The RssB/RssA two-component system regulates biosynthesis of the tripyrrole antibiotic, prodigiosin, in Serratia marcescens

Serratia marcescens CH-1 produces a red, cell-associated pigment, prodigiosin, synthesized by enzymes encoded in the pig operon. The underlying regulatory mechanism, especially its relationship with the RssAB two-component system signaling, remained uncharacterized. Here, we show that phosphorylated RssB (RssB-P) directly binds to the promoter region of the pig operon (pigA promoter), as observed using an electrophoretic mobility shift assay. Furthermore, we identify the RssB-P binding site located downstream of the -10 and -35 regions in pigA using a DNase I footprinting assay. A compilation of the RssB-P binding sites in flhDC, rssB and pigA promoter regions reveals the presence of a conserved core sequence, GAGATTTTAGCTAAATTAATBTTT (B=C, G, or T), which we believe is the RssB binding sequence. Site-specific mutation of conserved nucleotides within the conserved RssB binding sequence in the pigA promoter region leads to absence of retardation in the presence of RssB-P in vitro and elevated transcription of pigA in vivo. These data suggest that RssAB signaling negatively regulates prodigiosin production, and such inhibition is mediated through direct and specific repression of transcriptional activity of the pig operon.