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1.
Trends Microbiol ; 27(1): 86-87, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30459094

RESUMO

In this infographic we present the main tools available for the halophilic archaeon Haloferax volcanii, which have enabled successful research on its biology, including its genetics, proteostasis, cell surface structures, metabolic pathways, and adaptation to high salt environments. Isolated from the Dead Sea in 1975, Haloferax volcanii thrives in high salt environments and has emerged as an important archaeal model system. An extensive repertoire of genetic, molecular biological, and biochemical tools has been developed for this fast-growing, easily cultivated haloarchaeon, including expression vectors and gene-deletion strategies, including CRISPR. Its low mutation rate and ability to grow on defined media allow straightforward application of methods such as metabolic labeling, and the sequenced genome laid the foundation for transcriptomics and proteomics studies. These tools have allowed examination of key pathways such as transcription, noncoding RNAs, protein synthesis and degradation, protein glycosylation, motility, and biofilm formation. With the collaborative spirit of the H. volcanii community, this model system has become invaluable not only for enhancing our understanding of archaea but also for improving the development of biotech applications.


Assuntos
Genética Microbiana/métodos , Haloferax volcanii/genética , Haloferax volcanii/fisiologia , Biologia Molecular/métodos , Genômica/métodos , Haloferax volcanii/classificação , Haloferax volcanii/isolamento & purificação , Redes e Vias Metabólicas/genética , Proteômica/métodos
2.
Appl Environ Microbiol ; 82(2): 538-48, 2016 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-26546423

RESUMO

Soluble inorganic pyrophosphatases (PPAs) that hydrolyze inorganic pyrophosphate (PPi) to orthophosphate (Pi) are commonly used to accelerate and detect biosynthetic reactions that generate PPi as a by-product. Current PPAs are inactivated by high salt concentrations and organic solvents, which limits the extent of their use. Here we report a class A type PPA of the haloarchaeon Haloferax volcanii (HvPPA) that is thermostable and displays robust PPi-hydrolyzing activity under conditions of 25% (vol/vol) organic solvent and salt concentrations from 25 mM to 3 M. HvPPA was purified to homogeneity as a homohexamer by a rapid two-step method and was found to display non-Michaelis-Menten kinetics with a Vmax of 465 U · mg(-1) for PPi hydrolysis (optimal at 42°C and pH 8.5) and Hill coefficients that indicated cooperative binding to PPi and Mg(2+). Similarly to other class A type PPAs, HvPPA was inhibited by sodium fluoride; however, hierarchical clustering and three-dimensional (3D) homology modeling revealed HvPPA to be distinct in structure from characterized PPAs. In particular, HvPPA was highly negative in surface charge, which explained its extreme resistance to organic solvents. To demonstrate that HvPPA could drive thermodynamically unfavorable reactions to completion under conditions of reduced water activity, a novel coupled assay was developed; HvPPA hydrolyzed the PPi by-product generated in 2 M NaCl by UbaA (a "salt-loving" noncanonical E1 enzyme that adenylates ubiquitin-like proteins in the presence of ATP). Overall, we demonstrate HvPPA to be useful for hydrolyzing PPi under conditions of reduced water activity that are a hurdle to current PPA-based technologies.


Assuntos
Proteínas Arqueais/metabolismo , Haloferax volcanii/enzimologia , Pirofosfatase Inorgânica/metabolismo , Cloreto de Sódio/metabolismo , Sequência de Aminoácidos , Proteínas Arqueais/química , Proteínas Arqueais/genética , Estabilidade Enzimática , Haloferax volcanii/química , Haloferax volcanii/classificação , Haloferax volcanii/genética , Temperatura Alta , Pirofosfatase Inorgânica/química , Pirofosfatase Inorgânica/genética , Cinética , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência , Cloreto de Sódio/análise , Solventes/química , Solventes/metabolismo , Especificidade por Substrato
3.
PLoS One ; 9(3): e90763, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24637842

RESUMO

The haloarchaeon Haloferax volcanii was shown to contain 145 intergenic and 45 antisense sRNAs. In a comprehensive approach to unravel various biological roles of haloarchaeal sRNAs in vivo, 27 sRNA genes were selected and deletion mutants were generated. The phenotypes of these mutants were compared to that of the parent strain under ten different conditions, i.e. growth on four different carbon sources, growth at three different salt concentrations, and application of four different stress conditions. In addition, cell morphologies in exponential and stationary phase were observed. Furthermore, swarming of 17 mutants was analyzed. 24 of the 27 mutants exhibited a difference from the parent strain under at least one condition, revealing that haloarchaeal sRNAs are involved in metabolic regulation, growth under extreme conditions, regulation of morphology and behavior, and stress adaptation. Notably, 7 deletion mutants showed a gain of function phenotype, which has not yet been described for any other prokaryotic sRNA gene deletion mutant. Comparison of the transcriptomes of one sRNA gene deletion mutant and the parent strain led to the identification of differentially expressed genes. Genes for flagellins and chemotaxis were up-regulated in the mutant, in accordance with its gain of function swarming phenotype. While the deletion mutant analysis underscored that haloarchaeal sRNAs are involved in many biological functions, the degree of conservation is extremely low. Only 3 of the 27 genes are conserved in more than 10 haloarchaeal species. 22 of the 27 genes are confined to H. volcanii, indicating a fast evolution of haloarchaeal sRNA genes.


Assuntos
Deleção de Genes , Genes Arqueais , Haloferax volcanii/classificação , Haloferax volcanii/genética , Fenótipo , RNA Arqueal , Adaptação Biológica/genética , Metabolismo Energético , Evolução Molecular , Perfilação da Expressão Gênica , Haloferax volcanii/crescimento & desenvolvimento , Haloferax volcanii/metabolismo , Cloreto de Sódio/farmacologia , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genética , Transcriptoma
4.
BMC Mol Biol ; 7: 44, 2006 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-17132163

RESUMO

BACKGROUND: DNA ligases are required for DNA strand joining in all forms of cellular life. NAD+-dependent DNA ligases are found primarily in eubacteria but also in some eukaryotic viruses, bacteriophage and archaea. Among the archaeal NAD+-dependent DNA ligases is the LigN enzyme of the halophilic euryarchaeon Haloferax volcanii, the gene for which was apparently acquired by Hfx. volcanii through lateral gene transfer (LGT) from a halophilic eubacterium. Genetic studies show that the LGT-acquired LigN enzyme shares an essential function with the native Hfx. volcanii ATP-dependent DNA ligase protein LigA. RESULTS: To characterise the enzymatic properties of the LigN protein, wild-type and three mutant forms of the LigN protein were separately expressed in recombinant form in E.coli and purified to apparent homogeneity by immobilised metal ion affinity chromatography (IMAC). Non-isotopic DNA ligase activity assays using lambda DNA restriction fragments with 12 bp cos cohesive ends were used to show that LigN activity was dependent on addition of divalent cations and salt. No activity was detected in the absence of KCl, whereas maximum activity could be detected at 3.2 M KCl, close to the intracellular KCl concentration of Hfx. volcanii cells. CONCLUSION: LigN is unique amongst characterised DNA ligase enzymes in displaying maximal DNA strand joining activity at high (> 3 M) salt levels. As such the LigN enzyme has potential both as a novel tool for biotechnology and as a model enzyme for studying the adaptation of proteins to high intracellular salt levels.


Assuntos
DNA Ligases/metabolismo , Haloferax volcanii/enzimologia , Cloreto de Potássio/farmacologia , DNA Ligases/química , DNA Ligases/genética , DNA Ligases/isolamento & purificação , Haloferax volcanii/classificação , Haloferax volcanii/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Sais
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