Direct proteolytic control of an extracytoplasmic function RNA polymerase sigma factor

Streptomyces species are well known for harbouring a large number of extracytoplasmic function (ECF) RNA polymerase sigma (σ) factors; nearly all of regulated genes required for morphological differentiation and/or response to environmental stress. The activity of ECF σ factors is typically modulated by a cognate anti-σ factor protein co-encoded at the same locus. In previous work, we identified σAntA as a cluster-situated regulator of starter unit biosynthesis in the production of antimycin, an anticancer compound. Unlike a canonical ECF σ factor, whose activity is controlled by a cognate anti-σ factor, σAntA is an orphan, which raises intriguing questions about how its activity is controlled. Inspection of the σAntA amino acid sequence revealed a carboxyterminal di-alanine, which is the only motif of the Clp-protease recognition signal obligately required for proteolysis. Here, we show by immunoblotting that the abundance of 3xFLAG-σAntA in vivo is enhanced by alteration of the C-terminal Ala-Ala to Asp-Asp and that abundance of both variants is elevated in the absence of genes encoding the Clp-protease and its regulatory subunits ClpX and ClpA. We also show that (His)6-SUMO-σAntA, but not a variant lacking the C-terminal di-alanine motif, is degraded by the ClpXP protease in vitro. These data unambiguously establish direct proteolysis as an alternative control strategy for ECF RNA polymerase σ factors and expands the paradigmatic understanding microbial signal transduction regulation.