A plasmid-based vector system for the cloning and expression of Helicobacter pylori genes encoding outer membrane proteins

Helicobacter pylori produces a number of proteins associated with the outer membrane, including adhesins and the vacuolating cytotoxin. We observed that the functional expression of such proteins is deleterious to Escherichia coli, the host bacterium used for gene cloning. Therefore, a general method was developed for the functional expression of such genes on a shuttle vector in H. pylori, which has been termed SOMPES (Shuttle vector-based Outer Membrane Protein Expression System). The intact, active gene is reconstituted by recombination in H. pylori from partial gene sequences cloned on an E. coli-H. pylori shuttle vector. This system was established in an H. pylori strain carrying a precise, unmarked chromosomal deletion of the vacA gene, which was constructed by adapting the streptomycin sensitivity system to H. pylori. It is based on the expression of the H. pylori rpsL gene as a counterselectable marker in the genetic background of an rpsL mutant. The utility of this approach is demonstrated by the expression of a recombinant gene encoding vacuolating cytotoxin (vacA) and a recombinant gene encoding an adherence-associated outer membrane protein (alpA) in H. pylori. Received: 10 May 1999 / Accepted: 7 July 1999     

Nucleosome Spacing Generated by ISWI and CHD1 Remodelers Is Constant Regardless of Nucleosome Density

Arrays of regularly spaced nucleosomes are a hallmark of chromatin, but it remains unclear how they are generated. Recent genome-wide studies, in vitro and in vivo, showed constant nucleosome spacing even if the histone concentration was experimentally reduced. This counters the long-held assumption that nucleosome density determines spacing and calls for factors keeping spacing constant regardless of nucleosome density. We call this a clamping activity. Here, we show in a purified system that ISWI- and CHD1-type nucleosome remodelers have a clamping activity such that they not only generate regularly spaced nucleosome arrays but also generate constant spacing regardless of nucleosome density. This points to a functionally attractive nucleosome interaction that could be mediated either directly by nucleosome-nucleosome contacts or indirectly through the remodelers. Mutant Drosophila melanogaster ISWI without the HAND-SANT-SLIDE (HSS) domain had no detectable spacing activity even though it is known to remodel and slide nucleosomes. This suggests that the role of ISWI remodelers in generating constant spacing is not just to mediate nucleosome sliding; they actively contribute to the attractive interaction. Additional factors are necessary to set physiological spacing in absolute terms.     

Mechanically probing the folding pathway of single RNA molecules

We study theoretically the denaturation of single RNA molecules by mechanical stretching, focusing on signatures of the (un)folding pathway in molecular fluctuations. Our model describes the interactions between nucleotides by incorporating the experimentally determined free energy rules for RNA secondary structure, whereas exterior single-stranded regions are modeled as freely jointed chains. For exemplary RNA sequences (hairpins and the Tetrahymena thermophila group I intron), we compute the quasiequilibrium fluctuations in the end-to-end distance as the molecule is unfolded by pulling on opposite ends. Unlike the average quasiequilibrium force-extension curves, these fluctuations reveal clear signatures from the unfolding of individual structural elements. We find that the resolution of these signatures depends on the spring constant of the force-measuring device, with an optimal value intermediate between very rigid and very soft. We compare and relate our results to recent experiments by Liphardt et al. (2001).     

Differential control of cell cycle,proliferation, and survival of primary T lymphocytes by purine and pyrimidine nucleotides

Purine and pyrimidine nucleotides play critical roles in DNA and RNA synthesis as well as in membrane lipid biosynthesis and protein glycosylation. They are necessary for the development and survival of mature T lymphocytes. Activation of T lymphocytes is associated with an increase of purine and pyrimidine pools. However, the question of how purine vs pyrimidine nucleotides regulate proliferation, cell cycle, and survival of primary T lymphocytes following activation has not yet been specifically addressed. This was investigated in the present study by using well-known purine (mycophenolic acid, 6-mercaptopurine) and pyrimidine (methotrexate, 5-fluorouracil) inhibitors, which are used in neoplastic diseases or as immunosuppressive agents. The effect of these inhibitors was analyzed according to their time of addition with respect to the initiation of mitogenic activation. We showed that synthesis of both purine and pyrimidine nucleotides is required for T cell proliferation. However, purine and pyrimidine nucleotides differentially regulate the cell cycle since purines control both G(1) to S phase transition and progression through the S phase, whereas pyrimidines only control progression from early to intermediate S phase. Furthermore, inhibition of pyrimidine synthesis induces apoptosis whatever the time of inhibitor addition whereas inhibition of purine nucleotides induces apoptosis only when applied to already cycling T cells, suggesting that both purine and pyrimidine nucleotides are required for survival of cells committed into S phase. These findings reveal a hitherto unknown role of purine and pyrimidine de novo synthesis in regulating cell cycle progression and maintaining survival of activated T lymphocytes.     

Mycophenolic acid inhibits IL-2-dependent T cell proliferation,but not IL-2-dependent survival and sensitization to apoptosis

Mycophenolic acid (MPA), the active metabolite of the immunosuppressive drug mycophenolate mofetil, is a selective inhibitor of inosine 5'-monophosphate dehydrogenase type II, a de novo purine nucleotide synthesis enzyme expressed in T and B lymphocytes and up-regulated upon cell activation. In this study, we report that the blockade of guanosine nucleotide synthesis by MPA inhibits mitogen-induced proliferation of PBL, an effect fully reversed by addition of guanosine and shared with mizoribine, another inhibitor of inosine 5'-monophosphate dehydrogenase. Because MPA does not inhibit early TCR-mediated activation events, such as CD25 expression and IL-2 synthesis, we investigated how it interferes with cytokine-dependent proliferation and survival. In activated lymphoblasts that are dependent on IL-2 or IL-15 for their proliferation, MPA does not impair signaling events such as of the extracellular signal-regulated kinase 2 and Stat5 phosphorylation, but inhibits down-regulation of the cyclin-dependent kinase inhibitor p27(Kip1). Therefore, in activated lymphoblasts, MPA specifically interferes with cytokine-dependent signals that control cell cycle and blocks activated T cells in the mid-G(1) phase of the cell cycle. Although it blocks IL-2-mediated proliferation, MPA does not inhibit cell survival and Bcl-x(L) up-regulation by IL-2 or other cytokines whose receptors share the common gamma-chain (CD132). Finally, MPA does not interfere with IL-2-dependent acquisition of susceptibility to CD95-mediated apoptosis and degradation of cellular FLIP. Therefore, MPA has unique functional properties not shared by other immunosuppressive drugs interfering with IL-2R signaling events such as rapamycin and CD25 mAbs.     

Replication-guided nucleosome packing and nucleosome breathing expedite the formation of dense arrays

The first level of genome packaging in eukaryotic cells involves the formation of dense nucleosome arrays, with DNA coverage near 90% in yeasts. How cells achieve such high coverage within a short time, e.g. after DNA replication, remains poorly understood. It is known that random sequential adsorption of impenetrable particles on a line reaches high density extremely slowly, due to a jamming phenomenon. The nucleosome-shifting action of remodeling enzymes has been proposed as a mechanism to resolve such jams. Here, we suggest two biophysical mechanisms which assist rapid filling of DNA with nucleosomes, and we quantitatively characterize these mechanisms within mathematical models. First, we show that the ‘softness’ of nucleosomes, due to nucleosome breathing and stepwise nucleosome assembly, significantly alters the filling behavior, speeding up the process relative to ‘hard’ particles with fixed, mutually exclusive DNA footprints. Second, we explore model scenarios in which the progression of the replication fork could eliminate nucleosome jamming, either by rapid filling in its wake or via memory of the parental nucleosome positions. Taken together, our results suggest that biophysical effects promote rapid nucleosome filling, making the reassembly of densely packed nucleosomes after DNA replication a simpler task for cells than was previously thought.     

Autodisplay: functional display of active beta-lactamase on the surface of Escherichia coli by the AIDA-I autotransporter

Members of the protein family of immunoglobulin A1 protease-like autotransporters comprise multidomain precursors consisting of a C-terminal autotransporter domain that promotes the translocation of N-terminally attached passenger domains across the cell envelopes of gram-negative bacteria. Several autotransporter domains have recently been shown to efficiently promote the export of heterologous passenger domains, opening up an effective tool for surface display of heterologous proteins. Here we report on the autotransporter domain of the Escherichia coli adhesin involved in diffuse adherence (AIDA-I), which was genetically fused to the C terminus of the periplasmic enzyme beta-lactamase, leading to efficient expression of the fusion protein in E. coli. The beta-lactamase moiety of the fusion protein was presented on the bacterial surface in a stable manner, and the surface-located beta-lactamase was shown to be enzymatically active. Enzymatic activity was completely removed by protease treatment, indicating that surface display of beta-lactamase was almost quantitative. The periplasmic domain of the outer membrane protein OmpA was not affected by externally added proteases, demonstrating that the outer membranes of E. coli cells expressing the beta-lactamase AIDA-I fusion protein remained physiologically intact.