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Background: Rhizobium giardinii has been demonstrated to colonize the roots of a variety of legume species, including common beans, and to increase nitrogen fixation. This suggests that Rhizobium giardinii might be a beneficial tool for sustainable agriculture by lowering dependency on synthetic nitrogen fertilizers and enhancing soil fertility. Understanding the regulatory components in the R. giardinii A3AY_RS01 genes might also lead to the creation of innovative ways for increasing the effectiveness of nitrogen fixation in other agriculturally important bacteria. Therefore, this study was aimed to predict regulatory element of R. giardinii DNA-binding response regulator A3AY_RS01 genes., Results: The locations for 19 % of the Transcriptional start site (TSSs) were within -300 bp relative to the start codon and ten candidate motifs were identified that are shared by at least 50 % of the R. giardinii A3AY_RS01 promoter input sequences from both strands. Motif 1 was revealed as the common promoter motif for all of R. giardinii A3AY_RS01 genes that serves as binding sites for TFs involved in the expression regulation of these genes. Hence, it was revealed that Motif 1 may serve as the binding site chiefly for Ferric uptake regulator (Fur) transcription factor family to regulate expression of A3AY_RS01 genes. High CpG density in the promoter than body regions were observed for most of the genes except for A3AY_RS0102950, A3AY_RS0120195 and A3AY_RS0131150 genes. Nonetheless, promoter areas were richer than body regions in both techniques., Conclusions: MV1 motif can serve as a binding site for the Fur transcription factor gene family in R. giardinii to regulate the expression of R. giardinii A3AY_RS01 genes. R. giardinii A3AY_RS01 genes are rich in CpG Islands, and play an important role in the regulation of the gene expression of nitrogen fixing in this bacterium., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)