Role of RNPP and Agr quorum sensing in the regulation of stationary phase phenotypes in Clostridium acetobutylicum

Bean, Zak (2017) Role of RNPP and Agr quorum sensing in the regulation of stationary phase phenotypes in Clostridium acetobutylicum. PhD thesis, University of Nottingham.

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The Gram-positive, endospore forming, strictly anaerobic bacterium Clostridium acetobutylicum is well known for its ability to ferment sugars to organic acids and solvents, most notably butanol. In batch culture, following an initial phase of acid production, the organism undergoes a metabolic and physiological shift to solvent production, granulose accumulation and initiation of endospore formation, usually associated with entry into stationary phase. The regulatory control of this shift and the associated phenotypes is poorly understood and has previously been linked to the master regulator Spo0A, but also the RNPP-like quorum sensing regulators Ca_c0957 and Ca_c0958 and Agr quorum sensing. The main objective of this study was to provide a better understanding of these systems and their interactions with respect to shift-associated phenotypes.

The role of Ca_c0957 and Ca_c0958 as master regulators of stationary phase phenotypes were confirmed through in-frame deletion and chromosomal complementation and both were shown to be essential for solvent formation and sporulation. While previously assumed to act primarily through control of Spo0A phosphorylation, this study demonstrated that some Spo0A activity remained in the absence of Ca_c0957 and served to independently control granulose accumulation. Inactivation of Ca_c0082, a LanC-like peptide cyclase controlled by Ca_c0957, produced a granulose-negative phenotype with reduced sporulation but capable of producing the Agr signalling peptide, thus resembling a Spo0A mutant.

C-terminally tagged Ca_c0957 and Ca_c0958 proteins were constructed and confirmed to be active, allowing for their future structural characterisation, even though initial purification attempts remained unsuccessful.

Agr signalling, controlled by Ca_c0957/Ca_c0958, was shown to be more promiscuous than previously thought, with several synthetic peptides displaying biological activity, including one representing the putative Clostridium roseum AgrD signal. Agr signalling influenced sporulation primarily through granulose accumulation, as granulose-deficient mutants were shown to produce fewer spores but generate solvents earlier than the wild type, especially under glucose limitation.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Winzer, K.
Cockayne, A.
Keywords: Clostridium acetobutylicum, Quorum sensing, Shift-associated phenotypes
Subjects: Q Science > QR Microbiology
QS-QZ Preclinical sciences (NLM Classification) > QW Microbiology. Immunology
Faculties/Schools: UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences
Item ID: 41621
Depositing User: Bean, Zak
Date Deposited: 17 Jul 2017 04:40
Last Modified: 13 Oct 2017 01:18

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