The symbiotic interaction between leguminous and Bradyrhizobium sp. SUTN9-2 mainly relies on the nodulation process through Nod factors (NFs), while the type IV secretion system (T4SS) acts as an alternative pathway in this symbiosis. Two copies of T4SS (T4SS₁ and T4SS₂) are located on the chromosome of SUTN9-2. ΔT4SS₁ reduces both nodule number and nitrogenase activity in all SUTN9-2 nodulating legumes. The functions of three selected genes (copG₁, traG₁, and virD2₁) within the region of T4SS₁ were examined. We generated deleted mutants and tested them in Vigna radiata cv. SUT4. ΔtraG₁ and ΔvirD2₁ exhibited lower invasion efficiency at the early stages of root infection but could be recently restored. In contrast, ΔcopG₁ completely hindered nodule organogenesis and nitrogenase activity in all tested legumes. ΔcopG₁ showed low expression of the nodulation gene and ttsI but exhibited high expression levels of the T4SS genes, traG₁ and trbE₁. The secreted proteins from ΔT4SS₁ were down-regulated compared to the wild-type. Although ΔcopG₁ secreted several proteins after flavonoid induction, T3SS (nopP and nopX) and the C₄-dicarboxylate transporter (dct) were not detected. These results confirm the crucial role of the copG₁ gene as a novel key regulator in the symbiotic relationship between SUTN9-2 and legumes.

In this study, we investigate two RpoN homologs in Bradyrhizobium sp. DOA9—chromosomal RpoNc and megaplasmid-borne RpoNp—and their roles in free-living conditions and nitrogen fixation. Phylogenetic analysis showed that RpoNc clusters with RpoN proteins from symbiotic nitrogen-fixing strains, whereas RpoNp forms a distinct clade, consistent with a function in stress responses. RpoNc proved essential for free-living conditions: ΔrpoNc mutants displayed severe growth defects that RpoNp could not compensate for. Transcriptomic comparisons between wild type and mutant RpoN identified 541 differentially expressed genes (DEGs) grouped into three clusters: 100 downregulated, 175 upregulated, and 254 moderately downregulated (with a fold change > 2, and a q-value (FDR, padj) < 0.05). Affected pathways involved nitrogen metabolism, motility, and environmental adaptation. RpoNc controlled major nitrogen fixation genes (nif and fix) along with core growth and stress response functions, while RpoNp mainly influenced stress-adaptation pathways. Genome-wide promoter motif analysis predicted 68 putative RpoNc targets, mainly associated with nitrogen fixation and metabolism, compared with only 22 predicted RpoNp targets, indicating a more restricted regulon. Electrophoretic mobility shift assays (EMSAs) further confirmed that both RpoN proteins directly bind σ⁵⁴-dependent promoters identified from transcriptomic data, supporting their regulatory roles under free-living conditions. Two mutants (ΔrpoNc and ΔrpoNp::ΩrpoNc) showed broad transcriptional disruption across nitrogen fixation, metabolism, and stress responses, underscoring complementary regulation. Overall, RpoNc is the dominant regulator of nitrogen fixation and core metabolism during free-living conditions, whereas RpoNp fine-tunes stress responses, revealing new regulatory insights for DOA9 adaptation. These results clarify how RpoN systems optimize survival across fluctuating conditions.

The symbiotic interaction between leguminous and Bradyrhizobium sp. SUTN9-2 mainly relies on the nodulation process through Nod factors (NFs), while the type IV secretion system (T4SS) acts as an alternative pathway in this symbiosis. Two copies of T4SS (T4SS1 and T4SS2) are located on the chromosome of SUTN9-2. ΔT4SS1 reduces both nodule number and nitrogenase activity in all SUTN9-2 nodulating legumes. The functions of three selected genes (copG1, traG1, and virD21) within the region of the T4SS1 were examined. We generated deleted mutants and tested in Vigna radiata cv. SUT4. ΔtraG1 and ΔvirD21 exhibited lower invasion efficiency at the early stages of root infection but could be lately restored. In contrast, ΔcopG1 completely hindered nodule organogenesis and nitrogenase activity in all tested legumes. ΔcopG1 showed low expression of nodulation gene and ttsI, but exhibited high expression levels of T4SS genes, traG1 and trbE1. The secreted proteins from ΔT4SS1 were down-regulated compared to the wild type. Although ΔcopG1 secreted several proteins after flavonoid induction, T3SS (nopP and nopX) and C4-dicarboxylate transporter (dct) were not detected. These results confirm the crucial role of the copG1 gene as a novel key regulator in the symbiotic relationship between SUTN9-2 and legumes.