Autoregulator Protein PhaR for Biosynthesis of Polyhydroxybutyrate [P(3HB)] Possibly Has Two Separate Domains That Bind to the Target DNA and P(3HB): Functional Mapping of Amino Acid Residues Responsible for DNA Binding

Abstract

PhaR fromcoli were evaluated using a gel shift assay and a surface plasmon resonance analysis. These experiments revealed that basic amino acids and a tyrosine in the N-terminal region, which is highly conserved among PhaR homologs, are responsible for DNA binding. However, most of the mutants with decreased DNA-binding abilities were unaffected in their ability to bind P(3HB), strongly suggesting that PhaR has two separate domains capable of binding to the target DNA and P(3HB).

Morphological Change in Cellular Granule Formation of Poly[(R)-3-hydroxybutyrate] Caused by DNA-Binding-Related Mutations of an Autoregulated Repressor PhaR

“…In conclusion, the feasibility of our regulation model of P(3HB) biosynthesis was confirmed by in vivo anlysis as well as the previous in vitro analyses. 8,9) At the next stage, an in vitro system such as the QCM system can be reconstructed for quantitative analysis of intermolecular interactions by mimicking the in vivo system consisting of four molecules (PhaR, the target DNA, P(3HB), and PhaP).…”

“…[5][6][7] Most recently, the proposed regulation model has been further supported by direct evidence that PhaR has two separate domains binding to the target DNA (including the phaP promoter region) and P(3HB), as was discovered using a quartz crystal microbalance (QCM). 8,9) In addition, amino acid residues responsible for DNA binding of PhaR were addressed by deletion and polymerase chain reactionmediated random point mutation experiments coupled with a green fluorescent protein reporter-based monitoring assay system. 9) In this study, the reduced effect of the DNA-binding ability of PhaR on phaP expression was analyzed by microscopic observation of differences in the morphogenesis of P(3HB) granules caused by changes in granule-packing intensity associated with the number of PhaP molecules present.…”

“…8,9) In addition, amino acid residues responsible for DNA binding of PhaR were addressed by deletion and polymerase chain reactionmediated random point mutation experiments coupled with a green fluorescent protein reporter-based monitoring assay system. 9) In this study, the reduced effect of the DNA-binding ability of PhaR on phaP expression was analyzed by microscopic observation of differences in the morphogenesis of P(3HB) granules caused by changes in granule-packing intensity associated with the number of PhaP molecules present. The dissociation constant for wild type PhaR to the target DNA was calculated to be 1:82 Â 10 À7 M by a previous surface plasmon resonance analysis.…”

“…9) Recombinants of Escherichia coli DH5 were prepared for microscopic study by the introduction of two plasmid vector constructs, pBBRKmAB 5) (to supply the 3HB-CoA monomer) and pTVCPR (for regulatory synthesis of P(3HB)), as shown in Fig. 1B.…”

EliA is required for inducing the stearyl alcohol-mediated expression of secretory proteins and production of polyester in Ralstonia sp. NT80

“…(Tanadchangsaeng et al, 2009;Lopes et al, 2011;Raberg et al, 2011). This polymer is formed in the presence of excess supply of carbon and/or in the absence of one or more essential nutritional elements, and it can account for as much as 85 % of the cell dry weight under optimized growth conditions (Yamada et al, 2007;Jendrossek, 2009;Beeby et al, 2012). The synthesized PHA is reutilized when carbon and energy sources are depleted from the surrounding environment (Jendrossek & Handrick, 2002).…”