Regulatory divergence and functional diversification of a c-di-GMP-controlled sigma factor in Actinomycetota

Members of the σ²⁸ family of alternative σ factors typically regulate genes involved in flagellar biosynthesis. However, the only member of the σ²⁸ family present in the actinobacterial genus Streptomyces, WhiG, controls the differentiation of aerial hyphae into spores. WhiG activity is regulated by the second messenger c-di-GMP, which arms its cognate anti-σ, RsiG, to bind and sequester the σ factor. Understanding WhiG evolution can thus shed light on the diversity of processes regulated by c-di-GMP across Phylum Actinomycetota. Members of Actinomycetota comprise highly diverse filamentous and unicellular, flagellated and non-flagellated species, and the actinobacterial ancestor is predicted to have been motile. Here, our systematic analysis reveals that WhiG homologs are broadly distributed throughout the Actinomycetota and form two distinct clades: WhiG1, whose members have retained the ancestral association with the flagellar biosynthesis cluster and are not regulated by an RsiG anti-σ, and WhiG2, whose members are typically regulated by RsiG via c-di-GMP. Bioinformatic analysis of their target regulons suggests that this σ factor has significantly diversified in function to control various processes, including motility, chemotaxis, Type IV pili synthesis, sporulation, antibiotic biosynthesis, and c-di-GMP metabolism across diverse actinobacterial species. These findings highlight a phylogenetic split in the regulation and function of this key transcription factor throughout the phylum.