Streptomyces are filamentous soil-dwelling bacteria with a complex life cycle. Elucidating the signals that regulate morphological change in microbes is of fundamental importance for biotechnology applications. In the case of Streptomyces a development switch occurs with the concomitant production of secondary metabolites, many of which have pharmaceutical properties. For the industrially used strain S. lividans this switch is dependent on the bioavailability of copper (Cu) in the environment. This thesis has explored the relationship between Cu-chaperones and a haem peroxidase, part of the sco operon, with the maturation of a Cu-containing oxidase, GlxA. In S. lividans the GlxA gene is part of the cslA/glxA operon that contains genes encoding putative enzymes involved in glycan processing. Both these gene clusters are highly conserved in streptomycetes. In Chapter 2 the characterisation of GlxA is reported. It was found to be membrane associated with a mononuclear Cu site and possess a Cys-Tyr redox cofactor capable of housing a protein radical, comparable to the fungal galactose oxidase (Gox). The tertiary structure of GlxA revealed a unique domain arrangement, atypical spectroscopic properties compared to Gox and a lack of enzymatic activity with classical Gox substrates. Generation of the ΔglxA null mutant was found to stall aerial hyphae development on solid media and dramatically change the morphology in liquid cultures. This was ascribed to the absence of the oxidation of a glycan by GlxA produced by CslA (a cellulose-synthase), required for morphogenesis on solid and liquid cultures. The molecular nature of this glycan is unknown. A number of GlxA variants were created in Chapter 3 to elucidate the proteins unique spectroscopic properties. It was found that the second coordination sphere residue, Trp288, plays a major role in tuning the electronic properties of the buried Cu site in GlxA. Its removal abolishes the Cys-Tyr radical and perturbs the spectroscopic properties such that they resemble Gox. Monoclonal antibodies were used to follow the maturation of GlxA through observing mobility differences on denaturing PAGE gels based on the presence or absence of the Cys-Tyr cross-link. X-ray crystallography provided structural insight into the maturation process. A surprising outcome of Chapter 3 was that upon removal of the crosslinking Cys121, a new protein radical is formed as opposed to the expected abolition. Chapter 4 addresses another surprising finding in that a putative haem peroxidase (DtpA), part of the sco operon, plays a role in GlxA maturation and in the Cu-dependent morphological development. DtpA is shown through enzymology and structural analysis to be a member of the dye-decolourising peroxidase (DyP) family. Crucially, it is shown that DtpA functions as a peroxidase in the presence of GlxA using the GlxA substrate, glycolaldehyde. Synthesis and modification of the CslA/GlxA glycan will inevitably require degradation during the life cycle. As part of the cslA-glxA gene cluster are two genes encoding for putative polysaccharide degrading enzymes. One of these is a putative Cu lytic polysaccharide monooxygenase, SliLPMO10E. Chapter 5 structurally characterises SliLPMO10E and also investigates the kinetics of Cu-binding. The latter brings to the attention that LPMOs are able to bind Cu in two forms at a single site before relaxing into a final substrate active form. Importantly, SliLPMO10E is found to be active only with chitin via a C1 sugar ring oxidation mechanism. This hints at the possibility that the glycan produced by CslA and modified by GlxA is chitin-like possessing N-acetyl glucosamine moieties. By combining the in vitro results from this thesis together with the in vivo results obtained through the duration of this work from collaborators at Leiden University an overall model of the Cu-dependent morphogenesis and glycan processing in the hyphal tips of S. lividans is presented. Chapters 2 through to 5 focus on events that occur under Cu limitations, i.e. homeostasis. Chapter 6 extends on previous work that characterised the CsoR regulon. The Cu sensitive operon repressor (CsoR) protein determines the set point of Cu(I) concentration in the cytosol. Under Cu stress, Cu(I) binds to CsoR and de-represses genes under its transcriptional control. Chapter 6 explores the possibility of whether CopZ-like Cuchaperones can traffic Cu(I) to the DNA-bound CsoR, resulting in the up-regulation of control systems to return the cell to homeostasis. Size-exclusion and EMSA studies showed that Cu(I) was transferred from CopZ to CsoR in a unidirectional manner. Re-analysis of previous RNA-seq data using the S. lividans genome as input, enabled for a more complete model for the CsoR regulon in S. lividans to be proposed.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:681829 |
| Date | January 2016 |
| Creators | Chaplin, Amanda K. |
| Publisher | University of Essex |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://repository.essex.ac.uk/16310/ |
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