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Functional characterization and transcriptional repression by Lacticaseibacillus paracasei DinJ-YafQ. | LitMetric

AI Article Synopsis

  • DinJ-YafQ is a type II toxin-antitoxin system found in different strains of Lacticaseibacillus paracasei, where the toxin YafQ has a substitution compared to E. coli's version, affecting its efficiency.
  • Both YafQ versions can digest ribosomal RNA but their activity is neutralized by the antitoxin DinJ, which also binds to DNA to repress the promotor of their gene.
  • A mutation in DinJ (R13A) significantly impacts its ability to bind DNA, and in vivo experiments reveal that this system can cause toxicity related to gene expression levels, particularly with the gfp reporter gene.

Article Abstract

DinJ-YafQ is a bacterial type II TA system formed by the toxin RNase YafQ and the antitoxin protein DinJ. The activity of YafQ and DinJ has been rigorously studied in Escherichia coli, but little has been reported about orthologous systems identified in different microorganisms. In this work, we report an in vitro and in vivo functional characterization of YafQ and DinJ identified in two different strains of Lacticaseibacillus paracasei and isolated as recombinant proteins. While DinJ is identical in both strains, the two YafQ orthologs differ only for the D72G substitution in the catalytic site. Both YafQ orthologs digest ribosomal RNA, albeit with different catalytic efficiencies, and their RNase activity is neutralized by DinJ. We further show that DinJ alone or in complex with YafQ can bind cooperatively to a 28-nt inverted repeat overlapping the -35 element of the TA operon promoter. Atomic force microscopy imaging of DinJ-YafQ in complex with DNA harboring the cognate site reveals the formation of different oligomeric states that prevent the binding of RNA polymerase to the promoter. A single amino acid substitution (R13A) within the RHH DNA-binding motif of DinJ is sufficient to abolish DinJ and DinJ-YafQ DNA binding in vitro. In vivo experiments confirm the negative regulation of the TA promoter by DinJ and DinJ-YafQ and unveil an unexpected high expression-related toxicity of the gfp reporter gene. A model for the binding of two YafQ-(DinJ)-YafQ tetramers to the promoter inverted repeat showing the absence of protein-protein steric clash is also presented. KEY POINTS: • The RNase activity of L. paracasei YafQ toxin is neutralized by DinJ antitoxin. • DinJ and DinJ-YafQ bind to an inverted repeat to repress their own promoter. • The R13A mutation of DinJ abolishes DNA binding of both DinJ and DinJ-YafQ.

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Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9592637PMC
http://dx.doi.org/10.1007/s00253-022-12195-4DOI Listing

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