Identification of the Haloarchaeal Phasin (PhaP) That Functions in Polyhydroxyalkanoate Accumulation and Granule Formation in Haloferax mediterranei

Abstract

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...

Production of Polyhydroxyalkanoate (PHA) Biopolyesters by Extremophiles?

“…mediteranneimediated PHA production has been further optimized by researchers all around the world. Whereas some of these groups focused on the application of inexpensive carbon feedstocks to safe substrate costs, such as whey permeate from dairy industry [12,13,48], rice-based ethanol stillage [49], extruded rice bran [50], enzymatically extruded starch [51], crude glycerol phase [28], olive mill waste water [52], vinasse [53], etc., others clarified the enzymatic and genetic background of PHA-synthesis [54][55][56][57] and in vitro degradation [58] by this organisms. Mathematic models of PHA-production [59], and kinetic studies of PHA and by-product synthesis and degradation [60] were reported for Hfx.…”

Bacterial polyhydroxyalkanoates-eco-friendly next generation plastic: Production, biocompatibility, biodegradation, physical properties and applications

“…Phasins are inhibiting individual granules from coalescing and agglutinating with other granules and acting as a barrier between the polymer and other cellular components (123,124,145). In phasins mutants of R. eutropha, the cells accumulate less PHB, and they possess only one single large granule because separate granules coagule due to the "nacked" surface if the hydrophobic PHA molecules get in contact ( Figure 4) (140,142,146). It was also speculated that phasins might have a protective function to reduce the passive attachment of cytosolic proteins.…”

Complete Genome Sequence of the Metabolically Versatile Halophilic Archaeon Haloferax mediterranei, a Poly(3-Hydroxybutyrate- co -3-Hydroxyvalerate) Producer

“…In particular, H. mediterranei is capable of accumulating poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) from many cheap carbon sources (12,17,18). Two key enzymes (PhaEC and PhaB) and a structure protein (PhaP) involved in PHBV biosynthesis have been identified in H. mediterranei (2,6,9,17). A highly efficient genetic manipulation system (14) and the availability of its genome sequence would expand our understanding of the relationship between PHBV biosynthesis and central metabolism in H. mediterranei.…”