Identification and characterization of PhbF: A DNA binding protein with regulatory role in the PHB metabolism of Herbaspirillum seropedicae SmR1

Abstract

BackgroundHerbaspirillum seropedicae SmR1 is a nitrogen fixing endophyte associated with important agricultural crops. It produces polyhydroxybutyrate (PHB) which is stored intracellularly as granules. However, PHB metabolism and regulatory control is not yet well studied in this organism.ResultsIn this work we describe the characterization of the PhbF protein from H. seropedicae SmR1 which was purified and characterized after expression in E. coli. The purified PhbF protein was able to bind to eleven putative promoters of genes involved in PHB metabolism in H. seropedicae SmR1. In silico analyses indicated a probable DNA-binding sequence which was shown to be protected in DNA footprinting assays using purified PhbF. Analyses using lacZ fusions showed that PhbF can act as a repressor protein controlling the expression of PHB metabolism-related genes.ConclusionsOur results indicate that H. seropedicae SmR1 PhbF regulates expression of phb-related genes by acting as a transcriptional repressor. The knowledge of the PHB metabolism of this plant-associated bacterium may contribute to the understanding of the plant-colonizing process and the organism's resistance and survival in planta.

Identification of Proteins Associated with Polyhydroxybutyrate Granules from Herbaspirillum seropedicae SmR1 - Old Partners, New Players

“…The ability of the regulatory H. seropedicae PhbF protein to bind PHB granule has been shown previously using a recombinant expressed PhbF protein [11], and our results now confirm the ability of this transcriptional repressor protein in binding PHB granule in vivo . Since PhbF binds to the promoter region of phb -related genes and also to PHB granules, the regulation of phb genes expression most likely involves disruption of PhbF-DNA and formation of PhbF-granule complexes as previously proposed [8].…”

“…These proteins do not show any significant homology to known domains, with exception of a very low homology (e-value of 0.26) to the eukaryotic MACPF protein (membrane attack complex perforins). Analysis of the promoter region of Hsero_3471 indicates a potential PhbF-binding site [11] located at −190 position from the start codon, suggesting that its expression is regulated by PHB metabolism.…”

“…Herbaspirillum seropedicae is a diazotrophic β-Proteobacterium, which associates with graminae [9] and produces PHB [10], [11]. In H. seropedicae , thirteen genes potentially involved in PHB metabolism were identified [11], [12], including two phbC, phaZ and phaP genes with codes for synthases, depolymerases and phasins, respectively, and the phbF gene which encodes the regulatory protein [11].…”

“…In H. seropedicae , thirteen genes potentially involved in PHB metabolism were identified [11], [12], including two phbC, phaZ and phaP genes with codes for synthases, depolymerases and phasins, respectively, and the phbF gene which encodes the regulatory protein [11]. It has been suggested that PHB has an important role in nitrogen fixation and in plant-bacteria interaction [3], [13], however the importance of PHB production for H. seropedicae was not assessed.…”

Molecular adaptations of Herbaspirillum seropedicae during colonization of the maize rhizosphere

“…Herbaspirillum seropedicae expresses the phbABC genes and produces PHB during growth in log phase when cultured in NFbHPN medium, and stores it intracellularly as insoluble inclusion bodies called PHB granules (Kadowaki et al, 2011;Catalan et al, 2007). In both planktonic and epiphytic bacteria colonizing the maize rhizosphere, the expression of these PHB biosynthesis genes was three to ninefold lower 1 d.a.i.…”