Regulation and symbiotic significance of nodulation outer proteins secretion in Sinorhizobium fredii HH103

Departamento de Microbiología, Facultad de Biología, Universidad de Sevilla, Sevilla, Spain Correspondence Francisco Javier Ollero fjom{at}us.es In this work we show that the Sinorhizobium fredii HH103 ttsI gene is essential for the expression of the tts genes and secretion of nodulation outer proteins (Nops). Moreover, we demonstrate for the first time, to our knowledge, that the nod box preceding ttsI is necessary for Nops secretion. TtsI is responsible for the transcriptional activation of nopX , nopA , rhcJ and rhcQ . We confirm that the S. fredii HH103 ttsI gene is activated by NodD1 and repressed by NolR. In contrast, NodD2 is not involved in the regulation of ttsI expression. Despite the dependence of expression of both ttsI and nodA on NodD1 and flavonoids, clear differences in the capacity of some flavonoids to activate these genes were found. The expression of the ttsI and nodA genes was also sensitive to differences in the pH of the media. Secretion of Nops in the ttsI mutant could not be complemented with a DNA fragment containing the ttsI gene and its nod box, but it was restored when a plasmid harbouring the ttsI , rhcC2 and y4xK genes was transferred to the mutant strain. The symbiotic effect of Nops secretion was host-dependent but independent of the type of nodule formed by the host legume. Nops are beneficial in the symbiosis with Glycine max and Glycyrrhiza uralensis , and detrimental in the case of the tropical legume Erythrina variegata . Abbreviations: , Statistical significance of the differences observed using the Mann–Whitney non-parametric test; LCO, lipochitin oligosaccharide; Nops, nodulation outer proteins; T3SS, type III secretion system The GenBank/EMBL/DDBJ accession number for the ttsI sequence of Sinorhizobium fredii HH103 is AY184383.