TtsI regulates symbiotic genes in <i>Rhizobium</i> species NGR234 by binding to <i>tts</i> boxes

<jats:title>Summary</jats:title><jats:p>Infection of legumes by <jats:italic>Rhizobium</jats:italic> sp. NGR234 and subsequent development of nitrogen‐fixing nodules are dependent on the coordinated actions of Nod factors, proteins secreted by a type III secretion system (T3SS) and modifications to surface polysaccharides. The production of these signal molecules is dependent on plant flavonoids which trigger a regulatory cascade controlled by the transcriptional activators NodD1, NodD2, SyrM2 and TtsI. TtsI is known to control the genes responsible for T3SS function and synthesis of a symbiotically important rhamnose‐rich lipo‐polysaccharide, most probably by binding to <jats:italic>cis</jats:italic> elements termed <jats:italic>tts</jats:italic> boxes. Eleven <jats:italic>tts</jats:italic> boxes were identified in the promoter regions of target genes on the symbiotic plasmid of NGR234. Expression profiles of <jats:italic>lacZ</jats:italic> fusions to these <jats:italic>tts</jats:italic> boxes showed that they are part of a TtsI‐dependent regulon induced by plant‐derived flavonoids. TtsI was purified and demonstrated to bind directly to two of these <jats:italic>tts</jats:italic> boxes. DNase I footprinting revealed that TtsI occupied not only the <jats:italic>tts</jats:italic> box consensus sequence, but also upstream and downstream regions in a concentration‐dependent manner. Highly conserved bases of the consensus <jats:italic>tts</jats:italic> box were mutated and, although TtsI binding was still observed <jats:italic>in vitro</jats:italic>, <jats:italic>gfp</jats:italic> fusions were no longer transcribed <jats:italic>in vivo</jats:italic>. Random mutagenesis of a <jats:italic>tts</jats:italic> box‐containing promoter revealed more nucleotides critical for transcriptional activity outside of the consensus.</jats:p>