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...

dHaloferax mediterranei is able to accumulate the bioplastic poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) with more than 10 mol% 3-hydroxyvalerate (3HV) from unrelated carbon sources. However, the pathways that produce propionyl coenzyme A (propionyl-CoA), an important precursor of 3HV monomer, have not yet been determined. Bioinformatic analysis of H. mediterranei genome indicated that this strain uses multiple pathways for propionyl-CoA biosynthesis, including the citramalate/2-oxobutyrate pathway, the aspartate/2-oxobutyrate pathway, the methylmalonyl-CoA pathway, and a novel 3-hydroxypropionate pathway. Cofeeding of pathway intermediates and inactivating pathway-specific genes supported that these four pathways were indeed involved in the biosynthesis of 3HV monomer. The novel 3-hydroxypropionate pathway that couples CO 2 assimilation with PHBV biosynthesis was further confirmed by analysis of 13 C positional enrichment in 3HV. Notably, 13 C metabolic flux analysis showed that the citramalate/2-oxobutyrate pathway (53.0% flux) and the 3-hydroxypropionate pathway (30.6% flux) were the two main generators of propionyl-CoA from glucose. In addition, genetic perturbation on the transcriptome of the ⌬phaEC mutant (deficient in PHBV accumulation) revealed that a considerable number of genes in the four propionyl-CoA synthetic pathways were significantly downregulated. We determined for the first time four propionyl-CoA-supplying pathways for PHBV production in haloarchaea, particularly including a new 3-hydroxypropionate pathway. These results would provide novel strategies for the production of PHBV with controllable 3HV molar fraction. P olyhydroxyalkanoates (PHAs) are deposited as carbon and energy materials by many bacteria and haloarchaea under unbalanced growth conditions (1, 2). Due to their excellent biodegradability, biocompatibility, and mechanical properties, PHAs have received increased attention as excellent alternatives for petroleum-derived plastics (3). The physical and mechanical properties of PHAs are closely correlated to monomer composition. Among the various types of PHAs, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and poly-3-hydroxybutyrate (PHB) are the two most extensively studied ones. Due to the incorporation of 3-hydroxyvalerate (3HV) monomer, PHBV becomes more ductile and easier to process and thus possesses a wider range of industrial applications than PHB (4).PHBV is usually produced from acetyl coenzyme A (acetylCoA) and propionyl coenzyme A (propionyl-CoA) via a threestep process catalyzed by ␤-ketothiolase (PhaA/BktB), ␤-ketoacyl-CoA reductase (PhaB), and PHA synthase sequentially (5). Thus far, few bacteria, including Nocardia corallina (6), Rhodococcus spp. (7), a mutant of Ralstonia eutropha (8), several purple nonsulfur bacteria (9), and Bacillus circulans (10) were found to be able to produce propionyl-CoA for PHBV biosynthesis from single unrelated carbon sources. Most bacteria require the addition of propionate in the media to produce PHBV (11). Since propion...

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...

The use of multiple origins for chromosome replication has been demonstrated in archaea. Similar to the dormant origins in eukaryotes, some potential origins in archaea appear to be inactive during genome replication. We have comprehensively explored the origin utilization in Haloferax mediterranei. Here we report three active chromosomal origins by genome-wide replication profiling, and demonstrate that when these three origins are deleted, a dormant origin becomes activated. Notably, this dormant origin cannot be further deleted when the other origins are already absent and vice versa. Interestingly, a potential origin that appears to stay dormant in its native host H. volcanii lacking the main active origins becomes activated and competent for replication of the entire chromosome when integrated into the chromosome of origin-deleted H. mediterranei. These results indicate that origin-dependent replication is strictly required for H. mediterranei and that dormant replication origins in archaea can be activated if needed.

Although polyhydroxyalkanoate (PHA) accumulation and mobilization are one of the most general mechanisms for haloarchaea to adapt to the hypersaline environments with changeable carbon sources, the PHA mobilization pathways are still not clear for any haloarchaea. In this study, the functions of five putative (R)-specific enoyl-CoA hydratases (R-ECHs) in Haloferax mediterranei, named PhaJ1 to PhaJ5, respectively, were thoroughly investigated. Through gene deletion and complementation, we demonstrated that only certain of these ECHs had a slight contribution to poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) biosynthesis. But significantly, PhaJ1, the only R-ECH that is associated with PHA granules, was shown to be involved in PHA mobilization in this haloarchaeon. PhaJ1 catalyzes the dehydration of (R)-3-hydroxyacyl-CoA, the common product of PHA degradation, to enoyl-CoA, the intermediate of the β-oxidation cycle, thus could link PHA mobilization to β-oxidation pathway in H. mediterranei. This linkage was further indicated from the up-regulation of the key genes of β-oxidation under the PHA mobilization condition, as well as the obvious inhibition of PHA degradation upon inhibition of the β-oxidation pathway. Interestingly, 96% of phaJ-containing haloarchaeal species possess both phaC (encoding PHA synthase) and the full set genes of β-oxidation, implying that the mobilization of carbon storage in PHA through the β-oxidation cycle would be general in haloarchaea.

cThe key enzymes and pathways involved in polyhydroxyalkanoate (PHA) biosynthesis in haloarchaea have been identified in recent years, but the haloarchaeal enzymes for PHA degradation remain unknown. In this study, a patatin-like PHA depolymerase, PhaZh1, was determined to be located on the PHA granules in the haloarchaeon Haloferax mediterranei. PhaZh1 hydrolyzed the native PHA (nPHA) [including native polyhydroxybutyrate (nPHB) and native poly(3-hydroxybutyrateco-3-hydroxyvalerate) (nPHBV) in this study] granules in vitro with 3-hydroxybutyrate (3HB) monomer as the primary product. The site-directed mutagenesis of PhaZh1 indicated that Gly 16 , Ser 47 (in a classical lipase box, G-X-S 47 -X-G), and Asp 195 of this depolymerase were essential for its activity in nPHA granule hydrolysis. Notably, phaZh1 and bdhA (encoding putative 3HB dehydrogenase) form a gene cluster (HFX_6463 to _6464) in H. mediterranei. The 3HB monomer generated from nPHA degradation by PhaZh1 could be further converted into acetoacetate by BdhA, indicating that PhaZh1-BdhA may constitute the first part of a PHA degradation pathway in vivo. Interestingly, although PhaZh1 showed efficient activity and was most likely the key enzyme in nPHA granule hydrolysis in vitro, the knockout of phaZh1 had no significant effect on the intracellular PHA mobilization, implying the existence of an alternative PHA mobilization pathway(s) that functions effectively within the cells of H. mediterranei. Therefore, identification of this patatin-like depolymerase of haloarchaea may provide a new strategy for producing the high-value-added chiral compound (R)-3HB and may also shed light on the PHA mobilization in haloarchaea. P olyhydroxyalkanoate (PHA) is accumulated in the form of granules and serves as storage compound of carbon and energy in bacteria (1) and archaea (2) during growth in the presence of excess carbon sources. Several proteins, which are known as PHA granule-associated proteins (PGAPs), are embedded on or attached to the PHA granules. These include PHA synthases, phasins, regulatory proteins, and depolymerases (3). A PHA-accumulating host may utilize the accumulated PHA for growth and survival under conditions of carbon starvation (4). PHA depolymerase (PhaZ) is the key enzyme that functions in PHA mobilization.In bacteria, PHA depolymerases are grouped into two classes: intracellular (catalyzing the degradation of endogenous PHA) (iPhaZ) and extracellular (catalyzing the degradation of exogenous PHA) (ePhaZ) PHA depolymerases (5). The extracellular PHA depolymerase degradation process is well known, but the mechanism underlying the metabolic pathway and regulation of PHA degradation in vivo remains poorly understood (5). Native polyhydroxybutyrate (nPHB) is degraded by iPhaZs in vitro to 3-hydroxybutyrate (3HB) (6, 7) or 3-hydroxybutyryl-coenzyme A (3HB-CoA) in the presence of CoA (8, 9). The PHB degradation product, 3HB-CoA, is the precursor of PHB synthesis; hence, the simultaneous synthesis and mobilization of PHB (10, 11) (8). Notably, th...

Halophilic archaea (haloarchaea) inhabit hypersaline environments, tolerating extreme salinity, low oxygen and nutrient availability, and in some cases, high pH (soda lakes) and irradiation (saltern ponds). Membrane-associated proteins of haloarchaea, such as surface layer (S-layer) proteins, transporters, retinal proteins, and internal organellar membrane proteins including intracellular gas vesicle proteins and those associated with polyhydroxyalkanoate (PHA) granules, contribute greatly to their environmental adaptations. This review focuses on these haloarchaeal cellular and organellar membrane-associated proteins, and provides insight into their physiological significance and biotechnological potential.