domain comprises approximately half of the extracytoplasmic region, and has a unique three-dimensional fold. The most C-terminal domain, the γ domain, is relatively unstructured. This protein has been proposed to play a role in septal peptidoglycan cross-linking or lengthening. The primary aims of these studies were to further characterise the structure and function of the bacterial cell division protein DivIB as well as investigate the interactions this protein has with the other divisomal proteins. It was anticipated that the knowledge gained should aid in the development of antimicrobials that target this protein’s function or protein-protein interactions. A molecular dissection approach was used to determine which of DivIB’s domains are essential for its recruitment to incipient division sites and for its cell division functions. It was determined that DivIB has three molecular epitopes that mediate its localisation to division septa; two epitopes are encoded within the extracytoplasmic region while the third is located in the transmembrane domain. It is proposed that these epitopes represent sites of interaction with other divisomal proteins, and this information was used to develop a model of the way in which DivIB and FtsQ are integrated into the divisome. Remarkably, two of the three DivIB localisation epitopes are dispensable for vegetative cell division; this suggests that the divisome is assembled using a complex network of protein-protein interactions, many of which are redundant and likely to be individually nonessential. Yeast and bacterial two-hybrid studies have revealed that most of these proteins have multiple binding partners, making it difficult to pinpoint epitopes that mediate the interaction between pairs of interacting proteins. Recently, a heterologous septal targeting approach was introduced to study the interaction between Bacillus subtilis divisomal proteins in E. coli. This technique allows the interaction between pairs of divisomal proteins to be studied in vivo without the complications caused by other interacting proteins. This approach was used to perform a molecular dissection of the interaction between B. subtilis DivIB and the divisomal transpeptidase PBP 2B. Although both proteins have septal localisation determinants in their transmembrane domains, it was found that these regions do not mediate their interaction. Rather, it was shown that DivIB interacts with PBP 2B through its extracytoplasmic region. Dissection studies revealed that all three extracytoplasmic domains of DivIB are necessary for interaction with PBP 2B, suggesting that the two proteins make multiple interactions, each of which is not strong enough in isolation to mediate formation of a stable complex. Finally, it was shown that E. coli FtsQ localises to the division septum in B. subtilis but cannot complement a divIB null. Multi-angle laser light scattering (MALLS) analysis revealed that the extracytoplasmic domain of Geobacillus stearothermophilus DivIB is predominantly monomeric at high concentrations. This indicated that if DivIB does exist as a dimer in vivo, it dimerises through its cytoplasmic or transmembrane region. In vitro observations suggest the C-terminal residues of DivIB may play a role in peptidoglycan binding. Finally, attempts were made to determine the three-dimensional structure of the complete extracytoplasmic domain of DivIB. Although it proved impossible to determine the structure using NMR spectroscopy, crystals were obtained under many different crystallisation conditions. Despite diffracting to 3.5 Å, we were unable to solve the protein structure using X-ray crystallography. However, this work has laid the groundwork for future attempts at solving the structure of this protein using X-ray crystallography.
Keyword Bacillus subtilis
Escherichia coli
cell division
Additional Notes Pages in colour: 18, 21, 23-24, 36, 54, 72, 90, 96, 109, 113, 129-130, 132, 149, 151-153

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Created: Wed, 24 Jun 2009, 21:09:28 EST by Ms Kimberly Wadsworth on behalf of Library - Information Access Service