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Reporting period: 2017-01-01 to 2018-12-31

Sporulation and germination in clostridia are poorly understood. These processes are key intervention points for the prevention of disease and food spoilage, for optimising biofuel and platform chemical production, and for refining clostridia as drug delivery systems. Progress has been hindered by a lack of a coordinated, focused effort in the European research community, an insufficient number of scientists with expertise in clostridial microbiology and an absence of robust genetic tools.

Clospore aimed to capitalise on recent advances in genomics, forwards and reverse genetics to identify and dissect the genes involved in sporulation and germination, and in commercial formation of biofuels. In the process, Clospore aimed to organise European research efforts in the field, both in pathogenic and commercially important clostridia, in order to bring about a coordinated and united effort to advance our understanding of these processes. The implementation of Clospore sought to create a critical mass of trained young researchers, and foster industry-academia relations to advance Europe as the leader in this field.

The development, optimisation and implementation of a suite of advanced genetic tools throughout Clospore, facilitated the identification of novel structural and regulatory genes involved in not only sporulation and germination, but also biological processes key to the pathogenesis and biotechnological suitability of many clostridia. In doing so, a plethora of previously uncharacterised genotype-phenotype characterisations have been uncovered, in particular, the central role of multiple sigma factors in the regulation of sporulation and germination.
Allelic exchange (AE) protocols for reverse genetics studies were developed and exemplified for C. botulinum and C. perfringens in-line with the initial objectives. Additionally, AE and ClosTron technologies were applied for mutagenesis in C. difficile, which has already been reported in the literature. A novel clustered-regularly interspaced short palindromic repeats (CRISPR-Cas9) protocol, was then developed for mutagenesis in C. botulinum, the utility of which has been further demonstrated by the effective mutagenesis of C. acetobutylicum, C. beijerinckii, C. lungdahlii and C. autoethanogenum (Cañadas et al 2019, ACS Synth Biol doi: 10.1021/acssynbio.9b00075). The availability of several robust gene tools for reverse genetics studies in clostridia of immense medical and biotechnological importance, will pave the way for fundamental and translation research aimed at eradicating debilitating diseases and exploiting untapped waste streams for the sustainable production of chemicals and fuels through clostridial fermentation. Forward genetics approaches, in particular, transposon-directed insertional sequencing (TraDIS), have also been optimised for random mutagenesis in Clostridium spp.

The abovementioned gene tools, coupled with bioinformatics analysis, were extensively applied in order to identify novel structural and regulatory genes involved in the sporulation and germination processes of clostridia. Doing so, has implicated the involvement of multiple sigma factors in the regulation of sporulation in C. botulinum, C. difficile (Kint et al 2019, Environ Microbiol doi: 10.1111/1462-2920.14642) C. perfringens and C. sporogenes. Meanwhile, novel structural sporulation genes have been identified such as cotL, encoding a morphogenetic spore coat protein in Clostridium difficile (Alves Feliciano et al 2019,Environ Microbiol doi:10.1111/1462-2920.14505).

For species of biotechnological importance, the co-regulation of sporulation and solventogenesis was investigated. In the case of C. beijerinckii, the regulation of sporulation and solvent production were shown to be intertwined (Kolek et al 2017, Appl Microbiol Biotechnol doi: 10.1007/s00253-017-8555-3). On the other hand, in the case of C. thermosuccinogenes, sporulation-defective mutants were not adversely affected in their abilities to grow under fermentation conditions or to generate the desired solvents during fermentation. Consequently, these strains are of immense commercial benefit to industry and should facilitate the development of application-specific mutants, genetically tailored for maximal production of desired products whilst behaving in a predictable and controllable manner to allow effective and efficient fermentation.

Clostridium sporogenes has potential as a cancer therapeutic agent through the Clostridium-directed enzyme prodrug therapy (CDEPT) principle. In order to develop CDEPT into a therapeutic application, an array of chromosomal alterations will be required to generate the fit-for-purpose application strain. This relies on effective conjugation of shuttle vectors from an E. coli conjugal donor. In order to enhance strain development, mutations were introduced which significantly enhance conjugation efficiency from E. coli to C. sporogenes. These new strains are not adversely affected in parameters relating to their therapeutic application and will consequently form the background strains for ongoing strain development studies.

One beneficial consequence of undertaking such a a large-scale study on sporulation, is the uncovering of novel phenotypes associated withe genes originally thought to be involved in sporulation, and side projects aimed at investigating other important biological processes for the clostridia chosen for investigation. Such undertakings have increased our understanding of metabolism in C. thermosuccinogenes (koendjbiharie et al 2018, Appl Environ Microbiol doi: 10.1128/AEM.00363-18) and C. beijerinckii (Dallo et al 2018, Appl Environ Microbiol doi: 10.1128/AEM.02656-18). In a similar fashion, Ser/Thr kinases were shown to be involved in cell well homeostasis in C. difficile, which as a consequence, alters antimicrobial resistance properties (Ceunot et al 2019, Infect Immun doi: 10.1128/IAI.00005-19).
The Clospore ITN has been operating at a pivotal time in clostridia research. The advent of CRISPR/Cas9 genome editing in Clostridium has drastically improved the genetic accessibility of this genus. Following this development, studying genes through reverse genetics has become significantly faster and more reliable, concomitant with reduced risk of single nucleotide polymorphism accumulation, which previous techniques have suffered from. Members of the Clospore ITN played a significant role in the development of this new tool.

The discovery of key genes involved in sporulation of C. botulinum, C. difficile and C. perfringens, uncovers potential therapeutic targets to eradicate the dissemination of pathogenic clostridia through their resilient endospores. Meanwhile, the identification of genes critical to sporulation of the biotechnologically relevant clostridia will facilitate the development of conditionally sporulating strains or the implementation of measures for their control in industry.