Tailoring of keV-ion beams by image charge when transmitting through rhombic and rectangular shaped nanocapillaries.

We report on an unexpected effect of tailoring transmission profiles of Ne(7+) ions through nanocapillaries of rhombic and rectangular cross sections in mica. We find that capillaries of rhombic cross sections produce rectangular shaped ion transmission profiles and, vice versa, that capillaries of rectangular geometry give a rhombic beam shape. This shaping effect only occurs for transmitted ions and is absent for the small fraction of neutralized particles. The experimental findings and simulations of the projectile trajectories give clear evidence that the observed effect is due to the image forces experienced by the transmitting ions. This novel beam shaping mechanism suggests applications for the guiding, focusing, and shaping of ion beams.

Evaporative cooling and coherent axial oscillations of highly charged ions in a penning trap.

Externally, in an electron beam ion trap, generated Ar16+ ions were retrapped in a Penning trap and evaporatively cooled in their axial motion. The cooling was observed by a novel extraction technique based on the excitation of a coherent axial oscillation which yields short ion bunches of well-defined energies. The initial temperature of the ion cloud was decreased by a factor of more than 140 within 1 s, while the phase-space density of the coldest extracted ion pulses was increased by a factor of up to about 9.

Prevalence of Bacillus anthracis-like organisms and bacteriophages in the intestinal tract of the earthworm Eisenia fetida.

Stable infection of Bacillus anthracis laboratory strains with environmental bacteriophages confers survival phenotypes in soil and earthworm intestinal niches (R. Schuch and V. A. Fischetti, PLoS One 4:e6532, 2009). Here, the natural occurrence of two such B. anthracis-infective bacteriophages, Wip1 and Wip4, was examined in the intestines of Eisenia fetida earthworms as part of a 6-year longitudinal study at a Pennsylvania forest site. The Wip1 tectivirus was initially dominant before being supplanted by the Wip4 siphovirus, which was then dominant for the next 3 years. In a host range analysis of a wide-ranging group of Bacillus species and related organisms, Wip1 and Wip4 were both infective only toward B. anthracis and certain B. cereus strains. The natural host of Wip4 remained constant for 3 years and was a B. cereus strain that expressed a B. anthracis-like surface polysaccharide at septal positions on the cell surface. Next, a novel metagenomic approach was used to determine the extent to which such B. cereus- and B. anthracis-like strains are found in worms from two geographical locations. Three different enrichment strategies were used for metagenomic DNA isolation, based either on the ability of B. cereus sensu lato to form heat-resistant spores, the sensitivity of B. anthracis to the PlyG lysin, or the selective amplification of environmental phages cocultured with B. anthracis. Findings from this work indicate that B. cereus sensu lato and its phages are common inhabitants of earthworm intestines.

Two-center double-capture interference in fast He2+ + H2 collisions.

We report the first observation of Young-type interference effects in a two-electron transfer process. These effects change strongly as the projectile velocity changes in fast (1.2 and 2.0 MeV) He(2+) + H(2) collisions as manifested in strong variations of the double-electron capture rates with the H(2) orientation. This is consistent with fully quantum mechanical calculations, which ignore sequential electron transfer, and a simple projectile de Broglie wave picture assuming that two-electron transfer probabilities are higher in collisions where the projectile passes close to either one of the H(2) nuclei.

Evidence of sequentially formed charge patches guiding ions through nanocapillaries.

We investigated the time evolution of the dynamically shifting distribution of 7 keV Ne7+ ions guided through nanocapillaries in SiO2. We present evidence for a small number of charge patches, formed sequentially in the charging-up process, guiding the ions. We show that the charge patches are distributed along the whole length of the capillaries and that they are maintained in the equilibrium state of transmission. The interpretations are supported by model calculations.

Evidence of wave-particle duality for single fast hydrogen atoms.

We report the direct observation of interference effects in a Young's double-slit experiment where the interfering waves are two spatially separated components of the de Broglie wave of single 1.3 MeV hydrogen atoms formed close to either target nucleus in H++H2 electron-transfer collisions. Quantum interference strongly influences the results even though the hydrogen atoms have a de Broglie wavelength, lambda_{dB}, as small as 25 fm.

New mass value for 7Li.

A high-accuracy mass measurement of 7Li was performed with the SMILETRAP Penning-trap mass spectrometer via a cyclotron frequency comparison of 7Li3+ and H2+. A new atomic-mass value of 7Li has been determined to be 7.016 003 425 6(45) u with a relative uncertainty of 0.63 ppb. It has uncovered a discrepancy as large as 14sigma (1.1 microu) deviation relative to the literature value given in the Atomic-Mass Evaluation AME 2003. The importance of the improved and revised 7Li mass value, for calibration purposes in nuclear-charge radii and atomic-mass measurements of the neutron halos 9Li and 11Li, is discussed.

Dielectronic resonance method for measuring isotope shifts.

Long standing problems in the comparison of very accurate hyperfine-shift measurements to theory were partly overcome by precise measurements on few-electron highly charged ions. Still the agreement between theory and experiment is unsatisfactory. In this Letter, we present a radically new way of precisely measuring hyperfine shifts, and demonstrate its effectiveness in the case of the hyperfine shift of 4s1/2 and 4p1/2 in 207Pb53+. It is based on the precise detection of dielectronic resonances that occur in electron-ion recombination at very low energy. This allows us to determine the hyperfine constant to around 0.6 meV accuracy which is on the order of 10%.

Double-to-single target ionization ratio for electron capture in fast p-He collisions.

We have used the ion storage ring CRYRING and its internal gas-jet target and recoil-ion-momentum spectrometer to measure absolute cross sections for transfer ionization (TI: p+He-->H0+He2++e(-)) in 2.5-4.5 MeV p-He collisions with separate Thomas (TTI) and kinematic (KTI) TI contributions. The probability for electron emission in kinematical capture decreases with increasing velocity and appears to approach the photoionization shakeoff value (1.63%) [T. Aberg, Phys. Rev. A 2, 1726 (1970)]]. The velocity dependence of the TTI cross section is consistent with the theoretically predicted v(-11) scaling [J. S. Briggs and K. Taulbjerg, J. Phys. B 12, 2565 (1979)]].

Electromagnetically induced nuclear-charge pickup observed in ultrarelativistic Pb collisions.

A strong increase of inclusive nuclear-charge pickup cross sections, forming 83Bi from 158A GeV 82Pb ions, is observed in comparison to similar measurements at 10.6A GeV. From the dependence of these cross sections on target atomic number, this increase is attributed to the electromagnetic process of pion production by equivalent photons. The observed cross sections can be reproduced quantitatively using the recently developed RELDIS code.

MxiM and MxiJ, base elements of the Mxi-Spa type III secretion system of Shigella, interact with and stabilize the MxiD secretin in the cell envelope.

The type III secretion pathway is broadly distributed across many parasitic bacterial genera and serves as a mechanism for delivering effector proteins to eukaryotic cell surface and cytosolic targets. While the effectors, as well as the host responses elicited, differ among type III systems, they all utilize a conserved set of 9 to 11 proteins that together form a bacterial envelope-associated secretory organelle or needle complex. The general structure of the needle complex consists of a transenvelope base containing at least three ring-forming proteins (MxiD, MxiJ, and MxiG in Shigella) that is connected to a hollow needle-like extension that projects away from the cell surface. Several studies have shown that the initial steps in needle complex assembly require interactions among the base proteins, although specific details of this process remain unknown. Here we identify a role for another base element in Shigella, MxiM, in interactions with the major outer-membrane-associated ring-forming protein, MxiD. MxiM affects several features of MxiD, including its stability, envelope association, and assembly into homomultimeric structures. Interestingly, many of the effects were also elicited by the inner-membrane-associated base element, MxiJ. We confirmed that MxiM-MxiD and MxiJ-MxiD interactions occur in vivo in the cell envelope, and we present evidence that together these base elements can form a transmembrane structure which is likely an important intermediary in the process of needle complex assembly.

Cadaverine prevents the escape of Shigella flexneri from the phagolysosome: a connection between bacterial dissemination and neutrophil transepithelial signaling.

Shigella flexneri causes bacillary dysentery in humans by invading epithelial cells of the colon, which is characterized by an acute polymorphonuclear leukocyte (PMNL)-rich inflammation. Our recent studies demonstrated that cadaverine, a polyamine, specifically acts to abrogate transepithelial signaling to PMNL induced by S. flexneri. Here, insight is provided into the cellular mechanisms by which cadaverine attenuates the ability of Shigella species to induce PMNL signaling. It was found that cadaverine retards the lysis of the Shigella species-containing vacuole, suggesting that a blockade is established, in which the pathogen is prevented from adequately interacting with the cytoskeleton. Furthermore, an IcsA mutant of S. flexneri that cannot interact with the cytoskeleton and spreads intercellularly fails to induce transmigration of PMNL. Results indicate that cadaverine-induced compartmentalization of Shigella species to the phagolysosome might be a protective response of the host that directly contributes to the diminished ability of PMNL to transmigrate across model intestinal epithelia.

QED effects in Cu-like Pb recombination resonances near threshold.

In an electron-ion recombination study with Pb53+ dielectronic recombination resonances are found for as low as approximately 10(-3)-10(-4) eV relative energy. The resonances have been calculated by relativistic many-body perturbation theory and through comparison with experiment the Pb53+(4p(1/2)-4s(1/2)) energy splitting of approximately 118 eV is determined with an accuracy comparable to the position of the first few resonances, i.e., approximately 10(-3) eV. Such a precision provides a test of QED in a many-body environment at a level which can still not be reached in calculations.

Spa33, a cell surface-associated subunit of the Mxi-Spa type III secretory pathway of Shigella flexneri, regulates Ipa protein traffic.

The Mxi-Spa type III secretion system of Shigella flexneri directs the host cell contact-induced secretion of a set of invasins, referred to as Ipas. In this study, we examined the role of Spa33 in Ipa secretion. A spa33-null mutant was both noninvasive and unable to translocate the Ipas from inner membrane to outer membrane (OM) positions of the Mxi-Spa transmembrane channel. Spa33 was found to be a Mxi-Spa substrate that is translocated to the bacterial cell surface upon the induction of Ipa secretion. This mobility may serve to drive Ipa translocation within Mxi-Spa toward OM positions. Consistent with a second distinct role in regulating Ipa traffic, the overexpression of Spa33 also blocked Ipa secretion and resulted in Ipa accumulation at the OM. Co-overexpression of Spa33 and another OM-associated element, Spa32, did not disrupt Ipa secretion, suggesting an interaction between the two proteins and an effect on the mechanism which serves to regulate Ipa release from the OM. These findings indicate that Spa33 is a mobile element within Mxi-Spa, which is required to control Ipa translocation into and out of OM positions of the secretory structure.

Unexpected x-ray emission due to formation of bound doubly excited states.

A strong emission of characteristic M x rays is observed, without an M vacancy initially present, when slow highly charged ions (Ta(q+), q = 39--48) capture a single electron in single collisions with rare gas atoms (He). This is explained by the formation of bound doubly excited states through electron correlation. An elaborate theoretical treatment shows that bound doubly excited states are mixed with states where a Rydberg electron is bound in the core of a highly charged ion. It is striking that this occurs with a large probability (close to unity), and one needs to assume that higher Rydberg states are populated than predicted by the overbarrier model in order to explain the experimental results.

Reaction mechanism of recombinant 3-oxoacyl-(acyl-carrier-protein) synthase III from Cuphea wrightii embryo, a fatty acid synthase type II condensing enzyme.

A unique feature of fatty acid synthase (FAS) type II of higher plants and bacteria is 3-oxoacyl-[acyl-carrier-protein (ACP)] synthase III (KAS III), which catalyses the committing condensing reaction. Working with KAS IIIs from Cuphea seeds we obtained kinetic evidence that KAS III catalysis follows a Ping-Pong mechanism and that these enzymes have substrate-binding sites for acetyl-CoA and malonyl-ACP. It was the aim of the present study to identify these binding sites and to elucidate the catalytic mechanism of recombinant Cuphea wrightii KAS III, which we expressed in Escherichia coli. We engineered mutants, which allowed us to dissect the condensing reaction into three stages, i.e. formation of acyl-enzyme, decarboxylation of malonyl-ACP, and final Claisen condensation. Incubation of recombinant enzyme with [1-(14)C]acetyl-CoA-labelled Cys(111), and the replacement of this residue by Ala and Ser resulted in loss of overall condensing activity. The Cys(111)Ser mutant, however, still was able to bind acetyl-CoA and to catalyse subsequent binding and decarboxylation of malonyl-ACP to acetyl-ACP. We replaced His(261) with Ala and Arg and found that the former lost activity, whereas the latter retained overall condensing activity, which indicated a general-base action of His(261). Double mutants Cys(111)Ser/His(261)Ala and Cys(111)Ser/His(261)Arg were not able to catalyse overall condensation, but the double mutant containing Arg induced decarboxylation of [2-(14)C]malonyl-ACP, a reaction indicating the role of His(261) in general-acid catalysis. Finally, alanine scanning revealed the involvement of Arg(150) and Arg(306) in KAS III catalysis. The results offer for the first time a detailed mechanism for a condensing reaction catalysed by a FAS type II condensing enzyme.

A system for identifying post-invasion functions of invasion genes: requirements for the Mxi-Spa type III secretion pathway of Shigella flexneri in intercellular dissemination.

Invasion and intercellular spread are hallmarks of Shigella pathogenicity. Invasion of the eukaryotic cell cytosol requires a type III secretion system (Mxi-Spa) and its cognate set of secreted Ipa invasins. Once intracellular, the IcsA protein directs a form of actin-based motility that helps to drive intracellular bacterial movement, formation of cellular protrusions and cell-to-cell spread. Work in our laboratory has focused on identifying additional factors required for this intercellular form of dissemination. In this study, we sought to identify novel contributions of the type III secretion pathway to post-invasion-specific processes, distinct from its previously characterized roles in invasion. Studies of post-invasion Ipa and Mxi-Spa functions are complicated by an absolute requirement for these virulence proteins in invasion. To circumvent this problem, we developed a system called TIER (for test of intracellular expression requirements), whereby specific ipa, mxi or spa loci are transiently expressed before infection of tissue culture cell monolayers (thus supporting invasion), but then repressed after invasion in the intracellular environment. Such invasive type III secretion mutants (called TIER mutants) were severely restricted in their ability to spread intercellularly and form plaques in confluent tissue culture cell monolayers. Intercellular spread defects were associated with the repression of most type III pathway components examined, including structural (MxiM and Spa33), secreted effector (IpaB, IpaC and IpaD) and regulatory elements (VirF and VirB). A kinetic analysis of bacterial growth in L2 cell monolayers showed that each of the TIER mutants was defective with respect to long-term intracellular proliferation and viability. Examination of TIER mutant-infected monolayers by electron microscopy revealed that the type III pathway was required for a late step in intercellular spread - bacterial escape from protrusion-derived, double-membrane-bound vacuoles. The TIER mutants were eventually degraded in a process involving vacuolar acidification. Based on these findings, we propose that Ipa secretion via Mxi-Spa is required in the protrusion vacuole for double-membrane lysis.

Nucleosides as a carbon source in Bacillus subtilis: characterization of the drm-pupG operon.

In Bacillus subtilis, nucleosides are readily taken up from the growth medium and metabolized. The key enzymes in nucleoside catabolism are nucleoside phosphorylases, phosphopentomutase, and deoxyriboaldolase. The characterization of two closely linked loci, drm and pupG, which encode phosphopentomutase (Drm) and guanosine (inosine) phosphorylase (PupG), respectively, is reported here. When expressed in Escherichia coli mutant backgrounds, drm and pupG confer phosphopentomutase and purine-nucleoside phosphorylase activity. Northern blot and enzyme analyses showed that drm and pupG form a dicistronic operon. Both enzymes are induced when nucleosides are present in the growth medium. Using mutants deficient in nucleoside catabolism, it was demonstrated that the low-molecular-mass effectors of this induction most likely were deoxyribose 5-phosphate and ribose 5-phosphate. Both Drm and PupG activity levels were higher when succinate rather than glucose served as the carbon source, indicating that the expression of the operon is subject to catabolite repression. Primer extension analysis identified two transcription initiation signals upstream of drm; both were utilized in induced and non-induced cells. The nucleoside-catabolizing system in B. subtilis serves to utilize the base for nucleotide synthesis while the pentose moiety serves as the carbon source. When added alone, inosine barely supports growth of B. subtilis. This slow nucleoside catabolism contrasts with that of E. coli, which grows rapidly on a nucleoside as a carbon source. When inosine was added with succinate or deoxyribose, however, a significant increase in growth was observed in B. subtilis. The findings of this study therefore indicate that the B. subtilis system for nucleoside catabolism differs greatly from the well-studied system in E. coli.

The mxi-Spa type III secretory pathway of Shigella flexneri requires an outer membrane lipoprotein, MxiM, for invasin translocation.

Invasion of epithelial cells by Shigella flexneri is mediated by a set of translocated bacterial invasins, the Ipa proteins, and its dedicated type III secretion system, called Mxi-Spa. We show here that mxiM, part of the mxi-spa locus in the S. flexneri virulence plasmid, encodes an indispensable type III secretion apparatus component, required for both Ipa translocation and tissue culture cell invasion. We demonstrated that mature MxiM, first identified as a putative lipoprotein, is lipidated in vivo. Consistent with features of known lipoproteins, MxiM (i) can be labeled with [3H]palmitate and [2-3H]glycerol, (ii) is associated with the cell envelope, (iii) is secreted independently of the type III pathway, and (iv) requires an intact lipoprotein modification and processing site for full activity. The lipidated form of MxiM was detected primarily in the outer membrane, where it establishes a peripheral association with the inner leaflet. Through analysis of subcellular Ipa distribution in a mxiM null mutant background, MxiM was found to be required for the assembly and/or function of outer, but not inner, membrane regions of Mxi-Spa. This function probably requires interactions with other Mxi-Spa subunits within the periplasmic space. We discuss implications of these findings with respect to the function of MxiM and the structure of Mxi-Spa as a whole.

The role of acyl carrier protein isoforms from Cuphea lanceolata seeds in the de-novo biosynthesis of medium-chain fatty acids.

To investigate the role of acyl carrier protein (ACP) in determining the fate of the acyl moieties linked to it in the course of de-novo fatty acid biosynthesis in higher plants, we carried out in vitro experiments to reconstitute the fatty acid synthase (FAS) reaction in extracts of spinach (Spinacia oleracea L.) leaves, rape (Brassica napus L.) seeds and Cuphea lanceolata Ait. seeds. The action of two major C. lanceolata ACP isoforms (ACP 1 and ACP 2) compared to ACP from Escherichia coli was monitored by saponification of the corresponding FAS products with subsequent analysis of the liberated fatty acids by high-performance liquid chromatography. In a second approach the preference of the medium-chain acyl-ACP-specific thioesterase (EC 3.1.2.14) of C. lanceolata seeds for the hydrolysis of acyl-ACPs prepared from the three ACP types was investigated. Both ACP isoforms from C. lanceolata seeds supported the synthesis of medium-chain fatty acids in a reconstituted FAS reaction of spinach leaf extracts. Compared to the isoform ACP 1, ACP 2 was more effective in supporting the synthesis of such fatty acids in the FAS reaction of rape seed extracts and caused a higher accumulation of FAS products in all experiments. No preference of the medium-chain thioesterase for one specific ACP isoform was observed. The results indicate that the presence of ACP 2 is essential for the synthesis of decanoic acid in C. lanceolata seeds, and its expression in the phase of accumulation of high levels of this fatty acid provides an additional and highly efficient cofactor for stimulating the FAS reaction.