Research Article: Monitoring and kinetic analysis of the molecular interactions by which a repressor protein, PhaR, binds to target DNAs and poly[(R)-3-hydroxybutyrate]

Date Published: January 27, 2013

Publisher: Springer

Author(s): Miwa Yamada, Shuntaro Takahashi, Yoshio Okahata, Yoshiharu Doi, Keiji Numata.


The repressor protein PhaR, which is a component of poly[(R)-3-hydroxybutyrate] granules, functions as a repressor of the gene expression of the phasin PhaP and of PhaR itself. We used a quartz crystal microbalance to investigate the binding behavior by which PhaR in Ralstonia eutropha H16 targets DNAs and amorphous poly[(R)-3-hydroxybutyrate] thin films. Binding rate constants, dissociation rate constants, and dissociation constants of the binding of PhaR to DNA and to amorphous poly[(R)-3-hydroxybutyrate] suggested that PhaR bind to both in a similar manner. On the basis of the binding rate constant values, we proposed that the phaP gene would be derepressed in harmony with the ratio of the concentration of the target DNA to the concentration of amorphous poly[(R)-3-hydroxybutyrate] at the start of poly[(R)-3-hydroxybutyrate] synthesis in R. eutropha H16.

Partial Text

Polyhydroxyalkanoate (PHA), an eco-friendly and biodegradable polyester, is synthesized by a variety of bacteria, as their intracellular storage material for carbon and energy (Doi et al. 1995; Steinbuchel and Fuchtenbusch 1998; Sudesh et al. 2000). In bacterial cells, PHA forms granules that are covered with a layer composed of proteins and phospholipids (Potter et al. 2002). The most abundant constituent of this layer is phasin (PhaP). The presence of PhaP on the surface of PHA granules contributes to the reduction in size of PHA granules as well as to the slight enhancement of PHA production (Kojima et al. 2006; Potter et al. 2002; Potter and Steinbuchel 2005). Recently, the ability of PhaP to bind to a hydrophobic surface was used to develop methods for protein purification, drug delivery, and tissue engineering applications in in vitro experiments (Backstrom et al. 2007; Banki et al. 2005; Wang et al. 2008). In the cells of microorganisms, a repressor protein PhaR regulates the expression of phaP and phaR. PhaR has also been reported to sense the presence of PHA and to interact with nascent PHA granules, resulting in the derepression of phaP expression (Potter et al. 2002; Potter and Steinbuchel 2005). The presence of genes homologous to PhaR and PhaP in the genomes of various PHA-producing bacteria suggests that a similar regulatory system by PhaR is likely to exist in PHA-producing bacteria (Eugenio et al. 2010; Kojima et al. 2006; Maehara et al. 2002; Yamada et al. 2007; Yamashita et al. 2006). This regulatory system of PHA production through phaR and phaP expression can be applied in a two-hybrid system for protein-protein interaction (Wang et al. 2011). Therefore, understanding of the regulatory system provides meaningful benefit to not only basic science but also applications in various fields such as industry and medicine.

In order to measure the binding behavior of PhaR to 5′-biotinylated dsDNAs (50 bp), the DNA fragments with phaR-binding sequences (the promoter regions of phaR and phaP) and a non-specific sequence (negative control) were immobilized on the electrode of a QCM by biotin-avidin linkage, according to methods outlined in previous papers (Matsuno et al. 2001; Okahata et al. 1998). PhaR was purified using the His-tag purification system, and the purity of PhaR was confirmed by SDS-PAGE (Figure 1). The binding behaviors of PhaR to the DNA fragments were monitored. Figure 2A shows a typical frequency decrease (mass increase) as a function of time, in response to the addition of PhaR. PhaR mainly bound to the DNA containing the phaP promoter region (curve a), and barely bound to the DNA containing the phaR promoter region (curve b) and the control DNA (curve c). Figure 2B shows that the amount of the bound PhaR (Δm) followed a saturation curve as a function of the PhaR concentration ([PhaR]). These binding curves formed a sigmoid curve.

In order to understand the regulatory system governing PHA production in detail, we investigated the binding behaviors of PhaR to the target DNA (containing the promoter region of phaP) and to P(3HB), using QCM measurements. Regarding PhaR-DNA binding, Figure 2A shows that PhaR mainly bound to the DNA containing the phaP promoter region (curve a), and barely bound to the DNA containing the phaR promoter region (curve b) or to the control DNA (curve c). The binding curve of PhaR to the phaP promoter region showed sigmoid curve, implying that PhaR binds to target DNA in a cooperative reaction. The SPR analysis of PhaR-DNA binding in previous studies was not capable of monitoring the initial binding of PhaR, because the concentration of PhaR (10 μM) was higher than in the present experimental conditions (2.5 to 10 nM) (Kojima et al. 2006; Maehara et al. 2002). The higher binding affinity of PhaR to the phaP promoter region accorded with the results of gel-mobility-shift assays (Maehara et al. 2002). The DNA fragments with the phaP promoter region shifted at a lower concentration of PhaR compared to the DNA fragments that contained the phaR promoter region (Potter et al. 2002).

(PHA): Polyhydroxyalkanoate; (PhaP): Phasin; [P(3HB)]: Poly(3-hydroxybutyrate); [cr-P(3HB)]: Poly[(R)-3-hydroxybutyrate]; (QCM): Quartz crystal microbalance; (kon): Binding rate constant; (koff): Dissociation rate constant; (Kd): Dissociation constant; [am-P(3HB)]: Amorphous poly(3-hydroxybutyrate); (SDS): Sodium dodecyl sulfate; (PAGE): Polyacrylamide gel electrophoresis.

The authors declare that they have no competing interests.




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