Novel glycoproteins of the halophilic archaeon Haloferax volcanii

Archaea possess many eukaryote-like properties, including the ability to glycosylate proteins. Using oligosaccharide staining and lectin binding, this study revealed the existence of several glycosylated Haloferax volcanii membrane proteins, besides the previously reported surface layer (S-layer) glycoprotein. While the presence of glycoproteins in archaeal S-layers and flagella is well-documented, few archaeal glycoproteins that are not part of these structures have been reported. The glycosylated 150, 98, 58 and 54 kDa protein species detected were neither precursors nor breakdown products of the 190 kDa S-layer glycoprotein. Furthermore, these novel glycoproteins were outwardly oriented and intimately associated with the membrane.     

A bioluminescent reporter for the halophilic archaeon Haloferax volcanii

Haloarchaea have evolved to thrive in hypersaline environments. Haloferax volcanii is of particular interest due to its genetic tractability; however, few in vivo reporters exist for halophiles. Haloarchaeal proteins evolved characteristics that promote proper folding and function at high salt concentrations, but many mesophilic reporter proteins lack these characteristics. Mesophilic proteins that acquire salt-stabilizing mutations, however, can lead to proper function in haloarchaea. Using laboratory-directed evolution, we developed and demonstrated an in vivo luciferase that functions in the hypersaline cytosol of H. volcanii.

     

Properties of the chaperonin complex from the halophilic archaeon Haloferax volcanii

The halophilic archaeon Haloferax volcanii has three genes encoding type II chaperonins, named cct1, cct2 and cct3. We show here that the three CCT proteins are all expressed but not to the same level. All three proteins are further induced on heat shock. The CCT proteins were purified by ammonium sulphate precipitation, sucrose gradient centrifugation and hydrophobic interaction chromatography. This procedure yields a high molecular mass complex (or complexes). The complex has ATPase activity, which is magnesium dependent, low salt-sensitive and stable to at least 75 degrees C. Activity requires high levels of potassium ions and was reduced in the presence of an increasing concentration of sodium ions.     

Defining the topology of the N-glycosylation pathway in the halophilic archaeon Haloferax volcanii

In Eukarya, N glycosylation involves the actions of enzymes working on both faces of the endoplasmic reticulum membrane. The steps of bacterial N glycosylation, in contrast, transpire essentially on the cytoplasmic side of the plasma membrane, with only transfer of the assembled glycan to the target protein occurring on the external surface of the cell. For Archaea, virtually nothing is known about the topology of enzymes involved in assembling those glycans that are subsequently N linked to target proteins on the external surface of the cell. To remedy this situation, subcellular localization and topology predictive algorithms, protease accessibility, and immunoblotting, together with cysteine modification following site-directed mutagenesis, were enlisted to define the topology of Haloferax volcanii proteins experimentally proven to participate in the N-glycosylation process. AglJ and AglD, involved in the earliest and latest stages, respectively, of assembly of the pentasaccharide decorating the H. volcanii S-layer glycoprotein, were shown to present their soluble N-terminal domain, likely containing the putative catalytic site of each enzyme, to the cytosol. The same holds true for Alg5-B, Dpm1-A, and Mpg1-D, proteins putatively involved in this posttranslational event. The results thus point to the assembly of the pentasaccharide linked to certain Asn residues of the H. volcanii S-layer glycoprotein as occurring within the cell.     

An unusual pattern of spontaneous mutations recovered in the halophilic archaeon Haloferax volcanii

Spontaneous mutations in the orotate:phosphoribosyl transferase (pyrE2) gene of the halophilic archaeon Haloferax volcanii were selected by 5-fluoroorotic acid plus uracil at a rate of approximately 2 x 10(-8)/cell division in fluctuation and null-fraction tests but approximately 6 x 10(-8)/cell division in mutation-accumulation tests. The corresponding genomic mutation rates were substantially lower than those observed for other mesophilic microbial DNA genomes on the basis of similar target genes. The mutational spectrum was dominated by indels adding or deleting multiples of 3 bp. Properties of the organism contributing to this unusual mutational pattern may include phenotypic lag caused by a high chromosomal copy number and efficient promotion of strand misalignments by short direct repeats.     

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.     

Novel polar lipids of halophilic eubacterium Planococcus H8 and archaeon Haloferax volcanii

As part of a study to identify novel lipids with immune adjuvant activity, a structural comparison was made between the polar lipids from two halophiles, an archaeon Haloferax volcanii and a eubacterium Planococcus H8. H. volcanii polar lipid extracts consisted of 44% archaetidylglycerol methylphosphate, 35% archaetidylglycerol, 4.7% of archaeal cardiolipin, 2.5% archaetidic acid, and 14% sulfated glycolipids 1 and 2. Nuclear magnetic resonance (NMR) and Fast atom bombardment mass spectrometry (FAB MS) data determined the glycolipids to be 6-HSO(3)-D-Man(p)-alpha1-2-D-Glc(p)-alpha1,1-[sn-2,3-di-O-phytanylglycerol] and a novel glycocardiolipin 6'-HSO(3)-D-Man(p)-alpha1-2-D-Glc(p)-alpha1,1-[sn-2,3-di-O-phytanylglycerol]-6-[phospho-sn-2,3-di-O-phytanylglycerol]. The polar lipids of Planococcus H8 consisted of 49% saturated phosphatidylglycerol and cardiolipin (9:1, w/w), and surprisingly 51% of the photosynthetic membrane lipid sulfoquinovosyldiacylglycerol (SQDG). This study documents archaeal cardiolipin and a novel glycocardiolipin in H. volcanii (lacking purple membrane), and is the first report of SQDG in a non-photosynthetic, halophilic bacterium.     

Effects of salt and temperature on plasmid topology in the halophilic archaeon Haloferax volcanii.

We report here the effect of environmental parameters, salinity, temperature, and an intercalating drug on plasmid topology in the halophilic archaeon Haloferax volcanii. We first studied the topological state of the plasmid pHV11 in media of different salt compositions and concentrations. The superhelical density of plasmid PHV11 varies in a way that depends on the kind of salt and on the concentrations of individual salts. With respect to growth temperature, the plasmid linking number increased at higher temperature in a linear way, contrary to what has been reported for Escherichia coli, in which the plasmid linking number decreased at higher temperature. These results suggest that some of the mechanisms that control DNA supercoiling in halophilic Archaea may be different from those described for E. coli. However, homeostatic control of DNA supercoiling seems to occur in haloarchaea, as in Bacteria, since we found that relaxation of DNA by chloroquine triggers an increase in negative supercoiling.     

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.     

Dihydrolipoamide dehydrogenase from the halophilic archaeon Haloferax volcanii: homologous overexpression of the cloned gene.

The gene encoding dihydrolipoamide dehydrogenase from the halophilic archaeon, Haloferax volcanii, has been subcloned and overexpressed in the parent organism by using the halophilic archaeal rRNA promoter. The recombinant protein has been purified to homogeneity and characterized with respect to its kinetic, molecular, and salt-dependent properties. A dihydrolipoamide dehydrogenase-minus mutant of H. volcanii has been created by homologous recombination with the subcloned gene after insertion of the mevinolin resistance determinant into the protein-coding region. To explore the physiological function of the dihydrolipoamide dehydrogenase, the growth properties of the mutant halophile have been examined.     

In vivo characterization of the homing endonuclease within the polB gene in the halophilic archaeon Haloferax volcanii

Inteins are parasitic genetic elements, analogous to introns that excise themselves at the protein level by self-splicing, allowing the formation of functional non-disrupted proteins. Many inteins contain a homing endonuclease (HEN) gene, and rely on its activity for horizontal propagation. In the halophilic archaeon, Haloferax volcanii, the gene encoding DNA polymerase B (polB) contains an intein with an annotated but uncharacterized HEN. Here we examine the activity of the polB HEN in vivo, within its natural archaeal host. We show that this HEN is highly active, and able to insert the intein into both a chromosomal target and an extra-chromosomal plasmid target, by gene conversion. We also demonstrate that the frequency of its incorporation depends on the length of the flanking homologous sequences around the target site, reflecting its dependence on the homologous recombination machinery. Although several evolutionary models predict that the presence of an intein involves a change in the fitness of the host organism, our results show that a strain deleted for the intein sequence shows no significant changes in growth rate compared to the wild type.     

Development of additional selectable markers for the halophilic archaeon Haloferax volcanii based on the leuB and trpA genes

Since most archaea are extremophilic and difficult to cultivate, our current knowledge of their biology is confined largely to comparative genomics and biochemistry. Haloferax volcanii offers great promise as a model organism for archaeal genetics, but until now there has been a lack of a wide variety of selectable markers for this organism. We describe here isolation of H. volcanii leuB and trpA genes encoding 3-isopropylmalate dehydrogenase and tryptophan synthase, respectively, and development of these genes as a positive selection system. DeltaleuB and DeltatrpA mutants were constructed in a variety of genetic backgrounds and were shown to be auxotrophic for leucine and tryptophan, respectively. We constructed both integrative and replicative plasmids carrying the leuB or trpA gene under control of a constitutive promoter. The use of these selectable markers in deletion of the lhr gene of H. volcanii is described.     

ATP- and NAD+-dependent DNA ligases share an essential function in the halophilic archaeon Haloferax volcanii

DNA ligases join the ends of DNA molecules during replication, repair and recombination. ATP-dependent ligases are found predominantly in the eukarya and archaea whereas NAD+-dependent DNA ligases are found only in the eubacteria and in entomopoxviruses. Using the genetically tractable halophile Haloferax volcanii as a model system, we describe the first genetic analysis of archaeal DNA ligase function. We show that the Hfx. volcanii ATP-dependent DNA ligase family member, LigA, is non-essential for cell viability, raising the question of how DNA strands are joined in its absence. We show that Hfx. volcanii also encodes an NAD+-dependent DNA ligase family member, LigN, the first such enzyme to be identified in the archaea, and present phylogenetic analysis indicating that the gene encoding this protein has been acquired by lateral gene transfer (LGT) from eubacteria. As with LigA, we show that LigN is also non-essential for cell viability. Simultaneous inactivation of both proteins is lethal, however, indicating that they now share an essential function. Thus the LigN protein acquired by LGT appears to have been co-opted as a back-up for LigA function, perhaps to provide additional ligase activity under conditions of high genotoxic stress.     

Development of a gene knockout system for the halophilic archaeon Haloferax volcanii by use of the pyrE gene

So far, the extremely halophilic archaeon Haloferax volcanii has the best genetic tools among the archaea. However, the lack of an efficient gene knockout system for this organism has hampered further genetic studies. In this paper we describe the development of pyrE-based positive selection and counterselection systems to generate an efficient gene knockout system. The H. volacanii pyrE1 and pyrE2 genes were isolated, and the pyrE2 gene was shown to code for the physiological enzyme orotate phosphoribosyl transferase. A DeltapyrE2 strain was constructed and used to isolate deletion mutants by the following two steps: (i) integration of a nonreplicative plasmid carrying both the pyrE2 wild-type gene, as a selectable marker, and a cloned chromosomal DNA fragment containing a deletion in the desired gene; and (ii) excision of the integrated plasmid after selection with 5-fluoroorotic acid. Application of this gene knockout system is described.     

Overexpression and purification of halophilic proteins in Haloferax volcanii

Halophilic enzymes function optimally at high salt concentrations and are active at low water availability. Such conditions are encountered at elevated concentrations of solutes such as salts and sugars, and at high concentrations of organic solvents. However, expression in heterologous hosts such as Escherichia coli can cause problems, since halophilic proteins typically misfold and aggregate in conditions of low ionic strength. We have harnessed the sophisticated genetic tools available for the haloarchaeon Haloferax volcanii, to develop a system for the overexpression and purification of halophilic proteins under native conditions.     

Evidence for post-translational membrane insertion of the integral membrane protein bacterioopsin expressed in the heterologous halophilic archaeon Haloferax volcanii

The gene coding for the integral membrane protein bacterioopsin (Bop), that is composed of seven transmembrane helices, was expressed in the halophilic archaeon Haloferax volcanii as a fusion protein with the halobacterial enzyme dihydrofolate reductase and with the cellulose binding domain of Clostridium thermocellum cellulosome. In each case, bacterioopsin was present both in the membrane and in the cytoplasmic fractions. Pulse-chase labeling experiments showed that the fusion protein in the cytoplasmic fraction is the precursor of the membrane-bound species. Bacterioopsin mutants that lack the seventh helix (BopDelta7) were found to accumulate only in the cytoplasmic fraction, whereas bacterioopsin mutants that lack either helices four and five (BopDelta4-5), or helices one and two (BopDelta1-2), were found in the cytoplasmic as well as in the membrane fractions. The seventh helix, when expressed alone, could target in trans the insertion of a separately expressed bacterioopsin mutant protein that has only the first six helices. These results support a model in which bacterioopsin is produced in H. volcanii as a soluble protein and in which its insertion into the membrane occurs post-translationally. According to this model, membrane insertion is directed by the seventh helix.     

Osmotically induced response in representatives of halophilic prokaryotes: the bacterium Halomonas elongata and the archaeon Haloferax volcanii.

Haloferax volcanii and Halomonas elongata have been selected as representatives of halophilic Archaea and Bacteria, respectively, to analyze the responses to various osmolarities at the protein synthesis level. We have identified a set of high-salt-related proteins (39, 24, 20, and 15.5 kDa in H. elongata; 70, 68, 48, and 16 kDa in H. volcanii) whose synthesis rates increased with increasing salinities. A different set of proteins (60, 42, 15, and 6 kDa for H. elongata; 63, 44, 34, 18, 17, and 6 kDa for H. volcanii), some unique for low salinities, was induced under low-salt conditions. For both organisms, and especially for the haloarchaeon, adaptation to low-salt conditions involved a stronger and more specific response than adaptation to high-salt conditions, indicating that unique mechanisms may have evolved for low-salinity adaptation. In the case of H. volcanii, proteins with a typical transient response to osmotic shock, induced by both hypo- and hyperosmotic conditions, probably corresponding to described heat shock proteins and showing the characteristics of general stress proteins, have also been identified. Cell recovery after a shift to low salinities was immediate in both organisms. In contrast, adaptation to higher salinities in both cases involved a lag period during which growth and general protein synthesis were halted, although the high-salt-related proteins were induced rapidly. In H. volcanii, this lag period corresponded exactly to the time needed for cells to accumulate adequate intracellular potassium concentrations, while extrusion of potassium after the down-shift was immediate. Thus, reaching osmotic balance must be the main limiting factor for recovery of cell functions after the variation in salinity.     

NADP-dependent isocitrate dehydrogenase from the halophilic archaeon Haloferax volcanii: cloning,sequence determination and overexpression in Escherichia coli

A gene encoding NADP-dependent Ds-threo-isocitrate dehydrogenase was isolated from Haloferax volcanii genomic DNA by using a combination of polymerase chain reaction and screening of a lambda EMBL3 library. Analysis of the nucleotide sequence revealed an open reading frame of 1260 bp encoding a protein of 419 amino acids with 45837 Da molecular mass. This sequence is highly similar to previously sequenced isocitrate dehydrogenases. In the alignment of the amino acid sequences with those from several archaeal and mesophilic NADP-dependent isocitrate dehydrogenases, the residues involved in dinucleotide binding and isocitrate binding are well conserved. We have developed methods for the expression in Escherichia coli and purification of the enzyme from H. volcanii. This expression was carried out in E. coli as inclusion bodies using the cytoplasmic expression vector pET3a. The enzyme was refolded by solubilisation in 8 M urea followed by dilution into a buffer containing EDTA, MgCl(2) and 3 M NaCl. Maximal activity was obtained after several hours incubation at room temperature.     

Post-translational secretion of fusion proteins in the halophilic archaea Haloferax volcanii

Although protein secretion occurs post-translationally in bacteria and is mainly a cotranslational event in Eukarya, the relationship between the translation and translocation of secreted proteins in Archaea is not known. To address this question, the signal peptide-encoding region of the surface layer glycoprotein gene from the Haloarchaea Haloferax volcanii was fused either to the cellulose-binding domain of the Clostridium thermocellum cellulosome or to the cytoplasmic enzyme dihydrofolate reductase from H. volcanii. Signal peptide-cleaved mature versions of both the cellulose-binding domain and dihydrofolate reductase could be detected in the growth medium of transformed H. volcanii cells. Immunoblot analysis revealed, however, the presence of full-length signal peptide-bearing forms of both proteins inside the cytoplasm of the transformed cells. Proteinase accessibility assays confirmed that the presence of cell-associated signal peptide-bearing proteins was not due to medium contamination. Moreover, the pulse-radiolabeled signal peptide cellulose-binding domain chimera could be chased from the cytoplasm into the growth medium even following treatment with anisomycin, an antibiotic inhibitor of haloarchaeal protein translation. Thus, these results provide evidence that, in Archaea, at least some secreted proteins are first synthesized inside the cell and only then translocated across the plasma membrane into the medium.     

Characterization of acetohydroxy acid synthase activity in the archaeon Haloferax volcanii

Whereas the biochemistry of acetohydroxy acid synthase has been extensively studied in bacteria and eukaryotes, relatively little is known about the enzyme in archaea, the third kingdom of life. The present study biochemically characterizes acetohydroxy acid synthase activity in the halophilic archaea Haloferax volcanii. In addressing ion requirements, enzyme inhibition and antibody labeling, the results reveal that, except for its elevated salt requirements, the haloarchaeal enzyme is remarkably similar to its bacterial counterpart.