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 the 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 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. These findings highlight a phylogenetic split in the regulation of this key transcription factor throughout the phylum. Bioinformatic analysis of WhiG 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.

Mounting global responses to dynamic environmental conditions is a crucial function that bacterial cells must perform. Global regulatory networks are most often studied in individual species, however, understanding how regulatory networks have evolved in distinct bacterial lineages remains an outstanding question. The alternative sigma factor WhiG, which is found in the actinobacterial genus Streptomyces, is a dedicated sporulation sigma, yet has long been known to have a close evolutionary relationship to sigma factors that are responsible for regulation of flagellar biosynthesis. Analysis of the distribution of WhiG in the phylum Actinomycetota reveals that homologs typically co-occur with either a flagellar cluster or the c-di-GMP-binding anti-sigma RsiG. Functional predictions of WhiG target genes further reveal that these transcription factors have been co-opted during the evolution of the Actinomycetota to control diverse biological processes.