AglQ Is a Novel Component of the Haloferax volcanii N-Glycosylation Pathway

N-glycosylation is a post-translational modification performed by members of all three domains of life. Studies on the halophile Haloferax volcanii have offered insight into the archaeal version of this universal protein-processing event. In the present study, AglQ was identified as a novel component of the pathway responsible for the assembly and addition of a pentasaccharide to select Asn residues of Hfx. volcanii glycoproteins, such as the S-layer glycoprotein. In cells deleted of aglQ, both dolichol phosphate, the lipid carrier used in Hfx. volcanii N-glycosylation, and modified S-layer glycoprotein Asn residues only presented the first three pentasaccharide subunits, pointing to a role for AglQ in either preparing the third sugar for attachment of the fourth pentasaccharide subunit or processing the fourth sugar prior to its addition to the lipid-linked trisaccharide. To better define the precise role of AglQ, shown to be a soluble protein, bioinformatics tools were recruited to identify sequence or structural homologs of known function. Site-directed mutagenesis experiments guided by these predictions identified residues important for AglQ function. The results obtained point to AglQ acting as an isomerase in Hfx. volcanii N-glycosylation.     

The Complete Genome Sequence of Haloferax volcanii DS2, a Model Archaeon

Background

Haloferax volcanii is an easily culturable moderate halophile that grows on simple defined media, is readily transformable, and has a relatively stable genome. This, in combination with its biochemical and genetic tractability, has made Hfx. volcanii a key model organism, not only for the study of halophilicity, but also for archaeal biology in general.

Methodology/Principal Findings

We report here the sequencing and analysis of the genome of Hfx. volcanii DS2, the type strain of this species. The genome contains a main 2.848 Mb chromosome, three smaller chromosomes pHV1, 3, 4 (85, 438, 636 kb, respectively) and the pHV2 plasmid (6.4 kb).

Conclusions/Significance

The completed genome sequence, presented here, provides an invaluable tool for further in vivo and in vitro studies of Hfx. volcanii.     

Biochemical characterisation of LigN, an NAD+-dependent DNA ligase from the halophilic euryarchaeon Haloferax volcanii that displays maximal in vitro activity at high salt concentrations

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.     

Substrate Promiscuity: AglB,the Archaeal Oligosaccharyltransferase,Can Process a Variety of Lipid-Linked Glycans

Across evolution, N-glycosylation involves oligosaccharyltransferases that transfer lipid-linked glycans to selected Asn residues of target proteins. While these enzymes catalyze similar reactions in each domain, differences exist in terms of the chemical composition, length and degree of phosphorylation of the lipid glycan carrier, the sugar linking the glycan to the lipid carrier, and the composition and structure of the transferred glycan. To gain insight into how oligosaccharyltransferases cope with such substrate diversity, the present study analyzed the archaeal oligosaccharyltransferase AglB from four haloarchaeal species. Accordingly, it was shown that despite processing distinct lipid-linked glycans in their native hosts, AglB from Haloarcula marismortui, Halobacterium salinarum, and Haloferax mediterranei could readily replace their counterpart from Haloferax volcanii when introduced into Hfx. volcanii cells deleted of aglB. As the four enzymes show significant sequence and apparently structural homology, it appears that the functional similarity of the four AglB proteins reflects the relaxed substrate specificity of these enzymes. Such demonstration of AglB substrate promiscuity is important not only for better understanding of N-glycosylation in Archaea and elsewhere but also for efforts aimed at transforming Hfx. volcanii into a glycoengineering platform.     

AglS,a Novel Component of the Haloferax volcanii N-Glycosylation Pathway,Is a Dolichol Phosphate-Mannose Mannosyltransferase

In Haloferax volcanii, a series of Agl proteins mediates protein N-glycosylation. The genes encoding all but one of the Agl proteins are sequestered into a single gene island. The same region of the genome includes sequences also suspected but not yet verified as serving N-glycosylation roles, such as HVO_1526. In the following, HVO_1526, renamed AglS, is shown to be necessary for the addition of the final mannose subunit of the pentasaccharide N-linked to the surface (S)-layer glycoprotein, a convenient reporter of N-glycosylation in Hfx. volcanii. Relying on bioinformatics, topological analysis, gene deletion, mass spectrometry, and biochemical assays, AglS was shown to act as a dolichol phosphate-mannose mannosyltransferase, mediating the transfer of mannose from dolichol phosphate to the tetrasaccharide corresponding to the first four subunits of the pentasaccharide N-linked to the S-layer glycoprotein.     

Crystal structure of the ubiquitin‐like small archaeal modifier protein 2 from Haloferax volcanii

The discovery of ubiquitin‐like small archaeal modifier protein 2 (SAMP2) that forms covalent polymeric chains in Haloferax volcanii has generated tremendous interest in the function and regulation of this protein. At present, it remains unclear whether the Hfx. volcanii modifier protein SAMP1 has such polyubiquitinating‐like activity. Although SAMP1 and SAMP2 use the same conjugation machinery to modify their target proteins, each can impart distinct functional consequences. To better understand the mechanism of SAMP2 conjugation, we have sought to characterize the biophysical and structural properties of the protein from Hfx. volcanii. SAMP2 is only partially structured under mesohalic solution conditions and adopts a well‐folded compact conformation in the presence of 2.5M of NaCl. Its 2.3‐Å‐resolution crystal structure reveals a characteristic α/β central core domain and a unique β‐hinge motif. This motif anchors an unusual C‐terminal extension comprising the diglycine tail as well as two lysine residues that can potentially serve to interlink SAMP2 moieties. Mutational alternation of the structural malleability of this β‐hinge motif essentially abolishes the conjugation activity of SAMP2 in vivo. In addition, NMR structural studies of the putative ubiquitin‐like protein HVO_2177 from Hfx. volcanii show that like SAMP1, HVO_2177 forms a classic β‐grasp fold in a salt‐independent manner. These results provide insights into the structure–function relationship of sampylating proteins of fundamental importance in post‐translational protein modification and environmental cues in Archaea.     

Genetic and physical mapping of DNA replication origins in Haloferax volcanii

The halophilic archaeon Haloferax volcanii has a multireplicon genome, consisting of a main chromosome, three secondary chromosomes, and a plasmid. Genes for the initiator protein Cdc6/Orc1, which are commonly located adjacent to archaeal origins of DNA replication, are found on all replicons except plasmid pHV2. However, prediction of DNA replication origins in H. volcanii is complicated by the fact that this species has no less than 14 cdc6/orc1 genes. We have used a combination of genetic, biochemical, and bioinformatic approaches to map DNA replication origins in H. volcanii. Five autonomously replicating sequences were found adjacent to cdc6/orc1 genes and replication initiation point mapping was used to confirm that these sequences function as bidirectional DNA replication origins in vivo. Pulsed field gel analyses revealed that cdc6/orc1-associated replication origins are distributed not only on the main chromosome (2.9 Mb) but also on pHV1 (86 kb), pHV3 (442 kb), and pHV4 (690 kb) replicons. Gene inactivation studies indicate that linkage of the initiator gene to the origin is not required for replication initiation, and genetic tests with autonomously replicating plasmids suggest that the origin located on pHV1 and pHV4 may be dominant to the principal chromosomal origin. The replication origins we have identified appear to show a functional hierarchy or differential usage, which might reflect the different replication requirements of their respective chromosomes. We propose that duplication of H. volcanii replication origins was a prerequisite for the multireplicon structure of this genome, and that this might provide a means for chromosome-specific replication control under certain growth conditions. Our observations also suggest that H. volcanii is an ideal organism for studying how replication of four replicons is regulated in the context of the archaeal cell cycle.     

Cu-NirK from Haloferax mediterranei as an example of metalloprotein maturation and exportation via Tat system

The green Cu-NirK from Haloferax mediterranei (Cu-NirK) has been expressed, refolded and retrieved as a trimeric enzyme using an expression method developed for halophilic Archaea. This method utilizes Haloferax volcanii as a halophilic host and an expression vector with a constitutive and strong promoter. The enzymatic activity of recombinant Cu-NirK was detected in both cellular fractions (cytoplasmic fraction and membranes) and in the culture media. The characterization of the enzyme isolated from the cytoplasmic fraction as well as the culture media revealed important differences in the primary structure of both forms indicating that Hfx. mediterranei could carry out a maturation and exportation process within the cell before the protein is exported to the S-layer. Several conserved signals found in Cu-NirK from Hfx. mediterranei sequence indicate that these processes are closely related to the Tat system. Furthermore, the N-terminal sequence of the two Cu-NirK subunits constituting different isoforms revealed that translation of this protein could begin at two different points, identifying two possible start codons. The hypothesis proposed in this work for halophilic Cu-NirK processing and exportation via the Tat system represents the first approximation of this mechanism in the Halobacteriaceae family and in Prokarya in general.     

Mre11-Rad50 Promotes Rapid Repair of DNA Damage in the Polyploid Archaeon Haloferax volcanii by Restraining Homologous Recombination

Polyploidy is frequent in nature and is a hallmark of cancer cells, but little is known about the strategy of DNA repair in polyploid organisms. We have studied DNA repair in the polyploid archaeon Haloferax volcanii, which contains up to 20 genome copies. We have focused on the role of Mre11 and Rad50 proteins, which are found in all domains of life and which form a complex that binds to and coordinates the repair of DNA double-strand breaks (DSBs). Surprisingly, mre11 rad50 mutants are more resistant to DNA damage than the wild-type. However, wild-type cells recover faster from DNA damage, and pulsed-field gel electrophoresis shows that DNA double-strand breaks are repaired more slowly in mre11 rad50 mutants. Using a plasmid repair assay, we show that wild-type and mre11 rad50 cells use different strategies of DSB repair. In the wild-type, Mre11-Rad50 appears to prevent the repair of DSBs by homologous recombination (HR), allowing microhomology-mediated end-joining to act as the primary repair pathway. However, genetic analysis of recombination-defective radA mutants suggests that DNA repair in wild-type cells ultimately requires HR, therefore Mre11-Rad50 merely delays this mode of repair. In polyploid organisms, DSB repair by HR is potentially hazardous, since each DNA end will have multiple partners. We show that in the polyploid archaeon H. volcanii the repair of DSBs by HR is restrained by Mre11-Rad50. The unrestrained use of HR in mre11 rad50 mutants enhances cell survival but leads to slow recovery from DNA damage, presumably due to difficulties in the resolution of DNA repair intermediates. Our results suggest that recombination might be similarly repressed in other polyploid organisms and at repetitive sequences in haploid and diploid species.     

A predicted geranylgeranyl reductase reduces the ω-position isoprene of dolichol phosphate in the halophilic archaeon, Haloferax volcanii

In N-glycosylation in both Eukarya and Archaea, N-linked oligosaccharides are assembled on dolichol phosphate prior to transfer of the glycan to the protein target. However, whereas only the α-position isoprene subunit is saturated in eukaryal dolichol phosphate, both the α- and ω-position isoprene subunits are reduced in the archaeal lipid. The agents responsible for dolichol phosphate saturation remain largely unknown. The present study sought to identify dolichol phosphate reductases in the halophilic archaeon, Haloferax volcanii. Homology-based searches recognize HVO_1799 as a geranylgeranyl reductase. Mass spectrometry revealed that cells deleted of HVO_1799 fail to fully reduce the isoprene chains of H. volcanii membrane phospholipids and glycolipids. Likewise, the absence of HVO_1799 led to a loss of saturation of the ω-position isoprene subunit of C₅₅ and C₆₀ dolichol phosphate, with the effect of HVO_1799 deletion being more pronounced with C₆₀ dolichol phosphate than with C₅₅ dolichol phosphate. Glycosylation of dolichol phosphate in the deletion strain occurred preferentially on that version of the lipid saturated at both the α- and ω-position isoprene subunits.     

Control of teichoicand teichuronic acid biosynthesis in Bacillus subitlis 168trp−: Evidence for repression of enzyme synthesis and inhibition of enzyme activity

Phosphate starvatiion induced teichuronic acid synthesis in cells of Bacillus subtilis 168trp^? which had previously been grown with excess phophate. This induction was prevented when protein synthesis was inhibited immediately prior to phosphate starvation and under these conditions cells continued to form teichoic acid. The converse was true when phosphate was added to cells previously grown in phosphate-limited chemostat. The increase in teichoic acid synthesis normally following phosphate addition was prevented by chlorampehnicol or amino acid starvation and cells continued to make teichuronic acid. The suggestion that repression of enzyme synthesis is involved in controlling the type of wall polymer made was supported by the low levels of UDP-glucose dehydrogenase found in cells grown with excess phosphate and of CDP-glycerol pyrophosphorylase in phophate-limited cells. The greater amounts of teichoic acid made under phosphate limitation and of teichuronic acid with excess phosphate when protein synthesis was also inhibited indicated that modulation of enzyme activity occurs. Glycerol starvation of a glycerol-requiring mutant did not derepress teichuronic acid synthesis, indicating that glycerol-containing intermediates do not act as repressors.     

Detection of DNA Damage in Prokaryotes by Terminal Deoxyribonucleotide Transferase-Mediated dUTP Nick End Labeling

Numerous agents can damage the DNA of prokaryotes in the environment (e.g., reactive oxygen species, irradiation, and secondary metabolites such as antibiotics, enzymes, starvation, etc.). The large number of potential DNA-damaging agents, as well as their diverse modes of action, precludes a simple test of DNA damage based on detection of nucleic acid breakdown products. In this study, free 3′-OH DNA ends, produced by either direct damage or excision DNA repair, were used to assess DNA damage. Terminal deoxyribonucleotide transferase (TdT)-mediated dUTP nick end labeling (TUNEL) is a procedure in which 3′-OH DNA ends are enzymatically labeled with dUTP-fluorescein isothiocyanate using TdT. Cells labeled by this method can be detected using fluorescence microscopy or flow cytometry. TUNEL was used to measure hydrogen peroxide-induced DNA damage in the archaeon Haloferax volcanii and the bacterium Escherichia coli. DNA repair systems were implicated in the hydrogen peroxide-dependent generation of 3′-OH DNA ends by the finding that the protein synthesis inhibitors chloramphenicol and diphtheria toxin blocked TUNEL labeling of E. coli and H. volcanii, respectively. DNA damage induced by UV light and bacteriophage infection was also measured using TUNEL. This methodology should be useful in applications where DNA damage and repair are of interest, including mutant screening and monitoring of DNA damage in the environment.     

Haloferax volcanii N-Glycosylation: Delineating the Pathway of dTDP-rhamnose Biosynthesis

In the halophilic archaea Haloferax volcanii, the surface (S)-layer glycoprotein can be modified by two distinct N-linked glycans. The tetrasaccharide attached to S-layer glycoprotein Asn-498 comprises a sulfated hexose, two hexoses and a rhamnose. While Agl11-14 have been implicated in the appearance of the terminal rhamnose subunit, the precise roles of these proteins have yet to be defined. Accordingly, a series of in vitro assays conducted with purified Agl11-Agl14 showed these proteins to catalyze the stepwise conversion of glucose-1-phosphate to dTDP-rhamnose, the final sugar of the tetrasaccharide glycan. Specifically, Agl11 is a glucose-1-phosphate thymidylyltransferase, Agl12 is a dTDP-glucose-4,6-dehydratase and Agl13 is a dTDP-4-dehydro-6-deoxy-glucose-3,5-epimerase, while Agl14 is a dTDP-4-dehydrorhamnose reductase. Archaea thus synthesize nucleotide-activated rhamnose by a pathway similar to that employed by Bacteria and distinct from that used by Eukarya and viruses. Moreover, a bioinformatics screen identified homologues of agl11-14 clustered in other archaeal genomes, often as part of an extended gene cluster also containing aglB, encoding the archaeal oligosaccharyltransferase. This points to rhamnose as being a component of N-linked glycans in Archaea other than Hfx. volcanii.     

Conditional identification of phosphate-starvation-response mutants in Arabidopsis thaliana

Plants have evolved elaborate metabolic and developmental adaptations to low phosphorus availability. Biochemical responses to phosphate limitation include increased production and secretion of phosphate-acquisition proteins such as nucleases, acid phosphatases, and high-affinity phosphate transporters. However, the signal transduction pathways that sense phosphate availability and integrate the phosphate-starvation response in plants are unknown. We have devised a screen for conditional mutants in Arabidopsis thaliana (L.) Heynh. to dissect signaling of phosphate limitation. Our genetic screen is based on the facultative ability of wild-type Arabidopsis plants to metabolize exogenous DNA when inorganic phosphate is limiting. After screening 50,000 M2 seedlings, we isolated 22 confirmed mutant lines that showed severely impaired growth on medium containing DNA as the only source of phosphorus, but which recovered on medium containing soluble inorganic phosphate. Characterization of nine such mutant lines demonstrated an inability to utilize either DNA or RNA. One mutant line, psr1 (phosphate starvation response), had significantly reduced activities of phosphate-starvation-inducible isoforms of ribonuclease and acid phosphatase under phosphate-limiting conditions. The data suggest that a subset of the selected mutations impairs the expression of more than one phosphate-starvation-inducible enzyme required for utilization of exogenous nucleic acids, and may thus affect regulatory components of a Pi starvation response pathway in higher plants. Received: 23 September 1999 / Accepted: 10 November 1999     

OsCYCP1;1, a PHO80 homologous protein,negatively regulates phosphate starvation signaling in the roots of rice (Oryza sativa L.)

Phosphorus is one of the most essential and limiting nutrients in all living organisms, thus the organisms have evolved complicated and precise regulatory mechanisms for phosphorus acquisition, storage and homeostasis. In the budding yeast, Saccharomyces cerevisiae, the modification of PHO4 by the PHO80 and PHO85 complex is a core regulation system. However, the existence and possible functions in phosphate signaling of the homologs of the PHO80 and PHO85 components in plants has yet to be determined. Here we describe the identification of a family of seven PHO80 homologous genes in rice named OsCYCPs. Among these, the OsCYCP1;1 gene was able to partially rescue the pho80 mutant strain of yeast. The OsCYCP1;1 protein was predominantly localized in the nucleus, and was ubiquitously expressed throughout the whole plant and during the entire growth period of rice. Consistent with the negative role of PHO80 in phosphate signaling in yeast, OsCYCP1;1 expression was reduced by phosphate starvation in the roots. This reduction was dependent on PHR2, the central regulator of phosphate signaling in rice. Overexpression and suppression of the expression of OsCYCP1;1 influenced the phosphate starvation signaling response. The inducible expression of phosphate starvation inducible and phosphate transporter genes was suppressed in the OsCYCP1;1 overexpression lines and was relatively enhanced in the OsCYCP1;1 RNAi plants by phosphate starvation. Together, these results demonstrate the role of PHO80 homologs in the phosphate starvation signaling pathway in rice.     

Changes in Ribosomal Activity of Escherichia coli Cells during Prolonged Culture in Sea Salts Medium

The activity of ribosomes from a clinical isolate of Escherichia coli, exposed to starvation for 7 days in sea salts medium, was investigated by measuring the kinetic parameters of ribosomal peptidyltransferase, by using the puromycin reaction as a model reaction. No alterations in the extent of peptide bond formation were observed during starvation. In contrast, a 50% reduction in the k_max/K_s ratio could be seen after 24 h of starvation; an additional 6 days of starvation resulted in a progressive but less abrupt decline in the k_max/K_s value. {k_max is the apparent catalytic rate constant of peptidyl transferase, and K_s is the dissociation constant of the encounter complex between acetyl (Ac)[³H]Phe-tRNA-poly(U)-ribosome and puromycin.} Although the distribution of ribosomal particles remained constant, a substantial decrease in the number of ribosomes per starved cell and a clear decline in the ability of ribosomes to bind AcPhe-tRNA were observed, particularly during the first day of starvation. Further analysis indicated that rRNA in general, but especially 23S rRNA, was rapidly degraded during the starvation period. In addition, the L12/L7 molar ratio decreased from 1.5 to 1 during the initial phase of starvation (up to 24 h) but remained constant during the subsequent starvation period. Ribosomes isolated from 24-h-starved cells, when artificially depleted of L7/L12 protein and reconstituted with L7/L12 protein from mid-logarithmic-phase cells, regenerated an L12/L7 molar ratio of 1.5 and restored the peptidyltransferase activity to a substantial level. An analogous effect of reconstitution on the efficiency of ribosomes in binding AcPhe-tRNA was evident not only during the initial phase but throughout the starvation period.