CLOSPORE

CLOSPORE PROJECTS

These roles will all involve experimental laboratory research, with opportunities to join network wide training events and international collaboration. In addition, the roles will include the preparation of scientific publications, assistance in student supervision and general tasks in the research group.

Synthetic Biology Research Centre

ESR 1:

The fellow will work within the BBSRC/EPSRC Synthetic Biology Research Centre (SBRC) on a project concerned with the spores of the benign species Clostridium sporogenes, which could be used for treating cancer. Intravenously injected spores localise to and selectively germinate in the hypoxic centres of solid tumours, a property that can be used to deliver anti-tumour agents. This strategy is known as Clostridia-Directed Enzyme Prodrug Therapy, or CDEPT.

ESR 2:

The fellow will work within the BBSRC/EPSRC Synthetic Biology Research Centre (SBRC). The aim of this project is to develop novel reporter systems to facilitate the quantitative assessment of gene expression in real foods. Such a system will detect food spoilage by clostridial species by measuring the growth and the expression of toxin and spore processes.

ESR 15:

The fellow will work within the BBSRC/EPSRC Synthetic Biology Research Centre (SBRC) to understand the mechanisms by which clostridial spores become resistant to chemical and physical agents using a molecular approach. The role will involve experimental laboratory research, including the production and testing of mutants to study spore resistance, and the use of transcriptomics to determine the location of different proteins within the spore.

NIZO Food Research BV

ESR 3:

The fellow will work within NIZO food research on sporulation and germination of Clostridium perfringens, the third most common cause of bacterial foodborne illness in the Western world. The role will involve implementing genetic tools to study key genes expressed during the sporulation process, which in turn determine toxin production and spore properties, including spore germination. The production and characterisation of mutants will allow for functional assignment of novel sporulation and germination genes.

ESR 4:

The fellow will work within NIZO food research on Clostridium perfringens on two groups of strains with the potential to cause food poisoning but that are phylogenetically distant and occupy different niches, namely the gut or food. The aim of this project is to gain improved understanding of the sporulation/toxigenesis process and spore properties, including heat resistance, ultimately to improve strategies aimed at preventing C. perfringens diarrhoea.

University of Helsinki

ESR 5 and 6:

The projects will use random and targeted mutagenesis and fluorescent single-cell biology to identify and characterize novel sporulation genes and to study the key links between C. botulinum sporulation, key metabolic pathways, and toxin production. The projects will involve secondments in CLOSPORE partner institutions.

Corbion-Purac

ESR 7:

The PhD project will be executed at Corbion’s lab facilities in Wageningen, The Netherlands. The aim of this project is to assess genetic accessibility of the thermophilic Clostridium thermosuccinogenes and related species that can be used for production of commodity chemicals such as organic acids. The mechanism of sporulation and the effect of its regulation on central metabolism will be studied.

ESR 8:

The PhD project will be executed at Corbion’s lab facilities in Wageningen, The Netherlands. The aim of this project is to apply thermophilic Clostridium thermosuccinogenes or related species for production of organic acids such as dicarboxylic acids. The metabolism will be studied to reveal what makes this organism a dedicated dicarboxylic acid producer. This knowledge will be applied for metabolic engineering strategies to improve its performance.

Universidade Nova de Lisboa

ESR 9: Cell-cell signaling during spore development

Cell-cell communication during spore development is important for the coordination of the forespore and mother cell-specific programs of gene expression. Previous work has shown that novel pathways of cell-cell signaling are likely to operate in C. difficile, relative to the B. subtilis model and thus there is new Biology to be learned here. These pathways involve the cell type-specific production of signaling proteins that are either membrane-embedded or secreted to the inter-membrane space that separates the two cells involved in sporulation and influence gene expression and spore morphogenesis in the adjacent cell. Proper signaling is required for the fidelity of the developmental process and will impact on the ability of spores to colonize and infect their hosts. The project involves the characterization of the genes coding for the proteins involved in the cell-cell signaling pathways, and their function, using a combination of genetics, cell biology, biochemical and structural biology approaches. The project will also assess the effects of altered signaling on the morphogenesis of the spore and its properties during colonization and infection. In particular, we now know that C. difficile is able to form different spore morphotypes, and the decision process is most likely traceable to key regulatory events during spore morphogenesis and to the cell-cell communication pathways.

ESR 10: Spore morphogenesis

Proper assembly of the spore surface layers is a pre-requisite for spore resistance and hence for its persistence and survival in the environment. It is also a pre-requisite for proper interaction of the spore with its immediate environment, including its interactions with host cells. The spore surface structures comprise the coat and exosporium layers, as well as additional adhesion structures. Recent work has lead to the identification of key morphogenetic proteins that govern assembly of the spore surface structures. However, how these proteins function to control the spore morphogenesis is not known. At least some of these proteins appear to function as hubs, interacting with several other proteins and directing them to their subcellular address. With what proteins do the hubs interact with can be approached by defining localized proteomes. Other spore surface proteins are enzymes that act by buffering its immediate environment and endow the spore with resistance to several insults, including, importantly, oxidative stress. Other enzymes are part of sensory systems that will lead to triggering of germination under favorable conditions. Yet others are required for post-translational modifications that enforce or maintain the correct pattern of protein-protein interactions leading to the assembly of the surface structures. The project involves dissecting the function of key morphogenetic effectors through genetics, cell biology, biochemistry and structural studies. A key aspect is to use cell biology methodologies, in combination with biochemical assays, to unravel the pattern of protein-protein interactions governing the subcellular localization of the proteins and other factors that participate in spore assembly. We will also aim at characterizing the effects of manipulating the assembly of the spore surface structures on colonization and infection.

Institut Pasteur

ESR 11:

The fellow will work within the LBPA laboratory at the Institut Pasteur on a project focused on the molecular mechanisms involved in the complex regulatory network of sporulation in Clostridium difficile. This pathogen is the leading cause of intestinal nosocomial post-antibiotic infections in adults. Spores have a central role in the C. difficile infectious cycle and in dissemination. However, the complex developmental program of sporulation/germination remains poorly characterized. The role of transcriptional regulators, noncoding RNAs and post-translational modification of key actors of the regulatory cascade as well as key components of inter-compartmental communication will be analyzed in details using genetic and genomic approaches coupled to single cell gene expression analysis.

ESR 12:

The fellow will work within the LBPA laboratory at the Institut Pasteur on a project focused on the characterization and the role of C. difficile spore surface components in colonization, relapse and resistance outside the host. The spores of this important enteropathogen are crucial for persistence in gut and transmission of the disease in hospital. The work will include the localization of spore-associated proteins and the construction of mutants to evaluate their impact of gene inactivation on signaling, resistance to harsh conditions, germination, adhesion to epithelial cells and colonization. This might allow the development of biomarkers for C. difficile spores and of new tools to target spores, the vector of dissemination of this pathogen.

Green Biologics Ltd

ESR 13:

The fellow will be based at Green Biologics’ labs in Abingdon, Oxfordshire, UK. As part of the strain development team they will use random mutagenesis and recombinant DNA technologies to generate sporulation defective strains, and will use molecular microbiology and small scale fermentation techniques to characterise them. This project aims to understand the link between feedstock, sporulation and solvent production in Clostridium sp. and will transfer that knowledge to the industrial ABE fermentation process.

Wageningen UR

ESR 14:

In this PhD project, the main tasks are:
(1) Use of random mutagenesis techniques to generate and isolate strains carrying mutations in key sporulation genes;
(2) Use targeted mutagenesis to recreate specific Spo0A mutants;
(3) Fermentation testing and characterisation of asporogenic mutants for butanol production, identification of metabolites produced by the WT and mutant strains;
(4) Molecular characterisation of strains

Publications in peer-reviewed international journals are expected, as well as participation in international conferences, meetings, etc. The candidate is obliged to perform 3 secondments (20 ECTS) at selected partner institutions. The candidate will join the research school VLAG