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Identifying interactome of paralogous SSB proteins in the multicellular prokaryote Streptomyces coelicolor

Principal investigator

Category
Projekti Hrvatske zaklade za znanost
Finance value
998500
HRK
Start date
Sep 15th 2019
End date
Sep 14th 2022
Status
Active

Identifying interactome of paralogous SSB proteins in the multicellular prokaryote Streptomyces coelicolor

Identifying interactome of paralogous SSB proteins in the multicellular prokaryote Streptomyces coelicolor

Streptomycetes are best known as producers of clinically important antibiotics and for their complex life cycle. Single-strand DNA-binding (SSB) proteins have a key role in DNA replication, repair and recombination in all domains of life. Various bacteria possess paralogous SSBs, but their biological role is poorly understood. We reported that two S. coelicolor SSBs have adopted different cellular functions through evolution. While SsbA is indispensable for survival, SsbB has a key role in chromosome segregation during spore formation.

Recently, we found that a specific mutant with deleted ssbB increases antibiotic production. To get a better insight into biological functions of paralogous SSB proteins, this project aims to identify SSB-interactomes, to analyse their binding domains and to elucidate the importance of disulphide bridges for SsbB functionality. Once identified by B2H system or TAP technology, the most promising interactants will be analysed by applying in vivo and in vitro techniques. Genetic constructs carrying mutations in gene encoding protein partner (if not essential) or double mutants with ssbB gene will be examined phenotypically and morphologically. The co-localization of selected SSB partners will be examined by fluorescence microscopy.

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To obtain information on the binding mechanisms of SSB-protein interactions, the affinity of the interacting proteins will be analysed by several approaches, such as semi-quantitative assays (EMSA), spectroscopic techniques (CD, fluorescence, MST), and calorimetric techniques like ITC. Comparative proteomic analysis will be applied to examine temporal synthesis of SsbB and its protein partners during early log and programmed cell death stage of growth in S. coelicolor and S. rimosus with elevated antibiotic production. Expecting that most of the SSB partners are conserved in Streptomyces spp., proposed analyses could uncover time-specific interaction, potentially important for antibiotic production.

Fig. 2 A) Crystal structure of the SsbA protein (Stefanic, Vujaklija et al. 2009). B) Superposition of SsbA (grey) and SsbB (green) (Paradzik, Ivic et a...

Fig. 2 A) Crystal structure of the SsbA protein (Stefanic, Vujaklija et al. 2009). B) Superposition of SsbA (grey) and SsbB (green) (Paradzik, Ivic et al. 2013). Disulphide bridges are marked in red and SsbA clamp in blue.

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