Potential and limitations of nucleon transfer experiments with radioactive beams at REX-ISOLDE

As a tool for studying the structure of nuclei far off stability the technique of gamma-ray spectroscopy after low-energy single-nucleon transfer reactions with radioactive nuclear beams in inverse kinematics was investigated. Modules of the MINIBALL germanium array and a thin position-sensitive parallel plate avalanche counter (PPAC) to be employed in future experiments at REX-ISOLDE were used in a test experiment performed with a stable 36S beam on deuteron and 9Be targets. It is demonstrated that the Doppler broadening of gamma lines detected by the MINIBALL modules is considerably reduced by exploiting their segmentation, and that for beam intensities up to 10^6 particles/s the PPAC positioned around zero degrees with respect to the beam axis allows not only to significantly reduce the gamma background by requiring coincidences with the transfer products but also to control the beam and its intensity by single particle counting. The predicted large neutron pickup cross sections of neutron-rich light nuclei on 2H and 9Be targets at REX-ISOLDE energies of 2.2 MeV A are confirmed.Comment: 11 pages, 8 figure

Excited bands and signature dependent electromagnetic decay properties in neutron-rich 159,161,163Dy

14 págs.; 12 figs.; 3 tabs. ; PACS number(s): 23.20.Lv, 27.70.1q, 21.10.ReHigh-spin states of the neutron-rich odd nuclei 159,161,163Dy have been studied using the incomplete fusion reactions 158,160Gd(7Li,(p,d,t)xn). In 159Dy, the band crossing in the 11/2-[505] band has been observed for the first time. Moreover, 11 E1 transitions connecting both signatures of the 3/2-[521] band to the 5/2+[642] band have been observed in this nucleus; the deduced B(E1)/B(E2) ratios as well as the B(M1)/B(E2) ratios for transitions within the 3/2-[521] band show a pronounced signature dependence. In 161Dy and 163Dy, rotational bands have been extended to significantly higher spin values. In 161Dy, the sequences built on the neutron 5/2-[523] and 3/2-[521] states have been followed up to spin 49/2- and 33/2-, respectively, and in both cases upbends have been observed around hℏ ω ≈0.26 MeV. In addition, a new band most probably built on the 11/2-[505] single-particle state has been identified in this isotope. In 163Dy, both the 5/2-[523] ground state band and the structure built on the 5/2+[642] neutron orbit have been extended up to the 45/2- and 49/2+ states, respectively. However, no band crossing has been observed in this nucleus. The properties of the observed bands in 159,161,163Dy are discussed and compared to calculations performed within the projected shell model. ©2003 The American Physical SocietyThis work was supported by the Deutsches Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie (BMBF). A.J. acknowledges support by the Deutsche Forschungsgemeinschaft (DFG) within the Heisenberg program.Peer Reviewe

Backbending region study in 160,162Dy using incomplete fusion reactions

18 págs.; 17 figs.; 3 tabs. ; PACS number(s): 23.20.Lv, 23.20.En, 27.70.1q, 21.60.CsThe incomplete fusion reactions 7Li→158,160Gd at beam energies of 8 MeV/nucleon have been used to study the first band crossing region in the heavy stable Dy isotopes 160,162Dy. The γ rays were detected in the GASP spectrometer in coincidence with fast charged particles detected in the ISIS silicon ball. We succeeded to observe the first backbending in 162Dy at a crossing frequency of ℏ ω ≈ 350 keV, a value much higher than expected from other nuclei in this mass region. Moreover, for the first time in a nucleus with a very large interaction strength, the yrare band in 160Dy could be established up to rather high spin (I= 20ℏ) allowing for a precise determination of the interaction strength between the ground state and the Stockholm band, |Vg-S| = 219(2) keV. Together with |Vg-S| = 14(2) kev determined for the corresponding interaction in 162Dy, a full oscillation of the strengths from one node to the next could be observed within an isotopic chain. In addition to the ground state and Stockholm bands, many other known bands in the two nuclei were considerably extended to higher spin and the experimental results are compared to calculations within the projected shell model. ©2002 The American Physical SocietyThis work has been supported by Deutsches Bundesministerium fur Bildung, Wissenschaft, Forschung und Technologie (BMBF). A.J. acknowledges support by the Deutsche Forschungsgemeinschaft (DFG).Peer Reviewe

Structural basis for cooperativity of human monoclonal antibodies to meningococcal factor H-binding protein

Monoclonal antibody (mAb) cooperativity is a phenomenon triggered when mAbs couples promote increased bactericidal killing compared to individual partners. Cooperativity has been deeply investigated among mAbs elicited by factor H-binding protein (fHbp), a Neisseria meningitidis surface-exposed lipoprotein and one of the key antigens included in both serogroup B meningococcus vaccine Bexsero and Trumenba. Here we report the structural and functional characterization of two cooperative mAbs pairs isolated from Bexsero vaccines. The 3D electron microscopy structures of the human mAb-fHbp-mAb cooperative complexes indicate that the angle formed between the antigen binding fragments (fAbs) assume regular angle and that fHbp is able to bind simultaneously and stably the cooperative mAbs pairs and human factor H (fH) in vitro. These findings shed light on molecular basis of the antibody-based mechanism of protection driven by simultaneous recognition of the different epitopes of the fHbp and underline that cooperativity is crucial in vaccine efficacy

Investigating the basis of substrate recognition in the pC221 relaxosome

The nicking of the origin of transfer (oriT) is an essential initial step in the conjugative mobilization of plasmid DNA. In the case of staphylococcal plasmid pC221, nicking by the plasmid-specific MobA relaxase is facilitated by the DNA-binding accessory protein MobC; however, the role of MobC in this process is currently unknown. In this study, the site of MobC binding was determined by DNase I footprinting. MobC interacts with oriT DNA at two directly repeated 9 bp sequences, mcb1 and mcb2, upstream of the oriT nic site, and additionally at a third, degenerate repeat within the mobC gene, mcb3. The binding activity of the conserved sequences was confirmed indirectly by competitive electrophoretic mobility shift assays and directly by Surface Plasmon Resonance studies. Mutation at mcb2 abolished detectable nicking activity, suggesting that binding of this site by MobC is a prerequisite for nicking by MobA. Sequential site-directed mutagenesis of each binding site in pC221 has demonstrated that all three are required for mobilization. The MobA relaxase, while unable to bind to oriT DNA alone, was found to associate with a MobC–oriT complex and alter the MobC binding profile in a region between mcb2 and the nic site. Mutagenesis of oriT in this region defines a 7 bp sequence, sra, which was essential for nicking by MobA. Exchange of four divergent bases between the sra of pC221 and the related plasmid pC223 was sufficient to swap their substrate identity in a MobA-specific nicking assay. Based on these observations we propose a model of layered specificity in the assembly of pC221-family relaxosomes, whereby a common MobC:mcb complex presents the oriT substrate, which is then nicked only by the cognate MobA

Transfer origins in the conjugative Enterococcus faecalis plasmids pAD1 and pAM373: identification of the pAD1 nic site, a specific relaxase and a possible TraG-like protein

The Enterococcus faecalis conjugative plasmids pAD1 and pAM373 encode a mating response to the peptide sex pheromones cAD1 and cAM373 respectively. Sequence determination of both plasmids has recently been completed with strong similarity evident over many of the structural genes related to conjugation. pAD1 has two origins of transfer, with oriT1 being located within the repA determinant, whereas the more efficiently utilized oriT2 is located between orf53 and orf57 , two genes found in the present study to be essential for conjugation. We have found a similarly located oriT to be present in pAM373. oriT2 corresponds to about 285 bp based on its ability to facilitate mobilization by pAD1 when ligated to the shuttle vector pAM401; however, it was not mobilized by pAM373. In contrast, a similarly ligated fragment containing the oriT of pAM373 did not facilitate mobilization by pAD1 but was efficiently mobilized by pAM373. The oriT sites of the two plasmids each contained a homologous large inverted repeat (spanning about 140 bp) adjacent to a series of non-homologous short (6 bp) direct repeats. A hybrid construction containing the inverted repeat of pAM373 and direct repeats of pAD1 was mobilized efficiently by pAD1 but not by pAM373, indicating a significantly greater degree of specificity is associated with the direct repeats. Mutational (deletion) analyses of the pAD1 oriT2 inverted repeat structure suggested its importance in facilitating transfer or perhaps ligation of the ends of the newly transferred DNA strand. Analyses showed that Orf57 (to be called TraX) is the relaxase, which was found to induce a specific nick in the large inverted repeat inside oriT ; the protein also facilitated site-specific recombination between two oriT2 sites. Orf53 (to be called TraW) exhibits certain structural similarities to TraG-like proteins, although there is little overall homology.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72536/1/j.1365-2958.2002.03007.x.pd

Exploring host-pathogen interactions through genome wide protein microarray analysis

During bacterial pathogenesis extensive contacts between the human and the bacterial extracellular proteomes take place. The identification of novel host-pathogen interactions by standard methods using a case-by-case approach is laborious and time consuming. To overcome this limitation, we took advantage of large libraries of human and bacterial recombinant proteins. We applied a large-scale protein microarray-based screening on two important human pathogens using two different approaches: (I) 75 human extracellular proteins were tested on 159 spotted Staphylococcus aureus recombinant proteins and (II) Neisseria meningitidis adhesin (NadA), an important vaccine component against serogroup B meningococcus, was screened against ∼2300 spotted human recombinant proteins. The approach presented here allowed the identification of the interaction between the S. aureus immune evasion protein FLIPr (formyl-peptide receptor like-1 inhibitory protein) and the human complement component C1q, key players of the offense-defense fighting; and of the interaction between meningococcal NadA and human LOX-1 (low-density oxidized lipoprotein receptor), an endothelial receptor. The novel interactions between bacterial and human extracellular proteins here presented might provide a better understanding of the molecular events underlying S. aureus and N. meningitidis pathogenesis

Adaptive modulation of antibiotic resistance through intragenomic coevolution

Bacteria gain antibiotic resistance genes by horizontal acquisition of mobile genetic elements (MGEs) from other lineages. Newly acquired MGEs are often poorly adapted causing intragenomic conflicts; these are resolved by either compensatory adaptation - of the chromosome or the MGE - or reciprocal coadaptation. The footprints of such intragenomic coevolution are present in bacterial genomes, suggesting an important role promoting genomic integration of horizontally acquired genes, but direct experimental evidence of the process is limited. Here we show adaptive modulation of tetracycline resistance via intragenomic coevolution between Escherichia coli and the multidrug resistant plasmid RK2. Tetracycline treatments, including monotherapy or combination therapies with ampicillin, favoured de novo chromosomal resistance mutations coupled with mutations on RK2 impairing the plasmid-encoded tetracycline efflux pump. These mutations together provided increased tetracycline resistance at reduced cost. Additionally, the chromosomal resistance mutations conferred cross-resistance to chloramphenicol. Reciprocal coadaptation was not observed under ampicillin-only or no antibiotic selection. Intragenomic coevolution can create genomes comprising multiple replicons that together provide high-level, low-cost resistance, but the resulting co-dependence may limit the spread of coadapted MGEs to other lineages

Characterization of the Partitioning System of Myxococcus Plasmid pMF1

pMF1 is the only autonomously replicating plasmid that has been recently identified in myxobacteria. This study characterized the partitioning (par) system of this plasmid. The fragment that significantly increased the retaining stability of plasmids in Myxococcus cells in the absence of selective antibiotics contained three open reading frames (ORFs) pMF1.21-pMF1.23 (parCAB). The pMF1.22 ORF (parA) is homologous to members of the parA ATPase family, with the highest similarity (56%) to the Sphingobium japonicum ParA-like protein, while the other two ORFs had no homologs in GenBank. DNase I footprinting and electrophoretic mobility shift assays showed that the pMF1.23 (parB) product is a DNA-binding protein of iteron DNA sequences, while the product of pMF1.21 (parC) has no binding activity but is able to enhance the DNA-binding activity of ParB to iterons. The ParB protein autogenously repressed the expression of the par genes, consistent with the type Ib par pattern, while the ParC protein has less repressive activity. The ParB-binding iteron sequences are distributed not only near the partitioning gene loci but also along pMF1. These results indicate that the pMF1 par system has novel structural and functional characteristics

Neisserial adhesin A (NadA) binds human Siglec-5 and Siglec-14 with high affinity and promotes bacterial adhesion/invasion

ABSTRACT Neisserial adhesin A (NadA) is a meningococcal surface protein included as recombinant antigen in 4CMenB, a protein-based vaccine able to induce protective immune responses against Neisseria meningitidis serogroup B (MenB). Although NadA is involved in the adhesion/invasion of epithelial cells and human myeloid cells, its function in meningococcal physiology is still poorly understood. To clarify the role played by NadA in the host-pathogen interaction, we sought to identify its cellular receptors. We screened a protein microarray encompassing 2,846 human and 297 mouse surface/secreted recombinant proteins using recombinant NadA as probe. Efficient NadA binding was revealed on the paired sialic acid-binding immunoglobulin-type lectins receptors 5 and 14 (Siglec-5 and Siglec-14), but not on Siglec-9 therein used as control. The interaction was confirmed by biochemical tools with the determination of the K D value in the order of nanomolar and the identification of the NadA binding site by hydrogen-deuterium exchange coupled to mass spectrometry. The N-terminal domain of the Siglec-5 that recognizes the sialic acid was identified as the NadA binding domain. Intriguingly, exogenously added recombinant soluble Siglecs, including Siglec-9, were found to decorate N. meningitidis surface in a NadA-dependent manner. However, Siglec-5 and Siglec-14 transiently expressed in CHO-K1 cells endorsed NadA binding and increased N. meningitidis adhesion/invasion while Siglec-9 did not. Taken together, Siglec-5 and Siglec-14 satisfy all features of NadA receptors suggesting a possible role of NadA in the acute meningococcal infection. IMPORTANCE Bacteria have developed several strategies for cell colonization and immune evasion. Knowledge of the host and pathogen factors involved in these mechanisms is crucial to build efficacious countermoves. Neisserial adhesin A (NadA) is a meningococcal surface protein included in the anti-meningococcus B vaccine 4CMenB, which mediates adhesion to and invasion of epithelial cells. Although NadA has been shown to bind to other cell types, like myeloid and endothelial cells, it still remains orphan of a defined host receptor. We have identified two strong NadA interactors, Siglec-5 and Siglec-14, which are mainly expressed on myeloid cells. This showcases that NadA is an additional and key player among the Neisseria meningitidis factors targeting immune cells. We thus provide novel insights on the strategies exploited by N. meningitidis during the infection process, which can progress to a severe illness and death. </jats:sec