The polyhydroxyalkanoate (PHA) granule-associated proteins (PGAPs) are important for PHA synthesis and granule formation, but currently little is known about the haloarchaeal PGAPs. This study focused on the identification and functional analysis of the PGAPs in the haloarchaeon Haloferax mediterranei. These PGAPs were visualized with two-dimensional gel electrophoresis (2-DE) and identified by matrix-assisted laser desorption ionization-tandem time of flight mass spectrometry (MALDI-TOF/ TOF MS). The most abundant protein on the granules was identified as a hypothetical protein, designated PhaP. A genome-wide analysis revealed that the phaP gene is located upstream of the previously identified phaEC genes. Through an integrative approach of gene knockout/complementation and fermentation analyses, we demonstrated that this PhaP is involved in PHA accumulation. The ⌬phaP mutant was defective in both PHA biosynthesis and cell growth compared to the wild-type strain. Additionally, transmission electron microscopy results indicated that the number of PHA granules in the ⌬phaP mutant cells was significantly lower, and in most of the ⌬phaP cells only a single large granule was observed. These results demonstrated that the H. mediterranei PhaP was the predominant structure protein (phasin) on the PHA granules involved in PHA accumulation and granule formation. In addition, BLASTp and phylogenetic results indicate that this type of PhaP is exclusively conserved in haloarchaea, implying that it is a representative of the haloarchaeal type PHA phasin. P olyhydroxyalkanoates (PHAs) are biodegradable polyesters present in most genera of bacteria (19,38) and in Halobacteriaceae members of archaea (7,20). PHAs usually accumulate when cells are cultivated in an environment with an excess of carbon sources and limited nitrogen, phosphorus, or oxygen (1). Serving as carbon and energy storage compounds, PHAs are often found in the cytoplasm as insoluble inclusions that are also known as PHA granules (1).There has been a considerable amount of research carried out on PHA granules in bacteria. Early investigations indicated that isolated native PHA granules contain approximately 97.5% PHA, 2% proteins, and small amounts of lipids (9). Many proteins coated onto PHA granules have been shown to be responsible for PHA synthesis and granule formation. Previously, these PHA granule-associated proteins (PGAPs) were placed into four categories: (i) PHA synthases, (ii) PHA depolymerases and 3-hydroxybutyrate oligomer hydrolases, (iii) the major structure proteins (phasins), and (iv) the regulatory proteins (29). A recent review gave a broader overview of PGAPs and listed many new types of PGAPs, such as the newly found acyl-coenzyme A (CoA) synthetase (14, 33). In recent years, the major proteins present on PHA granules, the phasins (PhaPs), which may constitute approximately 5% (wt/wt) of total cellular proteins (41), have attracted more attention. In addition to their main roles in preventing PHA granules from aggregating and in preventin...

Polyhydroxyalkanoates (PHAs) are accumulated as intracellular carbon and energy storage polymers by various bacteria and a few haloarchaea. In this study, 28 strains belonging to 15 genera in the family Halobacteriaceae were investigated with respect to their ability to synthesize PHAs and the types of their PHA synthases. Fermentation results showed that 18 strains from 12 genera could synthesize polyhydroxybutyrate (PHB) or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). For most of these haloarchaea, selected regions of the phaE and phaC genes encoding PHA synthases (type III) were cloned via PCR with consensusdegenerate hybrid oligonucleotide primers (CODEHOPs) and were sequenced. The PHA synthases were also examined by Western blotting using haloarchaeal Haloarcula marismortui PhaC (PhaC Hm ) antisera. Phylogenetic analysis showed that the type III PHA synthases from species of the Halobacteriaceae and the Bacteria domain clustered separately. Comparison of their amino acid sequences revealed that haloarchaeal PHA synthases differed greatly in both molecular weight and certain conserved motifs. The longer C terminus of haloarchaeal PhaC was found to be indispensable for its enzymatic activity, and two additional amino acid residues (C143 and C190) of PhaC Hm were proved to be important for its in vivo function. Thus, we conclude that a novel subtype (IIIA) of type III PHA synthase with unique features that distinguish it from the bacterial subtype (IIIB) is widely distributed in haloarchaea and appears to be involved in PHA biosynthesis.Haloarchaea are a distinct evolutionary branch of the domain Archaea, and they usually comprise the majority of the prokaryotic population in hypersaline environments (31). Most haloarchaea are able to utilize glucose as a carbon source. However, Halobacterium (15) and some Natrialba (42) and Natronomonas (6, 7) strains cannot. In the presence of excess carbon substrates, certain haloarchaea synthesize polyhydroxyalkanoates (PHAs) and deposit them as intracellular granules (27), which has been proposed as an optional standard for describing new haloarchaeal species (32). Compared with members of the domain Bacteria, haloarchaea have several advantages as PHA producers; e.g., they utilize unrelated cheap carbon sources, strict sterilization is not needed, and isolation of PHAs from haloarchaea is much easier (11,13,27,34). Thus, haloarchaea have regained attention in biotechnology lately, especially as an alternative source for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) production (11,20,28). Although the family Halobacteriaceae includes 30 genera, currently, only a few haloarchaeal strains belonging to the genera Haloferax, Haloarcula, Haloquadratum, Haloterrigena, Halorhabdus, Halobiforma, and Halopiger are found to accumulate short-chain-length PHAs (scl-PHAs), such as polyhydroxybutyrate (PHB) and PHBV (2, 11-14, 22, 27, 36, 41).In the pathway of PHA biosynthesis, PHA synthases play a key role by catalyzing the polymerization of (R)-3-hydroxyalkanoyl coenzym...