A Genome-Wide Screen for Bacterial Envelope Biogenesis Mutants Identifies a Novel Factor Involved in Cell Wall Precursor Metabolism

The cell envelope of Gram-negative bacteria is a formidable barrier that is difficult for antimicrobial drugs to penetrate. Thus, the list of treatments effective against these organisms is small and with the rise of new resistance mechanisms is shrinking rapidly. New therapies to treat Gram-negative bacterial infections are therefore sorely needed. This goal will be greatly aided by a detailed mechanistic understanding of envelope assembly. Although excellent progress in the identification of essential envelope biogenesis systems has been made in recent years, many aspects of the process remain to be elucidated. We therefore developed a simple, quantitative, and high-throughput assay for mutants with envelope biogenesis defects and used it to screen an ordered single-gene deletion library of Escherichia coli. The screen was robust and correctly identified numerous mutants known to be involved in envelope assembly. Importantly, the screen also implicated 102 genes of unknown function as encoding factors that likely impact envelope biogenesis. As a proof of principle, one of these factors, ElyC (YcbC), was characterized further and shown to play a critical role in the metabolism of the essential lipid carrier used for the biogenesis of cell wall and other bacterial surface polysaccharides. Further analysis of the function of ElyC and other hits identified in our screen is likely to uncover a wealth of new information about the biogenesis of the Gram-negative envelope and the vulnerabilities in the system suitable for drug targeting. Moreover, the screening assay described here should be readily adaptable to other organisms to study the biogenesis of different envelope architectures

Structure and dynamics of an archetypal DNA nanoarchitecture revealed via cryo-EM and molecular dynamics simulations

DNA can be folded into rationally designed, unique, and functional materials. To fully realise the potential of these DNA materials, a fundamental understanding of their structure and dynamics is necessary, both in simple solvents as well as more complex and diverse anisotropic environments. Here we analyse an archetypal six-duplex DNA nanoarchitecture with single-particle cryo-electron microscopy and molecular dynamics simulations in solvents of tunable ionic strength and within the anisotropic environment of biological membranes. Outside lipid bilayers, the six-duplex bundle lacks the designed symmetrical barrel-type architecture. Rather, duplexes are arranged in non-hexagonal fashion and are disorted to form a wider, less elongated structure. Insertion into lipid membranes, however, restores the anticipated barrel shape due to lateral duplex compression by the bilayer. The salt concentration has a drastic impact on the stability of the inserted barrel-shaped DNA nanopore given the tunable electrostatic repulsion between the negatively charged duplexes. By synergistically combining experiments and simulations, we increase fundamental understanding into the environment-dependent structural dynamics of a widely used nanoarchitecture. This insight will pave the way for future engineering and biosensing applications

NES and NNES Teachers: A Cross-cultural Comparison of Teaching Styles

The paper investigates the differences and similarities in teaching styles from a cross-cultural perspective. Although the dichotomy between native and non-native English speaking teachers has been the focus of numerous publications in the field, various elements of their teaching styles, in terms of both similarities and differences, have received little attention. For this purpose, authors surveyed teachers in the U.S. and the Czech Republic to analyze their general modes of classroom behavior, teaching methods, and self-image. Empirical evidence received through the survey does not fully support the idea that cultural factors influence certain aspects of classroom practices and teaching style

NES and NNES Teachers: A Cross-cultural Comparison of Teaching Styles

The paper investigates the differences and similarities in teaching styles from a cross-cultural perspective. Although the dichotomy between native and non-native English speaking teachers has been the focus of numerous publications in the field, various elements of their teaching styles, in terms of both similarities and differences, have received little attention. For this purpose, authors surveyed teachers in the U.S. and the Czech Republic to analyze their general modes of classroom behavior, teaching methods, and self-image. Empirical evidence received through the survey does not fully support the idea that cultural factors influence certain aspects of classroom practices and teaching style

NES and NNES Teachers: A Cross-cultural Comparison of Teaching Styles

The paper investigates the differences and similarities in teaching styles from a cross-cultural perspective. Although the dichotomy between native and non-native English speaking teachers has been the focus of numerous publications in the field, various elements of their teaching styles, in terms of both similarities and differences, have received little attention. For this purpose, authors surveyed teachers in the U.S. and the Czech Republic to analyze their general modes of classroom behavior, teaching methods, and self-image. Empirical evidence received through the survey does not fully support the idea that cultural factors influence certain aspects of classroom practices and teaching style

Structure and dynamics of an archetypal DNA nanoarchitecture revealed via cryo-EM and molecular dynamics simulations

Abstract DNA can be folded into rationally designed, unique, and functional materials. To fully realise the potential of these DNA materials, a fundamental understanding of their structure and dynamics is necessary, both in simple solvents as well as more complex and diverse anisotropic environments. Here we analyse an archetypal six-duplex DNA nanoarchitecture with single-particle cryo-electron microscopy and molecular dynamics simulations in solvents of tunable ionic strength and within the anisotropic environment of biological membranes. Outside lipid bilayers, the six-duplex bundle lacks the designed symmetrical barrel-type architecture. Rather, duplexes are arranged in non-hexagonal fashion and are disorted to form a wider, less elongated structure. Insertion into lipid membranes, however, restores the anticipated barrel shape due to lateral duplex compression by the bilayer. The salt concentration has a drastic impact on the stability of the inserted barrel-shaped DNA nanopore given the tunable electrostatic repulsion between the negatively charged duplexes. By synergistically combining experiments and simulations, we increase fundamental understanding into the environment-dependent structural dynamics of a widely used nanoarchitecture. This insight will pave the way for future engineering and biosensing applications

A screen for mutants defective in cell envelope assembly.

<p>A. Shown is a schematic illustrating the logic of the screen. Wild-type (WT) cells are unable to cleave CPRG because the enzyme (LacZ) is separated from its substrate (CPRG) by the intact cell envelope and thus remain white. Mutants that lyse or have defects in envelope permeability are able to cleave CPRG and turn red. B. Picture of CPRG agar with colonies from an <i>E. coli</i> transposon mutant library. Most colonies are “white”, with occasional red (single arrow) or pink (double arrow) colonies identifying mutants likely to have envelope defects. The particular “red” mutant shown also has a growth defect. The plate was incubated overnight at 30°C. Note that we were unable to carry out the screen at 37°C due to excessive background color development on the CPRG agar. C–D. Micrographs show cells from a colony of WT (MG1655) (C) or an Ely mutant (Ely7) (D) grown on LB agar prepared with 1% NaCl. The transposon in Ely7 was mapped to the <i>nhaA</i> gene coding for a sodium-proton antiporter. The phenotype shown was observed on LB with 1% NaCl, but not on LB lacking added salt (data not shown). Cells were imaged on 1.2% agarose pads with a Nikon 50i microscope with a 100× phase-contrast objective.</p

Suppression of the ElyC⁻ phenotypes.

<p>A. Diagram of the PG synthesis pathway. See text for details. M, MurNAc; G, GlcNAc. Colored circles represent amino acids in the PG stem peptide. B. Cells of EM1 [Δ<i>elyC</i>] and CB3 [Δ<i>mrcB</i>] containing multicopy plasmids with the indicated genes were patched onto CPRG indicator agar and grown as described for <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004056#pgen-1004056-g003" target="_blank">Figure 3B</a>. Plasmids were selected from an ordered ORF library set <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004056#pgen.1004056-Saka1" target="_blank">[47]</a>. C. A subset of the strains from B was grown on LB (1% NaCl) agar at room temperature and plates were photographed. D. The indicated subset of these strains was also grown in liquid and monitored as described for <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004056#pgen-1004056-g004" target="_blank">Figure 4A</a> except that the medium was supplemented with 100 µM IPTG. In B–D, the WT strain did not contain any plasmid. Antibiotics were not included in the plates or in the liquid medium to select for the plasmid.</p

Genetic interactions between <i>elyC</i> and enterobacterial common antigen biosynthesis genes revealed by high-throughput GIANT coli analysis.

<p>^a Growth score reflects the colony size of the double mutant clone in question divided by the average colony size of library clones. Scores greater than one indicate suppressive interactions and those less than one indicate negative interactions.</p

Using the CPRG assay in high-throughput.

<p>A. Picture of indicator agar (1% NaCl) with pin-transferred cells of the ordered library converted to Lac⁺ by conjugation. Plate was incubated for 23 hrs at room temperature. B. Output of the image analysis software (Iris) for the plate shown in (A). C. CPRG assay score distribution for the screen carried out at room temperature on agar prepared with 1% NaCl. Positions of genes of interest and/or known importance for envelope integrity are indicated by the red lines. Genes with scores above the cut-off (10^3.7 units) were designated as CPRG+ hits. D. Venn diagram comparing the hits identified in the different growth conditions.</p