New toxins homologous to ParE belonging to three-component toxin-antitoxin systems in Escherichia coli O157:H7

Abstract

SummaryType II toxin-antitoxin (TA) systems are considered as protein pairs in which a specific toxin is associated with a specific antitoxin. We have identified a novel antitoxin family (paaA) that is associated with parE toxins. The paaA-parE gene pairs form an operon with a third component (paaR) encoding a transcriptional regulator. Two paralogous paaR-paaA-parE systems are found in E. coli O157:H7. Deletions of the paaAparE pairs in O157:H7 allowed us to show that these systems are expressed in their natural host and that PaaA antitoxins specifically counteract toxicity of their associated ParE toxin. For the paaR2-paaA2-parE2 system, PaaR2 and Paa2-ParE2 complex are able to regulate the operon expression and both are necessary to ensure complete repression. The paaR2-paaA2-parE2 system mediates ClpXP-dependent post-segregational killing. The PaaR2 regulator appears to be essential for this function, most likely by maintaining an appropriate antitoxin : toxin ratio in steady-state conditions. Ectopic overexpression of ParE2 is bactericidal and is not resuscitated by PaaA2 expression. ParE2 colocalizes with the nucleoid, while it is diffusely distributed in the cytoplasm when PaaA2 is coexpressed. This indicates that ParE2 interacts with DNA-gyrase cycling on DNA and that coexpression of PaaA2 antitoxin sequesters ParE2 away from its target by protein-protein complex formation.

The ParE2–PaaA2 toxin–antitoxin complex fromEscherichia coliO157 forms a heterodocecamer in solution and in the crystal

“…The biological function of this entity still remains speculative. In vivo, this toxinantitoxin complex has been shown to partially repress the expression of the paaR2-paaA2-parE2 operon and to enhance the repression activity of PaaR2 on its own (Hallez et al, 2010). This suggests that the ParE2-PaaA2 complex possesses some DNA-binding activity.…”

“…NP_288008 and NP_310308) was picked up out of E. coli O157:H7 EDL933 as described by Hallez et al (2010) and was introduced into the pET21b vector (Novagen) via the EcoRI and XhoI sites using the primers 5 0 -GAATTCAGGAGGGAGTAATGGATTATAAAGATG-ACGATGACAAAAATAGAGCCCTTTCACCA-3 0 (forward) and 5 0 -CGCAAGACGCCAGTTTCCCCTCGAG-3 0 (reverse). This provides PaaA2 with an N-terminal FLAG tag (DYKDDDDK) and ParE2 with a C-terminal His tag (LEHHHHHH) attached to their native sequences.…”

“…Recently, two three-component ParE-containing TA operons have been identified on the chromosome of E. coli O157 (Hallez et al, 2010). These operons contain a ParE homologue downstream of a transcription regulator of the DicA transcriptional repressor family (PaaR) and an antitoxin (PaaA) which is shorter than the classic ParD antitoxins and does not display any significant sequence identity to known ParD proteins.…”

“…These operons contain a ParE homologue downstream of a transcription regulator of the DicA transcriptional repressor family (PaaR) and an antitoxin (PaaA) which is shorter than the classic ParD antitoxins and does not display any significant sequence identity to known ParD proteins. Interestingly, regulation of the system requires all three components (Hallez et al, 2010). In this paper, we describe the overexpression, purification, preliminary characterization and crystallization of the ParE2-PaaA2 complex encoded by the paaR2-paaA2-parE2 operon on the chromosome of E. coli O157.…”

Regulatory crosstalk between type I and type II toxin-antitoxin systems in the human pathogen Enterococcus faecalis

“…Parental E. coli K12 strains were DJ624Dara, 60 MG1655D10, 61 and DH5a (Invitrogen), and E. faecalis strains were V583, and OG1RF. 44,62 Plasmid pCRII-TOPO (Invitrogen) was used for direct cloning of amplicons.…”