Novel Glutamate-Putrescine Ligase Activity in Haloferax mediterranei: A New Function for glnA-2 Gene.

The genome of the halophilic archaea Haloferax mediterranei contains three ORFs that show homology with glutamine synthetase (GS) (glnA-1, glnA-2, and glnA-3). Previous studies have focused on the role of GlnA-1, suggesting that proteins GlnA-2 and GlnA-3 could play a different role to that of GS. Glutamine synthetase (EC 6.3.1.2) belongs to the class of ligases, including 20 subclasses of other different enzymes, such as aspartate-ammonia ligase (EC 6.3.1.1), glutamate-ethylamine ligase (EC 6.3.1.6), and glutamate-putrescine ligase (EC 6.3.1.11). The reaction catalyzed by glutamate-putrescine ligase is comparable to the reaction catalyzed by glutamine synthetase (GS). Both enzymes can bind a glutamate molecule to an amino group: ammonium (GS) or putrescine (glutamate-putrescine ligase). In addition, they present the characteristic catalytic domain of GS, showing significant similarities in their structure. Although these proteins are annotated as GS, the bioinformatics and experimental results obtained in this work indicate that the GlnA-2 protein (HFX_1688) is a glutamate-putrescine ligase, involved in polyamine catabolism. The most significant results are those related to glutamate-putrescine ligase's activity and the analysis of the transcriptional and translational expression of the glnA-2 gene in the presence of different nitrogen sources. This work confirms a new metabolic pathway in the Archaea domain which extends the knowledge regarding the utilization of alternative nitrogen sources in this domain.

In Silico Analysis of the Enzymes Involved in Haloarchaeal Denitrification.

During the last century, anthropogenic activities such as fertilization have led to an increase in pollution in many ecosystems by nitrogen compounds. Consequently, researchers aim to reduce nitrogen pollutants following different strategies. Some haloarchaea, owing to their denitrifier metabolism, have been proposed as good model organisms for the removal of not only nitrate, nitrite, and ammonium, but also (per)chlorates and bromate in brines and saline wastewater. Bacterial denitrification has been extensively described at the physiological, biochemical, and genetic levels. However, their haloarchaea counterparts remain poorly described. In previous work the model structure of nitric oxide reductase was analysed. In this study, a bioinformatic analysis of the sequences and the structural models of the nitrate, nitrite and nitrous oxide reductases has been described for the first time in the haloarchaeon model Haloferax mediterranei. The main residues involved in the catalytic mechanism and in the coordination of the metal centres have been explored to shed light on their structural characterization and classification. These results set the basis for understanding the molecular mechanism for haloarchaeal denitrification, necessary for the use and optimization of these microorganisms in bioremediation of saline environments among other potential applications including bioremediation of industrial waters.

Unusual Phosphoenolpyruvate (PEP) Synthetase-Like Protein Crucial to Enhancement of Polyhydroxyalkanoate Accumulation in Haloferax mediterranei Revealed by Dissection of PEP-Pyruvate Interconversion Mechanism.

Phosphoenolpyruvate (PEP)/pyruvate interconversion is a major metabolic point in glycolysis and gluconeogenesis and is catalyzed by various sets of enzymes in different Archaea groups. In this study, we report the key enzymes that catalyze the anabolic and catabolic directions of the PEP/pyruvate interconversion in Haloferax mediterranei The in silico analysis showed the presence of a potassium-dependent pyruvate kinase (PYKHm [HFX_0773]) and two phosphoenol pyruvate synthetase (PPS) candidates (PPSHm [HFX_0782] and a PPS homolog protein named PPS-like [HFX_2676]) in this strain. Expression of the pykHm gene and ppsHm was induced by glycerol and pyruvate, respectively; whereas the pps-like gene was not induced at all. Similarly, genetic analysis and enzyme activities of purified proteins showed that PYKHm catalyzed the conversion from PEP to pyruvate and that PPSHm catalyzed the reverse reaction, while PPS-like protein displayed no function in PEP/pyruvate interconversion. Interestingly, knockout of the pps-like gene led to a 70.46% increase in poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) production. The transcriptome sequencing (RNA-Seq) and quantitative reverse transcription-PCR (qRT-PCR) results showed that many genes responsible for PHBV monomer supply and for PHBV synthesis were upregulated in a pps-like gene deletion strain and thereby improved PHBV accumulation. Additionally, our phylogenetic evidence suggested that PPS-like protein diverged from PPS enzyme and evolved as a distinct protein with novel function in haloarchaea. Our findings attempt to fill the gaps in central metabolism of Archaea by providing comprehensive information about key enzymes involved in the haloarchaeal PEP/pyruvate interconversion, and we also report a high-yielding PHBV strain with great future potentials.IMPORTANCEArchaea, the third domain of life, have evolved diversified metabolic pathways to cope with their extreme habitats. Phosphoenol pyruvate (PEP)/pyruvate interconversion during carbohydrate metabolism is one such important metabolic process that is highly differentiated among Archaea However, this process is still uncharacterized in the haloarchaeal group. Haloferax mediterranei is a well-studied haloarchaeon that has the ability to produce polyhydroxyalkanoates (PHAs) under unbalanced nutritional conditions. In this study, we identified the key enzymes involved in this interconversion and discussed their differences with their counterparts from other members of the Archaea and Bacteria domains. Notably, we found a novel protein, phosphoenolpyruvate synthetase-like (PPS-like), which exhibited high homology to PPS enzyme. However, PPS-like protein has evolved some distinct sequence features and functions, and strikingly the corresponding gene deletion helped to enhance poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) synthesis significantly. Overall, we have filled the gap in knowledge about PEP/pyruvate interconversion in haloarchaea and reported an efficient strategy for improving PHBV production in H. mediterranei.

Purification and characterization of an organic solvent-tolerant and detergent-stable lipase from Haloferax mediterranei CNCMM 50101.

The present study investigates the purification and biochemical characterization of an extracellular lipase (HML) from Haloarchaea Haloferax mediterranei strain ATS1, isolated from the Sebkha (Medea, Algeria). The pure protein was obtained with ammonium sulfate precipitation (40-70%)-dialysis and UNO Q-6 FPLC, and characterized biochemically. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis revealed that the purified enzyme was a monomer, with a molecular mass of 45,011.09 Da. It showed optimum lipase activity at pH 7 and 60 °C. HML showed a higher specific activity on triacylglycerols with long-chains fatty acids, indicating that HML is a true lipase. This enzyme was completely inhibited by phenylmethanesulfonyl fluoride (PMSF) and diiodopropyl fluorophosphates (DFP), which suggested its belonging to the serine lipase family. The Km and Vmax for HML toward olive oil were 1.01 mM and 1195 U/mg, respectively. Compared to Lipolase, HML displayed an elevated organic solvent tolerance, an outstanding stability to surfactants, oxidizing, and auxiliary agents, a considerable compatibility with various commercialized laundry detergents, and wash performance analysis revealed that it could remove oil-stains effectively. Overall, HML has a number of attractive properties that make it a potential promising candidate for the synthesis of non-aqueous peptides and detergent formulations.

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.

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.

Propionyl coenzyme A (propionyl-CoA) carboxylase in Haloferax mediterranei: Indispensability for propionyl-CoA assimilation and impacts on global metabolism.

Propionyl coenzyme A (propionyl-CoA) is an important intermediate during the biosynthesis and catabolism of intracellular carbon storage of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) in haloarchaea. However, the haloarchaeal propionyl-CoA carboxylase (PCC) and its physiological significance remain unclear. In this study, we identified a PCC that catalyzed propionyl-CoA carboxylation with an acetyl-CoA carboxylation side activity in Haloferax mediterranei. Gene knockout/complementation demonstrated that the PCC enzyme consisted of a fusion protein of a biotin carboxylase and a biotin-carboxyl carrier protein (PccA [HFX_2490]), a carboxyltransferase component (PccB [HFX_2478]), and an essential small subunit (PccX [HFX_2479]). Knockout of pccBX led to an inability to utilize propionate and a higher intracellular propionyl-CoA level, indicating that the PCC enzyme is indispensable for propionyl-CoA utilization. Interestingly, H. mediterranei DBX (pccBX-deleted strain) displayed multiple phenotypic changes, including retarded cell growth, decreased glucose consumption, impaired PHBV biosynthesis, and wrinkled cells. A propionyl-CoA concentration equivalent to the concentration that accumulated in DBX cells was demonstrated to inhibit succinyl-CoA synthetase of the tricarboxylic acid cycle in vitro. Genome-wide microarray analysis showed that many genes for glycolysis, pyruvate oxidation, PHBV accumulation, electron transport, and stress responses were affected in DBX. This study not only identified the haloarchaeal PCC for the metabolism of propionyl-CoA, an important intermediate in haloarchaea, but also demonstrated that impaired propionyl-CoA metabolism affected global metabolism in H. mediterranei.

Characterization of genes for chitin catabolism in Haloferax mediterranei.

Chitin is the second most abundant natural polysaccharide after cellulose. But degradation of chitin has never been reported in haloarchaea. In this study, we revealed that Haloferax mediterranei, a metabolically versatile haloarchaeon, could utilize colloidal or powdered chitin for growth and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) accumulation, and the gene cluster (HFX_5025-5039) for the chitin catabolism pathway was experimentally identified. First, reverse transcription polymerase chain reaction results showed that the expression of the genes encoding the four putative chitinases (ChiAHme, ChiBHme, ChiCHme, and ChiDHme, HFX_5036-5039), the LmbE-like deacetylase (DacHme, HFX_5027), and the glycosidase (GlyAHme, HFX_5029) was induced by colloidal or powdered chitin, and chiA Hme, chiB Hme, and chiC Hme were cotranscribed. Knockout of chiABC Hme or chiD Hme had a significant effect on cell growth and PHBV production when chitin was used as the sole carbon source, and the chiABCD Hme knockout mutant lost the capability to utilize chitin. Knockout of dac Hme or glyA Hme also decreased PHBV accumulation on chitin. These results suggested that ChiABCDHme, DacHme, and GlyAHme were indeed involved in chitin degradation in H. mediterranei. Additionally, the chitinase assay showed that each chitinase possessed hydrolytic activity toward colloidal or powdered chitin, and the major product of colloidal chitin hydrolysis by ChiABCDHme was diacetylchitobiose, which was likely further degraded to monosaccharides by DacHme, GlyAHme, and other related enzymes for both cell growth and PHBV biosynthesis. Taken together, this study revealed the genes and enzymes involved in chitin catabolism in haloarchaea for the first time and indicated the potential of H. mediterranei as a whole-cell biocatalyst in chitin bioconversion.

Ferredoxin-dependent glutamate synthase: involvement in ammonium assimilation in Haloferax mediterranei.

Glutamate synthase (GOGAT) is one of the two important enzymes involved in the ammonium assimilation pathway glutamine synthetase (GS)/GOGAT, which enables Hfx. mediterranei to thrive in media with low ammonium concentration or containing just nitrate as single nitrogen source. The gene coding for this enzyme, gltS, has been sequenced, analysed and compared with other GOGATs from different organisms from the three domains of life. According to its amino acid sequence, Hfx. mediterranei GOGAT displays high homology with those from other archaeal halophilic organisms and with the bacterial alpha-like subunit. Hfx. mediterranei GOGAT and GS expression was induced under conditions of ammonium restriction. The GOGAT protein was found to be a monomer with a molecular mass of 163.78 kDa, which is consistent with that estimated by gel filtration, 198 ± 30 kDa. The enzyme is highly ferredoxin dependent: activity was only observed with one of the two different 2Fe-2S ferredoxins chromatographically isolated from Hfx. mediterranei. The enzyme also displayed typical halophilic behaviour, being fully stable, and producing maximal activity, at salt concentrations from 3 to 4 M NaCl, pH 7.5 and a temperature of 50 °C.

Haloarchaeal-type ß-ketothiolases involved in Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) synthesis in Haloferax mediterranei.

The key enzymes for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) biosynthesis in haloarchaea have been identified except the ß-ketothiolase(s), which condense two acetyl coenzyme A (acetyl-CoA) molecules to acetoacetyl-CoA, or one acetyl-CoA and one propionyl-CoA to 3-ketovaleryl-CoA. Whole-genome analysis has revealed eight potential ß-ketothiolase genes in the haloarchaeon Haloferax mediterranei, among which the PHBV-specific BktB and PhaA were identified by gene knockout and complementation analysis. Unlike all known bacterial counterparts encoded by a single gene, the haloarchaeal PhaA that was involved in acetoacetyl-CoA generation, was composed of two different types of subunits (PhaAα and PhaAß) and encoded by the cotranscribed HFX_1023 (phaAα) and HFX_1022 (phaAß) genes. Similarly, the BktB that was involved in generation of acetoacetyl-CoA and 3-ketovaleryl-CoA, was also composed of two different types of subunits (BktBα and BktBß) and encoded by cotranscribed HFX_6004 (bktBα) and HFX_6003 (bktBß). BktBα and PhaAα were the catalytic subunits and determined substrate specificities of BktB and PhaA, respectively. Their catalytic triad "Ser-His-His" was distinct from the bacterial "Cys-His-Cys." BktBß and PhaAß both contained an oligosaccharide-binding fold domain, which was essential for the ß-ketothiolase activity. Interestingly, BktBß and PhaAß were functionally interchangeable, although PhaAß preferred functioning with PhaAα. In addition, BktB showed biotechnological potential for the production of PHBV with the desired 3-hydroxyvalerate fraction in haloarchaea. This is the first report of the haloarchaeal type of PHBV-specific ß-ketothiolases, which are distinct from their bacterial counterparts in both subunit composition and catalytic residues.

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.

Role of the denitrifying Haloarchaea in the treatment of nitrite-brines

Haloferax mediterranei is a denitrifying halophilic archaeon able to reduce nitrate and nitrite under oxic and anoxic conditions. In the presence of oxygen, nitrate and nitrite are used as nitrogen sources for growth. Under oxygen scarcity, this haloarchaeon uses both ions as electron acceptors via a denitrification pathway. In the present work, the maximal nitrite concentration tolerated by this organism was determined by studying the growth of H. mediterranei in minimal medium containing 30, 40 and 50 mM nitrite as sole nitrogen source and under initial oxic conditions at 42 °C. The results showed the ability of H. mediterranei to withstand nitrite concentrations up to 50 mM. At the beginning of the incubation, nitrate was detected in the medium, probably due to the spontaneous oxidation of nitrite under the initial oxic conditions. The complete removal of nitrite and nitrate was accomplished in most of the tested conditions, except in culture medium containing 50 mM nitrite, suggesting that this concentration compromised the denitrification capacity of the cells. Nitrite and nitrate reductases activities were analyzed at different growth stages of H. mediterranei. In all cases, the activities of the respiratory enzymes were higher than their assimilative counterparts; this was especially the case for NirK. The denitrifying and possibly detoxifying role of this enzyme might explain the high nitrite tolerance of H. mediterranei. This archaeon was also able to remove 60 % of the nitrate and 75 % of the nitrite initially present in brine samples collected from a wastewater treatment facility. These results suggest that H. mediterranei, and probably other halophilic denitrifying Archaea, are suitable candidates for the bioremediation of brines with high nitrite and nitrate concentrations (AU)

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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.

Cyclodextrin glycosyltransferase: a key enzyme in the assimilation of starch by the halophilic archaeon Haloferax mediterranei.

A cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) was successfully isolated and characterized from the halophilic archaeon Haloferax mediterranei. The enzyme is a monomer with a molecular mass of 77 kDa and optimum activity at 55°C, pH 7.5 and 1.5 M NaCl. The enzyme displayed many activities related to the degradation and transformation of starch. Cyclization was found to be the predominant activity, yielding a mixture of cyclodextrins, mainly α-CD, followed by hydrolysis and to a lesser extent coupling and disproportionation activities. Gene encoding H. mediterranei CGTase was cloned and heterologously overexpressed. Sequence analysis revealed an open reading frame of 2142 bp that encodes a protein of 713 amino acids. The amino acid sequence displayed high homology with those belonging to the α-amylase family. The CGTase is secreted to the extracellular medium by the Tat pathway. Upstream of the CGTase gene, four maltose ABC transporter genes have been sequenced (malE, malF, malG, malK). The expression of the CGTase gene yielded a fully active CGTase with similar kinetic behavior to the wild-type enzyme. The H. mediterranei CGTase is the first halophilic archaeal CGTase characterized, sequenced and expressed.

Role of the denitrifying Haloarchaea in the treatment of nitrite-brines.

Haloferax mediterranei is a denitrifying halophilic archaeon able to reduce nitrate and nitrite under oxic and anoxic conditions. In the presence of oxygen, nitrate and nitrite are used as nitrogen sources for growth. Under oxygen scarcity, this haloarchaeon uses both ions as electron acceptors via a denitrification pathway. In the present work, the maximal nitrite concentration tolerated by this organism was determined by studying the growth of H. mediterranei in minimal medium containing 30, 40 and 50 mM nitrite as sole nitrogen source and under initial oxic conditions at 42 degrees C. The results showed the ability of H. mediterranei to withstand nitrite concentrations up to 50 mM. At the beginning of the incubation, nitrate was detected in the medium, probably due to the spontaneous oxidation of nitrite under the initial oxic conditions. The complete removal of nitrite and nitrate was accomplished in most of the tested conditions, except in culture medium containing 50 mM nitrite, suggesting that this concentration compromised the denitrification capacity of the cells. Nitrite and nitrate reductases activities were analyzed at different growth stages of H. mediterranei. In all cases, the activities of the respiratory enzymes were higher than their assimilative counterparts; this was especially the case for NirK. The denitrifying and possibly detoxifying role of this enzyme might explain the high nitrite tolerance of H. mediterranei. This archaeon was also able to remove 60% of the nitrate and 75% of the nitrite initially present in brine samples collected from a wastewater treatment facility. These results suggest that H. mediterranei, and probably other halophilic denitrifying Archaea, are suitable candidates for the bioremediation of brines with high nitrite and nitrate concentrations.

In vitro proof of direct regulation of glutamine synthetase by GlnK proteins in the extreme halophilic archaeon Haloferax mediterranei.

Haloferax mediterranei is an extreme halophilic micro-organism belonging to the Archaea domain that was isolated from the Santa Pola solar salterns (Alicante, Spain) in 1983. The biochemistry of the proteins involved in nitrogen metabolism is being studied, but the knowledge of their regulation is very scarce at present. The PII superfamily is constituted by major regulators of nitrogen metabolism, which are widespread in prokaryotic and eukaryotic organisms. These trimeric proteins (12 kDa per subunit) have in Escherichia coli long been known to regulate GS (glutamine synthetase) activity via its adenylyltransferase/adenylyl-removing enzyme and, more recently, to be able to interact directly with this enzyme in methanogenic archaea. We have tested the possible role of PII proteins in the regulation of ammonium assimilation in our model organism and the results clearly indicate that the direct influence of GS by PII proteins can also take place in halophilic archaea, starting with the comprehension of nitrogen regulation in those organisms.

[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.

Synthesis and production of polyhydroxyalkanoates by halophiles: current potential and future prospects.

Biodegradable materials with plastic or elastomeric properties are in great demand for a variety of applications. Polyhydroxyalkanoates (PHAs), polyesters synthesized by microorganisms, possess such desired features. Industrial production of PHAs is currently achieved using recombinant Escherichia coli. Nevertheless, recent research on halophiles, salt requiring microorganisms, has shown a remarkable potential for biotechnological production of PHAs. The halophilic archaeon Haloferax mediterranei accumulates a co-polymer, i.e., poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in large amounts using glucose, starch, and hydrolyzed whey as carbon sources. Chemical composition and molecular weight of PHAs produced by H. mediterranei can be modified depending on the substrate utilized as precursor. Phylogenetic studies on haloarchaeal enzymes able to polymerize the components of PHAs (i.e., PHA synthases) reveal a novel cluster, with a close relationship with PHA polymerases of bacteria and archaea found in marine-related niches. On the other hand, sequences of PHA synthases of two halophilic bacteria are more closely affiliated to synthases of Proteobacteria. Several bacterial species of the family Halomonadaceae accumulate PHAs. Halomonas boliviensis reached PHA yields and volumetric productivities close to the highest reported so far. Furthermore, H. boliviensis and other Halomonas species are able to co-produce PHA and osmolytes, i.e., ectoines and hydroxyectoine, in one process.

Crystallization and preliminary X-ray analysis of D-2-hydroxyacid dehydrogenase from Haloferax mediterranei.

D-2-hydroxyacid dehydrogenase (D2-HDH) from Haloferax mediterranei has been overexpressed in Escherichia coli, solubilized in 8 M urea and refolded by rapid dilution. The protein was purified and crystallized by the hanging-drop vapour-diffusion method using ammonium sulfate or PEG 3350 as precipitant. Two crystal forms representing the free enzyme and the nonproductive ternary complex with alpha-ketohexanoic acid and NAD(+) grew under these conditions. Crystals of form I diffracted to beyond 3.0 A resolution and belonged to the monoclinic space group P2(1), with unit-cell parameters a = 66.0, b = 119.6, c = 86.2 A, beta = 96.3 degrees . Crystals of form II diffracted to beyond 2.0 A resolution and belonged to the triclinic space group P1, with unit-cell parameters a = 66.5, b = 75.2, c = 77.6 A, alpha = 109.1, beta = 107.5, gamma = 95.9 degrees. The calculated values for V(M) and analysis of the self-rotation and self-Patterson functions suggest that the asymmetric unit in both crystal forms contains two dimers related by pseudo-translational symmetry.

Active site dynamics in the zinc-dependent medium chain alcohol dehydrogenase superfamily.

Despite being the subject of intensive investigations, many aspects of the mechanism of the zinc-dependent medium chain alcohol dehydrogenase (MDR) superfamily remain contentious. We have determined the high-resolution structures of a series of binary and ternary complexes of glucose dehydrogenase, an MDR enzyme from Haloferax mediterranei. In stark contrast to the textbook MDR mechanism in which the zinc ion is proposed to remain stationary and attached to a common set of protein ligands, analysis of these structures reveals that in each complex, there are dramatic differences in the nature of the zinc ligation. These changes arise as a direct consequence of linked movements of the zinc ion, a zinc-bound bound water molecule, and the substrate during progression through the reaction. These results provide evidence for the molecular basis of proton traffic during catalysis, a structural explanation for pentacoordinate zinc ion intermediates, a unifying view for the observed patterns of metal ligation in the MDR family, and highlight the importance of dynamic fluctuations at the metal center in changing the electrostatic potential in the active site, thereby influencing the proton traffic and hydride transfer events.