Diverse flavonoids stimulate NodD1 binding to nod gene promoters in Sinorhizobium meliloti

Abstract

NodD1 is a member of the NodD family of LysR-type transcriptional regulators that mediates the expression of nodulation (nod) genes in the soil bacterium Sinorhizobium meliloti. Each species of rhizobia establishes a symbiosis with a limited set of leguminous plants. This host specificity results in part from a NodD-dependent upregulation of nod genes in response to a cocktail of flavonoids in the host plant's root exudates. To demonstrate that NodD is a key determinant of host specificity, we expressed nodD genes from different species of rhizobia in a strain of S. meliloti lacking endogenous NodD activity. We observed that nod gene expression was initiated in response to distinct sets of flavonoid inducers depending on the source of NodD. To better understand the effects of flavonoids on NodD, we assayed the DNA binding activity of S. meliloti NodD1 treated with the flavonoid inducer luteolin. In the presence of luteolin, NodD1 exhibited increased binding to nod gene promoters compared to binding in the absence of luteolin. Surprisingly, although they do not stimulate nod gene expression in S. meliloti, the flavonoids naringenin, eriodictyol, and daidzein also stimulated an increase in the DNA binding affinity of NodD1 to nod gene promoters. In vivo competition assays demonstrate that noninducing flavonoids act as competitive inhibitors of luteolin, suggesting that both inducing and noninducing flavonoids are able to directly bind to NodD1 and mediate conformational changes at nod gene promoters but that only luteolin is capable of promoting the downstream changes necessary for nod gene induction.

FIG. 1.

Genetic evidence that NodD is the flavonoid sensor. nodC′-′lacZ reporter activity was assayed in S. meliloti A2105 expressing the indicated nodD. Cultures were grown in the absence or presence of 3 μM flavonoid in DMF. (A) Vector control (pRF771). (B) S. meliloti NodD1. (C) R. leguminosarum bv. viciae NodD. (D) R. leguminosarum bv. trifolii NodD. Data are an average of at least eight independent assays and are plotted as means ± standard deviations.

FIG. 2.

Luteolin stimulates the DNA binding activity of NodD1. Increasing amounts of affinity-purified NodD1 containing GroEL isolated from cells grown in the absence (open boxes) or presence (shaded boxes) of 3 μM luteolin were incubated with labeled nodF nod box DNA as described in Materials and Methods. Data are presented as a box plot (see Materials and Methods). Data represent ≥10 independent protein purifications and ≥14 independent DNA binding reactions for all samples except 5.9 μM NodD1+L (n = 5 independent protein purifications and 5 independent DNA binding reactions). A P value of <0.02 was found at each protein concentration tested.

FIG. 3.

Luteolin stimulates the DNA binding activity of SEC-HPLC-purified NodD1. Increasing amounts of SEC-HPLC-purified NodD1 containing GroEL were incubated with GroES and ATP for 10 min prior to the addition of DMF (−, □) or 10 μM luteolin (+, ▪). The samples were then incubated with labeled nodF nod box DNA and analyzed as described in Materials and Methods. n ≥ 8 for each concentration tested. (A) Representative experiment of NodD1 with or without luteolin binding to the nodF nod box. Arrows indicate the NodD1-DNA complex (bound DNA) and free DNA. (B) Quantitation of NodD1 binding to the nodF nod box from the experiment shown in panel A.

FIG. 4.

Luteolin does not significantly alter the footprint of affinity-purified NodD1 at the nodF nod box. (A) Free DNA fragments and NodD1-nod box complexes were subjected to DNase I footprinting within the polyacrylamide gel slice as previously described (16). G+A and G chemical sequencing reactions are labeled. The position of the nod box is indicated schematically to the left of each panel. The lines to the right of each panel indicate segments protected from cleavage; a • indicates hypersensitive cleavage sites. The left panel of each pair shows the results when the top strand shown in panel B was labeled, and the right panel of each pair shows the same when the bottom strand was labeled. − is the product of DNase I cleavage of the free DNA fragment, and + is the product of cleavage of the NodD1-nod box shifted complex. (B) Summary of footprinting reactions shown in panel A in the absence (-L) and presence (+L) of luteolin. The consensus nod box sequence is boxed. The thick lines above and below the sequence indicate segments protected from cleavage, and a • indicates hypersensitive cleavage sites. An asterisk indicates changes in the footprint in the presence versus absence of luteolin.

FIG. 5.

Noninducing flavonoids stimulate the DNA binding activity of NodD1. (A) Structures of flavonoids used in these assays. (B and C) Increasing concentrations of affinity-purified NodD1 isolated from cells that had been grown in the absence (open boxes) or presence (shaded boxes) of 3 μM daidzein (B) or eriodictyol (C) were incubated with labeled nodF nod box and analyzed as described in Materials and Methods. Data are presented as a box plot (see Materials and Methods). Each point represents at least four independent experiments. A P value of <0.05 was found at each protein concentration except at 1.5 and 2.2 μM eriodictyol, where P was <0.10.

FIG. 6.

Noninducing flavonoids stimulate the DNA binding activity of SEC-HPLC-purified NodD1. SEC-HPLC-purified NodD1 containing GroEL was preincubated with GroES and ATP for 10 min prior to the addition of DMF or 10 μM flavonoid. The sample was then mixed with labeled nodF nod box DNA (1.3 fmol) as described in Materials and Methods. (A) Representative gel shift analysis of 0.44 μM SEC-HPLC-purified NodD1 incubated with DMF, daidzein, luteolin, or naringenin. Arrows indicate the NodD1-DNA complex (bound DNA) and free DNA. (B) Increasing amounts of SEC-HPLC-purified NodD1 were incubated with DMF (□), luteolin (▪), eriodictyol (▴), or naringenin (•) and assayed for nodF nod box binding. A representative assay is shown.