Interactions of the Streptococcus pneumoniae Toxin-Antitoxin RelBE Proteins with Their Target DNA

Abstract

Type II bacterial toxin-antitoxin (TA) systems are found in most bacteria, archaea, and mobile genetic elements. TAs are usually found as a bi-cistronic operon composed of an unstable antitoxin and a stable toxin that targets crucial cellular functions like DNA supercoiling, cell-wall synthesis or mRNA translation. The type II RelBE system encoded by the pathogen Streptococcus pneumoniae is highly conserved among different strains and participates in biofilm formation and response to oxidative stress. Here, we have analyzed the participation of the RelB antitoxin and the RelB:RelE protein complex in the self-regulation of the pneumococcal relBE operon. RelB acted as a weak repressor, whereas RelE performed the role of a co-repressor. By DNA footprinting experiments, we show that the proteins bind to a region that encompasses two palindromic sequences that are located around the −10 sequences of the single promoter that directs the synthesis of the relBE mRNA. High-resolution footprinting assays showed the distribution of bases whose deoxyriboses are protected by the bound proteins, demonstrating that RelB and RelB:RelE contacted the DNA backbone on one face of the DNA helix and that these interactions extended beyond the palindromic sequences. Our findings suggest that the binding of the RelBE proteins to its DNA target would lead to direct inhibition of the binding of the host RNA polymerase to the relBE promoter.

Type II Toxin–Antitoxin Systems in Pseudomonas aeruginosa

“…In the pfiTA TA system, pfiT and pfiA are co-transcribed, and the PfiTA complex binds to the palindrome sequence (5′-AATTCN 5 GTTAA-3′) overlapping the −35 region of the pfiTA promoter, thereby repressing the expression of the pfiTA operon [ 28 ]. Interestingly, the relBE operon is regulated by both the antitoxin RelB and the TA complex RelBE; antitoxin RelB is a weak repressor, whereas RelE is an efficient co-repressor to further increase transcriptional regulation [ 30 , 31 ]. However, the transcriptional regulatory mechanisms of the pumAB , pacTA , parDE , and hicAB TA systems in P. aeruginosa are unclear.…”

Type II bacterial toxin–antitoxins: hypotheses, facts, and the newfound plethora of the PezAT system

“…Configurations other than heterohexamers have been detected for TA proteins, as in the case of the recently solved structures of the complex between antitoxin VPA0769 and toxin VPA0770 from Vibrio parahaemolyticus in which W-shaped heterohexamers and doughnut-shaped heterododecamers were observed depending upon protein concentration (Zhang et al 2023 ). In the above cases, the antitoxin wraps the toxin and occludes the amino acid residues of the active site of the toxin, and thus the lethal effect of the toxin is counteracted (Sterckx et al 2012 , 2016 , Xue et al 2020 , Moreno-Córdoba et al 2021 , Zhang et al 2023 ). Under stressful conditions (like temperature increases, lack of nutrients, presence of antibiotics, and so on), the antitoxins are selectively cleaved by intracellular ATPases associated with diverse cellular activities (AAA + ) proteases (Lon, ClpX), probably because the antitoxins appear to be partially unfolded and, consequently, more prone to degradation (Van Melderen et al 1996 , Loris et al 2003 , Cherny et al 2005 , Loris and Garcia-Pino 2014 , Chan et al 2016 , Kędzierska and Hayes 2016b , Page and Peti 2016 ).…”

“…The DNA binding sites of the pneumococcal TAs, as determined by DNase I and high-resolution (hydroxyl radical) footprinting experiments (Nieto et al 2007 , Chan et al 2011 , Moreno-Córdoba et al 2012 , 2021 ), showed the existence of one or two inverted repeats placed within (i) the −10 region and the initiation of the transcription region ( relBE ); ii) the −35 region (promoter 2 of yefM–yoeB , and pezAT promoter), and (iii) between both regions ( phd-doc ). Upstream and downstream of these TAs, IS elements, genes involved in the mobilization of MGEs, and integrases are located (Chan et al 2012 ).…”

“…Upstream and downstream of these TAs, IS elements, genes involved in the mobilization of MGEs, and integrases are located (Chan et al 2012 ). We have shown that relBE, pezAT , and phd-doc exhibit a single promoter directing the synthesis of a single mRNA (Nieto et al 2006 , Chan et al 2014 , Moreno-Córdoba et al 2021 ). A different organization was observed in the yefM–yoeB pair, in which transcription was demonstrated to be directed by two promoters (Chan et al 2011 ).…”