Prophages need to tightly control their lifestyle to either be maintained within the host genome or enter the lytic cycle. The SPβ prophage present in the genome ofBacillus subtilis168 was recently shown to possess anarbitriumsystem defining its replication stage. Using an historicB. subtilisstrain harboring the heat-sensitive SPβ c2 mutant, we analyzed a key component of the lysis-lysogeny decision system called YopR, which is crucial for maintenance of lysogeny. Here, we demonstrate that the heat-sensitive SPβ c2 phenotype is due to a single nucleotide exchange in theyopRgene, rendering the encoded YopRG136Eprotein temperature sensitive. Structural characterization of YopR revealed that the protein is a DNA-binding protein with an overall fold like tyrosine recombinases. Biochemical and functional analyses indicate that YopR has lost the recombinase function and the G136E exchange impairs its higher order structure and DNA binding activity. We also show that the serine recombinase SprA and its accessory factor SprB are not required for the heat-dependent induction of the lytic cycle of the SPβ c2 prophage. Finally, an evolution experiment with aB. subtilisstrain carrying SPβ c2 identified YosL as a novel component of the lysis-lysogeny management system, as the presence ofyosLis crucial for the induction of the lytic cycle of SPβ.
BackgroundRecombinant production of amebic cysteine proteases using Escherichia coli cells as the bacterial system has become a challenging effort, with protein insolubility being the most common issue. Since many of these enzymes need a native conformation stabilized by disulfide bonds, an elaborate process of oxidative folding is usually demanded to get a functional protein. The cytoplasm of E. coli SHuffle Express cells owns an enhanced ability to properly fold proteins with disulfide bonds. Because of this cellular feature, it was possible to assume that this strain represents a reliable expression system and worthwhile been considered as an efficient bacterial host for the recombinant production of amebic cysteine proteases.ResultsUsing E. coli SHuffle Express cells as the bacterial system, we efficiently produce soluble recombinant EhCP1protein. Enzymatic and inhibition analyses revealed that it exhibits proper catalytic abilities, proceeds effectively over the substrate (following an apparent Michaelis-Menten kinetics), and displays a typical inhibition profile.ConclusionsWe report the first feasibility study of the recombinant production of amebic cysteine proteases using E. coli SHuffle Express as the bacterial host. We present a simple protocol for the recombinant expression and purification of fully soluble and active EhCP1 enzyme. We confirm the suitability of recombinant EhCP1 as a therapeutic target. We propose an approachable bacterial system for the recombinant production of amebic proteins, particularly for those with a need for proper oxidative folding.Electronic supplementary materialThe online version of this article (10.1186/s12896-018-0429-y) contains supplementary material, which is available to authorized users.
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