Method of mapping DNA replication origins.

We have developed a method which allows determination of the direction in which replication forks move through segments of chromosomal DNA for which cloned probes are available. The method is based on the facts that DNA restriction fragments containing replication forks migrate more slowly through agarose gels than do non-fork-containing fragments and that the extent of retardation of the fork-containing fragments is a function of the extent of replication. The procedure allows the identification of DNA replication origins as sites from which replication forks diverge. In this paper we demonstrate the feasibility of this procedure, with simian virus 40 DNA as a model, and we discuss its applicability to other systems.     

tRNA-like elements in Haloferax volcanii

All functional RNAs are generated from precursor molecules by a plethora of processing steps. The generation of mature RNA molecules by processing is an important layer of gene expression regulation catalysed by ribonucleases. Here, we analysed 5S rRNA processing in the halophilic Archaeon Haloferax volcanii. Earlier experiments showed that the 5S rRNA is cleaved at its 5' end by the endonuclease tRNase Z. Interestingly, a tRNA-like structure was identified upstream of the 5S rRNA that might be used as a processing signal. Here, we show that this tRNA-like element is indeed recognised as a processing signal by tRNase Z. Substrates containing mutations in the tRNA-like sequence are no longer processed, whereas a substrate containing a deletion in the 5S rRNA sequence is still cleaved. Therefore, an intact 5S rRNA structure is not required for processing. Further, we used bioinformatics analyses to identify additional sequences in Haloferax containing tRNA-like structures. This search resulted in the identification of all tRNAs, the tRNA-like structure upstream of the 5S RNA and 47 new tRNA-like structural elements. However, the in vitro processing of selected examples showed no cleavage of these newly identified elements. Thus, tRNA-like elements are not a general processing signal in Haloferax.     

Regulatory RNAs in Haloferax volcanii

In organisms of all three domains of life, a plethora of sRNAs (small regulatory RNAs) exists in addition to the well-known RNAs such as rRNAs, tRNAs and mRNAs. Although sRNAs have been well studied in eukaryotes and in bacteria, the sRNA population in archaea has just recently been identified and only in a few archaeal species. In the present paper, we summarize our current knowledge about sRNAs and their function in the halophilic archaeon Haloferax volcanii. Using two different experimental approaches, 111 intergenic and 38 antisense sRNAs were identified, as well as 42 tRFs (tRNA-derived fragments). Observation of differential expression under various conditions suggests that these sRNAs might be active as regulators in gene expression like their bacterial and eukaryotic counterparts. The severe phenotypes observed upon deletion and overexpression of sRNA genes revealed that sRNAs are involved in, and important for, a variety of biological functions in H. volcanii and possibly other archaea. Investigation of the Haloferax Lsm protein suggests that this protein is involved in the archaeal sRNA pathway.     

Cytochrome aa3 in Haloferax volcanii

A cytochrome in an extremely halophilic archaeon, Haloferax volcanii, was purified to homogeneity. This protein displayed a redox difference spectrum that is characteristic of a-type cytochromes and a CN(-) complex spectrum that indicates the presence of heme a and heme a(3). This cytochrome aa(3) consisted of 44- and 35-kDa subunits. The amino acid sequence of the 44-kDa subunit was similar to that of the heme-copper oxidase subunit I, and critical amino acid residues for metal binding, such as histidines, were highly conserved. The reduced cytochrome c partially purified from the bacterial membrane fraction was oxidized by the cytochrome aa(3), providing physiological evidence for electron transfer from cytochrome c to cytochrome aa(3) in archaea.     

Genomic stability in the archaeae Haloferax volcanii and Haloferax mediterranei.

Through hybridization of available probes, we have added nine genes to the macrorestriction map of the Haloferax mediterranei chromosome and five genes to the contig map of Haloferax volcanii. Additionally, we hybridized 17 of the mapped cosmid clones from H. volcanii to the H. mediterranei genome. The resulting 35-point chromosomal comparison revealed only two inversions and a few translocations. Forces known to promote rearrangement, common in the haloarchaea, have been ineffective in changing global gene order throughout the nearly 10(7) years of these species' divergent evolution.     

Genomic mapping of single-stranded DNA in hydroxyurea-challenged yeasts identifies origins of replication

During DNA replication one or both strands transiently become single stranded: first at the sites where initiation of DNA synthesis occurs (known as origins of replication) and subsequently on the lagging strands of replication forks as discontinuous Okazaki fragments are generated. We report a genome-wide analysis of single-stranded DNA (ssDNA) formation in the presence of hydroxyurea during DNA replication in wild-type and checkpoint-deficient rad53 Saccharomyces cerevisiae cells. In wild-type cells, ssDNA was first observed at a subset of replication origins and later 'migrated' bi-directionally, suggesting that ssDNA formation is associated with continuously moving replication forks. In rad53 cells, ssDNA was observed at virtually every known origin, but remained there over time, suggesting that replication forks stall. Telomeric regions seemed to be particularly sensitive to the loss of Rad53 checkpoint function. Replication origins in Schizosaccharomyces pombe were also mapped using our method.     

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.     

Shotgun proteomics of the haloarchaeon Haloferax volcanii

Haloferax volcanii, an extreme halophile originally isolated from the Dead Sea, is used worldwide as a model organism for furthering our understanding of archaeal cell physiology. In this study, a combination of approaches was used to identify a total of 1296 proteins, representing 32% of the theoretical proteome of this haloarchaeon. This included separation of (phospho)proteins/peptides by 2-dimensional gel electrophoresis (2-D), immobilized metal affinity chromatography (IMAC), metal oxide affinity chromatography (MOAC), and Multidimensional Protein Identification Technology (MudPIT) including strong cation exchange (SCX) chromatography coupled with reversed phase (RP) HPLC. Proteins were identified by tandem mass spectrometry (MS/MS) using nanoelectrospray ionization hybrid quadrupole time-of-flight (QSTAR XL Hybrid LC/MS/MS System) and quadrupole ion trap (Thermo LCQ Deca). Results indicate that a SCX RP HPLC fractionation coupled with MS/MS provides the best high-throughput workflow for overall protein identification.     

Isolation and characterization of dolichol-linked oligosaccharides from Haloferax volcanii

Biosynthesis of procaryotic glycoproteins has been studied insome detail only in the extreme halophile Halobacterium halobium.To extend these studies for a moderate halophile, dolichol phosphate-linkedoligosaccharides were isolated and characterized from Haloferaxvolcanii. Mannosyl (ß1     

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.     

d-Xylose Degradation Pathway in the Halophilic Archaeon Haloferax volcanii

The pathway of d-xylose degradation in archaea is unknown. In a previous study we identified in Haloarcula marismortui the first enzyme of xylose degradation, an inducible xylose dehydrogenase (Johnsen, U., and Schönheit, P. (2004) J. Bacteriol. 186, 6198–6207). Here we report a comprehensive study of the complete d-xylose degradation pathway in the halophilic archaeon Haloferax volcanii. The analyses include the following: (i) identification of the degradation pathway in vivo following ¹³C-labeling patterns of proteinogenic amino acids after growth on [¹³C]xylose; (ii) identification of xylose-induced genes by DNA microarray experiments; (iii) characterization of enzymes; and (iv) construction of in-frame deletion mutants and their functional analyses in growth experiments. Together, the data indicate that d-xylose is oxidized exclusively to the tricarboxylic acid cycle intermediate α-ketoglutarate, involving d-xylose dehydrogenase (HVO_B0028), a novel xylonate dehydratase (HVO_B0038A), 2-keto-3-deoxyxylonate dehydratase (HVO_B0027), and α-ketoglutarate semialdehyde dehydrogenase (HVO_B0039). The functional involvement of these enzymes in xylose degradation was proven by growth studies of the corresponding in-frame deletion mutants, which all lost the ability to grow on d-xylose, but growth on glucose was not significantly affected. This is the first report of an archaeal d-xylose degradation pathway that differs from the classical d-xylose pathway in most bacteria involving the formation of xylulose 5-phosphate as an intermediate. However, the pathway shows similarities to proposed oxidative pentose degradation pathways to α-ketoglutarate in few bacteria, e.g. Azospirillum brasilense and Caulobacter crescentus, and in the archaeon Sulfolobus solfataricus.d-Xylose, a constituent of the polymer xylan, is the major component of the hemicellulose plant cell wall material and thus one of the most abundant carbohydrates in nature. The utilization of d-xylose by microorganisms has been described in detail in bacteria and fungi, for which two different catabolic pathways have been reported. In many bacteria, such as Escherichia coli, Bacillus, and Lactobacillus species, xylose is converted by the activities of xylose isomerase and xylulose kinase to xylulose 5-phosphate as an intermediate, which is further degraded mainly by the pentose phosphate cycle or phosphoketolase pathway. Most fungi convert xylose to xylulose 5-phosphate via xylose reductase, xylitol dehydrogenase, and xylulose kinase. Xylulose 5-phosphate is also an intermediate of the most common l-arabinose degradation pathway in bacteria, e.g. of E. coli, via activities of isomerase, kinase, and epimerase (1).Recently, by genetic evidence, a third pathway of xylose degradation was proposed for the bacterium Caulobacter crescentus, in analogy to an alternative catabolic pathway of l-arabinose, reported for some bacteria, including species of Azospirillum, Pseudomonas, Rhizobium, Burkholderia, and Herbasprillum (2, 3). In these organisms l-arabinose is oxidatively degraded to α-ketoglutarate, an intermediate of the tricarboxylic acid cycle, via the activities of l-arabinose dehydrogenase, l-arabinolactonase, and two successive dehydration reactions forming 2-keto-3-deoxy-l-arabinoate and α-ketoglutarate semialdehyde; the latter compound is further oxidized to α-ketoglutarate via NADP⁺-specific α-ketoglutarate semialdehyde dehydrogenase (KGSADH).² In a few Pseudomonas and Rhizobium species, a variant of this l-arabinose pathway was described involving aldolase cleavage of the intermediate 2-keto-3-deoxy-l-arabinoate to pyruvate and glycolaldehyde, rather than its dehydration and oxidation to α-ketoglutarate (4). Because of the presence of some analogous enzyme activities in xylose-grown cells of Azosprillum and Rhizobium, the oxidative pathway and its variant was also proposed as a catabolic pathway for d-xylose. Recent genetic analysis of Caulobacter crecentus indicates the presence of an oxidative pathway for d-xylose degradation to α-ketoglutarate. All genes encoding xylose dehydrogenase and putative lactonase, xylonate dehydratase, 2-keto-3-deoxylonate dehydratase, and KGSADH were found to be located on a xylose-inducible operon (5). With exception of xylose dehydrogenase, which has been partially characterized, the other postulated enzymes of the pathway have not been biochemically analyzed.The pathway of d-xylose degradation in the domain of archaea has not been studied so far. First analyses with the halophilic archaeon Haloarcula marismortui indicate that the initial step of d-xylose degradation involves a xylose-inducible xylose dehydrogenase (6) suggesting an oxidative pathway of xylose degradation to α-ketoglutarate, or to pyruvate and glycolaldehyde, in analogy to the proposed oxidative bacterial pentose degradation pathways. Recently, a detailed study of d-arabinose catabolism in the thermoacidophilic crenarchaeon Sulfolobus solfataricus was reported. d-Arabinose was found to be oxidized to α-ketoglutarate involving d-arabinose dehydrogenase, d-arabinoate dehydratase, 2-keto-3-deoxy-d-arabinoate dehydratase, and α-ketoglutarate semialdehyde dehydrogenase (3).In this study, we present a comprehensive analysis of the complete d-xylose degradation pathway in the halophilic archaeon Haloferax volcanii. This halophilic archaeon was chosen because it exerts several suitable properties for the analyses. For example, it can be cultivated on synthetic media with sugars, e.g. xylose, an advantage for in vivo labeling studies in growing cultures. Furthermore, a shotgun DNA microarray of H. volcanii is available (7) allowing the identification of xylose-inducible genes, and H. volcanii is one of the few archaea for which an efficient protocol was recently described to generate in-frame deletion mutants.Accordingly, the d-xylose degradation pathway was elucidated following in vivo labeling experiments with [¹³C]xylose, DNA microarray analyses, and the characterization of enzymes involved and their encoding genes. The functional involvement of genes and enzymes was proven by constructing corresponding in-frame deletion mutants and their analysis by selective growth experiments on xylose versus glucose. The data show that d-xylose was exclusively degraded to α-ketoglutarate involving xylose dehydrogenase, a novel xylonate dehydratase, 2-keto-3-deoxyxylonate dehydratase, and α-ketoglutarate semialdehyde dehydrogenase.     

Genetic identification of three ABC transporters as essential elements for nitrate respiration in Haloferax volcanii.

More than 40 nitrate respiration-deficient mutants of Haloferax volcanii belonging to three different phenotypic classes were isolated. All 15 mutants of the null phenotype were complemented with a genomic library of the wild type. Wild-type copies of mutated genes were recovered from complemented mutants using two different approaches. The DNA sequences of 13 isolated fragments were determined. Five fragments were found to overlap; therefore nine different genomic regions containing genes essential for nitrate respiration could be identified. Three genomic regions containing genes coding for subunits of ABC transporters were further characterized. In two cases, genes coding for an ATP-binding subunit and a permease subunit were clustered and overlapped by four nucleotides. The third gene for a permease subunit had no additional ABC transporter gene in proximity. One ABC transporter was found to be glucose specific. The mutant reveals that the ABC transporter solely mediates anaerobic glucose transport. Based on sequence similarity, the second ABC transporter is proposed to be molybdate specific, explaining its essential role in nitrate respiration. The third ABC transporter is proposed to be anion specific. Genome sequencing has shown that ABC transporters are widespread in Archaea. Nevertheless, this study represents only the second example of a functional characterization.     

The 2-oxoacid dehydrogenase multienzyme complex of Haloferax volcanii

Those aerobic archaea whose genomes have been sequenced possess four adjacent genes that, by sequence comparisons with bacteria and eukarya, appear to encode the component enzymes of a 2-oxoacid dehydrogenase multienzyme complex. However, no catalytic activity of any such complex has ever been detected in the archaea. In Thermoplasma acidophilum, evidence has been presented that the heterologously expressed recombinant enzyme possesses activity with the branched chain 2-oxoacids and, to a lesser extent, with pyruvate. In the current paper, we demonstrate that in Haloferax volcanii the four genes are transcribed as an operon in vivo. However, no functional complex or individual enzyme, except for the dihydrolipoamide dehydrogenase component, could be detected in this halophile grown on a variety of carbon sources. Dihydrolipoamide dehydrogenase is present at low catalytic activities, the level of which is increased three to fourfold when Haloferax volcanii is grown on the branched-chain amino acids valine, leucine and isoleucine.     

High-throughput mapping of origins of replication in human cells

Mapping origins of replication has been challenging in higher eukaryotes. We have developed a rapid, genome-wide method to map origins of replication in asynchronous human cells by combining the nascent strand abundance assay with a highly tiled microarray platform, and we validated the technique by two independent assays. We applied this method to analyse the enrichment of nascent DNA in three 50-kb regions containing known origins of replication in the MYC, lamin B2 (LMNB2) and haemoglobin beta (HBB) genes, a 200-kb region containing the rare fragile site, FRAXA, and a 1,075-kb region on chromosome 22; we detected most of the known origins and also 28 new origins. Surprisingly, the 28 new origins were small in size and located predominantly within genes. Our study also showed a strong correlation between origin replication timing and chromatin acetylation.     

Physical and genetic mapping of mammalian replication origins.

The neutral/neutral and neutral/alkaline two-dimensional gel electrophoretic techniques are sensitive physical mapping methods that have been used successfully to identify replication initiation sites in genomes of widely varying complexity. We present detailed methodology for the preparation of replication intermediates from mammalian cells and their analysis by both neutral/neutral and neutral/alkaline two-dimensional gel approaches. The methods described allow characterization of the replication pattern of single-copy loci, even in mammalian cells. When applied to metazoans, initiation is found to occur at multiple sites scattered throughout zones that can be as long as 50 kb, with some subregions being preferred. Although these observations do not rule out the possibility of genetically defined replicators, they offer the alternative or additional possibility that chromosomal context may play an important role in defining replication initiation sites in complex genomes. We discuss novel recombination strategies that can be used to test for the presence of sequence elements critical for origin function if the origin lies in the vicinity of a selectable gene. Application of this strategy to the DHFR locus shows that loss of sequences more than 25 kb from the local initiation zone can markedly affect origin activity in the zone.     

Pseudouridine formation in archaeal RNAs: The case of Haloferax volcanii

Pseudouridine (Ψ), the isomer of uridine, is commonly found at various positions of noncoding RNAs of all organisms. Ψ residues are formed by a number of single- or multisite specific Ψ synthases, which generally act as stand-alone proteins. In addition, in Eukarya and Archaea, specific ribonucleoprotein complexes, each containing a distinct box H/ACA guide RNA and four core proteins, can produce Ψ at many sites of different cellular RNAs. Cbf5 is the core Ψ synthase in these complexes. Using Haloferax volcanii as an archaeal model organism, we show that, contrary to eukaryotes, the Cbf5 homolog (HVO_2493) is not essential in this archaeon. The Cbf5-deleted strain of H. volcanii completely lacks Ψ at positions 1940, 1942, 2605, and 2591 (Escherichia coli positions 1915, 1917, 2572, and 2586) of its 23S rRNA, and contains reduced steady-state levels of some box H/ACA RNAs. Archaeal Cbf5 is known to have tRNA Ψ55 synthase activity in vitro but we could not confirm this activity in vivo in H. volcanii. Conversely, the Pus10 (previously PsuX) homolog (HVO_1979), which can produce tRNA Ψ55, as well as Ψ54 in vitro, is shown here to be essential in H. volcanii, whereas the corresponding tRNA Ψ55 synthases, Pus4 and TruB, are not essential in yeast and E. coli, respectively. Finally, we demonstrate that HVO_1852, the TruA/Pus3 homolog, is responsible for the pseudouridylation of position 39 in H. volcanii tRNAs and that the corresponding gene is not essential.