Characterisation of the conserved and essential wblE gene in Streptomyces venezuelae

Balis, Lucas (2024) Characterisation of the conserved and essential wblE gene in Streptomyces venezuelae. Doctoral thesis, University of East Anglia.

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Abstract

The Wbl (WhiB-like) family of proteins is phyletically confined to the Actinobacteria where they primarily act as transcription factors. WblE is an ancient protein and its homologue WhiB1 is essential in Mycobacterium tuberculosis, but its function is unknown and before this work it had not been comprehensively studied in Streptomyces species. Using CRISPR-Cas9, this work confirms that WblE is an essential [4Fe-4S] protein that functions as a global transcription factor: Distinct from its relatives, ChIP-seq shows that WblE predominantly binds promoters of ‘bld’ developmental genes; a characteristic that also pervaded Co-immunoprecipitation-MS CoIPMS) experiments. Importantly, WblE bound to the promoter of dnaA, the chromosomal replication initiator throughout development, which was confirmed in vitro by SPR; the response regulator MtrA, which also binds to the dnaA promoter, binds to the wblE promoter in a sequence-specific manner. This work posits that WblE is a major transcriptional activator of dnaA, which drives constitutive replication events in tight coordination with metabolism and the onset of development via other cell-cycle and developmental regulators, in addition to nitric oxide. Moreover, this alone could account for the essentiality demonstrated for wblE and its [4Fe-4S] cluster. CoIP-MS in tandem with bacterial two hybrid analysis shows that WblE mediates at least part of its function via a direct interaction with region 4.2 of σHrdB, akin to other WhiB-like proteins. However, a range of proteins with roles beyond transcription were consistently enriched and allude to a much broader functionality for the protein; this approach revealed at least one novel partner for WblE in S. venezuelae strain NRRL B-65442, that is conserved in the genus. These functions are discussed in the context of •NO signalling, WhiB-like cluster biochemistry and how this may link developmental signalling pathways that define Streptomyces spp. differentiation and the decision to initiate secondary metabolism.

Item Type:	Thesis (Doctoral)
Faculty \ School:	Faculty of Science > School of Biological Sciences
Depositing User:	Chris White
Date Deposited:	23 Apr 2025 12:19
Last Modified:	23 Apr 2025 12:19
URI:	https://ueaeprints.uea.ac.uk/id/eprint/99052
DOI:

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