TtsI regulates symbiotic genes in Rhizobium species NGR234 by binding to tts boxes

Abstract

SummaryInfection of legumes by Rhizobium 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 cis elements termed tts boxes. Eleven tts boxes were identified in the promoter regions of target genes on the symbiotic plasmid of NGR234. Expression profiles of lacZ fusions to these tts 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 tts boxes. DNase I footprinting revealed that TtsI occupied not only the tts box consensus sequence, but also upstream and downstream regions in a concentration-dependent manner. Highly conserved bases of the consensus tts box were mutated and, although TtsI binding was still observed in vitro, gfp fusions were no longer transcribed in vivo. Random mutagenesis of a tts box-containing promoter revealed more nucleotides critical for transcriptional activity outside of the consensus.

Hijacking of leguminous nodulation signaling by the rhizobial type III secretion system

“…This suggests that concerted expression, rather than simple copossession, of T3SS and nodulation genes is required for the formation of effective nodules. Such a requirement might be why most known rhizobial T3SS genes are situated downstream of flavonoid-responsive signaling (6,7). Host-derived flavonoid induction is found in most T3SS-harboring rhizobia, except for the β-rhizobium Cupriavidus taiwanensis, which incorporates a T3SS similar to that of the β-proteobacterium Burkholderia, and its expression is more likely to be glutamatedependent.…”

“…Flavonoids induce NodD to activate the expression of ttsI, a transcriptional activator of the type III secretion gene cluster (tts). TtsI subsequently activates the tts gene cluster, and proteins are secreted via T3SS (6,7). This cascade confines the expression of rhizobial T3SS during host-plant infection.…”

Roles of flavonoids and the transcriptional regulator TtsI in the activation of the type III secretion system of Bradyrhizobium elkanii SEMIA587

“…NodD1 activates the expression of the transcriptional activator TtsI by binding to the nod box in the promoter region of its gene (ttsI). A nod box element has been identified in the promoters of all ttsI genes studied (Viprey et al, 1998;Krause et al, 2002;Marie et al, 2003Marie et al, , 2004Ló pez-Baena et al, 2008;Wassem et al, 2008). No nod box sequences are found in the promoter regions of other genes encoding T3SSs; instead, a different cis element has been identified and named the tts box by Krause et al (2002).…”

“…No nod box sequences are found in the promoter regions of other genes encoding T3SSs; instead, a different cis element has been identified and named the tts box by Krause et al (2002). TtsI activates the transcription of other T3SS-related genes by binding to the consensus tts box sequence nnntcGTCAGcttntcGaaAGctagnccnntannnnn (Krause et al, 2002;Marie et al, 2004;Ló pez-Baena et al, 2008;Wassem et al, 2008;Zehner et al, 2008). Copies of this consensus sequence have been found in the promoter regions of the T3SS genes of NGR234 (Marie et al, 2004;Wassem et al, 2008), Bradyrhizobium japonicum USDA110 (Krause et al, 2002;Zehner et al, 2008), Sinorhizobium fredii HH103 and S. fredii USDA257 (Ló pez- Baena et al, 2008;Krause et al, 2002).…”

“…TtsI has homology to the DNA-binding response regulators of two-component regulatory systems (Viprey et al, 1998;Marie et al, 2004;Wassem et al, 2008). Such regulators are usually activated by their cognate sensor kinase, which transfers a phosphoryl group to an aspartate residue in the regulator, leading to a conformational change that activates the latter protein.…”