Shuangfeng Cai

Wide Distribution among Halophilic Archaea of a Novel Polyhydroxyalkanoate Synthase Subtype with Homology to Bacterial Type III Synthases

ABSTRACT 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 consensus-degenerate hybrid oligonucleotide primers (CODEHOPs) and were sequenced. The PHA synthases were also examined by Western blotting using haloarchaeal Haloarcula marismortui PhaC (PhaCHm) 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 PhaCHm 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.

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 ΔphaP 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 ΔphaP mutant cells was significantly lower, and in most of the ΔphaP 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.

Multiple Propionyl Coenzyme A-Supplying Pathways for Production of the Bioplastic Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) in Haloferax mediterranei

ABSTRACT Haloferax 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 CO2 assimilation with PHBV biosynthesis was further confirmed by analysis of 13C positional enrichment in 3HV. Notably, 13C 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 ΔphaEC 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.

Cellular and organellar membrane-associated proteins in haloarchaea: Perspectives on the physiological significance and biotechnological applications

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.

Activation of a dormant replication origin is essential for Haloferax mediterranei lacking the primary origins.

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.

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 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.

Analysis of the Transcriptional Regulator GlpR, Promoter Elements, and Posttranscriptional Processing Involved in Fructose-Induced Activation of the Phosphoenolpyruvate-Dependent Sugar Phosphotransferase System in Haloferax mediterranei

ABSTRACT Among all known archaeal strains, the phosphoenolpyruvate-dependent phosphotransferase system (PTS) for fructose utilization is used primarily by haloarchaea, which thrive in hypersaline environments, whereas the molecular details of the regulation of the archaeal PTS under fructose induction remain unclear. In this study, we present a comprehensive examination of the regulatory mechanism of the fructose PTS in the haloarchaeon Haloferax mediterranei. With gene knockout and complementation, microarray analysis, and chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR), we revealed that GlpR is the indispensable activator, which specifically binds to the PTS promoter (P PTS ) during fructose induction. Further promoter-scanning mutation indicated that three sites located upstream of the H. mediterranei P PTS , which are conserved in most haloarchaeal P PTS s, are involved in this induction. Interestingly, two PTS transcripts (named T8 and T17) with different lengths of 5′ untranslated region (UTR) were observed, and promoter or 5′ UTR swap experiments indicated that the shorter 5′ UTR was most likely generated from the longer one. Notably, the translation efficiency of the transcript with this shorter 5′ UTR was significantly higher and the ratio of T8 (with the shorter 5′ UTR) to T17 increased during fructose induction, implying that a posttranscriptional mechanism is also involved in PTS activation. With these insights into the molecular regulation of the haloarchaeal PTS, we have proposed a working model for haloarchaea in response to environmental fructose.

Enoyl-CoA hydratase mediates polyhydroxyalkanoate mobilization in Haloferax mediterranei

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.

Proteome reference map of Haloarcula hispanica and comparative proteomic and transcriptomic analysis of polyhydroxyalkanoate biosynthesis under genetic and environmental perturbations.

Many haloarchaea are known as polyhydroxyalkanoates (PHAs) producers, but a global and integrated view of the PHA biosynthesis is still lacking in this group of archaea. In this study, a combined proteomic and transcriptomic approach was employed in Haloarcula hispanica, a model haloarchaeon that accumulates poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) under nutrient-limiting conditions with excess carbon source. First, a comprehensive proteome reference map was established for H. hispanica. A total of 936 spots representing 839 unique proteins (21.7% of the predicted proteome) were identified by MALDI-TOF/TOF PMF and MS/MS. The map was further utilized to reconstruct central metabolic pathways to facilitate functional genomic analysis in H. hispanica. The results from the proteomic and transcriptomic analysis indicated that active PHA production coordinated with the TCA cycle to maintain balanced growth in wild-type H. hispanica, which was grown in nutrient-limited medium (PHA-accumulating conditions) versus nutrient-rich medium (non-PHA-accumulating conditions). Under nutrient-limiting conditions with excess carbon source, the PHA biosynthetic genes including phaEC, phaB, and phaP were upregulated at the transcriptional level, whereas the TCA cycle and respiratory chain were downregulated. Thus, acetyl-CoA could be fed into the PHA biosynthetic pathway, leading to the accumulation of PHA granules in the cell. Simultaneously, the large amount of NADPH required during PHA accumulation was likely supplied by the C3 (pyruvate) and C4 (malate) pathway coupled with the urea cycle. When PHA biosynthesis was blocked, that is, in the PHA synthase mutant (ΔphaEC) versus wild type grown in nutrient-limited medium, the mutant might direct additional carbon and energy to the TCA cycle, but without obvious contribution to biomass accumulation. The combined approaches of proteomic and transcriptomic analysis were highly complementary, extending the physiological understanding of PHA biosynthesis and its regulation. This is the first integrated proteome and transcriptome investigation of PHA biosynthesis and regulation in haloarchaea. It has provided basic information for future systemic engineering of haloarchaea to meet industrial needs.

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

ABSTRACT The 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-hydroxybutyrate-co-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 Gly16, Ser47 (in a classical lipase box, G-X-S47-X-G), and Asp195 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.