Sweet taste receptors play roles in artificial sweetener-induced enhanced urine output in mice.

Sweet taste receptors found in oral and extra oral tissues play important roles in the regulation of many physiological functions. Studies have shown that urine volume increases during the lifetime exposure to artificial sweeteners. However, the detailed molecular mechanism and the general effects of different artificial sweeteners exposure on urine volume remain unclear. In this study, we investigated the relationship between urinary excretion and the sweet taste receptor expression in mice after three artificial sweeteners exposure in a higher or lower concentration via animal behavioral studies, western blotting, and real-time quantitative PCR experiment in rodent model. Our results showed that high dose of acesulfame potassium and saccharin can significantly enhance the urine output and there was a positive correlation between K+ and urination volume. The acesulfame potassium administration assay of T1R3 knockout mice showed that artificial sweeteners may affect the urine output directly through the sweet taste signaling pathway. The expression of T1R3 encoding gene can be up-regulated specifically in bladder but not in kidney or other organs we tested. Through our study, the sweet taste receptors, distributing in many tissues as bladder, were indicated to function in the enhanced urine output. Different effects of long-term exposure to the three artificial sweeteners were shown and acesulfame potassium increased urine output even at a very low concentration.

Study on the production of high 3HV content PHBV via an open fermentation with waste silkworm excrement as the carbon source by the haloarchaeon Haloferax mediterranei.

Silkworm excrement is hard to be degraded or bio-utilized by environmental microorganisms due to its high content of heavy metals and antimicrobial biomacromolecules in mulberry leaves. In traditional Chinese silk industry, the silkworm excrement results in environmental problems. In this study, the silkworm excrement after chlorophyll ethanol-extraction was researched. An open fermentation strategy was developed using the silkworm excrement as the sole or partial carbon source by haloarchaea to accumulate polyhydroxyalkanoates. As a haloarchaeon with strong carbon source utilization ability, Haloferax mediterranei was found to accumulate a certain amount of poly(3-hydroxybutyrate-co-3-hydroxyvalerate; PHBV) using waste silkworm excrement. The results showed that the addition of silkworm excrement into glucose based fermentation medium can significantly improve the production of PHBV. Using a mixture carbon source including the extract of silkworm excrement and glucose (with a 1:1 carbon content ratio), the yield of PHBV was 1.73 ± 0.12 g/l, which showed a 26% increase than that of fermentation without the silkworm excrement addition. When the NaCl content of medium was set to approximately 15%, fermentation without sterilization was performed using silkworm excrement as the carbon source. Moreover, the addition of the silkworm excrement extract could increase the 3-hydroxyvalerate (3 HV) content of PHBV regardless of the sterilization or non-sterilization fermentation conditions. When using silkworm excrement as the sole carbon source, the 3 HV content was as high as 16.37 ± 0.54 mol %. The real-time quantitative PCR results showed that the addition of the silkworm excrement could specifically enhance the expression of genes involved in the aspartate/2-ketobutyric acid pathway related to 3 HV synthesis in H. mediterranei, and further analysis of the amino acid of the silkworm excrement suggested that the high content of threonine in the silkworm excrement might be the reason for the increase of 3 HV content. Taken together, the success of non-sterile fermentation in hypersaline condition using haloarchaea implied a novel way to reuse the silkworm excrement, which not only reduces the production costs of PHBV, but also is conducive to environmental protection.

Production of Polyhydroxyalkanoates in Unsterilized Hyper-Saline Medium by Halophiles Using Waste Silkworm Excrement as Carbon Source.

The chlorophyll ethanol-extracted silkworm excrement was hardly biologically reused or fermented by most microorganisms. However, partial extremely environmental halophiles were reported to be able to utilize a variety of inexpensive carbon sources to accumulate polyhydroxyalkanoates. In this study, by using the nile red staining and gas chromatography assays, two endogenous haloarchaea strains: Haloarcula hispanica A85 and Natrinema altunense A112 of silkworm excrement were shown to accumulate poly(3-hydroxybutyrate) up to 0.23 g/L and 0.08 g/L, respectively, when using the silkworm excrement as the sole carbon source. The PHA production of two haloarchaea showed no significant decreases in the silkworm excrement medium without being sterilized compared to that of the sterilized medium. Meanwhile, the CFU experiments revealed that there were more than 60% target PHAs producing haloarchaea cells at the time of the highest PHAs production, and the addition of 0.5% glucose into the open fermentation medium can largely increase both the ratio of target haloarchaea cells (to nearly 100%) and the production of PHAs. In conclusion, our study demonstrated the feasibility of using endogenous haloarchaea to utilize waste silkworm excrement, effectively. The introduce of halophiles could provide a potential way for open fermentation to further lower the cost of the production of PHAs.

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 ß-oxidation cycle, thus could link PHA mobilization to ß-oxidation pathway in H. mediterranei. This linkage was further indicated from the up-regulation of the key genes of ß-oxidation under the PHA mobilization condition, as well as the obvious inhibition of PHA degradation upon inhibition of the ß-oxidation pathway. Interestingly, 96% of phaJ-containing haloarchaeal species possess both phaC (encoding PHA synthase) and the full set genes of ß-oxidation, implying that the mobilization of carbon storage in PHA through the ß-oxidation cycle would be general in haloarchaea.

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 patatin-like protein associated with the polyhydroxyalkanoate (PHA) granules of Haloferax mediterranei acts as an efficient depolymerase in the degradation of native PHA.

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.

A novel DNA-binding protein, PhaR, plays a central role in the regulation of polyhydroxyalkanoate accumulation and granule formation in the haloarchaeon Haloferax mediterranei.

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.

[Development of PhaP-tagged protein expression and purification systems for extremely halophilic archaea ].

[ OBJECTIVE] To establish a convenient halophilic protein expression and purification system based on the haloarchaeal-type PhaP and polyhydroxyalkanoate (PHA) granule. [METHODS] We cloned a strong haloarchaeal promoter and the phaP-tag into the haloarchaea- Escherichia coli shuttle vector pWL502, and then used the constructed vector to express the PhaP-tagged haloarchaeal proteins in the phaP-deleted strain Haloferax mediterranei AphaP. We purified the PhaP-fusion proteins, which were associated with PHA granules, by sucrose density gradient centrifugation. We also inserted a haloarchaeal intein-containing fragment between phaP and multiple cloning sites, and modulated the intein splicing activity by site-directed mutagenesis. [RESULTS] We successfully constructed two expression vectors, pPM and pIP, in which PhaP was used as N-terminal and C-terminal fusion tag, respectively. The haloarchaeal proteins were effectively expressed by both vectors. The PhaP-tagged proteins were easily purified through the strategy of PHA granulemediated protein purification. In addition, we found that the intein-containing fragment Hbt21 from Halobacterium sp. NRC-1 had maintained splicing activity in H. mediterranei, and its C-terminal cleavage could be blocked or attenuated by mutating the conserved asparagine ( N182) or serine (S183) , respectively. [ CONCLUSION] We have established a convenient and economical halophilic protein expression and purification system. We have also identified the splicing active sites of a haloarchaeal intein, which showed potential for removing the PhaP-tag from the purified proteins.

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.

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 (PPTS) during fructose induction. Further promoter-scanning mutation indicated that three sites located upstream of the H. mediterranei PPTS, which are conserved in most haloarchaeal PPTSs, 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.

Multiple propionyl coenzyme A-supplying pathways for production of the bioplastic poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in Haloferax mediterranei.

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 (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 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.

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.

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.

Identification of the haloarchaeal phasin (PhaP) that functions in polyhydroxyalkanoate accumulation and granule formation in Haloferax mediterranei.

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.

[Identification of the phaB genes and analysis of the PHBV precursor supplying pathway in Haloferax mediterranei].

OBJECTIVE: Identification and characterization of the genes involved in precursor supplying for poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) biosynthesis in the haloarchaeon Haloferax mediterranei. METHODS: By using BLAST (Basic Local Alignment Search Tool) search methodology, we obtained five genes (phaB1, phaB2, phaJ1, phaJ2 and phaJ3) that were possibly involved in the 3-hydroxyacyl-CoA precursor supplying for PHBV biosynthesis in H. mediterranei. Firstly, we proved that these five genes were all transcribed under the PHBV-accumulating condition in H. mediterranei. Then, we knocked out these genes individually or in combination, by double-crossover homologous recombination, resulting in the following mutants: deltaphaB1, deltaphaB2, AphaJ1, deltaphaJ2, deltaphaJ3, deltaphaB1phaB2, deltaphaJ1phaJ2 and deltaphaJ1phaJ2phaJ3. Finally, we performed the complementation analysis of the deltaphaB1phaB2 strain, with the phaB1 and phaB2 genes, respectively. RESULTS: Whenever the three phaJ genes were knocked out individually or in combination, there was no obvious influence on PHBV accumulation in H. mediterranei. Knockout of phaB1 also did not affect the PHBV accumulation obviously. However, when phaB2 was knocked out, the yield of PHBV and the fraction of the 3-HV monomer decreased significantly. Notably, when the phaB1 and phaB2 were knocked out in combination, the CONCLUSIONS: The PHBV-specific acetoacetyl-CoA reductases mutant deltaphaB1phaB2 no longer produced PHBV. (PhaB) involved in the precursor supplying for PHBV biosynthesis are encoded by phaB1 and phaB2 in H. mediterranei.

Wide distribution among halophilic archaea of a novel polyhydroxyalkanoate synthase subtype with homology to bacterial type III synthases.

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 (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.