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María José Bonete - One of the best experts on this subject based on the ideXlab platform.
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analysis of Haloferax Mediterranei lrp transcriptional regulator
Genes, 2021Co-Authors: Laura Matarredona, Monica Camacho, J. Esclapez, Basilio Zafrilla, Mariajose Garciabonete, Belen Esquerra, María José BoneteAbstract:Haloferax Mediterranei is an extremely halophilic archaeon, able to live in hypersaline environments with versatile nutritional requirements, whose study represents an excellent basis in the field of biotechnology. The transcriptional machinery in Archaea combines the eukaryotic basal apparatus and the bacterial regulation mechanisms. However, little is known about molecular mechanisms of gene expression regulation compared with Bacteria, particularly in Haloarchaea. The genome of Hfx. Mediterranei contains a gene, lrp (HFX_RS01210), which encodes a transcriptional factor belonging to Lrp/AsnC family. It is located downstream of the glutamine synthetase gene (HFX_RS01205), an enzyme involved in ammonium assimilation and amino acid metabolism. To study this transcriptional factor more deeply, the lrp gene has been homologously overexpressed and purified under native conditions by two chromatographic steps, namely nickel affinity and gel filtration chromatography, showing that Lrp behaves asa tetrameric protein of approximately 67 kDa. Its promoter region has been characterized under different growth conditions using bgaH as a reporter gene. The amount of Lrp protein was also analyzed by Western blotting in different nitrogen sources and under various stress conditions. To sum up, regarding its involvement in the nitrogen cycle, it has been shown that its expression profile does not change in response to the nitrogen sources tested. Differences in its expression pattern have been observed under different stress conditions, such as in the presence of hydrogen peroxide or heavy metals. According to these results, the Lrp seems to be involved in a general response against stress factors, acting as a first-line transcriptional regulator.
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functional analysis of lsm protein under multiple stress conditions in the extreme haloarchaeon Haloferax Mediterranei
Biochimie, 2021Co-Authors: Gloria Paya, María José Bonete, Monica Camacho, Vanesa Bautista, J. EsclapezAbstract:Abstract The Sm, like-Sm, and Hfq proteins belonging to the Sm superfamily of proteins are represented in all domains of life. These proteins are involved in several RNA metabolism pathways. The functions of bacterial Hfq and eukaryotic Sm proteins have been described, but knowledge about the in vivo functions of archaeal Sm proteins remains limited. This study aims to improve the understanding of Lsm proteins and their role using the haloarchaeon Haloferax Mediterranei as a model microorganism. The Haloferax Mediterranei genome contains one lsm gene that overlaps with the rpl37e gene. To determine the expression of lsm and rpl37e genes and the co-transcription of both, reverse transcription-polymerase chain reaction (RT-PCR) analyses were performed under different standard and stress conditions. The results suggest that the expression of lsm and rpl37e is constitutive. Co-transcription occurs at sub-optimal salt concentrations and temperatures, depending on the growth phase. The halophilic Lsm protein contains two Sm motifs, Sm1 and Sm2, and the sequence encoding the Sm2 motif also constitutes the promoter of the rpl37e gene. To investigate their biological functions, the lsm deletion mutant and the Sm1 motif deletion mutant, where the Sm2 motif remained intact, were generated and characterised. Comparison of the lsm deletion mutant, Sm1 deletion mutant, and the parental strain HM26 under standard and stress growth conditions revealed growth differences. Finally, swarming assays in complex and defined media showed greater swarming capacity in the deletion mutants.
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towards the elucidation of assimilative nasabc operon transcriptional regulation in Haloferax Mediterranei
Genes, 2021Co-Authors: Sandra Pastorsoler, María José Bonete, Monica Camacho, Vanesa Bautista, J. EsclapezAbstract:The assimilatory pathway of the nitrogen cycle in the haloarchaeon Haloferax Mediterranei has been well described and characterized in previous studies. However, the regulatory mechanisms involved in the gene expression of this pathway remain unknown in haloarchaea. This work focuses on elucidating the regulation at the transcriptional level of the assimilative nasABC operon (HFX_2002 to HFX_2004) through different approaches. Characterization of its promoter region using β-galactosidase as a reporter gene and site-directed mutagenesis has allowed us to identify possible candidate binding regions for a transcriptional factor. The identification of a potential transcriptional regulator related to nitrogen metabolism has become a real challenge due to the lack of information on haloarchaea. The investigation of protein-DNA binding by streptavidin bead pull-down analysis combined with mass spectrometry resulted in the in vitro identification of a transcriptional regulator belonging to the Lrp/AsnC family, which binds to the nasABC operon promoter (p.nasABC). To our knowledge, this study is the first report to suggest the AsnC transcriptional regulator as a powerful candidate to play a regulatory role in nasABC gene expression in Hfx. Mediterranei and, in general, in the assimilatory nitrogen pathway.
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the survival of Haloferax Mediterranei under stressful conditions
Microorganisms, 2021Co-Authors: Laura Matarredona, Monica Camacho, J. Esclapez, Basilio Zafrilla, Gloria Bravobarrales, María José BoneteAbstract:Haloarchaea can survive and thrive under exposure to a wide range of extreme environmental factors, which represents a potential interest to biotechnology. Growth responses to different stressful conditions were examined in the haloarchaeon Haloferax Mediterranei R4. It has been demonstrated that this halophilic archaeon is able to grow between 10 and 32.5% (w/v) of sea water, at 32–52 °C, although it is expected to grow in temperatures lower than 32 °C, and between 5.75 and 8.75 of pH. Moreover, it can also grow under high metal concentrations (nickel, lithium, cobalt, arsenic), which are toxic to most living beings, making it a promising candidate for future biotechnological purposes and industrial applications. Inductively Coupled Plasma Mass Spectrometry (ICP-MS) analysis quantified the intracellular ion concentrations of these four metals in Hfx. Mediterranei, concluding that this haloarchaeon can accumulate Li+, Co2+, As5+, and Ni2+ within the cell. This paper is the first report on Hfx. Mediterranei in which multiple stress conditions have been studied to explore the mechanism of stress resistance. It constitutes the most detailed study in Haloarchaea, and, as a consequence, new biotechnological and industrial applications have emerged.
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Essentiality of the glnA gene in Haloferax Mediterranei: gene conversion and transcriptional analysis
Extremophiles, 2020Co-Authors: V. Rodríguez-herrero, G. Payá, V. Bautista, A. Vegara, M. Cortés-molina, M. Camacho, J. Esclapez, María José BoneteAbstract:Glutamine synthetase is an essential enzyme in ammonium assimilation and glutamine biosynthesis. The Haloferax Mediterranei genome has two other glnA -type genes ( glnA2 and glnA3 ) in addition to the glutamine synthetase gene glnA. To determine whether the glnA2 and glnA3 genes can replace glnA in nitrogen metabolism, we generated deletion mutants of glnA . The glnA deletion mutants could not be generated in a medium without glutamine, and thus, glnA is an essential gene in H. Mediterranei . The glnA deletion mutant was achieved by adding 40 mM glutamine to the selective medium. This conditional HM26-Δ glnA mutant was characterised with different approaches in the presence of distinct nitrogen sources and nitrogen starvation. Transcriptomic analysis was performed to compare the expression profiles of the strains HM26-Δ glnA and HM26 under different growth conditions. The glnA deletion did not affect the expression of glnA2, glnA3 and nitrogen assimilation genes under nitrogen starvation. Moreover, the results showed that glnA, glnA2 and glnA3 were not expressed under the same conditions. These results indicated that glnA is an essential gene for H. Mediterranei and, therefore, glnA2 and glnA3 cannot replace glnA in the conditions analysed.
Hua Xiang - One of the best experts on this subject based on the ideXlab platform.
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halolysin r4 of Haloferax Mediterranei confers its host antagonistic and defensive activities
Applied and Environmental Microbiology, 2021Co-Authors: Shaoxing Chen, Siqi Sun, Rui Wang, Hongli Feng, Hua XiangAbstract:Halolysins, which are subtilisin-like serine proteases of haloarchaea, are usually secreted into the extracellular matrix via the twin-arginine translocation pathway. A small number of activated molecules can greatly affect cell growth owing to their proteolytic activity. It is, however, unclear as to whether this proteolysis-based growth inhibition by halolysins conveys antagonistic or defensive effects against other resident abd potentially competitive microorganisms. Here, we report that halolysin R4 (HlyR4), encoded by the hlyR4 gene, is the key enzyme in the initial steps of extracellular protein utilization in Haloferax Mediterranei HlyR4 shows significant antagonistic activity against other haloarchaeal strains. Deletion of hlyR4 completely halts the inhibition activity of Hfx. Mediterranei towards other haloarchaea, while correspondingly, complementation of hlyR4 almost completely restores the inhibition activity. Furthermore, Hfx. Mediterranei strains containing hlyR4 showed a certain amount of resistance to halocins and halolysins in milieu, and this function of hlyR4 is reproducible in Haloarcula hispanica The versatility of HlyR4 enables its host to outcompete other haloarchaea living in the same hypersaline environment. Intriguingly, unlike the growth phase-dependent halolysins SptA and Nep, it is likely that HlyR4 may be secreted independent of growth phase. This study provides a new peptide antibiotics candidate in haloarchaea, as well as new insight towards a better understanding of the ecological roles of halolysins.Importance: This study shows that halolysin R4 from Haloferax Mediterranei provides its host antagonistic and defensive activities against other haloarchaea, which expands our knowledge on the traditional function of haloarchaeal extracellular proteases. Haloarchaeal extracellular serine proteases have been previously discussed as growth-phase-dependent proteins, whereas our study reports constitutive expression of halolysin R4. This work also clearly reveals a hidden diversity of extracellular proteases from haloarchaea. Studies on multifunctional halolysins reveal that they play an important ecological role in shaping microbial community composition and provide a new perspective towards understanding the intricate interactions between haloarchaeal cells in hypersaline environments. HlyR4 can lyse competing cells living in the same environment, and the cell debris may probably be utilized as nutrients, which may constitute an important part of nutrient cycling in extremely hypersaline environments.
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a halocin promotes dna uptake in Haloferax Mediterranei
Frontiers in Microbiology, 2019Co-Authors: Shaoxing Chen, Siqi Sun, Gregory A Korfanty, Jingwen Liu, Hua XiangAbstract:Halocins are antimicrobial peptides or proteins that are produced by halophilic archaea. Although their function in inhibiting the growth of closely related haloarchaeal strains is well known, other physiological functions of halocins have also been proposed in recent years. To unveil the possible function and mechanism of halocins in DNA uptake, the halocin H4 producing strain Haloferax Mediterranei DF50-ΔEPS (incapable of EPS production) was used in this study. We found that deletion of the halH4 resulted in the strain DF50-ΔEPSΔhalH4 which exhibited loss of natural DNA uptake ability. Moreover, supernatants of the halocin producing strain were capable of inducing the ability to uptake DNA. Obviously, halocin is likely responsible for inducing DNA uptake. Cell surface ultrastructures of these strains are varied from strains DF50-ΔEPS to DF50-ΔEPSΔhalH4. The cell surface of strain DF50-ΔEPS is rough due to numerous pinholes, while that of the strain DF50-ΔEPSΔhalH4 is smooth without visible pinholes. The morphology of the halH4 complemented strain, DF50-ΔEPSΔhalH4::H4, shows an intermediate phenotype between strains DF50-ΔEPS and DF50-ΔEPSΔhalH4. We speculate that halocin H4 may accelerate DNA uptake by perforating the cell surface ultrastructure. The halocin H4 may represent a novel inducer or activator of DNA uptake in Hfx. Mediterranei.
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Table_1_Engineering Haloferax Mediterranei as an Efficient Platform for High Level Production of Lycopene.DOC
2018Co-Authors: Zhen-qiang Zuo, Jian Zhou, Jing Han, Dahe Zhao, Qiong Xue, Hua XiangAbstract:Lycopene attracts increasing interests in the pharmaceutical, food, and cosmetic industries due to its anti-oxidative and anti-cancer properties. Compared with other lycopene production methods, such as chemical synthesis or direct extraction from plants, the biosynthesis approach using microbes is more economical and sustainable. In this work, we engineered Haloferax Mediterranei, a halophilic archaeon, as a new lycopene producer. H. Mediterranei has the de novo synthetic pathway for lycopene but cannot accumulate this compound. To address this issue, we reinforced the lycopene synthesis pathway, blocked its flux to other carotenoids and disrupted its competitive pathways. The reaction from geranylgeranyl-PP to phytoene catalyzed by phytoene synthase (CrtB) was identified as the rate-limiting step in H. Mediterranei. Insertion of a strong promoter PphaR immediately upstream of the crtB gene, or overexpression of the heterologous CrtB and phytoene desaturase (CrtI) led to a higher yield of lycopene. In addition, blocking bacterioruberin biosynthesis increased the purity and yield of lycopene. Knock-out of the key genes, responsible for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) biosynthesis, diverted more carbon flux into lycopene synthesis, and thus further enhanced lycopene production. The metabolic engineered H. Mediterranei strain produced lycopene at 119.25 ± 0.55 mg per gram of dry cell weight in shake flask fermentation. The obtained yield was superior compared to the lycopene production observed in most of the engineered Escherichia coli or yeast even when they were cultivated in pilot scale bioreactors. Collectively, this work offers insights into the mechanism involved in carotenoid biosynthesis in haloarchaea and demonstrates the potential of using haloarchaea for the production of lycopene or other carotenoids.
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Engineering Haloferax Mediterranei as an Efficient Platform for High Level Production of Lycopene
Frontiers Media S.A., 2018Co-Authors: Jian Zhou, Hua Xiang, Jing Han, Dahe Zhao, Zhen-qiang Zuo, Qiong XueAbstract:Lycopene attracts increasing interests in the pharmaceutical, food, and cosmetic industries due to its anti-oxidative and anti-cancer properties. Compared with other lycopene production methods, such as chemical synthesis or direct extraction from plants, the biosynthesis approach using microbes is more economical and sustainable. In this work, we engineered Haloferax Mediterranei, a halophilic archaeon, as a new lycopene producer. H. Mediterranei has the de novo synthetic pathway for lycopene but cannot accumulate this compound. To address this issue, we reinforced the lycopene synthesis pathway, blocked its flux to other carotenoids and disrupted its competitive pathways. The reaction from geranylgeranyl-PP to phytoene catalyzed by phytoene synthase (CrtB) was identified as the rate-limiting step in H. Mediterranei. Insertion of a strong promoter PphaR immediately upstream of the crtB gene, or overexpression of the heterologous CrtB and phytoene desaturase (CrtI) led to a higher yield of lycopene. In addition, blocking bacterioruberin biosynthesis increased the purity and yield of lycopene. Knock-out of the key genes, responsible for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) biosynthesis, diverted more carbon flux into lycopene synthesis, and thus further enhanced lycopene production. The metabolic engineered H. Mediterranei strain produced lycopene at 119.25 ± 0.55 mg per gram of dry cell weight in shake flask fermentation. The obtained yield was superior compared to the lycopene production observed in most of the engineered Escherichia coli or yeast even when they were cultivated in pilot scale bioreactors. Collectively, this work offers insights into the mechanism involved in carotenoid biosynthesis in haloarchaea and demonstrates the potential of using haloarchaea for the production of lycopene or other carotenoids
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systematic analysis of lysine acetylation in the halophilic archaeon Haloferax Mediterranei
Journal of Proteome Research, 2017Co-Authors: Jingfang Liu, Jing Han, Dahe Zhao, Qian Wang, Xiongjian Jiang, Haibo Yang, Yuanming Luo, Hua XiangAbstract:Lysine acetylation is a reversible and highly regulated post-translational modification that plays a critical role in regulating many aspects of cellular processes, both in bacteria and in eukaryotes. However, this modification has not been systematically studied in archaea. Herein, we report the lysine acetylome of a model haloarchaeon, Haloferax Mediterranei. Using immunoaffinity enrichment and LC–MS/MS analysis, we identified 1017 acetylation sites in 643 proteins, accounting for 17.3% of the total proteins in this haloarchaeon. Bioinformatics analysis indicated that lysine acetylation mainly distributes in cytoplasm (94%) and participates in protein biosynthesis and carbon metabolism. Specifically, the acetylation of key enzymes in PHBV biosynthesis further suggested that acetylation plays a key role in the energy and carbon storage. In addition, a survey of the acetylome revealed a universal rule in acetylated motifs: a positively charged residue (K, R, or H) located downstream of acetylated lysine a...
Rosa Maria Martinezespinosa - One of the best experts on this subject based on the ideXlab platform.
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analysis of polyhydroxyalkanoates granules in Haloferax Mediterranei by double fluorescence staining with nile red and sybr green by confocal fluorescence microscopy
Polymers, 2021Co-Authors: Veronica Canovas, Carmen Pire, Salvador Garciachumillas, Fuensanta Monzo, Lorena Simocabrera, Carmen Fernandezayuso, Rosa Maria MartinezespinosaAbstract:HaloferaxMediterranei is a haloarchaeon of high interest in biotechnology because it produces and mobilizes intracellular polyhydroxyalkanoate (PHA) granules during growth under stress conditions (limitation of phosphorous in the culture media), among other interesting metabolites (enzymes, carotenoids, etc.). The capability of PHA production by microbes can be monitored with the use of staining-based methods. However, the staining of haloarchaea cells is a challenging task; firstly, due to the high ionic strength of the medium, which is inappropriate for most of dyes, and secondly, due to the low permeability of the haloarchaea S-layer to macromolecules. In this work, Haloferax Mediterranei is used as a halophilic archaeon model to describe an optimized protocol for the visualization and analysis of intracellular PHA granules in living cells. The method is based on double-fluorescence staining using Nile red and SYBR Green by confocal fluorescence microscopy. Thanks to this method, the capability of PHA production by new haloarchaea isolates could be easily monitored.
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Haloferax Mediterranei cells as c50 carotenoid factories
Marine Drugs, 2021Co-Authors: Micaela Giani, Zaida Monterolobato, Ines Garbayo, Carlos Vilchez, Jose M Vega, Rosa Maria MartinezespinosaAbstract:Haloarchaea produce C50 carotenoids such as bacterioruberin, which are of biotechnological in-terest. This study aimed to analyze the effect of different environmental and nutritional conditions on the cellular growth and dynamics of carotenoids accumulation in Haloferax Mediterranei. The maximum production of carotenoids (40 µg·mL−1) was obtained during the stationary phase of growth, probably due to nutrient-limiting conditions (one-step culture). By seven days of culture, 1 mL culture produced 22.4 mg of dry weight biomass containing 0.18 % (w/w) of carotenoids. On the other hand, carbon-deficient cultures (low C/N ratio) were observed to be optimum for C50 bacterioruberin production by Hfx. Mediterranei, but negatively affected the growth of cells. Thus, a two-steps process was evaluated for optimum carotenoids yield. In the first step, a nutri-ent-repleted culture medium enabled the haloarchaea to produce biomass, while in the second step, the biomass was incubated under osmotic stress and in a carbon-deficient medium. Under the conditions used, the obtained biomass contained 0.27% (w/w) of carotenoids after seven days, which accounts for 58.49 µg·mL−1 of carotenoids for a culture with turbidity 14.0.
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carotenoids as a protection mechanism against oxidative stress in Haloferax Mediterranei
Antioxidants, 2020Co-Authors: Micaela Giani, Rosa Maria MartinezespinosaAbstract:Haloarchaea are extremophilic microorganisms that in their natural ecosystem encounter several sources of oxidative stress. They have developed different strategies to cope with these harsh environmental conditions, among which bacterioruberin production is a very notable strategy. Bacterioruberin (BR) is a C50 carotenoid synthesized in response to different types of stress. Previous works demonstrated that it shows interesting antioxidant properties with potential applications in biotechnology. In this study, Haloferax Mediterranei strain R-4 was exposed to different concentrations of the oxidant compound H2O2 to evaluate the effect on carotenoid production focusing the attention on the synthesis of bacterioruberin. Hfx. Mediterranei was able to grow in the presence of H2O2 from 1 mM to 25 mM. Cells produced between 16% and 78% (w/v) more carotenoids under the induced oxidative stress compared to control cultures. HPLC-MS analysis detected BR as the major identified carotenoid and confirmed the gradual increase of BR content as higher concentrations of hydrogen peroxide were added to the medium. These results shed some light on the biological role of bacterioruberin in haloarchaea, provide interesting information about the increase of the cellular pigmentation under oxidative stress conditions and will allow the optimization of the production of this pigment at large scale using these microbes as biofactories.
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Haloferax Mediterranei an archaeal model for denitrification in saline systems characterized through integrated physiological and transcriptional analyses
Frontiers in Microbiology, 2020Co-Authors: Javier Torregrosacrespo, Carmen Pire, Linda Bergaust, Rosa Maria MartinezespinosaAbstract:Haloferax Mediterranei (R4) belongs to the group of halophilic archaea, one of the predominant microbial populations in hypersaline environments. In these ecosystems, the low availability of oxygen pushes the microbial inhabitants toward anaerobic pathways and the presence of N-oxyanions favor denitrification. In a recent study comparing three Haloferax species carrying dissimilatory N-oxide reductases, H. Mediterranei showed promise as a future model for archaeal denitrification. This work further explores the respiratory physiology of this haloarchaeon when challenged with ranges of nitrite and nitrate concentrations and at neutral or sub-neutral pH during the transition to anoxia. Moreover, to begin to understand the transcriptional regulation of N-oxide reductases, detailed gas kinetics was combined with gene expression analyses at high resolution. The results show that H. Mediterranei has an expression pattern similar to that observed in the bacterial Domain, well-coordinated at low concentrations of N-oxyanions. However, it could only sustain a few generations of exponential anaerobic growth, apparently requiring micro-oxic conditions for de novo synthesis of denitrification enzymes. This is the first integrated study within this field of knowledge in haloarchaea and Archaea in general, and it sheds lights on denitrification in salty environments.
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characterisation of chlorate reduction in the haloarchaeon Haloferax Mediterranei
Biochimica et Biophysica Acta, 2015Co-Authors: Rosa Maria Martinezespinosa, D J Richardson, María José BoneteAbstract:Abstract Background Haloferax Mediterranei is a denitrifying haloarchaeon using nitrate as a respiratory electron acceptor under anaerobic conditions in a reaction catalysed by pNarGH. Other ions such as bromate, perchlorate and chlorate can also be reduced. Methods Hfx. Mediterranei cells were grown anaerobically with nitrate as electron acceptor and chlorate reductase activity measured in whole cells and purified nitrate reductase. Results No genes encoding (per)chlorate reductases have been detected either in the Hfx. Mediterranei genome or in other haloarchaea. However, a gene encoding a chlorite dismutase that is predicted to be exported across the cytoplasmic membrane has been identified in Hfx. Mediterranei genome. Cells did not grow anaerobically in presence of chlorate as the unique electron acceptor. However, cells anaerobically grown with nitrate and then transferred to chlorate-containing growth medium can grow a few generations. Chlorate reduction by the whole cells, as well as by pure pNarGH, has been characterised. No clear chlorite dismutase activity could be detected. Conclusions Hfx. Mediterranei pNarGH has its active site on the outer-face of the cytoplasmic membrane and reacts with chlorate and perchlorate. Biochemical characterisation of this enzymatic activity suggests that Hfx. Mediterranei or its pure pNarGH could be of great interest for waste water treatments or to better understand biological chlorate reduction in early Earth or Martian environments. General significance Some archaea species reduce (per)chlorate. However, results here presented as well as those recently reported by Liebensteiner and co-workers [1] suggest that complete perchlorate reduction in archaea follows different rules in terms of biological reactions.
J. Esclapez - One of the best experts on this subject based on the ideXlab platform.
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novel glutamate putrescine ligase activity in Haloferax Mediterranei a new function for glna 2 gene
Biomolecules, 2021Co-Authors: Veronica Rodriguezherrero, Monica Camacho, J. Esclapez, Vanesa Bautista, Arnau Peris, Maria Jose BoneteAbstract: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.
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analysis of Haloferax Mediterranei lrp transcriptional regulator
Genes, 2021Co-Authors: Laura Matarredona, Monica Camacho, J. Esclapez, Basilio Zafrilla, Mariajose Garciabonete, Belen Esquerra, María José BoneteAbstract:Haloferax Mediterranei is an extremely halophilic archaeon, able to live in hypersaline environments with versatile nutritional requirements, whose study represents an excellent basis in the field of biotechnology. The transcriptional machinery in Archaea combines the eukaryotic basal apparatus and the bacterial regulation mechanisms. However, little is known about molecular mechanisms of gene expression regulation compared with Bacteria, particularly in Haloarchaea. The genome of Hfx. Mediterranei contains a gene, lrp (HFX_RS01210), which encodes a transcriptional factor belonging to Lrp/AsnC family. It is located downstream of the glutamine synthetase gene (HFX_RS01205), an enzyme involved in ammonium assimilation and amino acid metabolism. To study this transcriptional factor more deeply, the lrp gene has been homologously overexpressed and purified under native conditions by two chromatographic steps, namely nickel affinity and gel filtration chromatography, showing that Lrp behaves asa tetrameric protein of approximately 67 kDa. Its promoter region has been characterized under different growth conditions using bgaH as a reporter gene. The amount of Lrp protein was also analyzed by Western blotting in different nitrogen sources and under various stress conditions. To sum up, regarding its involvement in the nitrogen cycle, it has been shown that its expression profile does not change in response to the nitrogen sources tested. Differences in its expression pattern have been observed under different stress conditions, such as in the presence of hydrogen peroxide or heavy metals. According to these results, the Lrp seems to be involved in a general response against stress factors, acting as a first-line transcriptional regulator.
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functional analysis of lsm protein under multiple stress conditions in the extreme haloarchaeon Haloferax Mediterranei
Biochimie, 2021Co-Authors: Gloria Paya, María José Bonete, Monica Camacho, Vanesa Bautista, J. EsclapezAbstract:Abstract The Sm, like-Sm, and Hfq proteins belonging to the Sm superfamily of proteins are represented in all domains of life. These proteins are involved in several RNA metabolism pathways. The functions of bacterial Hfq and eukaryotic Sm proteins have been described, but knowledge about the in vivo functions of archaeal Sm proteins remains limited. This study aims to improve the understanding of Lsm proteins and their role using the haloarchaeon Haloferax Mediterranei as a model microorganism. The Haloferax Mediterranei genome contains one lsm gene that overlaps with the rpl37e gene. To determine the expression of lsm and rpl37e genes and the co-transcription of both, reverse transcription-polymerase chain reaction (RT-PCR) analyses were performed under different standard and stress conditions. The results suggest that the expression of lsm and rpl37e is constitutive. Co-transcription occurs at sub-optimal salt concentrations and temperatures, depending on the growth phase. The halophilic Lsm protein contains two Sm motifs, Sm1 and Sm2, and the sequence encoding the Sm2 motif also constitutes the promoter of the rpl37e gene. To investigate their biological functions, the lsm deletion mutant and the Sm1 motif deletion mutant, where the Sm2 motif remained intact, were generated and characterised. Comparison of the lsm deletion mutant, Sm1 deletion mutant, and the parental strain HM26 under standard and stress growth conditions revealed growth differences. Finally, swarming assays in complex and defined media showed greater swarming capacity in the deletion mutants.
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towards the elucidation of assimilative nasabc operon transcriptional regulation in Haloferax Mediterranei
Genes, 2021Co-Authors: Sandra Pastorsoler, María José Bonete, Monica Camacho, Vanesa Bautista, J. EsclapezAbstract:The assimilatory pathway of the nitrogen cycle in the haloarchaeon Haloferax Mediterranei has been well described and characterized in previous studies. However, the regulatory mechanisms involved in the gene expression of this pathway remain unknown in haloarchaea. This work focuses on elucidating the regulation at the transcriptional level of the assimilative nasABC operon (HFX_2002 to HFX_2004) through different approaches. Characterization of its promoter region using β-galactosidase as a reporter gene and site-directed mutagenesis has allowed us to identify possible candidate binding regions for a transcriptional factor. The identification of a potential transcriptional regulator related to nitrogen metabolism has become a real challenge due to the lack of information on haloarchaea. The investigation of protein-DNA binding by streptavidin bead pull-down analysis combined with mass spectrometry resulted in the in vitro identification of a transcriptional regulator belonging to the Lrp/AsnC family, which binds to the nasABC operon promoter (p.nasABC). To our knowledge, this study is the first report to suggest the AsnC transcriptional regulator as a powerful candidate to play a regulatory role in nasABC gene expression in Hfx. Mediterranei and, in general, in the assimilatory nitrogen pathway.
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the survival of Haloferax Mediterranei under stressful conditions
Microorganisms, 2021Co-Authors: Laura Matarredona, Monica Camacho, J. Esclapez, Basilio Zafrilla, Gloria Bravobarrales, María José BoneteAbstract:Haloarchaea can survive and thrive under exposure to a wide range of extreme environmental factors, which represents a potential interest to biotechnology. Growth responses to different stressful conditions were examined in the haloarchaeon Haloferax Mediterranei R4. It has been demonstrated that this halophilic archaeon is able to grow between 10 and 32.5% (w/v) of sea water, at 32–52 °C, although it is expected to grow in temperatures lower than 32 °C, and between 5.75 and 8.75 of pH. Moreover, it can also grow under high metal concentrations (nickel, lithium, cobalt, arsenic), which are toxic to most living beings, making it a promising candidate for future biotechnological purposes and industrial applications. Inductively Coupled Plasma Mass Spectrometry (ICP-MS) analysis quantified the intracellular ion concentrations of these four metals in Hfx. Mediterranei, concluding that this haloarchaeon can accumulate Li+, Co2+, As5+, and Ni2+ within the cell. This paper is the first report on Hfx. Mediterranei in which multiple stress conditions have been studied to explore the mechanism of stress resistance. It constitutes the most detailed study in Haloarchaea, and, as a consequence, new biotechnological and industrial applications have emerged.
Carmen Pire - One of the best experts on this subject based on the ideXlab platform.
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analysis of polyhydroxyalkanoates granules in Haloferax Mediterranei by double fluorescence staining with nile red and sybr green by confocal fluorescence microscopy
Polymers, 2021Co-Authors: Veronica Canovas, Carmen Pire, Salvador Garciachumillas, Fuensanta Monzo, Lorena Simocabrera, Carmen Fernandezayuso, Rosa Maria MartinezespinosaAbstract:HaloferaxMediterranei is a haloarchaeon of high interest in biotechnology because it produces and mobilizes intracellular polyhydroxyalkanoate (PHA) granules during growth under stress conditions (limitation of phosphorous in the culture media), among other interesting metabolites (enzymes, carotenoids, etc.). The capability of PHA production by microbes can be monitored with the use of staining-based methods. However, the staining of haloarchaea cells is a challenging task; firstly, due to the high ionic strength of the medium, which is inappropriate for most of dyes, and secondly, due to the low permeability of the haloarchaea S-layer to macromolecules. In this work, Haloferax Mediterranei is used as a halophilic archaeon model to describe an optimized protocol for the visualization and analysis of intracellular PHA granules in living cells. The method is based on double-fluorescence staining using Nile red and SYBR Green by confocal fluorescence microscopy. Thanks to this method, the capability of PHA production by new haloarchaea isolates could be easily monitored.
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Haloferax Mediterranei an archaeal model for denitrification in saline systems characterized through integrated physiological and transcriptional analyses
Frontiers in Microbiology, 2020Co-Authors: Javier Torregrosacrespo, Carmen Pire, Linda Bergaust, Rosa Maria MartinezespinosaAbstract:Haloferax Mediterranei (R4) belongs to the group of halophilic archaea, one of the predominant microbial populations in hypersaline environments. In these ecosystems, the low availability of oxygen pushes the microbial inhabitants toward anaerobic pathways and the presence of N-oxyanions favor denitrification. In a recent study comparing three Haloferax species carrying dissimilatory N-oxide reductases, H. Mediterranei showed promise as a future model for archaeal denitrification. This work further explores the respiratory physiology of this haloarchaeon when challenged with ranges of nitrite and nitrate concentrations and at neutral or sub-neutral pH during the transition to anoxia. Moreover, to begin to understand the transcriptional regulation of N-oxide reductases, detailed gas kinetics was combined with gene expression analyses at high resolution. The results show that H. Mediterranei has an expression pattern similar to that observed in the bacterial Domain, well-coordinated at low concentrations of N-oxyanions. However, it could only sustain a few generations of exponential anaerobic growth, apparently requiring micro-oxic conditions for de novo synthesis of denitrification enzymes. This is the first integrated study within this field of knowledge in haloarchaea and Archaea in general, and it sheds lights on denitrification in salty environments.
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transcriptional profiles of Haloferax Mediterranei based on nitrogen availability
Journal of Biotechnology, 2015Co-Authors: J. Esclapez, Carmen Pire, Monica Camacho, A. Vegara, Luis A Alcaraz, Vanesa Bautista, Basilio Zafrilla, R M Martinezespinosa, María José BoneteAbstract:The haloarchaeon Haloferax Mediterranei is able to grow in the presence of different inorganic and organic nitrogen sources by means of the assimilatory pathway under aerobic conditions. In order to identify genes of potential importance in nitrogen metabolism and its regulation in the halophilic microorganism, we have analysed its global gene expression in three culture media with different nitrogen sources: (a) cells were grown stationary and exponentially in ammonium, (b) cells were grown exponentially in nitrate, and (c) cells were shifted to nitrogen starvation conditions. The main differences in the transcriptional profiles have been identified between the cultures with ammonium as nitrogen source and the cultures with nitrate or nitrogen starvation, supporting previous results which indicate the absence of ammonium as the factor responsible for the expression of genes involved in nitrate assimilation pathway. The results have also permitted the identification of transcriptional regulators and changes in metabolic pathways related to the catabolism and anabolism of amino acids or nucleotides. The microarray data was validated by real-time quantitative PCR on 4 selected genes involved in nitrogen metabolism. This work represents the first transcriptional profiles study related to nitrogen assimilation metabolism in extreme halophilic microorganisms using microarray technology.
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effects of nitrogen sources on the nitrate assimilation in Haloferax Mediterranei growth kinetics and transcriptomic analysis
Fems Microbiology Letters, 2014Co-Authors: J. Esclapez, Carmen Pire, Monica Camacho, Vanesa Bautista, Gloria Bravobarrales, María José BoneteAbstract:The haloarchaeon Haloferax Mediterranei is able to grow in a defined culture media not only in the presence of inorganic nitrogen salt but also with amino acid as the sole nitrogen source. Assimilatory nitrate and nitrite reductases, respectively, catalyze the first and second reactions. The genes involved in this process are nasA, which encodes nitrate reductase and is found within the operon nasABC, and nasD, which encodes nitrite reductase. These genes are subjected to transcriptional regulation, being repressed in the presence of ammonium and induced with either nitrate or nitrite. This type of regulation has also been described when the amino acids are used as nitrogen source in the minimal media. Furthermore, it has been observed that the microorganism growth depends on nitrogen source, obtaining the lowest growth rate in the presence of nitrate and aspartate. In this paper, we present the results of a comparative study of microorganism growth and transcriptomic analysis of the operon nasABC and gene nasD in different nitrogen sources. The results are the first ever produced in relation to amino acids as nitrogen sources within the Halobacteriaceae family.
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identification of several intracellular carbohydrate degrading activities from the halophilic archaeon Haloferax Mediterranei
Extremophiles, 2009Co-Authors: Francisco Perezpomares, Carmen Pire, J. Esclapez, Vanesa Bautista, Basilio Zafrilla, Susana Díaz, G Bravo, María José BoneteAbstract:Three different amylolytic activities, designated AMY1, AMY2, and AMY3 were detected in the cytoplasm of the extreme halophilic archaeon Haloferax Mediterranei grown in a starch containing medium. This organism had also been reported to excrete an α-amylase into the external medium in such conditions. The presence of these different enzymes which are also able to degrade starch may be related to the use of the available carbohydrates and maltodextrins, including the products obtained by the action of the extracellular amylase on starch that may be transported to the cytoplasm of the organism. The behavior of these intracellular hydrolytic enzymes on starch is reported here and compared with their extracellular counterpart. Two of these glycosidic activities (AMY1, AMY3) have also been purified and further characterized. As with other halophilic enzymes, they were salt dependent and displayed maximal activity at 3 M NaCl, and 50°C. The purification steps and molecular masses have also been reported. The other activity (AMY2) was also detected in extracts from cells grown in media with glycerol instead of starch and in a yeast extract medium. This enzyme was able to degrade starch yielding small oligosaccharides and displayed similar halophilic behavior with salt requirement in the range 1.5–3 M NaCl.
