PhaQ, a New Class of Poly-β-Hydroxybutyrate (PHB)-Responsive Repressor, Regulates phaQ and phaP (Phasin) Expression in Bacillus megaterium through Interaction with PHB

Abstract

Bacillus megaterium can produce poly-␤-hydroxybutyrate (PHB) as carbon and energy storage materials. We now report that the phaQ gene, which is located upstream of the phasin-encoding phaP gene, codes for a new class of transcriptional regulator that negatively controls expression of both phaQ and phaP. A PhaQ binding site that plays a role in this control has been identified by gel mobility shift assays and DNase I footprinting analysis. We have also provided evidence that PhaQ could sense the presence of PHB in vivo and that artificial PHB granules could inhibit the formation of PhaQ-DNA complex in vitro by binding to PhaQ directly. These suggest that PhaQ is a PHB-responsive repressor.Poly-␤-hydroxybutyrate (PHB) and other polyhydroxyalkanoates (PHAs) are biodegradable polyesters that are produced by a wide variety of bacteria as intracellular carbon and energy storage materials (1, 13, 31). PHB synthases (PhaC) and phasins (PhaP) are proteins that play important roles in PHB production and granule formation. Phasin, an abundant granule-associated protein, forms a boundary layer on the PHB surface to sequester hydrophobic PHB from the cytoplasm. Thus, phasin can inhibit individual granules from coalescing and promotes PHB synthesis by regulating the ratio of surface area to volume of PHB granules (27)(28)(29). PhaP may also have a protective function to reduce the passive attachment of cytoplasmic proteins to PHB surface (16,27). It is generally thought that the synthesis of phasin proteins is highly regulated (8). In Ralstonia eutropha, expression of the phaP gene is negatively controlled by the autoregulated repressor PhaR (21,30). The accumulation of PhaP in the R. eutropha cells is strictly dependent on PHB production (28). After the onset of PHB biosynthesis, PhaR can sense the presence of PHB and bind to nascent PHB granules, leading to derepression of phaP. In Paracoccus denitrificans the phaR gene, which is located immediately downstream of phaP, encodes a repressor that regulates phaP expression. PhaR can sense the presence of PHA and interact with PHA granules (14,15).Although Bacillus megaterium is already known to be able to synthesize PHB and accumulate PHB granules, genes that are involved in PHB synthesis and encode PHB granule-associated proteins were not cloned until recently (17). Among a cluster of five pha genes of B. megaterium, phaP and phaQ are transcribed in one direction, whereas phaR, phaB, and phaC are divergently transcribed as a tricistronic operon. phaP codes for a phasin protein. phaB and phaC encode NADPH-dependent acetoacetyl coenzyme A reductase and a novel PHB synthase, respectively (17). This novel PHB synthase requires both PhaC and PhaR for activity (18). It should be noted that the designation of the B. megaterium phaR gene does not follow the conventional rule. Rather than encoding a transcriptional regulator like the PhaR protein of R. eutropha or P. denitrificans, the B. megaterium phaR gene probably encodes a subunit of a heterodimeric PHB synthase (18,25). Neverthel...

Rapid Identification of Polyhydroxyalkanoate Accumulating Members of Bacillales Using Internal Primers forphaCGene ofBacillus megaterium

“…PhaQ is a DNA binding protein, which represses its own synthesis. Once PHB is produced, it binds to PhaQ resulting in the removal of repression on transcription of phaQ and phaP [49]. The phaQ and phaP are transcribed independently as a single mRNA 1.2 kb that is presumably processed to give 0.7 kb transcript responsible synthesis of phasins protein-PhaP.…”

Phasins, Multifaceted Polyhydroxyalkanoate Granule-Associated Proteins

“…Pötter et al (33) and Pötter and Steinbüchel (34) proposed that during active PHA biosynthesis in R. eutropha, PhaR binds to PHA granules, allowing the transcription of phaP, so at the later stages of PHA accumulation, when PhaR is no longer bound to the granules, the transcription of phaP is again repressed (33,34). In B. megaterium, PhaQ was proposed to act in a manner similar to that of PhaR from R. eutropha, as it binds to PHB granules and suffers transcriptional autoregulation (35). This mechanism is analogous to the one proposed by Prieto et al for the regulation of the PHA biosynthesis genes in Pseudomonas oleovorans (currently known as P. putida) by phasin PhaF Pp (10).…”

A Novel DNA-Binding Protein, PhaR, Plays a Central Role in the Regulation of Polyhydroxyalkanoate Accumulation and Granule Formation in the Haloarchaeon Haloferax mediterranei

“…A similar regulation pattern was proposed previously in the phaQ/phaP system in Bacillus megaterium. However, a likely phaP transcript was observed, and the phasin regulator gene phaQ is located far upstream (168 bp) of the phaP gene (40). Since 2004, there has been no additional convincing evidence to refute the suggestion presented in 1999 that B. megaterium phaP (phaP Bm ) was also transcribed from a separate promoter (41).…”

“…Second, unlike the bacterial phasin regulators PhaR and PhaQ (13,40,43), which are predicted to have a helix-turn-helix motif for DNA binding, the H. mediterranei PhaR was shown to have a putative DNA-binding motif similar to that of the N-terminal domain of AbrB. AbrB is a small protein (10.2 to 10.6 kDa) that functions as a transition state regulator in Bacillus species (44).…”