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1.
Mol Genet Genomics ; 295(3): 775-785, 2020 May.
Artículo en Inglés | MEDLINE | ID: mdl-32170429

RESUMEN

The regulatory networks involved in the uptake and metabolism of different nitrogen sources in response to their availability are crucial in all organisms. Nitrogen metabolism pathways have been studied in detail in archaea such as the extreme halophilic archaeon Haloferax mediterranei. However, knowledge about nitrogen metabolism regulation in haloarchaea is very scarce, and no transcriptional regulators involved in nitrogen metabolism have been identified to date. Advances in the molecular biology field have revealed that many small RNAs (sRNAs) are involved in the regulation of a diverse metabolic pathways. Surprisingly, no studies on regulation mediated by sRNAs have focused on the response to environmental fluctuations in nitrogen in haloarchaea. To identify sRNAs involved in the transcriptional regulation of nitrogen assimilation genes in Haloferax mediterranei and, thus, propose a novel regulatory mechanism, RNA-Seq was performed using cells grown in the presence of two different nitrogen sources. The differential transcriptional expression analysis of the RNA-Seq data revealed differences in the transcription patterns of 102 sRNAs according to the nitrogen source, and the molecular functions, cellular locations and biological processes with which the target genes were associated were predicted. These results enabled the identification of four sRNAs that could be directly related to the regulation of genes involved in nitrogen metabolism. This work provides the first proposed regulatory mechanism of nitrogen assimilation-related gene expression by sRNAs in haloarchaea as an alternative to transcriptional regulation mediated by proteins.


Asunto(s)
Proteínas Arqueales/genética , Regulación de la Expresión Génica Arqueal , Haloferax mediterranei/genética , Haloferax mediterranei/metabolismo , Nitrógeno/metabolismo , ARN de Archaea/genética , ARN Pequeño no Traducido/genética , Perfilación de la Expresión Génica , Haloferax mediterranei/crecimiento & desarrollo
2.
Appl Environ Microbiol ; 81(2): 794-804, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-25398867

RESUMEN

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.


Asunto(s)
Acilcoenzima A/metabolismo , Haloferax mediterranei/enzimología , Haloferax mediterranei/crecimiento & desarrollo , Metilmalonil-CoA Descarboxilasa/metabolismo , Poliésteres/metabolismo , Carbono/metabolismo , Perfilación de la Expresión Génica , Técnicas de Inactivación de Genes , Prueba de Complementación Genética , Glucosa/metabolismo , Haloferax mediterranei/genética , Haloferax mediterranei/metabolismo , Redes y Vías Metabólicas/genética , Metilmalonil-CoA Descarboxilasa/genética , Subunidades de Proteína/genética
3.
Extremophiles ; 18(2): 463-70, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-24442255

RESUMEN

Haloferax mediterranei holds promise for competitive industrial-scale production of polyhydroxyalkanoate (PHA) because cheap carbon sources can be used thus lowering production costs. Although high salt concentration in production medium permits a non-sterile, low-cost process, salt disposal after process completion is a problem as current environmental standards do not allow total dissolved solids (TDS) above 2000 mg/l in discharge water. As the first objective of this work, the waste product of rice-based ethanol industry, stillage, was used for the production of PHA by H. mediterranei in shake flasks. Utilization of raw stillage led to 71 ± 2% (of dry cell weight) PHA accumulation and 16.42 ± 0.02 g/l PHA production. The product yield coefficient was 0.35 while 0.17 g/l h volumetric productivity was attained. Simultaneous reduction of BOD5 and COD values of stillage by 83% was accomplished. The PHA was isolated by osmotic lysis of cells, purification by sodium dodecyl sulfate and organic solvents. The biopolymer was identified as poly-3-(hydroxybutyrate-co-15.4 mol%-hydroxyvalerate) (PHBV). This first report on utilization of rice-based ethanol stillage for PHBV production by H. mediterranei is currently the most cost effective. As the second objective, directional properties of decanoic acid together with temperature dependence of water solubility in decanoic acid were applied for two-stage desalination of the spent stillage medium. We report for the first time, recovery and re-use of 96% of the medium salts for PHA production thus removing the major bottleneck in the potential application of H. mediterranei for industrial production of PHBV. Final discharge water had TDS content of 670 mg/l.


Asunto(s)
Biotecnología/métodos , Etanol/metabolismo , Haloferax mediterranei/metabolismo , Oryza/química , Poliésteres/metabolismo , Fermentación , Haloferax mediterranei/crecimiento & desarrollo , Poliésteres/química , Sales (Química)/metabolismo
4.
Appl Microbiol Biotechnol ; 98(3): 1185-94, 2014 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-23674154

RESUMEN

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.


Asunto(s)
Quitina/metabolismo , Haloferax mediterranei/enzimología , Haloferax mediterranei/genética , Redes y Vías Metabólicas/genética , Familia de Multigenes , Biotransformación , Carbono/metabolismo , Perfilación de la Expresión Génica , Técnicas de Inactivación de Genes , Haloferax mediterranei/crecimiento & desarrollo , Poliésteres/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
5.
Appl Microbiol Biotechnol ; 97(7): 3027-36, 2013 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-23015099

RESUMEN

Haloferax mediterranei is capable of producing large amounts of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) from many kinds of carbon sources, with exopolysaccharide (EPS) as a by-product. In this study, we identified a gene cluster involved in EPS biosynthesis in H. mediterranei. Knocking out the genes in this cluster encoding the putative UDP-N-acetylglucosamine 6-dehydrogenase (HFX_2145), glycosyltransferases (HFX_2146 and HFX_2147) and polysaccharide transporter (HFX_2148) eliminated EPS synthesis. The deficiency in EPS biosynthesis in the mutant strain remarkably decreased the viscosity of culture broth, and hence increased the dissolved oxygen content and decreased the foaming propensity. Compared with the wild-type (WT) strain, the PHBV production of the EPS-mutant strain was significantly enhanced (approximately 20%), whereas the cell growth rate remained similar under the same culture conditions. These results indicated that the carbon sources used for synthesizing EPS were shifted to PHBV production. Thus, a novel engineered H. mediterranei strain was developed, which would be favorable for future industrial production of PHBV.


Asunto(s)
Técnicas de Inactivación de Genes , Haloferax mediterranei/genética , Haloferax mediterranei/metabolismo , Polihidroxialcanoatos/biosíntesis , Haloferax mediterranei/crecimiento & desarrollo , Ingeniería Metabólica/métodos , Redes y Vías Metabólicas/genética , Familia de Multigenes , Polihidroxialcanoatos/genética
6.
Appl Environ Microbiol ; 78(6): 1946-52, 2012 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-22247127

RESUMEN

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.


Asunto(s)
Haloferax mediterranei/enzimología , Haloferax mediterranei/genética , Lectinas de Plantas/genética , Lectinas de Plantas/metabolismo , Polihidroxialcanoatos/metabolismo , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Gránulos Citoplasmáticos/ultraestructura , ADN de Archaea/química , ADN de Archaea/genética , Electroforesis en Gel Bidimensional , Técnicas de Inactivación de Genes , Prueba de Complementación Genética , Haloferax mediterranei/crecimiento & desarrollo , Haloferax mediterranei/metabolismo , Microscopía Electrónica de Transmisión , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción
7.
J Microbiol Biotechnol ; 31(2): 338-347, 2021 Feb 28.
Artículo en Inglés | MEDLINE | ID: mdl-33203825

RESUMEN

Polyhydroxyalkanoates (PHA) are a family of microbial polyesters that are used as biodegradable plastics in replacement of conventional plastics for various applications. However, the high production cost is the barrier for PHA market expansion. This study aimed to utilize food waste as low-cost feedstock to produce poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) by Haloferax mediterranei. The effects of acetate (Ac), propionate (Pr), butyrate (Bu), and the short-chain carboxylates derived from food waste were examined on the microbial growth and PHBV production. Results showed that a mixture of carboxylates provided a 55% higher PHBV yield than glucose. The food-waste-derived nutrients achieved the yields of 0.41 to 0.54 g PHBV/g Ac from initial loadings of 450 mg/l to 1,800 mg/l Ac of total carboxylates. And the consumption of individual carboxylate varied between different compositions of the carbon source. The present study demonstrates the potential of using food waste as feedstock to produce PHBV by Haloferax mediterranei, which can provide economic benefits to the current PHA industry. Meanwhile, it will also help promote organic waste reduction in landfills and waste management in general.


Asunto(s)
Plásticos Biodegradables/metabolismo , Haloferax mediterranei/metabolismo , Poliésteres/metabolismo , Residuos/análisis , Fermentación , Haloferax mediterranei/crecimiento & desarrollo , Eliminación de Residuos
8.
Appl Microbiol Biotechnol ; 85(6): 1687-96, 2010 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-20024541

RESUMEN

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.


Asunto(s)
Proteínas Arqueales/metabolismo , Proteínas Bacterianas/metabolismo , Haloferax mediterranei/enzimología , Halomonas/enzimología , Ligasas/metabolismo , Poliésteres/metabolismo , Proteínas Arqueales/genética , Proteínas Bacterianas/genética , Escherichia coli/enzimología , Escherichia coli/genética , Glucosa/metabolismo , Haloferax mediterranei/genética , Haloferax mediterranei/crecimiento & desarrollo , Halomonas/genética , Halomonas/crecimiento & desarrollo , Ligasas/genética , Filogenia , Poliésteres/química , Almidón/metabolismo
9.
Microbiologyopen ; 9(8): e1055, 2020 08.
Artículo en Inglés | MEDLINE | ID: mdl-32410392

RESUMEN

The extreme halophilic archaeon, Haloferax mediterranei can accumulate polyhydroxyalkanoate (PHA) from different renewable resources. To enhance the biosynthesis and quality of PHA, H. mediterranei cultivation media was optimized at different C/N ratios using glucose as the main carbon source. Three sets of media (yeast extract [YE], NH4 Cl and combination of YE and NH4 Cl) were prepared at different nitrogen concentrations to achieve C/N ratios of 9, 20, and 35, respectively. The media containing YE (organic nitrogen source) produced a higher growth rate of H. mediterranei than NH4 Cl (inorganic source) at all tested C/N ratios. The highest PHA accumulation (18.4% PHA/cell dry mass) was achieved in a media that combined YE with NH4 Cl at a C/N ratio of 20. Analysis of the produced polymers revealed the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBHV) with different 3-hydroxyvalerate (3HV) content. The polymers produced from YE and the combined media have greater 3HV content (10 mol%) than those polymers recovered from NH4 Cl (1.5 mol%). Resultingly, PHBHV from YE and the combined media displayed reduced melting points at 144°C. The nitrogen type/concentration was found to also have an impact on the molecular weights and polydispersity indices of the produced biopolymers. Furthermore, the tensile strengths were found to vary with the best tensile strength (14.4 MPa) being recorded for the polymer recovered from YE at C/N = 9. Interestingly, the tensile strength of PHBHV was significantly higher than petroleum-based polyethylene (13.5 MPa), making it a much more suitable bioplastic for industrial application.


Asunto(s)
Cloruro de Amonio/metabolismo , Haloferax mediterranei/metabolismo , Compuestos Orgánicos/metabolismo , Poliésteres/metabolismo , Contaminación Química del Agua/análisis , Extractos Celulares , Fermentación , Haloferax mediterranei/crecimiento & desarrollo , Nitrógeno/metabolismo , Polihidroxialcanoatos/metabolismo
10.
Extremophiles ; 13(5): 785-92, 2009 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-19593595

RESUMEN

The haloarchaeon Haloferax mediterranei is able to assimilate nitrate or nitrite using the assimilatory nitrate pathway. An assimilatory nitrate reductase (Nas) and an assimilatory nitrite reductase (NiR) catalyze the first and second reactions, respectively. The genes involved in this process are transcribed as two messengers, one polycistronic (nasABC; nasA encodes Nas) and one monocistronic (nasD; codes for NiR). Here we report the Hfx mediterranei growth as well as the Nas and NiR activities in presence of high nitrate, nitrite and salt concentrations, using different approaches such as physiological experiments and enzymatic activities assays. The nasA and nasD expression profiles are also analysed by real-time quantitative PCR. The results presented reveal that the assimilatory nitrate/nitrite pathway in Hfx mediterranei takes place even if the salt concentration is higher than those usually present in the environments where this microorganism inhabits. This haloarchaeon grows in presence of 2 M nitrate or 50 mM nitrite, which are the highest nitrate and nitrite concentrations described from a prokaryotic microorganism. Therefore, it could be attractive for bioremediation applications in sewage plants where high salt, nitrate and nitrite concentrations are detected in wastewaters and brines.


Asunto(s)
Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Genes Arqueales , Haloferax mediterranei/genética , Haloferax mediterranei/metabolismo , Nitrato-Reductasa/genética , Nitrato-Reductasa/metabolismo , Nitrito Reductasas/genética , Nitrito Reductasas/metabolismo , Secuencia de Bases , Cartilla de ADN/genética , ADN de Archaea/genética , Expresión Génica , Haloferax mediterranei/crecimiento & desarrollo , Nitratos/metabolismo , Nitritos/metabolismo , Salinidad
11.
Nat Microbiol ; 4(1): 177-186, 2019 01.
Artículo en Inglés | MEDLINE | ID: mdl-30478289

RESUMEN

CRISPR-Cas systems provide prokaryotes with sequence-specific immunity against viruses and plasmids based on DNA acquired from these invaders, known as spacers. Surprisingly, many archaea possess spacers that match chromosomal genes of related species, including those encoding core housekeeping genes. By sequencing genomes of environmental archaea isolated from a single site, we demonstrate that inter-species spacers are common. We show experimentally, by mating Haloferax volcanii and Haloferax mediterranei, that spacers are indeed acquired chromosome-wide, although a preference for integrated mobile elements and nearby regions of the chromosome exists. Inter-species mating induces increased spacer acquisition and may result in interactions between the acquisition machinery of the two species. Surprisingly, many of the spacers acquired following inter-species mating target self-replicons along with those originating from the mating partner, indicating that the acquisition machinery cannot distinguish self from non-self under these conditions. Engineering the chromosome of one species to be targeted by the other's CRISPR-Cas reduces gene exchange between them substantially. Thus, spacers acquired during inter-species mating could limit future gene transfer, resulting in a role for CRISPR-Cas systems in microbial speciation.


Asunto(s)
Sistemas CRISPR-Cas/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , ADN Intergénico/genética , Transferencia de Gen Horizontal/genética , Haloferax mediterranei/genética , Haloferax volcanii/genética , Especiación Genética , Haloferax mediterranei/crecimiento & desarrollo , Haloferax volcanii/crecimiento & desarrollo
12.
FEMS Microbiol Lett ; 277(1): 50-5, 2007 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-17986084

RESUMEN

Haloferax mediterranei is a halophilic archaeon that can grow using nitrate as the sole nitrogen source. A ferredoxin that serves as the physiological electron donor to the nitrate and nitrite reductases in this assimilatory process has been characterized. The ferredoxin was found to contain approximately two atoms of iron and two atoms of sulphur, indicative of the binding of a [2Fe-2S] cluster. The electron paramagnetic resonance spectrum of the reduced form of the protein displayed a rhombic signal, with g(x)=1.91, g(y)=1.98, g(z)=2.07, that shows considerable similarity to plant and algal [2Fe-2S] ferredoxins. UV-visible spectropotentiometric analysis determined a midpoint redox potential for the [2Fe-2S](2+/1+) transition of around -285 mV vs. SHE that was independent of salt concentration. UV-visible spectroscopy was also used to establish that the [2Fe-2S] cluster integrity of this protein was maintained over the pH range 5-11. Significantly, the Haloferax mediterranei ferredoxin was shown to be a highly thermostable protein. It was stable up to 60 degrees C in a low-salt (0.2 M) medium and this increased to 80 degrees C in a high-salt (4 M) medium. This thermostability at high salt concentration is an essential physiological characteristic because haloarchaea are mainly found in environments where high temperatures and concentrated salt water occur.


Asunto(s)
Espectroscopía de Resonancia por Spin del Electrón/métodos , Ferredoxinas/química , Ferredoxinas/metabolismo , Haloferax mediterranei/enzimología , Calor , Nitratos/metabolismo , Cloruro de Sodio/farmacología , Espectrofotometría Ultravioleta/métodos , Proteínas Arqueales/química , Proteínas Arqueales/metabolismo , Espectroscopía de Resonancia por Spin del Electrón/instrumentación , Transporte de Electrón , Estabilidad de Enzimas , Ferredoxinas/aislamiento & purificación , Haloferax mediterranei/crecimiento & desarrollo , Concentración de Iones de Hidrógeno , Oxidación-Reducción , Espectrofotometría Ultravioleta/instrumentación
13.
N Biotechnol ; 34: 47-53, 2017 Jan 25.
Artículo en Inglés | MEDLINE | ID: mdl-27224675

RESUMEN

Olive mill wastewater (OMW), a highly polluting waste from the olive oil industry, was utilized as sole carbon source for the production of polyhydroxyalkanoate (PHA) by extremely halophilic Haloferax Mediterranei (H. mediterranei) in a one stage cultivation step. H. mediterranei showed remarkable cell growth and tolerated the inhibitory effect of polyphenols present in medium containing 25% of OMW. H. mediterranei cultivation conditions were optimized in medium containing 15% OMW by investigating several parameters that affect the production of PHA. The highest polymer yield (0.2g/L) and PHA content (43% PHA/cell dry mass) were achieved at 37°C, 170rpm and 22% salt concentration. Analysis of the produced PHA revealed the production of copolyester poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBHV) containing 6.5mol% 3-hydroxyvalerate (3HV). The production of PHBHV was observed without the need for fermentation step or adding external carbon source. The PHBHV displayed reduced melting points at 140.1°C and 154.4°C when compared to homopolymer polyhydroxybutyrate.


Asunto(s)
Haloferax mediterranei/metabolismo , Poliésteres/metabolismo , Aguas Residuales/química , Reactores Biológicos/microbiología , Biotecnología , Haloferax mediterranei/crecimiento & desarrollo , Olea , Poliésteres/química , Polihidroxialcanoatos/biosíntesis , Polihidroxialcanoatos/química , Espectroscopía Infrarroja por Transformada de Fourier
14.
FEMS Microbiol Lett ; 264(1): 110-6, 2006 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-17020556

RESUMEN

The glutamine synthetase (EC 6.3.1.2) from the haloarchaeon Haloferax mediterranei has been purified and characterized in order to understand the ammonium assimilation in haloarchaea. Based on sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel-filtration chromatography, the enzyme consists of eight subunits of 51.7 kDa, suggesting that this enzyme belongs to the glutamine synthetase type II. The purified enzyme has been characterized with respect to its optimum temperature (45 degrees C) and pH value (8.0). The optimal NaCl or KCl concentrations for the reaction were 0.5 and 0.25 M, respectively. The effect of l-methionine-d, l-sulphoximine and different divalent metal ions has also been tested. The glutamine synthetase presented here is unusual; it shows the typical characteristic of eukaryotic and soil bacteria glutamine synthetases.


Asunto(s)
Glutamato-Amoníaco Ligasa/química , Haloferax mediterranei/enzimología , Subunidades de Proteína/química , Electroforesis en Gel de Poliacrilamida , Inhibidores Enzimáticos/farmacología , Estabilidad de Enzimas , Glutamato-Amoníaco Ligasa/antagonistas & inhibidores , Glutamato-Amoníaco Ligasa/aislamiento & purificación , Haloferax mediterranei/genética , Haloferax mediterranei/crecimiento & desarrollo , Metionina/farmacología , Metionina Sulfoximina/farmacología , Cloruro de Potasio/química , Estructura Cuaternaria de Proteína , Cloruro de Sodio/química , Temperatura
15.
Nucleic Acids Res ; 30(24): 5436-43, 2002 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-12490712

RESUMEN

The mc-gvp genes for gas vesicle formation in Haloferax mediterranei are transcribed from two promoters located in front of the mc-gvpA and mc-gvpD genes. The different transcripts originating from both promoters show different abundances dependent on salt concentration in the medium and growth phase. Here we show that the half-lives of these transcripts differ significantly and that the small gvp transcripts exhibit higher stabilities than the larger gvp transcripts. While the stability of most gvp transcripts is independent of the salt concentration in the medium, the gvpA mRNA decays about twice as fast in cultures grown at 18% salt compared to cultures grown at 25% salt. The stability of the 0.45 kb transcript population derived from the 5' part of the gvpD gene depends on the growth phase of the culture. Thus, differences in mRNA stability contribute to the salt-dependent and growth phase-dependent abundance of gvp transcripts. This implies that, like in bacteria and eukarya, mRNA processing contributes to regulated gene expression in archaea.


Asunto(s)
Genes Arqueales/genética , Haloferax mediterranei/genética , ARN Mensajero/metabolismo , Cloruro de Sodio/farmacología , Northern Blotting , División Celular/fisiología , Semivida , Haloferax mediterranei/crecimiento & desarrollo , Operón , Procesamiento Postranscripcional del ARN , Estabilidad del ARN , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , Factores de Tiempo , Transcripción Genética
16.
N Biotechnol ; 33(1): 224-30, 2016 Jan 25.
Artículo en Inglés | MEDLINE | ID: mdl-26134839

RESUMEN

Haloferax mediterranei was cultivated in highly saline medium using cheese whey as the substrate for the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate), P(3HB-co-3HV). Acid hydrolysis provided a simple inexpensive method to obtain a cheese whey hydrolysate that was used for cultivation of H. mediterranei. Batch bioreactor cultivation of H. mediterranei resulted in the production of an active biomass concentration of 7.54 g L(-1) with a polymer content of 53%, and a volumetric productivity of 4.04 g L(-1) day(-1). Supplementation of the cultivation medium with micronutrients favored galactose consumption that was used for polymer synthesis after exhaustion of the available glucose. P(3HB-co-3HV) with a 3-hydroxyvalerate content of 1.5 mol% was extracted from the biomass by hypo-osmotic shock. The polymer presented a molecular mass of 4.4×10(5), with a polydispersity index of 1.5. This work demonstrated the feasibility of using cheese whey for the production of a value-added biopolymer with high volumetric productivity, by using a glucose- and galactose-rich substrate obtained by acid hydrolysis of cheese whey. The use of H. mediterranei as the producing strain avoids the need for strict sterility due to the culture's high salinity requirements and, also, allows for polymer extraction by simply contacting the biomass with water.


Asunto(s)
Queso/microbiología , Haloferax mediterranei/metabolismo , Poliésteres/metabolismo , Suero Lácteo/metabolismo , Técnicas de Cultivo Celular por Lotes , Biomasa , Biopolímeros/metabolismo , Reactores Biológicos/microbiología , Galactosa/metabolismo , Glucosa/metabolismo , Haloferax mediterranei/crecimiento & desarrollo , Hidrolisados de Proteína
17.
FEMS Microbiol Lett ; 196(2): 113-8, 2001 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-11267765

RESUMEN

The nitrite reductase from the extreme halophilic archaeon, Haloferax mediterranei, has been purified and characterised. H. mediterranei is capable of growing in a minimal medium (inorganic salts and glucose as a carbon source) with nitrate as the only nitrogen source. The overall purification was 46-fold with about 4% recovery of activity. The enzyme is a monomeric protein of approximately 66 kDa. A pH of 7.5 and high temperatures up to 60 degrees C are necessary for optimum activity. Reduced methyl viologen has been found to be an electron donor as effective as ferredoxin. NADPH and NADH, which are electron donors in nitrite reductases from different non-photosynthetic bacteria, were not effective with nitrite reductase from H. mediterranei.


Asunto(s)
Haloferax mediterranei/enzimología , Nitrito Reductasas/aislamiento & purificación , Ferredoxinas , Haloferax mediterranei/crecimiento & desarrollo , Haloferax mediterranei/aislamiento & purificación , Cinética , Nitrito Reductasas/metabolismo , Paraquat
18.
FEMS Microbiol Lett ; 204(2): 381-5, 2001 Nov 13.
Artículo en Inglés | MEDLINE | ID: mdl-11731152

RESUMEN

Haloferax mediterranei can use nitrate as sole nitrogen source during aerobic growth. We report here the purification and biochemical characterisation of the assimilatory nitrate reductase (EC 1.6.6.2) from H. mediterranei. The enzyme, as isolated, was composed of two subunits (105+/-1.3 kDa and 50+/-1.3 kDa) and behaved as a dimer during gel filtration (132+/-6 kDa). A pH of 9 and elevated temperatures up to 80 degrees C (at 3.1 M NaCl) are necessary for optimum activity. The enzyme stability and activity of the enzyme depend upon the salt concentration. Reduced methyl viologen was as effective as the natural electron donor ferredoxin in the catalytic process. In contrast, NADPH and NADH, which are electron donors in nitrate reductases from different non-photosynthetic bacteria, were ineffective.


Asunto(s)
Haloferax mediterranei/enzimología , Nitrato Reductasas/aislamiento & purificación , Nitrato Reductasas/metabolismo , Nitratos/metabolismo , Ferredoxinas , Haloferax mediterranei/crecimiento & desarrollo , Concentración de Iones de Hidrógeno , Cinética , Nitrato-Reductasa , Nitrato Reductasas/antagonistas & inhibidores , Temperatura
19.
FEMS Microbiol Lett ; 359(2): 134-42, 2014 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-25132231

RESUMEN

Heterotrophic prokaryotic communities that inhabit saltern crystallizer ponds are typically dominated by two species, the archaeon Haloquadratum walsbyi and the bacterium Salinibacter ruber, regardless of location. These organisms behave as 'microbial weeds' as defined by Cray et al. (Microb Biotechnol 6: 453-492, 2013) that possess the biological traits required to dominate the microbiology of these open habitats. Here, we discuss the enigma of the less abundant Haloferax mediterranei, an archaeon that grows faster than any other, comparable extreme halophile. It has a wide window for salt tolerance, can grow on simple as well as on complex substrates and degrade polymeric substances, has different modes of anaerobic growth, can accumulate storage polymers, produces gas vesicles, and excretes halocins capable of killing other Archaea. Therefore, Hfx. mediterranei is apparently more qualified as a 'microbial weed' than Haloquadratum and Salinibacter. However, the former differs because it produces carotenoid pigments only in the lower salinity range and lacks energy-generating retinal-based, light-driven ion pumps such as bacteriorhodopsin and halorhodopsin. We discuss these observations in relation to microbial weed biology in, and the open-habitat ecology of, hypersaline systems.


Asunto(s)
Ecosistema , Haloferax mediterranei/fisiología , Salinidad , Microbiología del Agua , Anaerobiosis , Halobacteriales/crecimiento & desarrollo , Halobacteriales/fisiología , Haloferax mediterranei/crecimiento & desarrollo , Tolerancia a la Sal
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