Transcriptional activation of the Azotobacter vinelandii polyhydroxybutyrate biosynthetic genes phbBAC by PhbR and RpoS

Abstract

We previously showed that inAzotobacter vinelandii, accumulation of polyhydroxybutyrate (PHB) occurs mainly during the stationary phase, and that a mutation inphbR, encoding a transcriptional regulator of the AraC family, reduces PHB accumulation. In this study, we characterized the roles of PhbR and RpoS, a central regulator during stationary phase in bacteria, in the regulation of expression of the PHB biosynthetic operonphbBACandphbR.We showed that inactivation ofrpoSreduced PHB accumulation, similar to thephbRmutation, and inactivation of bothrpoSandphbRresulted in an inability to produce PHB. We carried out expression studies with the wild-type, and therpoS,phbRand doublerpoS-phbRmutant strains, using quantitative RT-PCR, as well asphbB : : gusAandphbR : : gusAgene fusions. These studies showed that both PhbR and RpoS act as activators ofphbBandphbR, and revealed a role for PhbR as an autoactivator. We also demonstrated that PhbR binds specifically to two almost identical 18 bp sites, TGTCACCAA-N4-CACTA and TGTCACCAA-N4-CAGTA, present in thephbBpromoter region. The activation ofphbBandphbRtranscription by RpoS reported here is in agreement with the observation that accumulation of PHB inA. vinelandiioccurs mainly during the stationary phase.