Roles and regulation of a Bdellovibrio bacteriovorus diguanylate cyclase, phosphodiesterase and a PilZ domain receptor.

Nottingham, Ruth (2017) Roles and regulation of a Bdellovibrio bacteriovorus diguanylate cyclase, phosphodiesterase and a PilZ domain receptor. PhD thesis, University of Nottingham.

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Abstract

Cyclic-di-GMP is a ubiquitous second messenger regulating lifestyles across a wide range of bacteria, most notably motility and virulence. The components of c-di-GMP signalling networks are made up of: diguanylate cyclases (DGCs) that contain a GGDEF domain and make c-di-GMP, phosphodiesterases (PDEs) that contain an EAL or HD-GYP domain and break-down c-di-GMP and PilZ domains (or degenerate DGCs/PDEs) which are receptors for c-di-GMP.

Bdellovibrio bacteriovorus HD100 is a predatory Gram-negative bacterium that preys upon other Gram-negative bacteria. B. bacteriovorus HD100 has a complex c-di-GMP signalling network, including four DGCs; one EAL domain protein, six HD-GYP proteins, 19 PilZ domain-proteins, a degenerate DGC and a proposed riboswitch all of which can act as cdi-GMP receptors. Bd0367, a DGC, regulates gliding motility and prey cell exit in B. bacteriovorus. Bd0367 has a GGDEF domain but also a response regulator domain, which suggests that c-di-GMP production by Bd0367 may be regulated by a phosphorylation event by a cognate sensor kinase.

This thesis investigated the Bd0367 signalling pathway through analysis of Bd0367 protein interaction networks, Bd0367 DGC c-di-GMP production and searched for the cognate histidine kinase for Bd0367. This study confirmed that a conserved aspartate residue is required for Bd0367 to be an active DGC via mutational studies and cellular cdi-GMP extractions. It also demonstrated that Bd0367 enzymatic activity is essential for prey cell exit, but is less important for gliding motility, this was tested by a Bd0367D63A mutation. Which suggests that localised protein interactions formed by Bd0367 (independent of its enzymatic role) may be more important for gliding motility than the Bd0367 specific c-di-GMP generation.

This concept was supported by other data as deletion of a gene encoding a PilZ domain protein that interacted with Bd0367 by bacterial two hybrid analysis, caused hyperreversals during gliding motility. Future work is needed to dissect the exact mechanism by which the PilZ domain protein influences the gliding machinery.

A PDE protein Bd1971 interacts by bacterial two hybrid assay with Bd0367. This study found that ∆bd1971 resulted in impaired gliding motility, suggesting that a high global level of c-di-GMP in B. bacteriovorus cells can negatively affect motility as seen in many other bacteria. The molecular reason for impaired gliding at high c-di-GMP levels in B. bacteriovorus is yet to be determined and requires further study

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Sockett, R.E.
Lambert, C.
Keywords: Cyclic guanylic acid, Second messengers, Cyclic GMP, Bdellovibrio bacteriovorus
Subjects: Q Science > QP Physiology
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: 40055
Depositing User: Nottingham, Ruth
Date Deposited: 05 Dec 2019 14:02
Last Modified: 06 May 2020 12:47
URI: http://eprints.nottingham.ac.uk/id/eprint/40055

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