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.     

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.     

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.     

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.     

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.     

Genes for tryptophan biosynthesis in the halophilic archaebacterium Haloferax volcanii: the trpDFEG cluster.

Tryptophan auxotrophs of the archaebacterium Haloferax volcanii define a cluster of overlapping genes homologous to eubacterial-eukaryotic trpD, -F, -E, and -G, linked in that order and each preceded by a possible ribosome binding site. Residues involved in feedback inhibition of eubacterial anthranilate synthetases are conserved.     

ABC transporters as differentiation markers in glioblastoma cells

Glioblastoma multiforme (GBM) is the most common primary malignant brain tumour, characterized by a high aggressivity, a huge heterogeneity attending a hierarchical model and resistance to therapy. Drug resistance has been correlated with the presence of the ABC efflux transporters which are able to exclude drugs for the cellular cytoplasm. In the nucleus of the GBM, initiating cells (ICs) can self-renew and give rise to cancer stem cells, which differ to the side population cells and the different cellular subtypes that form the mass around them. The ICs do not express or express ATP binding cassette (ABC) at very low levels, but this expression may increase with the differentiation process. We suggest that the differentiation process may be responsible of chemoresistance of the GBM cells. We compared three ABC transporters expression: ABCA1, MRP4 and MRP5, in the ICs obtained from 9 patients with GBM and their respective differentiated GBM cells. We show an overexpression of the three ABC transporters in the differentiated GBM cells in comparison to ICs. Implications of the hypothesis: The blockade of these ABC transporters could help to improve the drug effectivity and thus reduce the tumour growth and prevent the tumour recurrence.     

Primary structure and glycosylation of the S-layer protein of Haloferax volcanii.

The outer surface of the archaebacterium Haloferax volcanii (formerly named Halobacterium volcanii) is covered with a hexagonally packed surface (S) layer. The gene coding for the S-layer protein was cloned and sequenced. The mature polypeptide is composed of 794 amino acids and is preceded by a typical signal sequence of 34 amino acid residues. A highly hydrophobic stretch of 20 amino acids at the C-terminal end probably serves as a transmembrane domain. Clusters of threonine residues are located adjacent to this membrane anchor. The S-layer protein is a glycoprotein containing both N- and O-glycosidic bonds. Glucosyl-(1----2)-galactose disaccharides are linked to threonine residues. The primary structure and the glycosylation pattern of the S-layer glycoproteins from Haloferax volcanii and from Halobacterium halobium were compared and found to exhibit distinct differences, despite the fact that three-dimensional reconstructions from electron micrographs revealed no structural differences at least to the 2.5-nm level attained so far (M. Kessel, I. Wildhaber, S. Cohe, and W. Baumeister, EMBO J. 7:1549-1554, 1988).     

5-Methoxyindole-2-carboxylic acid is a potent inhibitor of respiration and potassium ion transport in the archaebacterium Haloferax volcanii

Abstract The chorioallantoic membrane (CAM) inoculated chick embryo model was used to study the effect of host lineage on the virulence of Campylobacter jejuni and Campylobacter coli . LD₅₀ values were used to compare the susceptibilities of chick embryos from eight inbred chicken lines to infection by four strains of C. jejuni and one strain of C. coli . Differences in susceptibility were found between inbred chicken lines. These were shown not to be due to maternal antibody status, not transfer of antibody to the developing embryo. Susceptibility to infection was also found to vary according to the Campylobacter strain used. These results indicate that both the bacterial strain and host lineage of the chicken line used affect resistance to infection in the CAM inoculated chick embryo model.     

Optimized generation of vectors for the construction of Haloferax volcanii deletion mutants

A method for the construction of in frame deletions in chromosomal genes of the archaeon Haloferax volcanii has been described recently (Allers et al. (2004), Appl. Environ. Microbiol. 70:943-953). Two steps of the procedure for deletion vector construction were optimized. First, the deletion version of the gene of interest was generated by fusion of two PCR products, which were comprised of the upstream and downstream regions of the gene, respectively, using a third PCR reaction. Second, the fusion PCR product was ligated with the optimized vector pMH101 in the presence of one of four possible restriction enzymes ("restriction selection cloning"). Taken together, the optimized procedure omits one cloning step and enhances both speed and efficiency of deletion vector construction considerably.     

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.