Boak, Lauren
(2024)
The role of Agr quorum sensing on sporulation and solvent formation in Clostridium beijerinckii.
PhD thesis, University of Nottingham.
Abstract
In recent decades there has been a growing demand for the development of biofuels, in particular bioethanol and biobutanol. Such solvents can be produced via acetone-butanol-ethanol (ABE) fermentation in solventogenic Clostridium species. For solventogenic Clostridium, it has been shown that the switch from acidogenesis to solventogenesis and sporulation along with improved ABE productivity occurs at high cell density in batch culture, such as industrial fermentations, suggesting quorum sensing is involved. Whilst regulation of metabolism and sporulation via QS has been investigated in other clostridia, its role remains unclear in the industrially important species Clostridium beijerinckii.
Studies into the C. beijerinckii NCIMB 8052 genome sequence suggested that the bacterium is capable of Agr-type QS, a QS system best studied in Staphylococcus aureus. Here, the agrD gene encodes the precursor for a signal molecule, the autoinducing peptide (AIP); agrB encodes the membrane protein which processes the AgrD precursor into the mature AIP, agrC encodes the membrane histidine kinase receptor which senses the AIP, and agrA encodes a dedicated response regulator. Accumulation of the AIP serves as a measure of population density and at a given threshold concentration triggers a response via the AgrC-AgrA two-component system. The aim of this study was to investigate and further understand if Agr QS has a role in the regulation of stationary phase phenotypes, primarily sporulation and solvent production, in C. beijerinckii.
An initial study into the genomes of various C. beijerinckii strains identified highly conserved Agr loci in this species. Through CRISPS- Cas9 based genome editing the agrB genes of the three most highly conserved systems, denoted as Agr2, Agr4 and Agr5, in the C. beijerinckii NCIMB 8052 genome were inactivated. Phenotypic characterisation showed a significant reduction in heat-resistant endospore formation in the agr2 and agr4 mutants, as well as reduced glucose consumption and solvent formation in all three mutants, however no observed change in granulose accumulation when compared to the WT. RNA-seq data comparing mutants to the WT, identified in the agrB2 and agrB4 mutants, later stage sporulation genes were hugely downregulated with spoIVB and sigK genes showing to be a key point of regulatory change. Various solvent related genes were downregulated in the mutants also, and alternative agr loci were shown to be differentially regulated in each of the agrB mutants, showing the multiple agr loci present in one genome influence the regulation of each other.
These findings show that specific agr loci do hold a regulatory role in the stationary phase phenotypes sporulation and solvent formation in C. beijerinckii NCIMB 8052. However, the depth of each agr loci and its role in such processes varies, with a complex agr hierarchical system being possible.
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