Abstract
Bacterial cultures, especially biofilms, produce a small number of persister cells, a genetically identical subpopulation of wild type cells that are metabolically dormant, exhibit multidrug tolerance, and are highly enriched in bacterial toxins. The gene most highly up-regulated in Escherichia coli persisters is mqsR, a ribonuclease toxin that, along with mqsA, forms a novel toxin·antitoxin (TA) system. Like all known TA systems, both the MqsR·MqsA complex and MqsA alone regulate their own transcription. Despite the importance of TA systems in persistence and biofilms, very little is known about how TA modules, and antitoxins in particular, bind and recognize DNA at a molecular level. Here, we report the crystal structure of MqsA bound to a 26-bp fragment from the mqsRA promoter. We show that MqsA binds DNA predominantly via its C-terminal helix-turn-helix domain, with direct binding of recognition helix residues Asn(97) and Arg(101) to the DNA major groove. Unexpectedly, the structure also revealed that the MqsA N-terminal domain interacts with the DNA phosphate backbone. This results in a more than 105° rotation of the N-terminal domains between the free and complexed states, an unprecedented rearrangement for an antitoxin. The structure also shows that MqsA bends the DNA by more than 55° in order to achieve symmetrical binding. Finally, using a combination of biochemical and NMR studies, we show that the DNA sequence specificity of MqsA is mediated by direct readout.
Highlights
Biofilms, complex communities of bacteria that are encased in an extracellular matrix and adhere to almost any surface, are responsible for more than 65– 80% of human infections
14 antitoxin structures have been solved to date, their primary sequences are highly divergent when compared with MqsA because the sequence identity between MqsA and the most closely related antitoxin, the E. coli protein HigA, is only 13%
We recently showed that MqsA is a dimer, with the MqsR1⁄7MqsA complex forming a heterotetramer of composition MqsR1⁄7MqsA21⁄7MqsR
Summary
Complex communities of bacteria that are encased in an extracellular matrix and adhere to almost any surface, are responsible for more than 65– 80% of human infections TA complexes regulate their own transcription, where the antitoxins bind to the promoter DNAs within their own operons and the cognate toxins enhance DNA binding (28 –31) Structure of the MqsA1⁄7PmqsRA DNA Complex believed that multiple factors play important roles in TA expression, stability, and transcription regulation, a detailed molecular picture of the structural transitions that are required for DNA binding and recognition by TA pairs, and antitoxins in particular, is largely missing. We determined the crystal structures of MqsA alone and the MqsR1⁄7MqsA complex and showed that the mqsRA operon defines a novel family of TA systems in E. coli, in which MqsR is the toxin and MqsA is the antitoxin (19). 14 antitoxin structures have been solved to date, their primary sequences are highly divergent when compared with MqsA (supplemental Table S1) because the sequence identity between MqsA and the most closely related antitoxin, the E. coli protein HigA, is only 13%
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