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

Abstract

c Polyhydroxyalkanoates (PHAs) are synthesized and assembled as PHA granules that undergo well-regulated formation in many microorganisms. However, this regulation remains unclear in haloarchaea. In this study, we identified a PHA granule-associated regulator (PhaR) that negatively regulates the expression of both its own gene and the granule structural gene phaP in the same operon (phaRP) in Haloferax mediterranei. Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) assays demonstrated a significant interaction between PhaR and the phaRP promoter in vivo. Scanning mutagenesis of the phaRP promoter revealed a specific cis-element as the possible binding position of the PhaR. The haloarchaeal homologs of the PhaR contain a novel conserved domain that belongs to a swapped-hairpin barrel fold family found in AbrB-like proteins. Amino acid substitution indicated that this AbrB-like domain is critical for the repression activity of PhaR. In addition, the phaRP promoter had a weaker activity in the PHA-negative strains, implying a function of the PHA granules in titration of the PhaR. Moreover, the H. mediterranei strain lacking phaR was deficient in PHA accumulation and produced granules with irregular shapes. Interestingly, the PhaR itself can promote PHA synthesis and granule formation in a PhaP-independent manner. Collectively, our results demonstrated that the haloarchaeal PhaR is a novel bifunctional protein that plays the central role in the regulation of PHA accumulation and granule formation in H. mediterranei. P olyhydroxyalkanoates (PHAs) are biodegradable polyesters synthesized by most genera of bacteria (1, 2) and some archaea (3-5). PHAs are accumulated as storage compounds of energy and carbon under imbalanced growth conditions (i.e., when nutrients such as nitrogen, phosphorus, or oxygen are limited but the carbon sources are in excess) (6).PHAs are often deposited in the cytoplasm as water-insoluble inclusions that are called PHA granules (6). Native PHA granules are found to be composed of 97.5% PHA, 2% proteins, and likely some amount of lipids (7). At least four types of proteins were found to be the PHA granule-associated proteins (PGAPs) in bacteria: PHA synthases, PHA depolymerases, regulators, and structural proteins (phasins [PhaPs]) (8, 9). In recent years, increasing new roles have been found for the PGAPs. Besides the classical phasin role of preventing PHA granules from coalescing, two distinct phasin-like proteins, PhaM and PhaF, have also been characterized as being crucial for granule distribution during cell division (10, 11).The PGAPs play important roles in PHA synthesis, PHA utilization, and granule formation and distribution (8, 9, 12), among which the regulatory proteins are responsible for ensuring the proper formation of PHA granules by influencing the expression of both phasins and themselves (13)(14)(15)(16)(17). A classic regulation model was presented in a poly(3-hydroxybutyrate) (PHB [a type of PHA])-accumulating bacterium, Ralstonia eutropha H16 (9). Briefly, the cy...

Phasins, Multifaceted Polyhydroxyalkanoate Granule-Associated Proteins

“…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). PhaR was also found in H. mediterranei, but the regulation pattern of phaP by PhaR in this archaeon is slightly different from that of its bacterial counterparts, as it was shown to repress the expression of both its own gene and phaP, which are transcribed from the same operon (phaRP) (36).…”

A Patatin-Like Protein Associated with the Polyhydroxyalkanoate (PHA) Granules of Haloferax mediterranei Acts as an Efficient Depolymerase in the Degradation of Native PHA

“…This result confirmed that Gly 16 , Ser 47 , and Asp 195 are indeed essential for nPHA granule hydrolysis. Ser 47 in the classical lipase box (G-X-S-X-G) may be the active-site serine (6,38,39) (18,21,22). However, no putative PHA depolymerase was found in this PHA granule proteome.…”

“…To further biochemically determine the hydrolysis activity of PhaZh1 with the nPHA granules, we expressed PhaZh1 with a C-terminal His 6 tag (His 6 -PhaZh1) in the haloarchaeon H. volcanii H1424 (26). The promoter of the expression plasmid pTA1228 (27) used in this study was replaced with a strong constitutive promoter of P.phaR in which its negative cis element has been deleted (22). The PHA depolymerase activity of His 6 -PhaZh1 (Fig.…”

“…The plasmids and primers used in this study are listed in Tables 1 and 2. For gene cloning and protein expression in H. volcanii H1424, several new expression vectors were developed, at first based on the plasmid pTA1228 (27), in which the tryptophanase promoter P.tnaA was replaced by the strong promoter P.phaR (22). Briefly, the P.phaR promoter was amplified with the primer pair P.phaR-F/R and was then digested with BamHI plus KpnI and inserted into pTA1228 to yield pTA01.…”

“…The PHA synthase (PhaEC) (18), acetoacetyl-CoA reductases (PhaB1 and PhaB2) (19), and haloarchaeal-type ␤-ketothiolases (PhaA and BktB) (20), which are indispensable for PHBV biosynthesis in H. mediterranei, have been identified. The PHA granule-associated phasin protein (PhaP) (21) and regulatory protein (PhaR) (22), which are important for PHA accumulation and granule formation, have also been characterized. However, the key enzymes of PHA degradation have not been identified yet for any haloarchaeal species.…”