A Novel Module Promotes Horizontal Gene Transfer in Azorhizobium caulinodans ORS571

Azorhizobium caulinodans ORS571 contains an 87.6 kb integrative and conjugative element (ICE(Ac)) that conjugatively transfers symbiosis genes to other rhizobia. Many hypothetical redundant gene fragments (rgfs) are abundant in ICE(Ac), but their potential function in horizontal gene transfer (HGT) is unknown. Molecular biological methods were employed to delete hypothetical rgfs, expecting to acquire a minimal ICE(Ac) and consider non-functional rgfs as editable regions for inserting genes related to new symbiotic functions. We determined the significance of rgf4 in HGT and identified the physiological function of genes designated rihF1a (AZC_3879), rihF1b (AZC_RS26200), and rihR (AZC_3881). In-frame deletion and complementation assays revealed that rihF1a and rihF1b work as a unit (rihF1) that positively affects HGT frequency. The EMSA assay and lacZ-based reporter system showed that the XRE-family protein RihR is not a regulator of rihF1 but promotes the expression of the integrase (intC) that has been reported to be upregulated by the LysR-family protein, AhaR, through sensing host’s flavonoid. Overall, a conservative module containing rihF1 and rihR was characterized, eliminating the size of ICE(Ac) by 18.5%. We propose the feasibility of constructing a minimal ICE(Ac) element to facilitate the exchange of new genetic components essential for symbiosis or other metabolic functions between soil bacteria.