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Characterization of Escherichia coli dinJ-yafQ toxin-antitoxin system using insights from mutagenesis data.

ABSTRACT: Escherichia coli dinJ-yafQ operon codes for a functional toxin-antitoxin (TA) system. YafQ toxin is an RNase which, upon overproduction, specifically inhibits the translation process by cleaving cellular mRNA at specific sequences. DinJ is an antitoxin and counteracts YafQ-mediated toxicity by forming a strong protein complex. In the present study we used site-directed mutagenesis of YafQ to determine the amino acids important for its catalytic activity. His50Ala, His63Ala, Asp67Ala, Trp68Ala, Trp68Phe, Arg83Ala, His87Ala, and Phe91Ala substitutions of the predicted active-site residues of YafQ abolished mRNA cleavage in vivo, whereas Asp61Ala and Phe91Tyr mutations inhibited YafQ RNase activity only moderately. We show that YafQ, upon overexpression, cleaved mRNAs preferably 5' to A between the second and third nucleotides in the codon in vivo. YafQ also showed RNase activity against mRNA, tRNA, and 5S rRNA molecules in vitro, albeit with no strong specificity. The endoribonuclease activity of YafQ was inhibited in the complex with DinJ antitoxin in vitro. DinJ-YafQ protein complex and DinJ antitoxin alone selectively bind to one of the two palindromic sequences present in the intergenic region upstream of the dinJ-yafQ operon, suggesting the autoregulation mode of this TA system.

SUBMITTER: Armalyte J

PROVIDER: S-EPMC3294823 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature

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Characterization of Escherichia coli dinJ-yafQ toxin-antitoxin system using insights from mutagenesis data.

Armalyte Julija J Jurenaite Milda M Beinoraviciūte Gina G Teiserskas Justinas J Suziedeliene Edita E

Journal of bacteriology 20120113 6

Escherichia coli dinJ-yafQ operon codes for a functional toxin-antitoxin (TA) system. YafQ toxin is an RNase which, upon overproduction, specifically inhibits the translation process by cleaving cellular mRNA at specific sequences. DinJ is an antitoxin and counteracts YafQ-mediated toxicity by forming a strong protein complex. In the present study we used site-directed mutagenesis of YafQ to determine the amino acids important for its catalytic activity. His50Ala, His63Ala, Asp67Ala, Trp68Ala, T ...[more]

PMID: 22247505

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Project description:?(E), an alternative ? factor that governs a major signaling pathway in envelope stress responses in Gram-negative bacteria, is essential for growth of Escherichia coli not only under stressful conditions, such as elevated temperature, but also under normal laboratory conditions. A mutational inactivation of the hicB gene has been reported to suppress the lethality caused by the loss of ?(E). hicB encodes the antitoxin of the HicA-HicB toxin-antitoxin (TA) system; overexpression of the HicA toxin, which exhibits mRNA interferase activity, causes cleavage of mRNAs and an arrest of cell growth, while simultaneous expression of HicB neutralizes the toxic effects of overproduced HicA. To date, however, how the loss of HicB rescues the cell lethality in the absence of ?(E) and, more specifically, whether HicA is involved in this process remain unknown. Here we showed that simultaneous disruption of hicA abolished suppression of the ?(E) essentiality in the absence of hicB, while ectopic expression of wild-type HicA, but not that of its mutant forms without mRNA interferase activity, restored the suppression. Furthermore, HicA and two other mRNA interferase toxins, HigB and YafQ, suppressed the ?(E) essentiality even in the presence of chromosomally encoded cognate antitoxins when these toxins were overexpressed individually. Interestingly, when the growth media were supplemented with low levels of antibiotics that are known to activate toxins, E. coli cells with no suppressor mutations grew independently of ?(E). Taken together, our results indicate that the activation of TA system toxins can suppress the ?(E) essentiality and affect the extracytoplasmic stress responses.?(E) is an alternative ? factor involved in extracytoplasmic stress responses. Unlike other alternative ? factors, ?(E) is indispensable for the survival of E. coli even under unstressed conditions, although the exact reason for its essentiality remains unknown. Toxin-antitoxin (TA) systems are widely distributed in prokaryotes and are composed of two adjacent genes, encoding a toxin that exerts harmful effects on the toxin-producing bacterium itself and an antitoxin that neutralizes the cognate toxin. Curiously, it is known that inactivation of an antitoxin rescues the ?(E) essentiality, suggesting a connection between TA systems and ?(E) function. We demonstrate here that toxin activation is necessary for this rescue and suggest the possible involvement of TA systems in extracytoplasmic stress responses.

Dataset Information

Characterization of Escherichia coli dinJ-yafQ toxin-antitoxin system using insights from mutagenesis data.

Publications

Characterization of Escherichia coli dinJ-yafQ toxin-antitoxin system using insights from mutagenesis data.

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