Clostridial strain degeneration: new approaches to an old problem

Humphreys, Jonathan Richard (2019) Clostridial strain degeneration: new approaches to an old problem. PhD thesis, University of Nottingham.

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Abstract

The genus Clostridium comprises a range of physiologically diverse species including notorious pathogens as well as industrially relevant organisms. Solventogenic Clostridia produce solvents via their unique acetone-butanol-ethanol (ABE) fermentation metabolism and encompass species of significance for future biofuel production. Typically these organisms undergo an initial acidogenic phase which is followed by a solventogenic phase as they enter stationary growth. The later phase also sees a morphological change in these organisms as they accumulate storage compounds and endospores. With an increased need for sustainable fuel production, solventogenic Clostridia offer an attractive alternative to produce biobutanol. An intrinsic problem with these organisms however is the phenomenon of spontaneous strain degeneration. This phenomenon is characterised by a reduced or complete loss in the production of solvents and the formation of spores which typically occurs when the bacteria are repeatedly subcultured in batch culture or grown in continuous fermentations.

The aim of this study was to gain a further understanding of the degeneration process by investigating the genetic, ecological and evolutionary forces behind this phenomenon. To do this, Clostridium beijerinckii NCIMB 8052 was selected as a model organism due to its tendency to degenerate more rapidly than other strains. Furthermore, this strain was progenitor to a hyper-butanol producing strain making it a relevant candidate for industrial use.

Through repeated subculturing of C. beijerinckii NCIMB 8052, four distinct colony morphologies emerged. A total of 71 degenerate isolates were obtained based on their colony appearance. These were phenotypically characterised in regards to their solvent, spore and granulose producing capabilities. The most severe of the colony types was unable to produce solvents or endospores.

Genetic comparisons between derived isolates and their parental ancestor revealed four regions that contained more mutations than anywhere else in the genome. These regions included the master regulator gene of solvent and spore formation spo0A and two notable histidine kinases. The role of these genes was proven via insertional disruption which showed a huge reduction in sporulation capacity.

Social interactions were studied by mixing degenerate isolates with the wild type in various starting frequencies. At low frequencies, isolates were much fitter than the wild type however this fitness rapidly declined as the starting frequencies increased. This frequency-dependent fitness is suggestive of social cheating.

These findings led to the hypothesis that degenerate isolates have gained an increased fitness compared to the wild type and this fitness is due to the loss of Spo0A activity.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Winzer, Klaus
Cockayne, Alan
Keywords: Solventogenic Clostridia, Clostridium beijerinckii, Strain degeneration, Whole genome sequencing, Comparative genomics, Hot spot regions, Evolution studies, Fitness assays
Subjects: Q Science > QR Microbiology
Faculties/Schools: UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences
Item ID: 56578
Depositing User: Humphreys, Jonathan
Date Deposited: 09 Aug 2019 13:09
Last Modified: 07 May 2020 10:46
URI: http://eprints.nottingham.ac.uk/id/eprint/56578

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