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

Abstract

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.

Structural basis for regulation of rhizobial nodulation and symbiosis gene expression by the regulatory protein NolR

“…The shift in position of Gln56 in each half-site of the oligo AT DNA, either to interact with A2 in the first site or away from T7' in the second site, suggested that movement of this residue may play a role in the previously observed recognition by NolR of variable operator sites (17)(18)(19)(20)(21)(22)(23)(24). To examine the possible role of Gln56 as a conformational switch in DNA binding, NolR was crystallized with oligo AA DNA (Fig.…”

“…1-3) more accurately define the interaction sequences in each asymmetric half site as ATTAG on the 5′ strand and CTTC on the 3′ strand. A remarkable feature of NolR is that the homodimer binds to an asymmetric operator site with variable sequences at two positions, one in each half site, which allows versatility in recognition of NolR operator sites of multiple target genes (19,20,(22)(23)(24).…”

“…Control of Nod factor production by NolR may also aid in optimization of nodulation specificity, as NolR binding sequences were later found in the promoter regions of nodABC, nodD1, ttsI-nodD2, nolR, hesB, and nodZ (23). Transcript levels of nolR are high in freeliving rhizobia and in the bacteroid but are down-regulated by luteolin, a nod gene inducer (24). Subsequent studies implicated NolR as a global regulatory factor that responds to environmental factors to fine-tune a range of symbiotic signals, not just the genes required for nodulation, and that the absence or presence of NolR affects symbiotic interactions with host plants (19,20,23,24).…”

“…Subsequent studies implicated NolR as a global regulatory factor that responds to environmental factors to fine-tune a range of symbiotic signals, not just the genes required for nodulation, and that the absence or presence of NolR affects symbiotic interactions with host plants (19,20,23,24). For example, NolR represses expression of the type III secretion system ttsI gene, which is required for secretion of nodulation outer proteins (nops) that are beneficial for symbiosis of Sinorhizobium fredii with some soybean cultivars (24). The molecular basis for NolR recognition of nonpalindromic DNA target sites and subsequent control of nodulation and symbiosis genes is unclear.…”

“…In rhizobia, NolR modulates expression of the NodD activator protein, the core nod genes, and multiple genes involved in symbiosis (17,(19)(20)(21)(22)(23)(24). Based on amino acid sequence homology, NolR was proposed to be a helix-turn-helix family member that binds to a nonpalindromic consensus motif -(A/T)TTAG-N 9 -A(T/A) (17).…”