Fluorescent D-amino-acids reveal bi-cellular cell wall modifications important for Bdellovibrio bacteriovorous predation

Modification of essential bacterial peptidoglycan (PG) containing cell walls can lead to antibiotic resistance, for example β-lactam resistance by L,D-transpeptidase activities. Predatory Bdellovibrio bacteriovorus are naturally antibacterial and combat infections by traversing, modifying and finally destroying walls of Gram-negative prey bacteria, modifying their own PG as they grow inside prey. Historically, these multi-enzymatic processes on two similar PG walls have proved challenging to elucidate. Here, with a PG labelling approach utilizing timed pulses of multiple fluorescent D-amino acids (FDAAs), we illuminate dynamic changes that predator and prey walls go through during the different phases of bacteria:bacteria invasion. We show formation of a reinforced circular port-hole in the prey wall; L,D-transpeptidaseBd mediated D-amino acid modifications strengthening prey PG during Bdellovibrio invasion and a zonal mode of predator-elongation. This process is followed by unconventional, multi-point and synchronous septation of the intracellular Bdellovibrio, accommodating odd- and even-numbered progeny formation by non-binary division

Feasibility study of Nb3Al Rutherford cable for high field accelerator magnet application

Feasibility study of Cu stabilized Nb{sub 3}Al strand and Rutherford cable for the application to high field accelerator magnets are being done at Fermilab in collaboration with NIMS. The Nb{sub 3}Al strand, which was developed and manufactured at NIMS in Japan, has a non-copper Jc of about 844 A/mm{sup 2} at 15 Tesla at 4.2 K, a copper content of 50%, and filament size of about 50 microns. Rutherford cables with 27 Nb{sub 3}Al strands of 1.03 mm diameter were fabricated and tested. Quench tests on a short cable were done to study its stability with only its self field, utilizing a high current transformer. A pair of 2 meter long Nb{sub 3}Al cables was tested extensively at CERN at 4.3 and 1.9 K up to 11 Tesla including its self field with a high transport current of 20.2 kA. In the low field test we observed instability near splices and in the central region. This is related to the flux-jump like behavior, because of excessive amount of Nb in the Nb{sub 3}Al strand. There is possibility that the Nb in Nb{sub 3}Al can cause instability below 2 Tesla field regions. We need further investigation on this problem. Above 8 Tesla, we observed quenches near the critical surface at fast ramp rate from 1000 to 3000 A/sec, with quench velocity over 100 m/sec. A small racetrack magnet was made using a 14 m of Rutherford cable and successfully tested up to 21.8 kA, corresponding to 8.7 T

Detection of Ligation Products of DNA Linkers with 5′-OH Ends by Denaturing PAGE Silver Stain

To explore if DNA linkers with 5′-hydroxyl (OH) ends could be joined by commercial T4 and E. coli DNA ligase, these linkers were synthesized by using the solid-phase phosphoramidite method and joined by using commercial T4 and E. coli DNA ligases. The ligation products were detected by using denaturing PAGE silver stain and PCR method. About 0.5–1% of linkers A–B and E–F, and 0.13–0.5% of linkers C–D could be joined by T4 DNA ligases. About 0.25–0.77% of linkers A–B and E–F, and 0.06–0.39% of linkers C–D could be joined by E. coli DNA ligases. A 1-base deletion (-G) and a 5-base deletion (-GGAGC) could be found at the ligation junctions of the linkers. But about 80% of the ligation products purified with a PCR product purification kit did not contain these base deletions, meaning that some linkers had been correctly joined by T4 and E. coli DNA ligases. In addition, about 0.025–0.1% of oligo 11 could be phosphorylated by commercial T4 DNA ligase. The phosphorylation products could be increased when the phosphorylation reaction was extended from 1 hr to 2 hrs. We speculated that perhaps the linkers with 5′-OH ends could be joined by T4 or E. coli DNA ligase in 2 different manners: (i) about 0.025–0.1% of linkers could be phosphorylated by commercial T4 DNA ligase, and then these phosphorylated linkers could be joined to the 3′-OH ends of other linkers; and (ii) the linkers could delete one or more nucleotide(s) at their 5′-ends and thereby generated some 5′-phosphate ends, and then these 5′-phosphate ends could be joined to the 3′-OH ends of other linkers at a low efficiency. Our findings may probably indicate that some DNA nicks with 5′-OH ends can be joined by commercial T4 or E. coli DNA ligase even in the absence of PNK

Quench tests and FEM analysis of Nb3Al Rutherford cables and small racetrack magnets

In collaboration between NIMS and Fermilab, we have made copper stabilized Nb{sub 3}Al Rutherford cables, using Nb-matrixed and Ta-matrixed strands. First these cables were investigated at high current in low self field using a flux pump. Using these Rutherford cables, we built and tested small racetrack magnets. The magnet made with the Nb-matrixed strand showed the flux jump instability in low field. The small racetrack magnet wound with the Ta-matrixed Nb{sub 3}Al Rutherford cable was very stable at 4.5 K operation without any instability, as well as at 2.2 K operation. With the successful operation of the small racetrack magnet up to its short sample data, the feasibility of the Nb{sub 3}Al strand and its Rutherford cable for their application to high field magnets is established. The characteristics of Nb{sub 3}Al Rutherford cable is compared with that of the Nb{sub 3}Sn Rutherford cable and the advantages of Nb{sub 3}Al Rutherford cable are discussed

Genome-Wide Identification of Ampicillin Resistance Determinants in Enterococcus faecium

Enterococcus faecium has become a nosocomial pathogen of major importance, causing infections that are difficult to treat owing to its multi-drug resistance. In particular, resistance to the β-lactam antibiotic ampicillin has become ubiquitous among clinical isolates. Mutations in the low-affinity penicillin binding protein PBP5 have previously been shown to be important for ampicillin resistance in E. faecium, but the existence of additional resistance determinants has been suggested. Here, we constructed a high-density transposon mutant library in E. faecium and developed a transposon mutant tracking approach termed Microarray-based Transposon Mapping (M-TraM), leading to the identification of a compendium of E. faecium genes that contribute to ampicillin resistance. These genes are part of the core genome of E. faecium, indicating a high potential for E. faecium to evolve towards β-lactam resistance. To validate the M-TraM results, we adapted a Cre-lox recombination system to construct targeted, markerless mutants in E. faecium. We confirmed the role of four genes in ampicillin resistance by the generation of targeted mutants and further characterized these mutants regarding their resistance to lysozyme. The results revealed that ddcP, a gene predicted to encode a low-molecular-weight penicillin binding protein with D-alanyl-D-alanine carboxypeptidase activity, was essential for high-level ampicillin resistance. Furthermore, deletion of ddcP sensitized E. faecium to lysozyme and abolished membrane-associated D,D-carboxypeptidase activity. This study has led to the development of a broadly applicable platform for functional genomic-based studies in E. faecium, and it provides a new perspective on the genetic basis of ampicillin resistance in this organism

Understanding the Origins of Bacterial Resistance to Aminoglycosides through Molecular Dynamics Mutational Study of the Ribosomal A-Site

Paromomycin is an aminoglycosidic antibiotic that targets the RNA of the bacterial small ribosomal subunit. It binds in the A-site, which is one of the three tRNA binding sites, and affects translational fidelity by stabilizing two adenines (A1492 and A1493) in the flipped-out state. Experiments have shown that various mutations in the A-site result in bacterial resistance to aminoglycosides. In this study, we performed multiple molecular dynamics simulations of the mutated A-site RNA fragment in explicit solvent to analyze changes in the physicochemical features of the A-site that were introduced by substitutions of specific bases. The simulations were conducted for free RNA and in complex with paromomycin. We found that the specific mutations affect the shape and dynamics of the binding cleft as well as significantly alter its electrostatic properties. The most pronounced changes were observed in the U1406C∶U1495A mutant, where important hydrogen bonds between the RNA and paromomycin were disrupted. The present study aims to clarify the underlying physicochemical mechanisms of bacterial resistance to aminoglycosides due to target mutations

Distinct Salmonella Enteritidis lineages associated with enterocolitis in high-income settings and invasive disease in low-income settings.

An epidemiological paradox surrounds Salmonella enterica serovar Enteritidis. In high-income settings, it has been responsible for an epidemic of poultry-associated, self-limiting enterocolitis, whereas in sub-Saharan Africa it is a major cause of invasive nontyphoidal Salmonella disease, associated with high case fatality. By whole-genome sequence analysis of 675 isolates of S. Enteritidis from 45 countries, we show the existence of a global epidemic clade and two new clades of S. Enteritidis that are geographically restricted to distinct regions of Africa. The African isolates display genomic degradation, a novel prophage repertoire, and an expanded multidrug resistance plasmid. S. Enteritidis is a further example of a Salmonella serotype that displays niche plasticity, with distinct clades that enable it to become a prominent cause of gastroenteritis in association with the industrial production of eggs and of multidrug-resistant, bloodstream-invasive infection in Africa.This work was supported by the Wellcome Trust. We would like to thank the members of the Pathogen Informatics Team and the core sequencing teams at the Wellcome Trust Sanger Institute (Cambridge, UK). We are grateful to D. Harris for work in managing the sequence data

Pair Correlations in a Bidisperse Ferrofluid in an External Magnetic Field:Theory and Computer Simulations

The pair distribution function g(r) for a ferrofluid modeled by a bidisperse system of dipolar hard spheres is calculated. The influence of an external uniform magnetic field and polydispersity on g(r) and the related structure factor is studied. The calculation is performed by diagrammatic expansion methods within the thermodynamic perturbation theory in terms of the particle number density and the interparticle dipole–dipole interaction strength. Analytical expressions are provided for the pair distribution function to within the first order in number density and the second order in dipole–dipole interaction strength. The constructed theory is compared with the results of computer (Monte Carlo) simulations to determine the range of its validity. The scattering structure factor is determined using the Fourier transform of the pair correlation func-tion g(r) – 1. The influence of the granulometric composition and magnetic field strength on the height and position of the first peak of the structure factor that is most amenable to an experimental study is analyzed. The data obtained can serve as a basis for interpreting the experimental small[1]angle neutron scattering results and determining the regularities in the behavior of the structure factor, its dependence on the fractional com-position of a ferrofluid, interparticle correlations, and external magnetic field. © Pleiades Publishing, Inc., 2014