Modular evolution and regulatory diversification of nodD-like LysR-type transcriptional regulators in...

The nodD gene encodes a LysR-type transcriptional regulator critical for nodulation gene expression in rhizobia, yet its evolutionary origin, structural plasticity, and regulatory reach beyond symbiosis remain incompletely resolved. Here we investigate the genomic organization, structural variation, and functional diversification of nodD and its homologs across α-proteobacteria with selected outgroups. Using orthogroup-based pangenome clustering, dated species trees, and gene-tree-species-tree reconciliation, we reconstruct the evolutionary trajectory of nodD, indicating emergence from ancient LTTRs deep in proteobacterial history. Reconciliation reveals widespread duplication and horizontal gene transfer (HGT), with several rhizobia showing notable duplication and exchange, and marine/non-rhizobial taxa contributing to a mosaic of nodD-like genes. Gene-neighborhood and operon analyses show conserved syntenic tendencies in classical rhizobia but extensive architectural divergence in free-living lineages, including frequent monocistronic anchors with extended upstream regions and, when polycistronic, enrichment for transporters and local metabolic enzymes within compact multi-regulator cassettes. Structural comparisons with AlphaFold and PyMOL confirm the canonical LTTR fold while uncovering species-specific deviations concentrated in effector-binding loops and interfaces. Motif discovery and genome-wide scanning identify targets involved in metabolism, stress responses, and transcriptional control, and network analysis reveals modular connectivity spanning core metabolism and accessory processes such as secondary metabolism, transport, and biofilm-associated functions. These findings portray nodD as a structurally conserved yet functionally flexible regulator repeatedly reshaped by duplication, HGT, and local genome context, extending nodD-like systems beyond symbiosis and broadening the regulatory landscape of bacterial LTTRs.