Dihydrolipoamide dehydrogenase from Haloferax volcanii: gene cloning, complete primary structure, and comparison to other dihydrolipoamide dehydrogenases.

We used the N-terminal amino acid sequence of dihydrolipoamide dehydrogenase from Haloferax volcanii, to design and synthesize two oligonucleotide probes that were used to identify and clone a 4.3 kilobase pair (kbp) fragment from MboI restriction endonuclease digestion of Hf. volcanii genomic DNA. The nucleotide sequence of a 1.5-kbp region of this clone was determined and this revealed an open reading frame that translated into a protein with good homology to dihydrolipoamide dehydrogenase from other sources. The first 48 amino acids were identical with the N-terminal sequence data obtained from the purified protein. The complete primary structure of the halophilic dihydrolipoamide dehydrogenase was analyzed in terms of its homologies to dihydrolipoamide dehydrogenases from other sources and its molecular adaptations to high intracellular ionic strength.     

Purification of a new dihydrolipoamide dehydrogenase from Escherichia coli.

I purified a new dihydrolipoamide dehydrogenase from a lpd mutant of Escherichia coli deficient in the lipoamide dehydrogenase (EC 1.6.4.3) common to the pyruvate dehydrogenase (EC 1.2.4.1) and 2-oxoglutarate dehydrogenase complexes. The occurrence of the new lipoamide dehydrogenase in lpd mutants, including a lpd deletion mutant and the immunological properties of the enzyme, showed that it is different from the lpd gene product. The new dihydrolipoamide dehydrogenase had a molecular weight of 46,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was expressed in low amounts. It catalyzed the NAD+-dependent reduction of dihydrolipoamide with a maximal activity of 20 mumol/min per mg of protein and exhibited a hyperbolic dependence of catalytic activity on the concentration of both dihydrolipoamide and NAD+. The possible implication of the new dihydrolipoamide in the function of 2-oxo acid dehydrogenase complexes is discussed, as is its relation to binding protein-dependent transport.     

Dihydrolipoamide dehydrogenase from the halophilic archaebacterium Haloferax volcanii: characterization and N-terminal sequence.

Dihydrolipoamide dehydrogenase, a flavin disulfide reductase, has been purified and characterized from Haloferax volcanii. The enzyme is a dimer of relative mass 128,000, with an optimal activity at pH 9.0 in 1 M NaCl. Following reduction with its substrate, dihydrolipoamide, the enzyme is inactivated through covalent bond formation with the trivalent arsenical p-aminophenyl arsenoxide. The amino acid composition and the amino acid sequence of the first 49 residues of the N-terminus have been determined.     

Molecular cloning and sequence determination of the lpd gene encoding lipoamide dehydrogenase from Pseudomonas fluorescens

The lpd gene encoding lipoamide dehydrogenase (dihydrolipoamide dehydrogenase; EC 1.8.1.4) was isolated from a library of Pseudomonas fluorescens DNA cloned in Escherichia coli TG2 by use of serum raised against lipoamide dehydrogenase from Azotobacter vinelandii. Large amounts (up to 15% of total cellular protein) of the P. fluorescens lipoamide dehydrogenase were produced by the E. coli clone harbouring plasmid pCJB94 with the lipoamide dehydrogenase gene. The enzyme was purified to homogeneity by a three-step procedure. The gene was subcloned from plasmid pCJB94 and the complete nucleotide sequence of the subcloned fragment (3610 bp) was determined. The derived amino acid sequence of P. fluorescens lipoamide dehydrogenase showed 84% and 42% homology when compared to the amino acid sequences of lipoamide dehydrogenase from A. vinelandii and E. coli, respectively. The lpd gene of P. fluorescens is clustered in the genome with genes for the other components of the 2-oxoglutarate dehydrogenase complex.     

The extremely halophilic archaeon Haloferax volcanii has two very different dihydrofolate reductases

The gene encoding dihydrofolate reductase, hdrA, from the extremely halophilic archaeon Haloferax volcanii was previously isolated from a spontaneous trimethoprim-resistant mutant in a DNA sequence that had undergone amplification. Here, we show that deletion of hdrA did not affect growth in minimal medium and that the strain carrying the deletion remained sensitive to trimethoprim. A spontaneous trimethoprim-resistant colony was isolated in the hdrA deletion strain and found to possess a new DNA amplification. Sequencing of the amplification revealed a second, substantially different, dihydrofolate reductase gene, hdrB, which was found to be located immediately downstream of the thymidylate synthase gene, hts. The physiological role of hDHFR-1 and hDHFR-2 was determined by generating Haloferax volcanii strains in which each gene, hdrA or hdrB, or both genes were deleted. It was found that hdrB alone can support growth of Haloferax volcanii in minimal medium, whereas hdrA alone can support growth of Haloferax volcanii in minimal medium only when the medium is supplemented with thymidine. It was also shown that, in contrast to Escherichia coli, the DeltahdrA, DeltahdrB double deletion mutant is viable in the presence of a functional thymidylate synthase gene. The hdrB gene was overexpressed in Escherichia coli and the enzyme purified to homogeneity. The biochemical properties of the new enzyme (hDHFR-2) are markedly different from those of hDHFR-1. The use of the dihydrofolate reductase and thymidylate synthase genes as stable selectable markers is described.     

Molecular cloning and sequencing of the gene for a halophilic alkaline serine protease (halolysin) from an unidentified halophilic archaea strain (172P1) and expression of the gene in Haloferax volcanii.

The gene of a halophilic alkaline serine protease, halolysin, from an unidentified halophilic archaea (archaebacterium) was cloned and its nucleotide sequence was determined. The deduced amino acid sequence showed that halolysin consists of 411 amino acids, with a molecular weight of 41,963. The highest homology was found with thermitase from Thermoactinomyces vulgaris. Halolysin has a long C-terminal extension of approximately 120 amino acids which has not been found in other extracellular subtilisin type serine proteases. The gene, hly, was expressed in another halophilic archaea, Haloferax volcanii, in a medium containing 18% salts by using a plasmid shuttle vector which has a novobiocin resistance determinant as a selectable marker.     

Identification of a thiolase gene essential for β-oxidation of the acyl side chain of the steroid compound cholate in Pseudomonas sp. strain Chol1

A method to grow the halophilic archaeon Haloferax volcanii in microtiter plates has been optimized and now allows the parallel generation of very reproducible growth curves. The doubling time in a synthetic medium with glucose is around 6 h. The method was used to optimize glucose and casamino acid concentrations, to clarify carbon source usage and to analyze vitamin dependence. The characterization of osmotolerance revealed that after a lag phase of 24 h, H. volcanii is able to grow at salt concentrations as low as 0.7 M NaCl, much lower than the 1.4 M NaCl described as the lowest concentration until now. The application of oxidative stresses showed that H. volcanii exhibits a reaction to paraquat that is delayed by about 10 h. Surprisingly, only one of two amino acid auxotrophic mutants could be fully supplemented by the addition of the respective amino acid. Analysis of eight sRNA gene deletion mutants exemplified that the method can be applied for bona fide phenotyping of mutant collections. This method for the parallel analysis of many cultures contributes towards making H. volcanii an archaeal model species for functional genomic approaches.     

Importance of the anticodon sequence in the aminoacylation of tRNAs by methionyl-tRNA synthetase and by valyl-tRNA synthetase in an Archaebacterium

The mode of recognition of tRNAs by aminoacyl-tRNA synthetases and translation factors is largely unknown in archaebacteria. To study this process, we have cloned the wild type initiator tRNA gene from the moderate halophilic archaebacterium Haloferax volcanii and mutants derived from it into a plasmid capable of expressing the tRNA in these cells. Analysis of tRNAs in vivo show that the initiator tRNA is aminoacylated but is not formylated in H. volcanii. This result provides direct support for the notion that protein synthesis in archaebacteria is initiated with methionine and not with formylmethionine. We have analyzed the effect of two different mutations (CAU-->CUA and CAU-->GAC) in the anticodon sequence of the initiator tRNA on its recognition by the aminoacyl-tRNA synthetases in vivo. The CAU-->CUA mutant was not aminoacylated to any significant extent in vivo, suggesting the importance of the anticodon in aminoacylation of tRNA by methionyl-tRNA synthetase. This mutant initiator tRNA can, however, be aminoacylated in vitro by the Escherichia coli glutaminyl-tRNA synthetase, suggesting that the lack of aminoacylation is due to the absence in H. volcanii of a synthetase, which recognizes the mutant tRNA. Archaebacteria lack glutaminyl-tRNA synthetase and utilize a two-step pathway involving glutamyl-tRNA synthetase and glutamine amidotransferase to generate glutaminyl-tRNA. The lack of aminoacylation of the mutant tRNA indicates that this mutant tRNA is not a substrate for the H. volcanii glutamyl-tRNA synthetase. The CAU-->GAC anticodon mutant is most likely aminoacylated with valine in vivo. Thus, the anticodon plays an important role in the recognition of tRNA by at least two of the halobacterial aminoacyl-tRNA synthetases.     

Threonyl-tRNA synthetase of archaea: importance of the discriminator base in the aminoacylation of threonine tRNA.

To investigate the contribution of the discriminator base of archaeal tRNA(Thr) in aminoacylation by threonyl-tRNA synthetase (ThrRS), cross-species aminoacylation between Escherichia coli and Haloferax volcanii, halophilic archaea, was studied. It was found that E. coli ThrRS threonylated the H. volcanii tRNA(Thr) but that E. coli threonine tRNA was not aminoacylated by H. volcanii ThrRS. Results of a threonylation experiment using in vitro mutants of E. coli threonine tRNA showed that only the mutant tRNA(Thr) having U73 was threonylated by H. volcanii ThrRS. These findings indicate that the discriminator base U73 of H. volcanii tRNA(Thr) is a strong determinant for the recognition by ThrRS.     

Characterization of a tightly controlled promoter of the halophilic archaeon Haloferax volcanii and its use in the analysis of the essential cct1 gene

A system where archaeal gene expression could be controlled by simple manipulation of growth conditions would enable the construction of conditional lethal mutants in essential genes, and permit the controlled overproduction of proteins in their native host. As tools for the genetic manipulation of Haloferax volcanii are well developed, we set out to identify promoters with a wide dynamic range of expression in this organism. Tryptophan is the most costly amino acid for the cell to make, so we reasoned that tryptophan-regulated promoters might be good candidates. Microarray analysis of H. volcanii gene expression in the presence and absence of tryptophan identified a tryptophanase gene (tna) that showed strong induction in the presence of tryptophan. qRT-PCR revealed a very fast response and an up to 100-fold induction after tryptophan addition. This result has been confirmed using three independent reporter genes (cct1, pyrE2 and bgaH). Vectors containing this promoter will be very useful for investigating gene function in H. volcanii and potentially in other halophilic archaea. To demonstrate this, we used the promoter to follow the consequences of depletion of the essential chaperonin protein CCT1, and to determine the ability of heterologous CCT proteins to function in H. volcanii.     

A plasmid vector with a selectable marker for halophilic archaebacteria.

A mutant resistant to the gyrase inhibitor novobiocin was selected from a halophilic archaebacterium belonging to the genus Haloferax. Chromosomal DNA from this mutant was able to transform wild-type cells to novobiocin resistance, and these transformants formed visible colonies in 3 to 4 days on selective plates. The resistance gene was isolated on a 6.7-kilobase DNA KpnI fragment, which was inserted into a cryptic multicopy plasmid (pHK2) derived from the same host strain. The recombinant plasmid transformed wild-type cells at a high efficiency (greater than 10(6)/micrograms), was stably maintained, and could readily be reisolated from transformants. It could also transform Halobacterium volcanii and appears to be a useful system for genetic analysis in halophilic archaebacteria.     

一种基于线性DNA片段同源重组的嗜盐古菌高效基因敲除系统

随着功能基因组学研究的深入发展,基因敲除技术日益成为基因功能研究的重要手段。嗜盐古菌Haloferax volcanii易于培养,是研究古菌基因功能的良好模式菌株。虽然现已开发了多种嗜盐古菌的遗传操作系统,但基因敲除成功率不十分理想。这些遗传操作方法基于pyrE筛选标记,利用携带同源片段的环状质粒与基因组同源片段间的两次同源重组,敲除目的基因。由于基于环状质粒和pyrE筛选标记的经典同源重组敲除方法在二次重组时,普遍存在回复到野生型菌株的可能,导致二次重组子中敲除目的基因的阳性菌株比例较低。为了克服传统同源重组技术的上述缺陷,文章建立了基于线性DNA片段的同源重组技术。该方法通过一次重组在目标基因的下游引入一段上游同源片段和pyrE标记,从而限定二次重组的发生部位只能在两段上游同源片段之间,发生二次重组的重组子理论上都敲除了目标基因。利用该方法,文章成功敲除了嗜盐古菌Haloferax volcanii的xpd2基因,阳性克隆率达65%。这种线性DNA片段重组法为嗜盐古菌的基因敲除提供了一种高效策略,便于嗜盐古菌的基因改造。     

Functional role for a 2-oxo acid dehydrogenase in the halophilic archaeon Haloferax volcanii

The archaeon Haloferax volcanii was previously shown to contain and transcribe the genes for a 2-oxo acid dehydrogenase (OADH) complex, but their presence remained a mystery because no enzymatic activity with any of the known OADH substrates could be found, and an inactivation of one of the genes did not lead to any phenotype. Here we report the identification of an additional oadh gene cluster in the genome of H. volcanii. In contrast to previously known oadh loci, it contains three genes, oadh2A1, oadh2A2, and oadh2ld, with coding capacity for the E1alpha and E1beta subunits and an unattached lipoyl domain, but it is devoid of the genes for a complete E2 and an E3. The genes were isolated by complementation of a nitrate respiration-deficient mutant of H. volcanii and therefore were shown to be functional in vivo. Phylogenetic analyses revealed that the deduced E1alpha and E1beta subunits of OADH2 group with bacterial acetoin dehydrogenases but not with the OADH1 subunits, and thus, H. volcanii has obtained the two gene groups independently. Comparison of the wild type and the mutant allowed us to exclude a function of OADH2 in the aerobic or anaerobic degradation of acetoin or glucose. Instead, it could be shown that OADH2 is important during nitrate-respirative growth on Casamino Acids. Many physiological and biochemical experiments failed to indicate that OADH2 uses any of the previously known OADH substrates. Growth potentials of the mutant were markedly different in media with a single carbon source versus media with mixed carbon sources.     

Molecular cloning of four tricarboxylic acid cyclic genes of Escherichia coli.

A fragment of DNA (3.1 kilobases [kb]) from a ColE1 Escherichia coli DNA hybrid plasmid containing the bacterial citrate synthase gene (gltA) was subcloned in both orientations into phage lambda vectors by in vitro recombination. The resulting phages were able to transduce gltA and, as prophages, complemented the lesion of a gltA mutant, showing that a functional gltA gene is contained in the 3.1-kb fragment. The segment of E. coli DNA cloned in these lambda gltA phages was extended in vivo by prophage integration and aberrant excision in the gltA region. Plaque-forming derivatives, carrying up to three additional tricarboxylic acid cycle genes, succinate dehydrogenase (sdh), 2-oxoglutarate dehydrogenase (sucA), and dihydrolipoamide succinyltransferase (sucB), were isolated and characterized by their transducing and complementing activities with corresponding mutants, and the order of the genes was confirmed as gltA-sdh-sucA-sucB. Physical maps of a variety of the transducing phages showed that the four tricarboxylic acid cycle genes are contained in a 12.8-kb segment of bacterial DNA. The four gene products, plus a possible succinate dehydrogenase small subunit, were identified in postinfection labeling studies, and the polarities of gene expression were defined as counterclockwise for gltA and clockwise for sdh, sucA, and sucB, relative to the E. coli linkage map.     

Membrane binding of SRP pathway components in the halophilic archaea Haloferax volcanii.

Across evolution, the signal recognition particle pathway targets extra-cytoplasmic proteins to membranous translocation sites. Whereas the pathway has been extensively studied in Eukarya and Bacteria, little is known of this system in Archaea. In the following, membrane association of FtsY, the prokaryal signal recognition particle receptor, and SRP54, a central component of the signal recognition particle, was addressed in the halophilic archaea Haloferax volcanii. Purified H. volcanii FtsY, the FtsY C-terminal GTP-binding domain (NG domain) or SRP54, were combined separately or in different combinations with H. volcanii inverted membrane vesicles and examined by gradient floatation to differentiate between soluble and membrane-bound protein. Such studies revealed that both FtsY and the FtsY NG domain bound to H. volcanii vesicles in a manner unaffected by proteolytic pretreatment of the membranes, implying that in Archaea, FtsY association is mediated through the membrane lipids. Indeed, membrane association of FtsY was also detected in intact H. volcanii cells. The contribution of the NG domain to FtsY binding in halophilic archaea may be considerable, given the low number of basic charges found at the start of the N-terminal acidic domain of haloarchaeal FtsY proteins (the region of the protein thought to mediate FtsY-membrane association in Bacteria). Moreover, FtsY, but not the NG domain, was shown to mediate membrane association of H. volcanii SRP54, a protein that did not otherwise interact with the membrane.     

Expression, reactivation, and purification of enzymes from Haloferax volcanii in Escherichia coli.

Enzymes from extreme halophiles have potential as catalysts in biotransformations. We have developed methods for the expression in Escherichia coli and purification of two enzymes from Haloferax volcanii: dihydrolipoamide dehydrogenase and citrate synthase. Both enzymes were expressed in E. coli using the cytoplasmic expression vectors, pET3a and pET3d. Citrate synthase was soluble and inactive, whereas dihydrolipoamide dehydrogenase was expressed as inclusion bodies. Citrate synthase was reactivated following overnight incubation in 2 M KCl, and dihydrolipoamide dehydrogenase was refolded by solubilisation in 8 M urea followed by dilution into a buffer containing 2 M KCl, 10 microM FAD, 1 mM NAD, and 0.3 mM GSSG/3 mM GSH. Maximal activity was obtained after 3 days incubation at 4 degrees C. Purification of the two active enzymes was carried out using high-resolution methods. Dihydrolipoamide dehydrogenase was purified using copper-based metal ion affinity chromatography in the presence of 2 M KCl. Citrate synthase was recovered using dye-affinity chromatography in the presence of salt. A high yield of active enzyme was obtained in both cases. Following purification, characterisation of both recombinant proteins showed that their kinetics and salt-dependence were comparable to those of the native enzymes. Expression of active protein was attempted both by growth of E. coli in the presence of salt and betaine, and also by using periplasmic expression vectors in combination with a high salt growth media. Neither strategy was successful.     

Redetermination of the molecular weights of the components of the pyruvate dehydrogenase complex from E. coli Kl2+.

The validity of molecular weight determination in SDS-polyacrylamide gels for the three components of the pyruvate dehydrogenase complex: pyruvate dehydrogenase, dihydrolipoamide transacetylase, and dihydrolipoamide dehydrogenase has been checked by measuring their free electrophoretic mobilities and their retardation coefficients. A linear relationship between these parameters has been found for all three enzymes as compared with standard proteins. This substantiates earlier molecular weight determinations in SDS-polyacrylamide gels for the components of the pyruvate dehydrogenase complex which are confirmed by this study for different acrylamide gel concentrations.     

A halocin-H4 mutant Haloferax mediterranei strain retains the ability to inhibit growth of other halophilic archaea

Many members of the Halobacteriaceae were found to produce halocins, molecules that inhibit the growth of other halophilic archaea. Halocin H4 that is produced by Haloferax mediterranei and inhibits the growth of Halobacterium salinarum is one of the best studied halocins to date. The gene encoding this halocin had been previously identified as halH4, located on one of Hfx. mediterranei megaplasmids. We generated a mutant of the halH4 gene and examined the killing ability of the Haloferax mediterranei halH4 mutant with respect to both Halobacterium salinarum and Haloferax volcanii. We showed that both wild-type Hfx. mediterranei and the halH4 mutant strain efficiently inhibited the growth of both species, indicating halocin redundancy. Surprisingly, the halH4 deletion mutant exhibited faster growth in standard medium than the wild type, and is likely to have a better response to several nucleotides, which could explain this phenotype.     

Cloning, expression, and nucleotide sequence of the alpha-amylase gene from the haloalkaliphilic archaeon Natronococcus sp. strain Ah-36.

The alpha-amylase gene of a Natronococcus sp. (1,512 bp) contained a signal peptide of 43 amino acids. Haloferax volcanii expressed the gene and cleaved the signal peptide accurately. The signal peptide shared an extremely high amino acid sequence identity with that of a protease from the halophilic archaeon 172P1.